Understanding how the nervous system orchestrates physiology across the body has long been a central question in neuroscience. While neural mechanisms underlying behavior have been extensively characterized, the cellular and circuit principles that mediate brain-body communication remain underexplored. In this thesis, I investigate how internal physiological signals are detected and translated into coordinated regulation of organ functions through specialized sensory and autonomic pathways.
\r\n\r\nUsing molecular, behavioral, and genetic perturbation approaches, I first examine how changes in body fluid balance are detected by central sensory neurons. I identify distinct neuronal populations within forebrain circumventricular regions that detect hyperosmotic and hypovolemic challenges and drive modality-specific fluid consumption behaviors. Then I show how water signals in the gut are encoded by a dedicated vagal afferent population, providing feed-forward inputs that contribute to thirst satiation. These studies demonstrate that internal states are monitored through specialized channels spanning central and peripheral circuits.
\r\n \r\nNext, I investigate the circuit logic of sympathetic regulation in the abdomen, identifying molecularly defined neuronal populations that project selectively to visceral organs and differentially regulate gastrointestinal transit and digestive processes. These results demonstrate that sympathetic outputs are organized into discrete pathways that enable precise and independent control of physiology. I then synthesize current knowledge of autonomic organization, highlighting its molecular diversity and modular architecture as key features enabling selective regulation of organ function.
\r\n \r\nTogether, these findings reveal that brain-body communication is mediated by structured sensory pathways and modular autonomic circuits to achieve precise yet flexible control of physiology. This work provides a framework for understanding how neural systems coordinate internal stability and offers insight into how disruptions of these processes may contribute to diseases.
", "doi": "10.7907/a8mn-0e75", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18707", "collection": "thesis", "collection_id": "18707", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302026-011912053", "primary_object_url": { "basename": "20260603-Cameron_Jackson_PhD_Dissertation_Final.pdf", "content": "final", "filesize": 1906763, "license": "other", "mime_type": "application/pdf", "url": "/18707/1/20260603-Cameron_Jackson_PhD_Dissertation_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Accessing the Developing CNS: Advancing AAV-Mediated Prenatal Gene Editing in the Brain", "author": [ { "family_name": "Jackson", "given_name": "Cameron Richard", "orcid": "0009-0008-7212-616X", "clpid": "Jackson-Cameron-Richard" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Targeted gene delivery to defined neural populations is a central challenge in neuroscience and a major barrier to gene therapy for neurodevelopmental and congenital disorders. In the developing central nervous system, this is further complicated by dynamic changes in accessibility, vector tropism, and cellular composition across developmental stages. This thesis develops new approaches for systemic prenatal CNS access, capsid engineering, and in utero genome editing in the embryonic brain.
\r\n\r\nWe introduce UseqFISH, a spatial transcriptomic platform that enables high-sensitivity, multiplexed detection of endogenous transcripts and barcoded viral genomes in intact tissue at single-cell resolution. This allows quantitative in situ mapping of AAV tropism and scalable evaluation of viral libraries in embryonic contexts. Using new and existing techniques, we characterize systemic AAV biodistribution across development and show that adult brain-biased serotypes do not maintain specificity in late-mid gestation embryos. We engineer modified capsids, including \u201cnull\u201d scaffolds with reduced native tropism, which can be reprogrammed via targeting motifs. High-throughput barcoded in vivo selection identifies sequence features associated with improved CNS enrichment during embryogenesis.
\r\n\r\nTo expand access to midgestational delivery, we develop a surgical method enabling systemic AAV administration at embryonic day 12.5 via uterine microdissection of the vitelline circulation, achieving widespread and viable transduction through gestational development. Finally, we established an in utero genome editing platform using AAV-mediated CRISPR and homology-directed repair. We demonstrate efficient gene disruption, epitope tagging, and precise genome modification, including endogenous tagging and introduction of disease-associated alleles across cortical cell types, enabling modeling of neurodevelopmental phenotypes.
\r\n\r\nTogether, this work provides a framework for engineering AAV-based gene delivery and genome editing in the developing CNS, advancing tools for studying brain development, modeling disease, and enabling early therapeutic strategies.
", "doi": "10.7907/881q-x239", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17694", "collection": "thesis", "collection_id": "17694", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09212025-192830865", "primary_object_url": { "basename": "20250920_PhD_thesis_draft_v21_submitted-version.pdf", "content": "final", "filesize": 33782320, "license": "other", "mime_type": "application/pdf", "url": "/17694/1/20250920_PhD_thesis_draft_v21_submitted-version.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Acoustically Targeted Gene Delivery for Non-Invasive Neuroengineering", "author": [ { "family_name": "Li", "given_name": "Hongyi Richard", "orcid": "0000-0001-6970-0230", "clpid": "Li-Hongyi-Richard" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Andersen", "given_name": "Richard A.", "orcid": "0000-0002-7947-0472", "clpid": "Andersen-R-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Tsao", "given_name": "Doris Y.", "orcid": "0000-0003-1083-1919", "clpid": "Tsao-D-Y" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Noninvasive, spatially targeted gene delivery to the brain holds tremendous promise for addressing some of the most pressing neurological and psychiatric conditions of our time, including Parkinson\u2019s disease, treatment-resistant epilepsy, obsessive-compulsive disorder, and addictions. While adeno-associated viruses (AAVs) are the leading vectors for gene therapy in the state of the art, their clinical translation is hindered by the need for invasive injections to achieve site-specific delivery in the brain. Over the past two decades, focused ultrasound blood-brain barrier opening (FUS-BBBO) has emerged as a compelling alternative \u2014 enabling targeted entry of biomolecules, nanoparticles, and even small viral vectors like AAVs from the bloodstream into the brain without surgical intervention. Yet, natural AAV serotypes have shown only modest success with this method, often displaying low transduction efficiency and undesirable off-target expression in peripheral organs.
\r\n\r\nTo overcome these limitations, we have developed a new framework for acoustically targeted gene delivery \u2014 a noninvasive, spatially and cell-type-specific approach for delivering genetic material to the brain. In this thesis, I will describe how we harnessed high-throughput in vivo directed evolution to engineer AAV variants optimized for neuronal transduction specifically at the site of ultrasound targeting. In rodent models, these newly evolved vectors demonstrate significantly improved performance \u2014 achieving efficient, localized gene delivery to neurons while minimizing peripheral expression. Building on these successes, we advanced the platform toward clinical relevance by extending our evolutionary screening to non-human primates (NHPs). This allowed us to identify AAV variants with enhanced translational potential and establish a strong foundation for future studies in human clinical trials.
\r\n\r\nIn the final part of this thesis, I will showcase how these engineered AAVs can be further empowered by combining them with acoustic reporter genes \u2014 specifically, gas vesicle (GV) proteins \u2014 enabling non-invasive imaging of molecular activity deep within the brain. Using this powerful platform, we have also developed a novel therapeutic strategy for treating opioid addiction, in which biomolecular ultrasound coalesces with chemogenetic neuromodulation. Taken together, I hope to convince you that the technique of ultrasound-based acoustically targeted gene delivery, paired with engineered delivery vectors, unlocks a new frontier in non-invasive neurotherapeutics and brings us one step closer to precise, personalized neuroengineering in interfacing the human brain.
", "doi": "10.7907/qbrx-4132", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:16751", "collection": "thesis", "collection_id": "16751", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09222024-230441454", "primary_object_url": { "basename": "HeatherLukas_PhDThesis.pdf", "content": "final", "filesize": 48618608, "license": "other", "mime_type": "application/pdf", "url": "/16751/1/HeatherLukas_PhDThesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Engineering Bioaffinity Sensors toward Continuous Electrochemical Biosensing", "author": [ { "family_name": "Lukas", "given_name": "Heather Lauren", "orcid": "0000-0002-8160-9066", "clpid": "Lukas-Heather-Lauren" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The rise of wearable sensing through smartwatches and continuous glucose monitors has made health data more widely accessible. Advances in machine learning have also been pivotal in identifying personalized health insights from biometric data streams. However, continuous biochemical data has been limited in sensor design by the availability of oxidoreductases (e.g., glucose oxidase, lactate dehydrogenase) to a given target. The challenge in engineering diverse oxidoreductase enzymes has led to the exploration of other generalized approaches to continuous electrochemical biosensing. To meet this need, we have explored a variety of bioaffinity sensing schemes using broad bioreceptor classes including antibodies, nucleic acids, and periplasmic binding proteins. We present a case study in electrochemical sensor design utilizing high-affinity antibodies for the rapid diagnosis of COVID-19 disease states. We then investigate the potential of nucleic acid-based electrochemical sensors for continuous sensing with a focus on structure-switching nucleic acid aptamers. The utility of aptamer sensors is demonstrated in the development of a serotonin aptamer sensor embedded in an ingestible capsule for continuous biosensing in the gastrointestinal tract. Applying the principles of electrochemical aptamer-based sensing, we explored the development of an electrochemical protein-based sensor for nicotine, which exploits the hinge-like binding motion of periplasmic binding proteins while also capitalizing on decades of protein evolution and characterization research. With the goal of continuous, noninvasive biochemical sensing, we evaluate the design considerations and translatability of these sensors for wearable sweat analysis. These biosensing techniques may enable the future hardware necessary to expand accessible biomedical data for the next wave of personalized health monitoring.", "doi": "10.7907/2c89-k924", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17070", "collection": "thesis", "collection_id": "17070", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03182025-023751137", "type": "thesis", "title": "RNA-Mediated Toxicity In Neurodegeneration: The Mechanistic Role Of The C9ORF72 Repeat Expansion In ALS Molecular Pathogenesis", "author": [ { "family_name": "Bhattacharya", "given_name": "Paulomi", "clpid": "Bhattacharya-Paulomi" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ichida", "given_name": "Justin K.", "orcid": "0000-0002-8827-8087", "clpid": "Ichida-Justin-K" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The G4C2 hexanucleotide repeat expansion in the first intron of the C9ORF72 gene is the most common genetic mutation linked to ALS, accounting for ~40 percent of familial and 10 percent of sporadic cases. Yet, its functional contribution to molecular pathogenesis remains unknown. The prevailing model is that this expansion leads to transcription of a novel RNA (C9-repeat RNA) that leads to disease either through its RNA product or translation of dipeptide repeat proteins it encodes (\u201cgain-of-function\u201d). However, recent attempts to degrade the C9-repeat RNA in several major clinical trials have failed to show any improvement in C9-ALS patients, raising questions about what role, if any, the C9-repeat RNA plays in ALS pathogenesis. Here, we demonstrate that the C9-repeat RNA is not detectable in C9-ALS patient-derived iPSNs or postmortem brain tissue. We show that transcription of the C9ORF72 gene initiates downstream of the G4C2 repeat sequence with the repeat expansion residing at a promoter-proximal region and displaying chromatin signatures of an enhancer. Because this region is GC-rich and has been reported to be preferentially methylated in C9-ALS patients, we explored whether this repeat expansion might lead to reduced C9ORF72 gene expression. We show that the C9-repeat is associated with reduced allele-specific expression of the C9ORF72 gene, consistent with the GC-rich features of the repeat expansion and previous reports of preferential DNA methylation in C9-ALS patients. Taken together, our findings challenge the prevailing gain-of-function models in C9-ALS and instead suggest that the repeat expansion region may function as a regulatory element that silences C9ORF72 expression from the mutant allele.", "doi": "10.7907/2ywx-7a47", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16454", "collection": "thesis", "collection_id": "16454", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312024-185303498", "type": "thesis", "title": "Customized and Modular Control of Gene Expression for Precision Gene Therapies", "author": [ { "family_name": "Mayfield", "given_name": "Acacia Michelle Hori", "orcid": "0000-0001-7308-6480", "clpid": "Mayfield-Acacia-Michelle-Hori" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Genetic disorders are caused by mutations in essential genes that disturb the abundance or function of proteins, tipping cells and tissues from homeostatic harmony into disorder. Developing treatment for genetic diseases involves precision approaches, as gene therapies target the root causes of highly specific pathologic processes at the level of gene replacement, editing, or downstream compensation for a harmful genetic change. Safe access to these cell populations, and the ability to control the behavior of therapeutic cargo after delivery to target tissues, will enable the field to develop safe and effective therapies with the potential to be curative. Systemically delivered AAVs can noninvasively target therapeutic genetic cargo to diverse disease loci throughout the body, but at high doses required for therapeutic penetrance of naturally occurring serotypes, these vectors can cause severe toxicity, emphasizing the need for both targeted, efficient gene delivery vectors, and other means of transgene expression control. This work describes three examples of AAV capsid and cargo design strategies that seek to control where, when, and at what level therapeutic transgene expression can be achieved in a preclinical context. First, we utilize native putative regulatory elements to encourage physiologic level of ectopic frataxin expression in a mouse model of Friedreich\u2019s Ataxia, finding that when delivered to both the brain and peripheral nervous system, treatment prevents progression of motor and coordination deficits. Next, we utilize the genetic incoherent feedforward loop circuit motif at the RNA level to decouple vector delivery level from transgene expression level of MeCP2 in a mouse model of Rett Syndrome, finding that when regulated to near endogenous healthy levels of RNA, AAV-MeCP2-IFFL enables behavioral rescue without overexpression toxicity. Lastly, we employ the mechanism for AAV-genome stability in vivo to modulate expression using a post-hoc AAV administration. Together, these methods and applications demonstrate that modular and custom approaches can improve the precision, safety and efficacy problems that the gene therapy field needs in order to advance more treatments for rare disorders.", "doi": "10.7907/d05v-0t68", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16494", "collection": "thesis", "collection_id": "16494", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042024-004047037", "type": "thesis", "title": "Neuropsychiatric Drug Biosensors in Organelles, Cells, Biofluids, & Behaving Animals", "author": [ { "family_name": "Muthusamy", "given_name": "Anand Kumar", "orcid": "0000-0003-1041-914X", "clpid": "Muthusamy-Anand-Kumar" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biology is distinguished in its several levels of spatial organization\u2014from molecular to whole body\u2014that give rise to coherent, goal-related behaviors. Cutting across these layers are definable circuits, each with its own dynamics. These systems should be studied in their natural context to preserve their structure and function. However, quantitative measurements typically demand invasive apparatuses or infrequent, ex vivo measurements. The advent of genetically encoded fluorescent biosensors solves this problem by detecting molecules in situ, read by a microscope or implantable optical probe. These biosensors typically are fusions of a conformational switch and a fluorescent protein. A naturally occurring protein that binds the target of interest typically provides an initial scaffold. However, several molecules, particularly various human-made drugs, do not have similar naturally occurring cognate conformational switches in nature robust enough for this approach.
\r\n\r\nThis work develops and applies the first genetically encoded drug biosensors in cellular and behavioral assays addressing substance abuse disorders. We term these biosensors intensity-based drug-sensing fluorescent reporters or \u201ciDrugSnFRs.\u201d These biosensors are based on a choline-binding periplasmic binding protein (PBP), OpuBC, interrupting a circularly permuted green fluorescent protein (GFP). This work reports a method of optimizing this construct toward the detection of several classes of neural drugs, including nicotinic, SSRIs, ketamine family drugs, and opioids.
\r\n\r\nThe opportunity for continuous monitoring is particularly prominent in brain-body-behavior relationships. For example, a core tenet of behavioral neuropharmacology is the existence of some stereotyped relationship between the time course of a drug and behavioral outcomes such as opioid use disorder. Interindividual variability in pharmacokinetics (PK) complicates the problem of optimized opioid dosing, especially outside the clinic. The problem of personalizing pharmacokinetics is severe in substance use disorders: the patient must receive opioid levels that relieve pain, minimize tolerance and other side effects, and remain within a therapeutic window to maximize adherence. That ideal window is a \u201cmoving target\u201d due to tolerance, changes in metabolism, and stressors.
\r\n\r\nAs an end-to-end study with a preclinical model, the final chapter reports the development of iOpioidSnFRs and their application to the continuous monitoring of fentanyl alongside a computer vision routine to quantify behavior. The fentanyl sensor, iFentanylSnFR2.0, was expressed in the ventral tegmental area of mice and reported [fentanyl] vs. time. This recording is the longest continuous measurement of the brain [drug] alongside behavior (4 hours). We found a stereotypic, repetitive motor pattern that tracked the entire fentanyl time course (2-3 hours) despite variable PK across individuals. This result challenges current models of cellular desensitization and acute tolerance timescales. In a separate experiment, we investigated if this stereotypical pattern impaired mice in a survival task where mice forage for water through a labyrinth maze. Like in the open arena, mice in the maze exhibited circling/stalling for approximately 3 h, to the complete exclusion of successful foraging. Critically, this paradigm offers a normative definition of a deficit, as mice should have a baseline level of successful foraging to survive. We introduce this task to the substance use disorder field as an additional metric for the deficits caused by opioid administration.
\r\n\r\nFinally, this work demonstrates the utility of iOpioidSnFRs in diagnostic tests owing to their suitable aqueous solubility, dynamic range, sensitivity, selectivity, kinetics, and stability after lyophilization. Plate reader assays using iFentanylSnFR2.0, iS-methadoneSnFR, iTapentadolSnFR, and iLevorphanolSnFR provided quantitation across the pharmacologically relevant concentration ranges. These biosensors were also used in a simulated field test using readily available parts: dark box, blue LED strips, band pass filter, and a cellphone camera. This test could be used to determine the presence of a health hazard in the environment (e.g., fentanyl) or determine the exposure level in a person. These results encourage diagnostic and continuous monitoring approaches to personalizing opioid regimens.
", "doi": "10.7907/1043-8k76", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16255", "collection": "thesis", "collection_id": "16255", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052023-185529151", "primary_object_url": { "basename": "Thesis - Jihong Min.pdf", "content": "final", "filesize": 9341850, "license": "other", "mime_type": "application/pdf", "url": "/16255/11/Thesis - Jihong Min.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Innovations in Wireless Bioelectronics for Precision Medicine, from Sustainable Sweat Sensing to Ingestible Gut Monitoring", "author": [ { "family_name": "Min", "given_name": "Jihong", "orcid": "0000-0002-5788-1473", "clpid": "Min-Jihong" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Emami", "given_name": "Azita", "orcid": "0000-0002-6945-9958", "clpid": "Emami-A" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Biofluids, constituting about 60% of the human body, serve as treasure troves of biomarkers such as metabolites and electrolytes, shedding light on individual health conditions. Although blood and urine tests have been routinely utilized, they are limited by their invasive and episodic nature. However, the promise of continuous and noninvasive access to other biofluids like sweat, GI fluids, and saliva paves the way for real-time, onsite health monitoring. This thesis delves into the untapped potential of wearable sensors and noninvasive biofluid analysis, emphasizing the importance of continuous and sustainable monitoring for predictive personal healthcare. Chapter 1 introduces the paradigm of biofluid sensing, focusing on sweat as a key candidate for personalized healthcare applications. Chapter 2 delves into the physiology of sweat glands, highlighting the composition of sweat and the mechanisms behind sweat extraction, either through natural exercise or iontophoretic stimulation. Chapter 3 embarks on the development of innovative sensors designed for detecting clinically pertinent biomarkers in sweat, a step forward in predictive health analytics. In Chapter 4, the spotlight is on system integration, as the study emphasizes the need for miniaturized and reliable wireless sensor devices that ensure minimal discomfort and maximum reliability. Chapters 5 and 6 delve into strategies for sustainably powering wearable devices from energy harvested from body motions and from ambient light, respectively. The final chapter, Chapter 7, extrapolates the aforementioned technologies for the realm of ingestible devices, adapting them for electrochemical sensing in alternate media, primarily gastrointestinal fluids. This allows for enhanced detection of gastrointestinal diseases and a deeper understanding of the intricate gut-brain axis. The ultimate vision of this research is to equip individuals with wearable and ingestible sensors that can seamlessly monitor a broad spectrum of clinically relevant biomarkers. This continuous monitoring, coupled with data analytics, will potentially catalyze a shift from reactive to predictive healthcare, ushering in an era of personalized therapeutic interventions. As wearable sweat and ingestible sensors become mainstream, a confluence of biosensing mechanisms, materials science, and flexible electronics is anticipated enable continuous and unobtrusive acquisition of clinically relevant biomarkers over prolonged periods and large populations, further refining the nexus between health monitoring and precision medicine.", "doi": "10.7907/kcm7-wz71", "publication_date": "2024-06-14", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16285", "collection": "thesis", "collection_id": "16285", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02042024-025331192", "primary_object_url": { "basename": "Griffiths_Thesis_2_3_2024.pdf", "content": "final", "filesize": 28536604, "license": "other", "mime_type": "application/pdf", "url": "/16285/2/Griffiths_Thesis_2_3_2024.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Bidirectional Interactions Between the Gut Microbiome and Nervous System", "author": [ { "family_name": "Griffiths", "given_name": "Jessica Anne", "orcid": "0000-0002-5586-1567", "clpid": "Griffiths-Jessica-Anne" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "There is roughly one microbe for every human cell in your body. Though some are inconsequential hitchhikers, and some are potentially harmful, many perform beneficial roles. This thesis focuses on the function and interaction of resident microbes within laboratory mice, with the hope that it may translate to us as humans. Chapter (1) highlights recent findings of microbiome involvement in neurologic disorders. Each subsequent chapter presents a different interaction between the mammalian nervous system and gut microbiome. (2) Excitatory signaling in the brain is partially regulated by a genetic factor (Shank3), which is further modulated by environmental interactions through presence or absence of the gut microbiome. This genetic factor implicated in brain and behavior also affects gastrointestinal function and inflammation susceptibility. (3) Applying powerful genetic tools developed for the brain to the enteric nervous system reveals the impact of different enteric neuron populations on gut motility and fluid secretion as well as the immune system, pancreatic activity, and microbial populations. (4) Common opinion has shifted from the belief that microbes are primarily pathogens to viewing them as symbiotic organisms. With this paradigm shift, the artificially clean laboratory mouse microbiome has been found to stunt the immune system, and is being reevaluated. Male mice with natural \u201cwild\u201d microbiomes have altered behavioral and neurological profiles, which may reflect a more physiological state.", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16384", "collection": "thesis", "collection_id": "16384", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152024-052419062", "type": "thesis", "title": "Acoustic Biosensors for Noninvasive Imaging of Molecular Processes", "author": [ { "family_name": "Jin", "given_name": "Zhiyang", "orcid": "0000-0002-4411-6991", "clpid": "Jin-Zhiyang" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Understanding biology in its native context has been a major scientific endeavor. Yet, it is challenging to visualize cellular dynamics at the molecular scale in the context of a living organism at the macroscopic scale. Ultrasound imaging represents a promising candidate to address this challenge, with its unique advantages of large imaging volume, deep penetration, and good spatiotemporal resolution. However, ultrasound was historically limited in retrieving molecular information that biology carries. Until very recently, the discovery of the first ultrasound-interacting biomolecules, gas vesicles (GVs), established a connection between connect cellular function and ultrasound signals, which later enabled ultrasound imaging of gene expression and thus the location of GV-expressing cells. Going beyond location tracking, this thesis describes the engineering of GV-based acoustic biosensors that made it possible to noninvasively image the dynamics of cellular signaling in living organisms.
\r\n \r\nGVs are genetically encoded intracellular air-filled \u201cballoons\u201d that are encapsulated by protein shells. The acoustic biosensor design leverages the GV surface protein GvpC, which controls GVs' ultrasound scattering by setting the stiffness of their protein shell. We developed the first acoustic biosensors by engineering GvpC to change its confirmation and thereby GVs\u2019 ultrasound contrast in response to the activity or concentration of specific molecules. Specifically, we first built the biosensors for three different types of enzymes and demonstrated noninvasive imaging of enzyme activity inside probiotic cells in the mouse colon in vivo. Next, we engineered the acoustic biosensors for calcium, a ubiquitous signaling molecule that is essential in many cellular processes (e.g., neural activity). With the first generation of this calcium sensor for ultrasound, we demonstrated imaging of receptor-specific calcium signaling deep inside the mouse brain through the intact skull noninvasively, which opened up the possibility of whole-brain neuroimaging that can lead to many breakthroughs in neuroscience. Last, we established a high-throughput engineering platform to develop all these GV-based imaging agents in a much shorter time frame. Collectively, this thesis presents the first demonstration of noninvasively imaging dynamic cellular signaling with acoustic biosensors and the feasibility of efficiently improving them for potential real-world applications with our engineering pipeline, opening up a new route towards understanding biology across scales.
", "doi": "10.7907/b51h-q307", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:15228", "collection": "thesis", "collection_id": "15228", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292023-213541270", "type": "thesis", "title": "Genetically Encoded Biosensors for Ketamine and Other Rapidly Acting Antidepressants in Zebrafish and Cell Culture", "author": [ { "family_name": "Blumenfeld", "given_name": "Zachary", "orcid": "0000-0002-4627-5582", "clpid": "Blumenfeld-Zachary" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" } ], "thesis_committee": [ { "family_name": "Adolphs", "given_name": "Ralph", "orcid": "0000-0002-8053-9692", "clpid": "Adolphs-R" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" }, { "family_name": "Hong", "given_name": "Elizabeth J.", "orcid": "0000-0003-3866-418X", "clpid": "Hong-Elizabeth-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Over the past century, the development and use of treatments for depression has been one of the most important projects in both neuroscience and medicine. Not only is relatively little known about the underlying pathophysiology of major depressive disorder (MDD), a mechanistic understanding of the ways in which common antidepressants \u2014 such as selective serotonin reuptake inhibitors (SSRIs) \u2014 contribute to symptomatic relief remains elusive. Furthermore, the delay until typical antidepressant treatments take effect (a 'therapeutic lag' of weeks to months) presents a series of challenges to researchers in chemistry, neuroscience, pharmacology, and medicine, as the connection between apparent physiological changes and clinical benefit has yet to be established. The recent advent of a new class of drugs \u2014 rapidly acting antidepressants (RAADs), including the multi-purpose compound ketamine \u2014 which ameliorate symptoms within hours to days provides a crucial (if perplexing) perspective on the treatment of MDD and neuropsychiatric disorders more broadly. To answer questions concerning how various kinds of antidepressants might exert their effects, where those interactions take place, and what sorts of physiological changes drive clinical response, we have designed genetically encoded drug-specific intensity-based sensing fluorescent reporters (iDrugSnFRs) which are engineered to detect drugs of interest in both in vitro and in vivo applications. We have successfully evolved iDrugSnFRs for an array of RAADs (iRAADSnFRs) which detect pharmacologically relevant concentrations of their target drugs sensitively and specifically in both cell culture as well as in the nervous tissue of larval zebrafish. Another set of iDrugSnFRs for SSRIs has provided novel insights into the potential reasons for the aforementioned 'therapeutic lag' as well as side effects, while yet another set has provided a pharmacokinetic basis for the evaluation of smoking cessation drugs. In all, our findings lead us to posit that iDrugSnFRs can aid in the elucidation of mechanisms by which a wide variety of orally active pharmaceutical compounds operate as well as provide a crucial basis for the development of better medicines.
", "doi": "10.7907/4wtg-qb69", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16075", "collection": "thesis", "collection_id": "16075", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032023-030758184", "type": "thesis", "title": "Neurotechnology for Multiplexed Interrogation of Brain Circuits and Synaptic Activity", "author": [ { "family_name": "Hsu", "given_name": "Alice", "orcid": "0000-0001-6609-2559", "clpid": "Hsu-Alice" } ], "thesis_advisor": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "thesis_committee": [ { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Tian", "given_name": "Lin", "orcid": "0000-0001-7012-6926", "clpid": "Tian Lin" }, { "family_name": "Shepard", "given_name": "Kenneth L.", "orcid": "0000-0003-0665-6775", "clpid": "Shepard-Kenneth-L" }, { "family_name": "Moreaux", "given_name": "Laurent C.", "orcid": "0000-0003-1276-5062", "clpid": "Moreaux-Laurent-C" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis describes the development of neural technologies for 1) multiplexed brain circuit electrophysiology (ephys) recordings and control of activity in optogenetic mice lines with concurrent recording paired with two-photon imaging and 2) multiplexed measurements of synaptic release events in microfluidic platforms. The first part of this thesis describes efforts to provide deterministic correlation of excited neuron action potential with resulting ephys recordings in vivo. This consisted of technological development of novel, high density multisite silicon probes for electrophysiology recordings in vivo. The probes consist of four columns of electrodes densely packed at the shank tip. This density of electrode arrays allowed for higher resolution isolation of more distinct waveforms than previous ephys probes and benchmarking measurements to triangulate the locations of emitting neurons. These measurements help benchmark the ability of existing silicon extracellular probes to capture surrounding extracellular activity. When combined with two-photon imaging, we can simultaneously record ephys activity, image the probe and surrounding brain, quantify brain damage during probe implantation, and control neural activity using optogenetic mouse lines.
\r\n\r\nThe second project described development of a microfluidic platform to monitor synaptic release of the neurotransmitter glutamate. Microfluidic devices were used to isolate synaptic processes expressing synaptic reporters and provide targeted recording of glutamate activity across the synapse. Synaptic glutamate release was monitored with a two part genetically encoded fluorescent reporter that detects glutamate released at the synapse, called split-iGluSnFR, developed in Professor Lin Tian\u2019s lab at UC Davis. We designed new microfluidic devices to better isolate neuron processes with split-iGluSnFR and be compatible with existing fluorescent complementary metal\u2013oxide\u2013semiconductor (CMOS) contact imagers. Using computational fluid dynamic simulations, we demonstrate efficient perfusion in the device. The form factor of this new device is designed to be compatible with CMOS contact imagers, and that when combined will help us achieve our ultimate goal to monitor the kinetics of simultaneous synaptic release events modulated by perfused neuromodulating drugs.
", "doi": "10.7907/tba9-sb03", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15194", "collection": "thesis", "collection_id": "15194", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05192023-001330664", "type": "thesis", "title": "Engineering of Second-Generation Acoustic Reporter Genes", "author": [ { "family_name": "Hurt", "given_name": "Robert Cooper", "orcid": "0000-0002-4347-6901", "clpid": "Hurt-Robert-Cooper" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 \u00b5m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.
\r\n\r\nThe goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the \u201cnature and nurture\u201d of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.
\r\n\r\nThis thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them\u2014one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.
\r\n\r\nThe approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.
", "doi": "10.7907/qs6v-5d67", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14996", "collection": "thesis", "collection_id": "14996", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08072022-205043055", "primary_object_url": { "basename": "Shan_Huang_Thesis_2023_final.pdf", "content": "final", "filesize": 1264391, "license": "other", "mime_type": "application/pdf", "url": "/14996/1/Shan_Huang_Thesis_2023_final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Guided Genome Manipulation in Escherichia coli", "author": [ { "family_name": "Huang", "given_name": "Shan", "orcid": "0000-0002-4436-3327", "clpid": "Huang-Shan" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Argonaute proteins (Agos) were initially discovered in eukaryotes as key players in RNA interference (RNAi) pathways and later found in prokaryotes. Some prokaryotic argonautes (pAgos) have been shown to mediate nucleic acid-guided cleavage of DNA targets, reminiscent of the nucleic acid-guided DNase activity of the CRISPR/Cas9 system. It has been postulated that pAgo variants might be used as a novel genome-editing tool. However, genome manipulation induced by pAgo-mediated DNA cleavage has never been established. To demonstrate that pAgo-mediated DNA cleavage can introduce genomic mutations in Escherichia coli, we first created a recombination system and showed that CbAgo, a pAgo from Clostridium butyricum, can be directed by plasmid-encoded guide sequences to cleave the genome target site and induce chromosome recombination between downstream direct repeat sequences. Results from testing different pAgo variants suggest that the recombination rate correlates well with pAgo DNA cleavage activity, and the mechanistic study suggests the recombination involves DSB generation and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. Furthermore, we created a dCbAgo-based deaminase and showed that it can not only act as a random mutagen in vivo but also has the potential to be directed by plasmid-encoded guide sequences. We anticipate the novel DNA-guided interference by pAgo only or by its fusion protein to be useful in broader genetic manipulation. My work of engineering fluorescent protein-based nicotine biosensors via computational design and experimental evolution is also described in the thesis.
", "doi": "10.7907/3yab-jg50", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15039", "collection": "thesis", "collection_id": "15039", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10112022-213905012", "primary_object_url": { "basename": "Caltech_Thesis_Yu_Li_Ni_20221019.pdf", "content": "final", "filesize": 6497485, "license": "other", "mime_type": "application/pdf", "url": "/15039/1/Caltech_Thesis_Yu_Li_Ni_20221019.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Developing Tools for Neurobiology: The Retina as a\u00a0Neuropharmacology Testbed & Electrode Pooling\u00a0to Boost Extracellular Array Recording", "author": [ { "family_name": "Ni", "given_name": "Yu-Li", "orcid": "0000-0003-1600-9854", "clpid": "Ni-Yu-Li" } ], "thesis_advisor": [ { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis presents two tool development projects for neurobiology and one explorative project to find organizing principles for autism.
\r\n\r\nThe first project (Chap 2, Retina as Probe) was conceived to tackle the problem that there hasn\u2019t been a reliable model system for system-level neuropharmacology. We introduce a testbed for this: the mammalian retina. The retina involves many of the known neurotransmitters and modulators. Yet its synaptic wiring is well understood, and quantitative models exist to explain its input-output functions. One can connect the systems-level effects to the underlying cellular and molecular causes. To demonstrate the retina\u2019s use, we explored the effects of a range of general anesthetics on the light responses of the mouse retina. At sub-anesthetic doses, we found that certain anesthetics exert a paradoxical effect: they increase the light response of some retinal neurons and suppress the response of others. Notably, this occurred for alcohols and ketamine but not for isoflurane. We traced these effects to transmitter release at a specific synapse and, in one case, to a specific presynaptic ion channel. All the anesthetics silenced the output of the retina completely at concentrations similar to their effective dose for anesthesia in humans. Sedatives reduced retinal sensitivity but did not silence it. Finally, we used specific drugs that target hypothesized molecular mechanisms to probe how much they each contribute to anesthesia of the retina.
\r\n\r\nThe second project which attempted to probe the principles of autism (Chap 3) was conceptually a direct extension of the retina as a testbed. Similar to the situation in seeking for what the mechanism of general anesthesia is, the field of autism research also lacks a good testbed but for systemically comparing gene mutation - circuit defect - behavior outcomes. Similarly, we utilized the retina as a platform to identify circuit defects in four different autism model mice and followed through the different mouse line\u2019s behavior readouts using our lab\u2019s maze navigation paradigm. We discovered that the different autism mouse lines varied in the retinal circuits and varied in their navigation preferences. Nevertheless, unlike the anesthetic project, there wasn\u2019t a simple mechanism to explain why or how these differences are coupled together.
\r\n\r\nThe last project, Electrode Pooling, (Chap 4) aimed to boost the yield of extracellular recording electrode arrays with a novel method we named electrode pooling. The per-implant yield of extracellular recording leaped significantly from the order of tens to the order of hundreds when engineers built multiple electrode arrays based on silicon technology to replace tetrode wires. Unfortunately, this yield-per-site is already maxed out with modern silicon technology. The constraint of the yield is mainly biological, as explained in the chapter, and thus could not be further advanced by improving the manufacturing processes of semiconductors. Our solution utilized an approach that multiplexed the array recording sites (not the bottleneck) onto the readout wires with accompanying filters (the actual bottleneck). Specifically, the method proposes intelligently choosing many recording sites that carry signals and connecting them to a single wire via manipulating the switches and later un-mixed with a spike-sorting algorithm. We demonstrated the first proof-of-principle study that shows that one could get more single-neuron recordings per implant site with electrode pooling, and made recommendations on the hardware design that could facilitate the advancement of probes that use pooling algorithms.
", "doi": "10.7907/jeh6-w316", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14302", "collection": "thesis", "collection_id": "14302", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07142021-175425546", "primary_object_url": { "basename": "Thesis Stephen Grant Final_v2.pdf", "content": "final", "filesize": 6628490, "license": "other", "mime_type": "application/pdf", "url": "/14302/1/Thesis Stephen Grant Final_v2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigation of Some Small Molecule-Protein and Protein-Protein Interactions in Nicotine Addiction, Opioid Use Disorder, and COVID-19", "author": [ { "family_name": "Grant", "given_name": "Stephen Nicholas", "orcid": "0000-0003-0923-8886", "clpid": "Grant-Stephen-Nicholas" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_chem" } ], "abstract": "Nicotine addiction, opioid use disorder, and COVID-19 have made lasting impacts on every aspect of society. These are complicated conditions, and studies in these fields will likely continue for decades, if not centuries. Here, we make contributions to each of these issues using electrophysiology and microscopy. The first chapter goes into the motivation behind this thesis and the major experiments I used in my graduate career. In the second chapter, we introduce a new amino acid into the mouse muscle nicotinic acetylcholine receptor in an attempt to understand the dynamics of receptor activation. In the third chapter, we continue the Lester lab\u2019s work on the neuroscientific effects of menthol and how it plays a role in nicotine addiction. We found the binding site for menthol on the \u03b14\u03b22 nicotinic acetylcholine receptor, which continues our hypothesis that the neuroscientific effects of menthol are detrimental to cigarette smokers. Fortunately, partly because of our studies, mentholated nicotine products are being phased out of the United States. The fourth and fifth chapters investigate \u03bc-opioid receptor trafficking, both the trafficking from the endoplasmic reticulum and endocytosis from the plasma membrane. Both of these events play a role in inducing opioid use disorder and increasing the danger of using opioids. We hope that these studies will help other researchers understand opioid use disorder and fight the opioid epidemic. Finally, we studied the effects of SARS-COV-2 proteins on epithelial sodium channels. These channels are important for regulating lung fluid levels where their improper function may cause pulmonary edema. Pulmonary edema has been observed in COVID-19 patients. Altogether, we believe that we have made meaningful impacts on these important health concerns in this thesis. We look forward to how the scientific communities continue to build on our results.
", "doi": "10.7907/pdtj-8238", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:13747", "collection": "thesis", "collection_id": "13747", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312020-153151158", "type": "thesis", "title": "Pezo-1 Function in Caenorhabditis elegans", "author": [ { "family_name": "Brugman", "given_name": "Katherine Irene", "orcid": "0000-0003-2625-2903", "clpid": "Brugman-Katherine-Irene" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The piezo class of mechanosensative ion channels is a recently discovered class of cation channels with orthologs found in every phylogenetic clade aside from yeast and bacteria. THey are large channels, both in gene length and in overall diameter, with the diameter of the human PIEZO2 protein measuring in at 280 \u00c5 in its full homotrimeric form. In addition, like many similar mechanosensitive channels, such as the DEG/ENaC channels, TRP channels, and TREK/TRAAK channels, they have been linked to a number of different functions within drosophila, zebrafish, and mice, including light touch, nociception, blood cell volume regulation, vascular development, and neuropathic pain. Structurally, this channel is intriguing as it possesses no previously categorized structural motifs and is organized into a central pore with a cap, surrounded by three \"propeller blade\" regions that are theorized to anchor the channel to the membrane and control gating through hydrophobic mismatch based on membrane curvature.
\r\n\r\nThe C. elegans piezo, pezo-1, has not yet been fully characterized, even though a crystal structure of part of this particular piezo was used to assist in the resolution of the first set of cryo-EM images. Here, I generated a number of GFP transcriptional fusions of non-coding potential promoter regions to track the expression of the pezo-1 gene in C. elegans, using these expression patterns to design further experiments. From these expression patterns, I identified expression in a number of neurons of the C. elegans male tail, the primary mating apparatus of the male, and identified these neurons as key neurons as relating to mating. In particular, I identified neurons HOB, PCB, PCC and various ray neurons as potential candidates, which are ciliated neurons theorized to have mechanosensitive properties. In addition, I also identified expression in the vulva muscle and spermatheca of the hermaphrodite, both theorized to be involved in ovulation and egg-laying processes.
\r\n\r\nFrom there, I designed CRISPR/Cas9 mutants with defects in pezo-1 in order to investigate the potential link between the pezo-1 expression in those neurons and mating behavior via a mating assay. Similarly, I devised a fecundity assay to investigate the link between pezo-1 expression in ovulation organs and progeny survival. I have discovered that pezo-1 has function in both of these areas, with pezo-1 mutant males demonstrating discrete mating defects that correlate with the expression pattern seen from the GFP transcriptional fusion mutants and with pezo-1 mutant hermaphrodites having much smaller brood sizes than wildtype hermaphrodites. However, I have also discovered that the processes this mechanotransducer is involved in are also more complex than I originally believed, as I discovered that pezo-1 appears to interact with another mechanotransducer, trp-4, illuminating some potentially novel pathway considerations for how these channels overlap in function.
", "doi": "10.7907/fz4z-c850", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13727", "collection": "thesis", "collection_id": "13727", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282020-000951837", "primary_object_url": { "basename": "Thesis SL.pdf", "content": "final", "filesize": 6504898, "license": "other", "mime_type": "application/pdf", "url": "/13727/1/Thesis SL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Neural Basis of Sodium Appetite", "author": [ { "family_name": "Lee", "given_name": "Sangjun", "orcid": "0000-0002-0846-8252", "clpid": "Lee-Sangjun" } ], "thesis_advisor": [ { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Fluid homeostasis, which maintains a stable internal environment, is critical for survival. Body fluid is tightly monitored and regulated through its main components, water and salt. Here, I focus on the aspect of sodium regulation when sodium is the main cation in the extracellular fluid and is also required for primary metabolism. The depletion of sodium induces the retention of sodium but also a central mechanism to obtain sodium from the external sources. This need for sodium specifically drives animals towards sodium consumption, called sodium appetite. Even though sodium appetite is specific for only sodium ion, sodium appetite observed as an innate behavior across the animal kingdom.
\r\n\r\nSodium appetite is strictly regulated by both peripheral sensory signals and central appetite signals. Due to the development of genetic tools, I was able to investigate the neural basis of sodium appetite from searching sodium appetite dedicated neurons. Here, I identify two genetically defined neural circuits in mice that control sodium intake. The activation of these neurons drives robust sodium intake in sated animals. Particularly, prodynorphin expressing neurons in the pre-locus coeruleus shown specific consumption to sodium compounds, including rock salt. In terms of loss-of-function, inhibition of these neurons selectively reduced sodium consumption. It was further shown that these neurons receive sodium depleted signals by aldosterone-sensitive neurons.
\r\n\r\nPreviously, it was suggested that taste signals have a central role in sodium satiation. I demonstrate that the oral detection of sodium rapidly suppresses sodium appetite neurons. The blockage of the sodium taste or gastric infusion of sodium abolished the sodium suppression in the sodium appetite neurons. Consistently, gastric infusion of sodium did not cause sodium satiation. Moreover, retrograde-viral methods showed that specific inhibitory neurons partially mediate sensory modulation in the bed nucleus of the stria terminalis.
\r\n\r\nTogether, I identified a specific neural population as a functional unit for sodium appetite. By knowing the dedicated circuits for sodium appetite, I demonstrated chemosensory and physiological signals regulate the neural circuits. The genetically defined neural population can be handle as an entry point of further investigation of the neural basis of sodium appetite.
", "doi": "10.7907/bcxa-s404", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13612", "collection": "thesis", "collection_id": "13612", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062020-004548466", "type": "thesis", "title": "Effects of Sensory Experience on Early Stages of Olfactory Processing in the Fruit Fly", "author": [ { "family_name": "Gugel", "given_name": "Zhannetta V.", "orcid": "0000-0003-1082-3281", "clpid": "Gugel-Zhannetta-V" } ], "thesis_advisor": [ { "family_name": "Hong", "given_name": "Elizabeth J.", "clpid": "Hong-Elizabeth-J" } ], "thesis_committee": [ { "family_name": "Dickinson", "given_name": "Michael H.", "orcid": "0000-0002-8587-9936", "clpid": "Dickinson-M-H" }, { "family_name": "Hong", "given_name": "Elizabeth J.", "clpid": "Hong-Elizabeth-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Plasticity is widely studied across different sensory systems and behavioral paradigms, but the underlying mechanisms are varied and incompletely understood. Previous work in the fruit fly Drosophila melanogaster reported changes in odor preference and walking behavior after chronic odor exposure during early adulthood. Here, we investigated the hypothesis that changes in behavior reflect changes in how odors are encoded in the first two layers of the fly olfactory circuit. We chronically exposed flies to naturalistic odor stimuli that selectively and robustly activate a single olfactory receptor neuron (ORN) class. We then performed targeted intracellular recordings from genetically identified second-order olfactory projection neurons (PNs) that either receive direct input from the activated ORN class, or receive indirect activity (via local lateral circuitry), during chronic odor exposure. In addition, we used existing reagents to create a novel optical method to characterize ORN-PN synaptic strength. We find that the fly antennal lobe is resistant to plasticity, with a few exceptions. Of the odors we tested, we find that rearing in trans-2-hexenal, a leaf aldehyde that selectively activates ab4a ORNs, weakly enhanced odor responses in some PNs. The effects of rearing on PNs were not explained by ORN odor responses or changes in ORN-PN synaptic strength. We find evidence that lateral excitation may increase across glomeruli following rearing, suggesting that some odors may alter PN responses globally. We discuss possible reasons for differences between our observations and prior work on olfactory plasticity in this circuit, which has been conducted primarily in the context of exposures to much higher, non-naturalistic concentrations of odor. Our results point to the stability of insect sensory circuits in the face of large perturbations in the sensory environment.
\r\n\r\n\u2028\u2028During our optical stimulation experiments, we find that driving Chrimson expression may abolish odor responses in some ORNs. We include sample data highlighting this observation in a population of pb1a olfactory neurons. Lastly, we include antennal local field potential recordings in response to a variety of odor concentrations to help guide future experiments seeking isointense odor panels.
", "doi": "10.7907/e39b-w024", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13612", "collection": "thesis", "collection_id": "13612", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062020-004548466", "type": "thesis", "title": "Effects of Sensory Experience on Early Stages of Olfactory Processing in the Fruit Fly", "author": [ { "family_name": "Gugel", "given_name": "Zhannetta V.", "orcid": "0000-0003-1082-3281", "clpid": "Gugel-Zhannetta-V" } ], "thesis_advisor": [ { "family_name": "Hong", "given_name": "Elizabeth J.", "clpid": "Hong-Elizabeth-J" } ], "thesis_committee": [ { "family_name": "Dickinson", "given_name": "Michael H.", "orcid": "0000-0002-8587-9936", "clpid": "Dickinson-M-H" }, { "family_name": "Hong", "given_name": "Elizabeth J.", "clpid": "Hong-Elizabeth-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Meister", "given_name": "Markus", "orcid": "0000-0003-2136-6506", "clpid": "Meister-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Plasticity is widely studied across different sensory systems and behavioral paradigms, but the underlying mechanisms are varied and incompletely understood. Previous work in the fruit fly Drosophila melanogaster reported changes in odor preference and walking behavior after chronic odor exposure during early adulthood. Here, we investigated the hypothesis that changes in behavior reflect changes in how odors are encoded in the first two layers of the fly olfactory circuit. We chronically exposed flies to naturalistic odor stimuli that selectively and robustly activate a single olfactory receptor neuron (ORN) class. We then performed targeted intracellular recordings from genetically identified second-order olfactory projection neurons (PNs) that either receive direct input from the activated ORN class, or receive indirect activity (via local lateral circuitry), during chronic odor exposure. In addition, we used existing reagents to create a novel optical method to characterize ORN-PN synaptic strength. We find that the fly antennal lobe is resistant to plasticity, with a few exceptions. Of the odors we tested, we find that rearing in trans-2-hexenal, a leaf aldehyde that selectively activates ab4a ORNs, weakly enhanced odor responses in some PNs. The effects of rearing on PNs were not explained by ORN odor responses or changes in ORN-PN synaptic strength. We find evidence that lateral excitation may increase across glomeruli following rearing, suggesting that some odors may alter PN responses globally. We discuss possible reasons for differences between our observations and prior work on olfactory plasticity in this circuit, which has been conducted primarily in the context of exposures to much higher, non-naturalistic concentrations of odor. Our results point to the stability of insect sensory circuits in the face of large perturbations in the sensory environment.
\r\n\r\n\u2028\u2028During our optical stimulation experiments, we find that driving Chrimson expression may abolish odor responses in some ORNs. We include sample data highlighting this observation in a population of pb1a olfactory neurons. Lastly, we include antennal local field potential recordings in response to a variety of odor concentrations to help guide future experiments seeking isointense odor panels.
", "doi": "10.7907/e39b-w024", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13686", "collection": "thesis", "collection_id": "13686", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282020-145500248", "primary_object_url": { "basename": "Thesis - Caltech - BBE - Bryan B. Yoo- FINAL - submit to library - compressed.pdf", "content": "final", "filesize": 9465428, "license": "other", "mime_type": "application/pdf", "url": "/13686/1/Thesis - Caltech - BBE - Bryan B. Yoo- FINAL - submit to library - compressed.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Host-Microbe Interactions Impacting and Mediated by Nervous Systems", "author": [ { "family_name": "Yoo", "given_name": "Bryan B.", "orcid": "0000-0003-1450-2696", "clpid": "Yoo-Bryan-B" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Animals and microbes coevolved, and thus it is not surprising that the trillions of microorganisms that harmoniously inhabit the mammalian gastrointestinal tract (GIT), collectively termed the gut microbiome, continue to be implicated in healthy and disease states. However, less is known about the mechanisms by which these states are maintained, and how deviations from homeostasis (i.e., dysbiosis) occurr. This thesis explores the relationship between host-microbe interactions and the central and peripheral nervous systems. Specifically, the first chapter of this thesis explores how the microbiome differs is patients with multiple sclerosis and how these differences alter diseases outcomes in a mouse model of the disease. Next, we introduce the enteric nervous system (ENS), the intrinsic nervous system of the GI tract which is supposed as a major conduit of the bidirectional communication between the gut and the brain. Lastly, by adopting biotechnologies in gene delivery and genetically encoded tools for neuroscience, we introduce a molecular toolkit to characterize the ENS in a robust and efficient manner and modulate the ENS to uncover novel mechanisms by which innervation of the GI mediates host-microbe interactions.", "doi": "10.7907/1zv5-ve82", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11185", "collection": "thesis", "collection_id": "11185", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09172018-140652131", "primary_object_url": { "basename": "Fowler_thesis_Final_20180924.pdf", "content": "final", "filesize": 66566036, "license": "other", "mime_type": "application/pdf", "url": "/11185/12/Fowler_thesis_Final_20180924.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Silicon Neural Probes for Stimulation of Neurons and the Excitation and Detection of Proteins in the Brain", "author": [ { "family_name": "Fowler", "given_name": "Trevor Michael", "clpid": "Fowler-Trevor-Michael" } ], "thesis_advisor": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" } ], "thesis_committee": [ { "family_name": "Faraon", "given_name": "Andrei", "orcid": "0000-0002-8141-391X", "clpid": "Faraon-A" }, { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Moreaux", "given_name": "Laurent C.", "orcid": "0000-0003-1276-5062", "clpid": "Moreaux-Laurent-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis describes the development of a number of novel microfabricated neural probes for a variety of specific neuroscience applications. These devices rely on single mode waveguides and grating couplers constructed from silicon nitride thin films, which allows the use of planar lightwave circuits to create advanced device geometries and functions. These probes utilize array waveguide gratings to select an individual emitter from a large array of emitters using the wavelength of incoming light, allowing for spatial multiplexing of optical stimulation. These devices were tested in the laboratory and in living tissue to verify their efficacy. This technology was then modified to create steerable beam forming for stimulation of neurons using optical phase arrays. This technology was also tested for use in fluoresence lifetime imaging microscopy and the first application of pulsed light through the photonic circuits. Finally, this technology was again modified to create laminar illumination patterns for light sheet fluorescence microscopy applications. These devices were further improved by adding embedded microfluidics to the probes. The process of creating embedded microfluidic channels by the dig and seal method is described in detail, including modifications to the procedure that were added to address potential pitfalls in the fabrication process. Next, two projects which combine microfluidics with the optical devices described in the previous chapter are detailed. One project involves combining the use of optical emitters with microfluidic injections containing caged neurotransmitters to stimulate neurons is described. The other project involves microfluidic sampling of the extracellular space for neuropeptides which are detected using ring resonator biosensors. The sensitivity of these biosensors was analyzed in detail, determining both the physical limit of detection and the effect of biological noise due to non-specific binding on the sensors.", "doi": "10.7907/2kvw-ad56", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11154", "collection": "thesis", "collection_id": "11154", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08222018-105935103", "primary_object_url": { "basename": "Catherine Schretter Thesis_8 22.pdf", "content": "final", "filesize": 3643354, "license": "other", "mime_type": "application/pdf", "url": "/11154/1/Catherine Schretter Thesis_8 22.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Microbial Modulation of Host Locomotion", "author": [ { "family_name": "Schretter", "given_name": "Catherine Elizabeth", "orcid": "0000-0002-3957-6838", "clpid": "Schretter-Catherine-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Coordinated locomotor behavior is critical for the survival and propagation of an individual and is modulated by internal and external sensory inputs. The microbiota regulates host metabolism, which is closely intertwined with motor behavior. However, little is known regarding influences by the gut microbiome on host locomotion, or the pathways involved. The work presented in this thesis examines microbial regulation of locomotor behavior from both bacterial and host perspectives. Removal of the microbiota results in hyperactivity in female D. melanogaster, which is reversible through colonization with specific bacteria or administration of bacterial-derived products, including xylose isomerase (Xi) from Lactobacillus brevis. We found that Xi modulates host speed via sugar metabolism and octopamine signaling in flies. Additionally, aspects of microbial regulation of host locomotion appear to be conserved in mice. This work suggests that microbial modulation of host physiology extends beyond local intestinal effects to locomotor behavior through alterations in energy-related pathways.", "doi": "10.7907/Z1DX-4J03", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11721", "collection": "thesis", "collection_id": "11721", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-163128348", "primary_object_url": { "basename": "Liu_Thesis_190527 complete.pdf", "content": "final", "filesize": 2365859, "license": "other", "mime_type": "application/pdf", "url": "/11721/1/Liu_Thesis_190527 complete.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Engineering and Application of cGAL, a GAL4 Bipartite Expression System for Caenorhabditis elegans", "author": [ { "family_name": "Liu", "given_name": "Jonathan C.", "orcid": "0000-0002-5761-8704", "clpid": "Liu-Jonathan-C" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The core objectives of genetics are to dissect and understand the function of genes, the consequence of their perturbation on an organism, and how their collective action influences an organism\u2019s biology. For genetic model organisms, transgenesis is a tool that allows researchers to introduce synthetic genetic constructs to determine where a gene acts, when it is required, and infer its function. Caenorhabditis elegans is a powerful genetic model organism, with a variety of transgenesis methods available to researchers. Each has its own advantages in speed, efficiency, control of copy number, and control of integration site. However, all methods suffer from issues of reproducibility, reusability, and labor cost. Bipartite systems offer solutions to these issues- they separate the promoter element from the gene product producing strains in which one sex contains the promoter (\u2018driver\u2019 strain) and the other contains the gene (\u2018effector\u2019 strain). Crossing driver and effector strains reunites promoter and gene in the progeny, which are assayed and analyzed for gene function. This separation of drivers from effectors allows for a variety of benefits. Driver and effector strains can be combinatorially reused, meaning less time-consuming strain construction. Reusing strains allows for more reproducibility and consistency between experiments and between laboratories. Additionally, novel genes and promoters can be crossed to existing strains for novel transgenic patterns requiring minimal effort. Thus, bipartite systems greatly increase the rigor and pace of genetic analysis. This thesis details the engineering of cGAL, a GAL4-based bipartite system for C. elegans. It uses a novel GAL4 gene from Saccharomyces cerevisiae, a yeast whose optimal growth temperature is similar to that of C. elegans. This thesis also describes an intein-based split bipartite system that offers more refined spatiotemporal control, by allowing two promoters to dictate gene expression instead of one. This split method is used to analyze rhythmic feeding in C. elegans. Finally, engineering of cGAL using single copy methodology is detailed, with a discussion of future improvements to, and usage of, single copy cGAL. This development of a new bipartite system will greatly accelerate genetic analysis for the C. elegans, improve reproducibility for the field, and generate a valuable resource for the community.", "doi": "10.7907/V1DD-9108", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:9891", "collection": "thesis", "collection_id": "9891", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182016-144756449", "primary_object_url": { "basename": "Aron_Kamajaya_2016_thesis_final.pdf", "content": "final", "filesize": 7119497, "license": "other", "mime_type": "application/pdf", "url": "/9891/1/Aron_Kamajaya_2016_thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Structural Study of Piezo Channel, a Unique Family of Eukaryotic Mechanosensitive Channel", "author": [ { "family_name": "Kamajaya", "given_name": "Aron", "clpid": "Kamajaya-Aron" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Piezo is a unique family of eukaryotic mechanosensitive (MS) channel. With over 2500 amino acids per subunit, intact Piezo channel is one of the largest ion channels known to date. Two versions of Piezo can be found in vertebrates, namely PIEZO1 and PIEZO2. PIEZO1 appears to play roles in processes which control physiological homeostasis, whereas PIEZO2 assumes roles in mechanical somatosensation. A number of mutations mapped onto PIEZO1 or PIEZO2 are found in several hereditary human diseases, such as Dehydrated Hereditary Stomatocytosis, Gordon syndrome, and Distal Arthrogryposis. Although biochemical and functional studies provided many insightful findings, structural study of Piezo was very minimal. Herein, I described the structural investigation of Piezo channel. In the first study, we isolated a conserved soluble domain of Piezo (C-terminal loop 2, CTL2) from the C. elegans homolog, and provided the first molecular glimpse into this enigmatic MS channel. Subsequently, I described challenges that are associated with the expression and protein preparation of the full length Piezo channel. Recently, the full length mouse PIEZO1 structure solved by single particle cryo-EM revealed trimeric arrangement of the intact channel. CTL2 domain forms an extracellular cap which makes up the central core in this Piezo model. Lastly, we isolated a stable C-terminal fragment of Piezo. This fragment corresponds to the entire central core of Piezo channel and a few upstream transmembrane helices. This fragment can be localized to the plasma membrane. Further investigation is needed to look at the functionality of this fragment.", "doi": "10.7907/Z9JQ0Z00", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10226", "collection": "thesis", "collection_id": "10226", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302017-222932176", "primary_object_url": { "basename": "Chan-Ken-Thesis-05-31-2017.pdf", "content": "final", "filesize": 6002407, "license": "other", "mime_type": "application/pdf", "url": "/10226/23/Chan-Ken-Thesis-05-31-2017.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineered Viral Vectors and Developed Tissue Clearing Methods for Single-cell Phenotyping in Whole Organs", "author": [ { "family_name": "Chan", "given_name": "Ken Yee", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Yee" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A central question in biology is how different cell types interact with each other and their native environment to form complex functional systems and networks. Although our ability to investigate this question has considerably expanded from the development of genetically encoded tools, some limitations still persist. For instance, we are limited in our ability to visualize the native three dimensional environments of whole organs. Additionally, it is challenging to efficiently deliver transgenes into difficult-to-target areas through direct-injections, such as the cardiac ganglia, or broadly distributed networks, such as the myenteric nervous system, which limits our ability to extensively study these areas. Therefore, tools and methods that overcome these limitations are needed. Towards this end, my thesis work has been focused on developing tools for single-cell resolution phenotyping in whole organs. I have been developing tissue clearing technologies to render whole organs transparent for optical interrogation and characterizing viral capsids and engineering viral vectors for noninvasive widespread gene delivery to the central and peripheral nervous system.
\r\n\r\nTissue clearing techniques for three dimensional optical interrogation were invented over a century ago. However, these earlier methods used harsh organic chemicals and failed to retain the tissue\u2019s native fluorescence or epitopes. These earlier methods eventually became incompatible to the hundreds of newly generated transgenic mouse lines that allowed for cell type-specific expression of fluorescent transgenes or to fluorescent labeling techniques, such as immunohistochemistry (IHC). The first part of my dissertation is aimed at addressing these limitations by further developing and standardizing a tissue clearing method that utilizes the vasculature to perfuse clearing reagents. This technique, called perfusion assisted agent release in situ (PARS) enables (i) whole organ clearing of soft tissue, (ii) preservation of native fluorescence, and (iii) preservation of epitopes compatible with IHC.
\r\n\r\nAlthough PARS allows us to optically investigate whole soft tissue organs, it is unsuitable for clearing bone tissue. The clearing of bone is important as it may provide optical access to delicate environments, such as the lymphatic vessels lining the dural sinuses beneath the skull that would otherwise be damaged through traditional methods. However, clearing bone tissue is challenging since it is composed of both soft (bone marrow) and hard (mineral) tissue. To overcome this challenge, I developed a clearing method that rendered intact bone tissue transparent by using EDTA to decalcify bones and by constructing a convective flow chamber to efficiently clear bones. This method, called Bone CLARITY, is able to preserve native fluorescence and epitopes. In order to demonstrate the utility of Bone CLARITY, I collaborated with colleagues to quantitatively access a rare and non-uniformly distributed population of osteoprogenitor cells in their native three dimensional environment. Bone CLARITY in conjunction with light-sheet microscope enabled the early detection of an increase to this osteoprogenitor population after administration of a novel anabolic drug, which may have been undetected with traditional techniques.
\r\n\r\nTowards my second goal, I have been working on characterizing adeno-associated viruses (AAVs) for non-invasive widespread gene delivery across the central or peripheral nervous system. Through systemic delivery, these novel AAVs are able to efficiently deliver transgenes to (i) difficult-to-target areas, such as the dorsal root ganglia; (ii) cellular populations that are widely distributed across the mouse body, such as neurons in the myenteric nervous system, and (iii) through highly selective barriers, such as the blood-brain barrier. These viruses enable rapid expression of transgenes for perturbing and monitoring cellular circuits, or for potentially treating neurological diseases. In addition, I worked on engineering or validating several different gene regulatory elements to achieve cell type restricted expression in transgenic and non-transgenic animals with AAVs. These viral vectors may prove useful in rapidly testing newly developed genetic tools. Finally, I developed and characterized two different two-component viral vector systems to control the density of labeling when systemically delivering genes with our novel engineered viruses. I utilized this two-component system to perform single-cell morphology studies in the CNS and PNS. Collectively, these capsids and vectors expand the AAV toolbox and enable efficient and versatile gene delivery into the CNS and PNS of transgenic and non-transgenic animals.
\r\n", "doi": "10.7907/Z9NC5Z7H", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10146", "collection": "thesis", "collection_id": "10146", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04262017-114441886", "type": "thesis", "title": "Compact Microscope System for Biomedical Applications", "author": [ { "family_name": "Kim", "given_name": "Jinho", "clpid": "Kim-Jinho" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Demands for an imaging system which has high space-bandwidth product (SBP) are increasing in modern biomedical research as the amount of information to be dealt with is increasing. However, conventional microscopy has a limited SBP of about 10 mega pixels, and as such if a user wants an image in high resolution, the field of view (FOV) of the image is reduced, or if a wide FOV is necessary, the user needs to give up the resolution of image. A common way of overcoming this SBP limit in the conventional microscopy is to use mechanical moving stages and scan through wide sample area, however, it is time consuming to image large area using a high numerical aperture (NA) objective lens. This thesis presents compact imaging systems based on Fourier ptychographic microscopy for biomedical applications which are able to increase SBP without having any mechanical moving parts: one imaging system for an incubator embedded imaging system to be used in in-vitro cell culture monitoring, and the other for a high throughput 96 well plate imaging system for fast drug screening.
", "doi": "10.7907/Z9H9937R", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10227", "collection": "thesis", "collection_id": "10227", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302017-225018783", "type": "thesis", "title": "Biophysical Studies of Ligand-gated Ion Channels", "author": [ { "family_name": "Wong", "given_name": "Betty Ko", "clpid": "Wong-Betty-Ko" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes building a methodology for and the biophysical studies of ligand-gated ion channels (LGICs).
\r\n\r\nThe primary focus of the first half of this dissertation is on developing a fluorescence-based assay to broadly study LGICs. Chapter 2 describes the site-selective incorporation of a turn-on fluorophore via unnatural amino acid mutagenesis on the mouse muscle-type nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes as a proof-of-principle study. This method has proven to yield very low levels of undesired fluorescent background, which was a problem for previous incorporation techniques. Chapter 3 describes efforts towards imaging this in vivo system using lifetime imaging with efforts hampered by the inability to detect a clear signal. Chapter 4 describes efforts to apply the lifetime imaging approach towards a different system involving 5-HT3 proteins fused to fluorescent proteins in COS-7 cells.
\r\n\r\nThe second half of this dissertation focuses on studies of menthol, a flavorant added to cigarettes that contributes to smoking addiction, as a negative allosteric modulator of the \u03b1\u03b242 nAChR. Chapter 5 reveals the stereochemical effects, or rather lack of, of menthol on the two stoichiometries of the \u03b1\u03b242 receptor. Chapter 6 seeks to identify the residue interactions with menthol of the \u03b1\u03b242 receptor using a combination of computational and experimental studies.
\r\n", "doi": "10.7907/Z9TT4P0Q", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9703", "collection": "thesis", "collection_id": "9703", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052016-123724002", "primary_object_url": { "basename": "Chaubard_Jean-Luc_Thesis_2016.pdf", "content": "final", "filesize": 18091047, "license": "other", "mime_type": "application/pdf", "url": "/9703/1/Chaubard_Jean-Luc_Thesis_2016.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Development of Chemoenzymatic Labeling Approaches for the Detection of Fucosylated Biomarkers", "author": [ { "family_name": "Chaubard", "given_name": "Jean-Luc", "clpid": "Chaubard-Jean-Luc" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein fucosylation regulates a diverse set of physiological functions such as memory and learning, development, and disease pathogenesis. However, our current understanding of these processes is far behind that of other post-translational modifications, such as phosphorylation. This is, in part, due to the lack of tools available for the study of this important protein modification. To address this need, I have developed novel chemoenzymatic methods that enable the labeling and detection of unique forms of fucosylation, specifically fucose-\u03b1(1-2)-galactose (Fuc\u03b1(1-2)Gal) and core fucose. Additionally, novel glycosyltransferase assays were developed in-house to aid in the future development of both new and existing chemoenzymatic approaches.
\r\n\r\nI have demonstrated that the approach to detect Fuc\u03b1(1-2)Gal is highly selective for this disaccharide motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of this motif on live cells, discriminating malignant from normal cells. I have also shown that the chemoenzymatic detection of core fucose exhibits superior specificity towards this glycan on a variety of complex N-glycans and when compared to current fucose-specific lectins. Further, the approach is amenable to detection of core fucosylated glycans from multiple biological settings, can be exploited as an antibody-conjugation method, and can be integrated into a diagnostic platform for the profiling of protein specific core fucosylation levels. These approaches represent new potential strategies for biomarker identification and expand the technologies available for understanding the role of these important fucosylated glycans in physiology and disease.
\r\n", "doi": "10.7907/Z9K35RN1", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9090", "collection": "thesis", "collection_id": "9090", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08082015-121950812", "primary_object_url": { "basename": "Thesis - Shijia Chen.pdf", "content": "final", "filesize": 5401589, "license": "other", "mime_type": "application/pdf", "url": "/9090/1/Thesis - Shijia Chen.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Light Dependent Regulation of Sleep/Wake States by Prokineticin 2 in Larval Zebrafish", "author": [ { "family_name": "Chen", "given_name": "Shijia", "clpid": "Chen-Shijia" } ], "thesis_advisor": [ { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Sleep is an evolutionarily conserved behavior and essential to survival. The classic two process model of sleep regulation proposes that sleep results from the interaction between circadian and homeostatic processes, but the details remain elusive. Most sleep research is performed using nocturnal rodents, and diurnal vertebrates are under-represented. It is unclear whether circadian regulatory mechanisms of sleep in nocturnal animals can be directly translated into diurnal animals. In this thesis, I first briefly describe sleep behavior and the two process model of sleep regulation, focusing on the circadian process, and then discuss the advantages of using larval zebrafish as a model to study sleep behavior in diurnal vertebrates. In Chapter 2, I characterize the role of Prokineticin 2, a proposed circadian output factor in nocturnal animals, in sleep/wake regulation in larval zebrafish. I show that, similar to nocturnal rodents, Prok2 is both necessary for daytime sleep/wake behavior and sufficient to modulate sleep/wake states in a light dependent manner. However, unlike nocturnal rodents and similar to humans, Prok2 is not required for maintaining circadian rhythmicity in larval zebrafish after removing external light cue. This result demonstrates the potential functional difference of circadian output factors in different chronotypes, and establishes larval zebrafish as an alternative model for studying circadian regulation of sleep and possibly other behaviors in humans. In Chapter 3, I describe the adaptation and development of TRP channels to manipulate neuronal activity in larval zebrafish, in an effort to expand the existing repertoire of genetic tools for studying behavior in zebrafish. I show that three TRP channels, TRPV1, TRPM8 and TRPA1, can inducibly activate specific populations of neurons in larval zebrafish by using their appropriate agonists. At high agonist concentrations, TRPV1, can rapidly induce cell ablation. Adaptation of TRP channels for use in larval zebrafish expands the variety of behavioral experiments and combinatorial manipulation of neuronal activity that can be performed in zebrafish. In summary, this work deepens our understanding of sleep regulation, establishes larval zebrafish as an appropriate model for studying circadian regulation of sleep in diurnal vertebrates, and presents novel genetic tools for studying behavior in larval zebrafish.", "doi": "10.7907/Z99P2ZNR", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8720", "collection": "thesis", "collection_id": "8720", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11072014-145237990", "primary_object_url": { "basename": "NStiles_Thesis_v94.pdf", "content": "final", "filesize": 33068256, "license": "other", "mime_type": "application/pdf", "url": "/8720/1/NStiles_Thesis_v94.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Behavioral and fMRI Measures of Crossmodal Plasticity Induced by Auditory Sensory Substitution", "author": [ { "family_name": "Stiles", "given_name": "Noelle Rebecca Barry", "clpid": "Stiles-Noelle-Rebecca-Barry" } ], "thesis_advisor": [ { "family_name": "Shimojo", "given_name": "Shinsuke", "clpid": "Shimojo-S" } ], "thesis_committee": [ { "family_name": "O'Doherty", "given_name": "John P.", "clpid": "O'Doherty-J-P" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Tsao", "given_name": "Doris Y.", "clpid": "Tsao-D-Y" }, { "family_name": "Shimojo", "given_name": "Shinsuke", "clpid": "Shimojo-S" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Thirty nine million people are blind worldwide. Sensory Substitution (SS) attempts to aid the blind by translating images into sound and thereby restoring visual function. Previous studies have found that training on SS generates crossmodal neural changes allowing for activation in early visual regions in response to SS sounds. Unfortunately, training on auditory sensory substitution to become proficient at basic visual tasks takes 1 week to 3 months and even then is slow, inaccurate, and attention-intensive. In this thesis it was studied if SS interpretation could be performed by entirely naive users automatically, and if the crossmodal plasticity engendered through training could be engaged automatically. In contrast to the top-down SS interpretation, we have found that SS interpretation can be effortless and automatic in entirely naive individuals when crossmodally intuitive stimuli that contain crossmodal mappings are used. Crossmodal mappings are pre-existing associations in all individuals of images and sounds that were found to be used for entirely naive interpretation of SS. This result indicates that SS could potentially be made more useful to the blind with appropriate training and translation algorithms. We also studied if the crossmodal plasticity generated by SS training can also be activated automatically in trained blind and sighted device users. We found that crossmodal plasticity engendered through a week of training could be triggered automatically by SS stimuli. This indicates that crossmodal plasticity does not require an attention-intensive task be used and therefore is not entirely top-down cognitive. It might be possible to tap into this automatic processing in visual cortex of SS stimuli to make SS interpretation less effortful and more perceptual following the appropriate training. Overall, this thesis attempts to use SS to understand crossmodal neural processing and plasticity, and to through this broadened knowledge restore some visual function to the entirely blind.", "doi": "10.7907/Z9N29TZP", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8528", "collection": "thesis", "collection_id": "8528", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06292014-232713483", "primary_object_url": { "basename": "Rell_Parker_Complete_Thesis_2015.pdf", "content": "final", "filesize": 27520331, "license": "other", "mime_type": "application/pdf", "url": "/8528/1/Rell_Parker_Complete_Thesis_2015.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Lynx1 Modulation of Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Parker", "given_name": "Rell Lin", "clpid": "Parker-Rell-Lin" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Meister", "given_name": "Markus", "clpid": "Meister-M" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Nicotinic receptors are the target of nicotine in the brain. They are pentameric ion channels. The pentamer structure allows many combinations of receptors to be formed. These various subtypes exhibit specific properties determined by their subunit composition. Each brain region contains a fixed complement of nicotinic receptor subunits. The midbrain region is of particular interest because the dopaminergic neurons of the midbrain express several subtypes of nicotinic receptors, and these dopaminergic neurons are important for the rewarding effects of nicotine. The \u03b16 nicotinic receptor subunit has garnered intense interest because it is present in dopaminergic neurons but very few other brain regions. With its specific and limited presence in the brain, targeting this subtype of nicotinic receptor may prove advantageous as a method for smoking cessation. However, we do not fully understand the trafficking and membrane localization of this receptor or its effects on dopamine release in the striatum. We hypothesized that lynx1, a known modulator of other nicotinic receptor subtypes, is important for the proper function of \u03b16 nicotinic receptors. lynx1 has been found to act upon several classes of nicotinic receptors, such as \u03b14\u03b22 and \u03b17, the two most common subtypes in the brain. To determine whether lynx1 affects \u03b16 containing nicotinic receptors we used biochemistry, patch clamp electrophysiology, fast scan cyclic voltammetry, and mouse behavior. We found that lynx1 has effects on \u03b16 containing nicotinic receptors, but the effects were subtle. This thesis will detail the observed effects of lynx1 on \u03b16 nicotinic receptors.", "doi": "10.7907/Z9HM56DT", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8954", "collection": "thesis", "collection_id": "8954", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012015-124819012", "primary_object_url": { "basename": "Thesis_Justin Liu_Final.pdf", "content": "final", "filesize": 16744102, "license": "other", "mime_type": "application/pdf", "url": "/8954/1/Thesis_Justin Liu_Final.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Development and Function of Sleep Regulatory Circuits in Zebrafish", "author": [ { "family_name": "Liu", "given_name": "Justin", "clpid": "Liu-Justin" } ], "thesis_advisor": [ { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Sleep is widely accepted as an essential behavior for optimum mental and physical health, yet the genetic and neural circuits that govern sleep remain poorly understood. In this thesis, I briefly introduce the behavioral criteria that define sleep, currently known sleep regulatory mechanisms, and the distinct advantages of the zebrafish, Danio rerio, as a simple animal model for studying sleep. I then investigate two factors previously implicated in sleep behavior: epidermal growth factor receptor and hypocretin. First, I show that epidermal growth factor receptor signaling is both necessary and sufficient for normal sleep behavior in zebrafish, just as it is in invertebrates. This demonstrates that sleep regulatory mechanisms can be conserved over large evolutionary distances, and is the first genetic study showing that the epidermal growth factor receptor signaling is necessary for normal sleep behavior in a vertebrate. Second, I capitalize upon the rapid external development of zebrafish embryos to screen for developmental factors that specify hypocretin neurons, which are known to promote arousal and consolidate sleep/wake bouts. I identify the LIM homeobox 9 transcription factor as necessary for hypocretin neuronal development in zebrafish and sufficient to specify additional hypocretin neurons in both zebrafish and mice. This is the first time any factor has been shown to induce hypocretin neurons in vivo and may be an important step towards curing narcolepsy, a debilitating sleep disorder caused by the selective loss of hypocretin neurons. These studies deepen our understanding of how sleep is regulated at a genetic and cellular level and underscore the potential for zebrafish to make future contributions to sleep research.", "doi": "10.7907/Z9WM1BDC", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8805", "collection": "thesis", "collection_id": "8805", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03262015-100311493", "primary_object_url": { "basename": "Teagan R Wall Complete Thesis 2015.pdf", "content": "final", "filesize": 2366199, "license": "other", "mime_type": "application/pdf", "url": "/8805/1/Teagan R Wall Complete Thesis 2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Wall", "given_name": "Teagan Rose", "clpid": "Wall-Teagan-Rose" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Camerer", "given_name": "Colin F.", "clpid": "Camerer-C-F" }, { "family_name": "Adolphs", "given_name": "Ralph", "clpid": "Adolphs-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated, cation channels found throughout the central and peripheral nervous system, whose endogenous ligand is acetylcholine, but which can also be acted upon by nicotine. The subunit compositions of nAChR determine their physiological and pharmacological properties, with different subunits expressed in different combinations or areas throughout the brain. The behavioral and physiological effects of nicotine are elicited by its agonistic and desensitizing actions selectively on neuronal nAChRs. The midbrain is of particular interest due to its population of nAChRs expressed on dopaminergic neurons, which are important for reward and reinforcement, and possibly contribute to nicotine dependence. The \u03b16-subunit is found on dopaminergic neurons but very few other regions of the brain, making it an interesting drug target. We assayed a novel nicotinic agonist, called TI-299423 or TC299, for its possible selectivity for \u03b16-containing nAChRs. Our goal was to isolate the role of \u03b16-containing nAChRs in nicotine reward and reinforcement, and provide insight into the search for more effective smoking cessation compounds. This was done using a variety of in vitro and behavioral assays, aimed dually at understanding TI-299423\u2019s exact mechanism of action and its downstream effects. Additionally, we looked at the effects of another compound, menthol, on nicotine reward. Understanding how reward is generated in the cholinergic system and how that is modulated by other compounds contributes to a better understand of our complex neural circuitry and provides insight for the future development of therapeutics.", "doi": "10.7907/Z9JD4TQ5", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8742", "collection": "thesis", "collection_id": "8742", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12152014-153232034", "primary_object_url": { "basename": "Idigo_2015.pdf", "content": "final", "filesize": 5222567, "license": "other", "mime_type": "application/pdf", "url": "/8742/21/Idigo_2015.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MsCL)", "author": [ { "family_name": "Idigo", "given_name": "Chinenye Abiodun", "clpid": "Idigo-Chinenye-Abiodun" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.
\r\n\r\nIt has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 \u00b0C \u2013 60 \u00b0C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.
\r\n\r\nExtensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.
\r\n\r\nMscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.
", "doi": "10.7907/Z9542KJ3", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8537", "collection": "thesis", "collection_id": "8537", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07142014-132743727", "type": "thesis", "title": "Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors", "author": [ { "family_name": "Miles", "given_name": "Timothy Francis", "clpid": "Miles-Timothy-Francis" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This dissertation primarily describes studies of serotonin type 3 (5-HT3) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT3A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand\u2019s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.
\r\n\r\nThe third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT3AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A3B2 stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT3AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT3AB receptor from these heteromeric sites.
\r\n\r\nFinally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RAR\u03b1 of the nuclear receptor family of transcription factors.
", "doi": "10.7907/Z9T151MC ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8246", "collection": "thesis", "collection_id": "8246", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202014-055842097", "primary_object_url": { "basename": "SchomburgThesis_May18.pdf", "content": "final", "filesize": 20835356, "license": "other", "mime_type": "application/pdf", "url": "/8246/1/SchomburgThesis_May18.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Biophysical and Network Mechanisms of High Frequency Extracellular Potentials in the Rat Hippocampus", "author": [ { "family_name": "Schomburg", "given_name": "Erik W.", "clpid": "Schomburg-Erik-W" } ], "thesis_advisor": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "thesis_committee": [ { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Buzsaki", "given_name": "Gyorgy", "clpid": "Buzsaki-G" }, { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "A fundamental question in neuroscience is how distributed networks of neurons communicate and coordinate dynamically and specifically. Several models propose that oscillating local networks can transiently couple to each other through phase-locked firing. Coherent local field potentials (LFP) between synaptically connected regions is often presented as evidence for such coupling. The physiological correlates of LFP signals depend on many anatomical and physiological factors, however, and how the underlying neural processes collectively generate features of different spatiotemporal scales is poorly understood. High frequency oscillations in the hippocampus, including gamma rhythms (30-100 Hz) that are organized by the theta oscillations (5-10 Hz) during active exploration and REM sleep, as well as sharp wave-ripples (SWRs, 140-200 Hz) during immobility or slow wave sleep, have each been associated with various aspects of learning and memory. Deciphering their physiology and functional consequences is crucial to understanding the operation of the hippocampal network.
\r\n\r\nWe investigated the origins and coordination of high frequency LFPs in the hippocampo-entorhinal network using both biophysical models and analyses of large-scale recordings in behaving and sleeping rats. We found that the synchronization of pyramidal cell spikes substantially shapes, or even dominates, the electrical signature of SWRs in area CA1 of the hippocampus. The precise mechanisms coordinating this synchrony are still unresolved, but they appear to also affect CA1 activity during theta oscillations. The input to CA1, which often arrives in the form of gamma-frequency waves of activity from area CA3 and layer 3 of entorhinal cortex (EC3), did not strongly influence the timing of CA1 pyramidal cells. Rather, our data are more consistent with local network interactions governing pyramidal cells' spike timing during the integration of their inputs. Furthermore, the relative timing of input from EC3 and CA3 during the theta cycle matched that found in previous work to engage mechanisms for synapse modification and active dendritic processes. Our work demonstrates how local networks interact with upstream inputs to generate a coordinated hippocampal output during behavior and sleep, in the form of theta-gamma coupling and SWRs.
", "doi": "10.7907/7TXG-7M48", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8415", "collection": "thesis", "collection_id": "8415", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-200718606", "primary_object_url": { "basename": "Crystal N Dilworth Thesis Final Upload.pdf", "content": "final", "filesize": 8253852, "license": "other", "mime_type": "application/pdf", "url": "/8415/25/Crystal N Dilworth Thesis Final Upload.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Fluorescence Microscopy of Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Dilworth", "given_name": "Crystal Noelle", "clpid": "Dilworth-Crystal-Noelle" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson\u2019s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent \u03b15 subunit and subsequent experiments to elucidate the cellular mechanisms through which \u03b15 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of \u03b15 are also examined.
\r\n\r\nAdditionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.
", "doi": "10.7907/Z96D5QZB", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8430", "collection": "thesis", "collection_id": "8430", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292014-190146869", "primary_object_url": { "basename": "2014-5-27 Weston Nichols Caltech Thesis Final.pdf", "content": "final", "filesize": 4749813, "license": "other", "mime_type": "application/pdf", "url": "/8430/1/2014-5-27 Weston Nichols Caltech Thesis Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Lynxl and the \u03b22V287L Mutation Affect the Stoichiometry of the \u03b14\u03b22 Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Nichols", "given_name": "Weston A.", "clpid": "Nichols-Weston-A" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on \u03b14\u03b22 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent \u03b14 and \u03b22 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.", "doi": "10.7907/2S9B-R910", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8225", "collection": "thesis", "collection_id": "8225", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072014-160207088", "primary_object_url": { "basename": "OC Loson Thesis 2014.pdf", "content": "final", "filesize": 6805019, "license": "other", "mime_type": "application/pdf", "url": "/8225/1/OC Loson Thesis 2014.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Regulation of Mitochondrial Division by the Drp1 Receptors", "author": [ { "family_name": "Loson", "given_name": "Oliver Calvin", "clpid": "Loson-Oliver-Calvin" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.
\r\n\r\nThe functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins\u2014Mff, MiD49, and MiD51\u2014play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.
\r\n\r\nWe have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.
\r\n\r\nTo better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.
\r\n", "doi": "10.7907/J23G-KQ18", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8246", "collection": "thesis", "collection_id": "8246", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202014-055842097", "primary_object_url": { "basename": "SchomburgThesis_May18.pdf", "content": "final", "filesize": 20835356, "license": "other", "mime_type": "application/pdf", "url": "/8246/1/SchomburgThesis_May18.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Biophysical and Network Mechanisms of High Frequency Extracellular Potentials in the Rat Hippocampus", "author": [ { "family_name": "Schomburg", "given_name": "Erik W.", "clpid": "Schomburg-Erik-W" } ], "thesis_advisor": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "thesis_committee": [ { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Buzsaki", "given_name": "Gyorgy", "clpid": "Buzsaki-G" }, { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "A fundamental question in neuroscience is how distributed networks of neurons communicate and coordinate dynamically and specifically. Several models propose that oscillating local networks can transiently couple to each other through phase-locked firing. Coherent local field potentials (LFP) between synaptically connected regions is often presented as evidence for such coupling. The physiological correlates of LFP signals depend on many anatomical and physiological factors, however, and how the underlying neural processes collectively generate features of different spatiotemporal scales is poorly understood. High frequency oscillations in the hippocampus, including gamma rhythms (30-100 Hz) that are organized by the theta oscillations (5-10 Hz) during active exploration and REM sleep, as well as sharp wave-ripples (SWRs, 140-200 Hz) during immobility or slow wave sleep, have each been associated with various aspects of learning and memory. Deciphering their physiology and functional consequences is crucial to understanding the operation of the hippocampal network.
\r\n\r\nWe investigated the origins and coordination of high frequency LFPs in the hippocampo-entorhinal network using both biophysical models and analyses of large-scale recordings in behaving and sleeping rats. We found that the synchronization of pyramidal cell spikes substantially shapes, or even dominates, the electrical signature of SWRs in area CA1 of the hippocampus. The precise mechanisms coordinating this synchrony are still unresolved, but they appear to also affect CA1 activity during theta oscillations. The input to CA1, which often arrives in the form of gamma-frequency waves of activity from area CA3 and layer 3 of entorhinal cortex (EC3), did not strongly influence the timing of CA1 pyramidal cells. Rather, our data are more consistent with local network interactions governing pyramidal cells' spike timing during the integration of their inputs. Furthermore, the relative timing of input from EC3 and CA3 during the theta cycle matched that found in previous work to engage mechanisms for synapse modification and active dendritic processes. Our work demonstrates how local networks interact with upstream inputs to generate a coordinated hippocampal output during behavior and sleep, in the form of theta-gamma coupling and SWRs.
", "doi": "10.7907/7TXG-7M48", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7710", "collection": "thesis", "collection_id": "7710", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-213725125", "type": "thesis", "title": "Single-Cell Analysis of the Physiology of Mechanosensation in Bacteria", "author": [ { "family_name": "Bialecka-Fornal", "given_name": "Maja I.", "clpid": "Bialecka-Fornal-Maja-I" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.", "doi": "10.7907/7SRD-WS94", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7195", "collection": "thesis", "collection_id": "7195", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09012012-181931478", "primary_object_url": { "basename": "pham_anh_2013_thesis.pdf", "content": "final", "filesize": 51244271, "license": "other", "mime_type": "application/pdf", "url": "/7195/1/pham_anh_2013_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Tools for Assessing Mitochondrial Dynamics in Mouse Tissues and Neurodegenerative Models", "author": [ { "family_name": "Pham", "given_name": "Anh Hoang", "clpid": "Pham-Anh-Hoang" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Mitochondria are dynamic organelles that undergo membrane fusion and fission and transport. The dynamic properties of mitochondria are important for regulating mitochondrial function. Defects in mitochondrial dynamics are linked to neurodegenerative diseases and affect the development of many tissues. To investigate the role of mitochondrial dynamics in diseases, versatile tools are needed to explore the physiology of these dynamic organelles in multiple tissues. Current tools for monitoring mitochondrial dynamics have been limited to studies in cell culture, which may be inadequate model systems for exploring the network of tissues. Here, we have generated mouse models for monitoring mitochondrial dynamics in a broad spectrum of tissues and cell types. The photoactivatable mitochondria (PhAMfloxed) line enables Cre-inducible expression of a mitochondrial targeted photoconvertible protein, Dendra2 (mito-Dendra2). In the PhAMexcised line, mito-Dendra2 is ubiquitously expressed to facilitate broad analysis of mitochondria at various developmental processes.
\r\n\r\nWe have utilized these models to study mitochondrial dynamics in the nigrostriatal circuit of Parkinson\u2019s disease (PD) and in the development of skeletal muscles. Increasing evidences implicate aberrant regulation of mitochondrial fusion and fission in models of PD. To assess the function of mitochondrial dynamics in the nigrostriatal circuit, we utilized transgenic techniques to abrogate mitochondrial fusion. We show that deletion of the Mfn2 leads to the degeneration of dopaminergic neurons and Parkinson\u2019s-like features in mice. To elucidate the dynamic properties of mitochondria during muscle development, we established a platform for examining mitochondrial compartmentalization in skeletal muscles. This model system may yield clues to the role of mitochondrial dynamics in mitochondrial myopathies.
", "doi": "10.7907/E61Z-9C26", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7255", "collection": "thesis", "collection_id": "7255", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11052012-192506907", "primary_object_url": { "basename": "wlimapichat_complete_thesis.pdf", "content": "final", "filesize": 7469835, "license": "other", "mime_type": "application/pdf", "url": "/7255/1/wlimapichat_complete_thesis.pdf", "version": "v11.0.0" }, "type": "thesis", "title": "Probing the Roles of Receptor Structure, Drug-Receptor Interactions, and Receptor Crosstalk in Ligand-Gated Ion Channel Function", "author": [ { "family_name": "Limapichat", "given_name": "Walrati", "clpid": "Limapichat-Walrati" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ligand-gated ion channels are multi-subunit transmembrane proteins that play crucial roles in synaptic transmission in the nervous system. These include the Cys-loop receptor superfamily, the ionotropic glutamate receptor (iGluR) family, and the purinergic P2X receptor family. Binding of specific neurotransmitters at the ligand-binding site triggers a series of conformational changes that ultimately leads to ion channel opening. This dissertation describes three molecular-scale functional studies on these receptors.
\r\n\r\nThe first project (Chapter 2) describes structure-function studies of the conserved Phe-Pro motif in the Cys loop of the nicotinic acetylcholine receptor (nAChR) of the Cys-loop superfamily. Both residues were substituted with natural and unnatural amino acids. A strong interaction between the Phe and Pro residues is evident, as is a preference for aromaticity at the Phe site. Hydrophobicity is preferred at both sites. A correlation between receptor function and the cis bias at the proline backbone suggests a significant role for the cis proline conformer in receptor function.
\r\n\r\nThe second project (Chapter 3) concerns the key binding interaction of memantine, a prescribed drug for Alzheimer\u2019s disease, on the N-methyl-D-aspartate (NMDA) receptor of the iGluR family. The data suggest that the special property of memantine as an NMDA receptor blocker stems from the presence of the two methyl groups and a proper shape-matching to the binding site. Comparing affinities of memantine and amantadine, a structurally related drug, in response to pore mutations allows an identification of the methyl group binding pockets on the NMDA channel pore.
\r\n\r\nThe final project (Chapter 4) involves a study of inhibitory crosstalk between two families of ion channels: \u03b16\u03b24-containing nAChRs and P2X receptors. When these two distinct receptors are co-expressed, their properties are modulated from their normal behavior when expressed alone. The effect is constitutive and does not require channel activation. When they are co-activated by their respective agonists, the observed current is smaller than the sum of the currents evoked by individual application of their agonists. This functional interaction between these nicotinic and purinergic receptors in dorsal root ganglion neurons is proposed to be involved in pain sensation.
\r\n", "doi": "10.7907/YPMW-9E79", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6826", "collection": "thesis", "collection_id": "6826", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02172012-141718925", "primary_object_url": { "basename": "NPuskar_Title.pdf", "content": "", "filesize": 131293, "license": "other", "mime_type": "application/pdf", "url": "/6826/1/NPuskar_Title.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids", "author": [ { "family_name": "Puskar", "given_name": "Nyssa Leigh", "clpid": "Puskar-Nyssa-Leigh" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes structure-function studies of the nicotinic acetylcholine receptors (nAChRs). These studies use a combination of unnatural amino acid mutagenesis and electrophysiology to determine the specific molecular interactions required for neurotransmitter binding to nAChRs. Chapter 2 examines the mode of agonist activation for the \u03b14\u03b22 nAChR, the receptor responsible for nicotine addiction. This study investigates the molecular interactions that differentiate the \u03b14\u03b22 receptor from other receptor subtypes and endow it with the ability to mediate nicotine addiction. We report that the high affinity for nicotine at the \u03b14\u03b22 receptor is a result of a strong cation-\u03c0 interaction and a strengthened backbone hydrogen bond to a conserved tryptophan (TrpB) of this receptor. We also establish that a point mutation just four residues away from TrpB appears to influence the shape of the agonist binding site, such that it can differentiate the agonist binding mode of the \u03b14\u03b22 and muscle-type receptors. Chapter 3 extends studies of the point mutation near TrpB, termed the \u201cloop B glycine.\u201d We examine the muscle-type, \u03b14\u03b22, and \u03b17 subtypes and show that the identity of this residue strongly correlates with agonist potency. Low-potency receptor subtypes have a glycine at the loop B site, while high-potency receptors have a lysine at this site. We establish that mutation of this residue can to convert a low-potency receptor to a high-potency receptor and vice versa. Chapter 4 investigates the agonist binding mechanism of the \u03b14\u03b24 receptor. We show both ACh and nicotine make a strong cation-\u03c0 interaction to TrpB, and nicotine makes a strong hydrogen bond to the backbone carbonyl of TrpB. Additionally, chimeric \u03b2 subunits are used to examine the influence of the complementary binding component on receptor pharmacology for the \u03b14\u03b22 and \u03b14\u03b24 receptors. Last, chapter 5 is a methodology-based project focused on optimizing the incorporation of unnatural amino acids into mammalian cells. Using HEK293T cells, we successfully suppressed an amber stop codon using HSAS, an in vivo aminoacylated tRNA. Additional studies will pursue the viability of in vitro aminoacylated tRNAs for nonsense suppression in mammalian cells.", "doi": "10.7907/YK2P-K088", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7007", "collection": "thesis", "collection_id": "7007", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05062012-022516031", "primary_object_url": { "basename": "Liu_CY_Thesis_2012-05-06.pdf", "content": "final", "filesize": 10603885, "license": "other", "mime_type": "application/pdf", "url": "/7007/8/Liu_CY_Thesis_2012-05-06.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Characterization of an Unusual Collection of Olfactory Neurons in the Nose", "author": [ { "family_name": "Liu", "given_name": "Cambrian Yangshao", "clpid": "Liu-Cambrian-Yangshao" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "We have used a combination of histochemical, electrophysiological, and behavioral approaches to study signal transduction, membrane biophysics, and chemosensory function in the neurons of the mouse Grueneberg ganglion (GG) olfactory subsystem. The GG is a recently appreciated collection of ~1,000 clustered primary olfactory neurons located at the anterior tip of the mammalian nasal cavity. Despite their far-forward position, GG neurons are fully trapped beneath a keratinized epithelium and are wrapped by glial cells. This raises the question of how they contribute to the sense of smell. We found that GG neurons have key components of cGMP signal transduction pathway and are molecularly similar to GC-D neurons, which project to the enigmatic necklace glomeruli in the olfactory bulb. In electrophysiological analyses, individual GG neurons spontaneously discharged action potentials in one of three distinct temporal patterns that were stable for >20 min. An auxiliary fast-inactivating Na+ current accounted for the various discharge patterns in computer simulations of the neuronal ionic currents. Despite differences in baseline activity, the majority of GG neurons responded to specific mammalian pheromones. In behavioral experiments, we found that the weaning of adolescent mice induced GG activity; however, the effects did not depend on ambient temperature or the presence of other animals. Because GG neurons reside on a dense vascular bed, have specialized access to serum contents, and directly responded to pressure ejections of serum, their activity can likely be modulated by internally circulating hormones or proteins associated with specific physiological states such as stress. Taken together, our results demonstrate unusual molecular and functional aspects of a morphologically and anatomically atypical olfactory nerve.", "doi": "10.7907/47P6-2H66", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7078", "collection": "thesis", "collection_id": "7078", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252012-121406958", "type": "thesis", "title": "Optimization of the GluC1/IVM Neuronal Silencing Tool via Protein Engineering", "author": [ { "family_name": "Frazier", "given_name": "Shawnalea Jimee", "clpid": "Frazier-Shawnalea-Jimee" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A variety of genetically encoded tools have been developed for deciphering the neural circuitry of the brain. Such tools allow physical manipulation of neuronal excitability in a reversible, cell-specific manner, enabling researchers to establish how electrical activity and connectivity facilitate the information processing that mediates perception and drives behavior. An expanding toolkit of engineered neuroreceptors, particularly those actuated by orthogonal pharmacological ligands, provide noninvasive manipulation of regional or disperse neuronal populations with adequate spatiotemporal precision and great potential for multiplexing. We previously engineered an invertebrate glutamate-gated chloride channel (GluCl \u03b1\u03b2) that enabled pharmacologically induced silencing of electrical activity in targeted CNS neurons in vivo by the anthelmintic drug compound ivermectin (IVM; Lerchner et al., 2007). With this receptor, GluCl opt \u03b1-CFP + opt \u03b2-YFP Y182F, the concentration of IVM necessary to elicit a consistent silencing phenotype was higher than expected, raising concern about its potential side effects. Considerable variability in the extent of spike suppression was also apparent and was attributed to variable co-expression levels of \u03b1 and \u03b2 subunits. Thus, a rational protein engineering strategy was employed to optimize the GluCl/IVM tool. To increase agonist sensitivity, a gain-of-function gating mutation involving the highly conserved leucine 9\u2019 residue of the \u03b1 pore-lining M2 transmembrane domain was introduced. Various mutations at this position facilitate channel opening in the absence and presence of ligand. Analysis of side chain properties revealed that helix-destabilizing energy correlated with increases in agonist sensitivity. One mutation, L9\u2019F, enhances \u03b2 subunit incorporation to substantially increase IVM sensitivity without permitting unliganded channel opening. Removal of an arginine-based ER retention motif (RSR_AAA) from the intracellular loop of \u03b2 promoted plasma membrane expression of heteromeric GluCl \u03b1\u03b2 by preventing ER-associated degradation of the \u03b2 subunit. An additional monomeric XFP mutation complements these effects. The newly engineered GluCl opt \u03b1-mXFP L9\u2019F + opt \u03b2-mXFP Y182F RSR_AAA receptor significantly increases conductance and reduces variability in evoked spike generation in vitro using a lower concentration of IVM. This receptor, dubbed \u2018GluClv2.0\u2019, is an improved tool for IVM-induced silencing.", "doi": "10.7907/4AGK-FP05", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6509", "collection": "thesis", "collection_id": "6509", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082011-080856211", "primary_object_url": { "basename": "JAPS_Thesis.pdf", "content": "final", "filesize": 9905191, "license": "other", "mime_type": "application/pdf", "url": "/6509/1/JAPS_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Single-Molecule Studies of Ion Channels Expressing Unnatural Amino Acids", "author": [ { "family_name": "Shanata", "given_name": "Jai Anand Pattur", "clpid": "Shanata-Jai-Anand-Pattur" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission in the brain and peripheral nervous system. After an introduction (Chapter 1), Chapter 2 describes my development of techniques to combine single-channel and whole-cell recording with nonsense suppression. Having established the feasibility of the combined use of single-channel and whole-cell recording, in Chapter 3 we developed a method to identify the functional interactions of amino acids that are physically far apart in a protein. This is fundamentally a whole-cell recording method to find allosteric interactions in ion channels. The significance of this method is strongly supported by single-channel measurements. Additionally, the relationship between the single-channel gating equilibrium constant, theta, and the whole-cell measurement of EC50 is considered.
\r\n\r\nIn Chapter 4, I describe my progress towards measuring the channel opening rate of the fetal and adult muscle-type nicotinic acetylcholine receptors. Multiple different agonists are used, including acetylcholine, choline, and tetramethylammonium. Single-channel data are reported for the wild-type receptors as well as for receptors with the unnatural amino acid 5-F-Trp (monofluoro-Trp). Data are reported for multiple concentrations for a mutated fetal nAChR, and QuB is used to fit various possible models and estimate theta for this mutant.
\r\n\r\nA major aim of this dissertation was to use single-molecule studies of ion channels expressing unnatural amino acids to provide even more convincing evidence for cation-pi interactions at the binding sites of ligand-gated ion channels, specifically the neuronal nicotinic acetylcholine receptor. Chapter 5 describes the combined application of single-channel, whole-cell, and unnatural amino acid mutagenesis to the specific question of how two molecules\u2014nicotine and Chantix\u00ae (varenicline)\u2014bind to the alpha4beta2 brain receptor. In Chapter 6, I describe single-channel experiments that establish a method for distinguishing between the two known stoichiometries of the wild type alpha4beta2 brain receptor. Specifically, I identify a difference in the rectification properties of the high and low affinity receptors.
\r\n", "doi": "10.7907/2Y28-RP86", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6027", "collection": "thesis", "collection_id": "6027", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09102010-145946974", "primary_object_url": { "basename": "13_Complete_Thesis_LWade.pdf", "content": "final", "filesize": 21198331, "license": "other", "mime_type": "application/pdf", "url": "/6027/15/13_Complete_Thesis_LWade.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "An Evanescent Perspective on Cells", "author": [ { "family_name": "Wade", "given_name": "Lawrence A.", "clpid": "Wade-Lawrence-A" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "We have optically sectioned living cells to a maximum depth of ~250 nm using a Variable Angle-Total Internal Reflection Fluorescence Microscope (VA-TIRFM). This yields 3D images of cell membranes and nearby organelles similar to that gained by confocal microscopes but with at least an order-of-magnitude greater depth resolution. It also enables cellular membranes to be imaged in near isolation from cell organelles. Key to achieving this resolution was integration of a controllable excitation laser micropositioner into a standard through-the-lens TIRF illuminator and development of a custom culture dish for re-use of expensive high index of refraction cover slips. Images are acquired at several penetration depths by varying the excitation laser illumination angles. At the shallowest penetration depth (~46 nm) just the membrane and a few internal puncta are imaged. As the penetration depth is increased up to 250 nm organelles near the membrane, such as the ER, are imaged as well. The sequence of images from shallow deep is processed to yield a z-stack of images of approximately constant thickness at increasing distance from the coverslip. We employ this method to distinguish membrane-localized fluorophores (\u03b14 GFP \u03b22 nicotinic acetylcholine receptors and pCS2:lyn-mCherry) at the plasma membrane (PM) from those in near-PM endoplasmic reticulum (ERTracker green, \u03b14 GFP \u03b22 nicotinic acetylcholine receptors), on a z-axis distance scale of ~45 to ~250 nm in N2a cells. In doing so we observe occasional smooth ER structures that cannot be resolved as being distinct from the membrane.
\r\n\r\nIn a second project substantial progress has been made towards developing a Tip Enhanced Fluorescence Microscope (TEFM) capable of imaging wet biological samples with ~10 nm resolution. A TEFM combines a TIRFM with an Atomic Force Microscope (AFM) to modulate sample fluorescence through near-field dipole-dipole coupling.
\r\n\r\nIn the third project the capability to consistently produce high quality nanotube AFM probes was developed and a technique for chemically functionalizing the tip of a nanotube AFM probe was invented.
\r\n", "doi": "10.7907/AGAJ-PE93", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5823", "collection": "thesis", "collection_id": "5823", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212010-144632355", "primary_object_url": { "basename": "HCB_thesis.pdf", "content": "final", "filesize": 4167824, "license": "other", "mime_type": "application/pdf", "url": "/5823/1/HCB_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Synaptic Signal Transduction and Transcriptional Control", "author": [ { "family_name": "Beale", "given_name": "Holly C.", "clpid": "Beale-Holly-C" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Synaptic signal transduction regulates synaptic plasticity, and, on a larger scale, memory itself. The aim of this dissertation is to elucidate some of the mechanisms that control synaptic plasticity in the short term by modulating synaptic morphology and in the long term by controlling gene expression.
\r\n\r\nOne modification associated with synaptic plasticity is the change in the size of the spine, the micron-scale structure on the dendrite which supports the synapse. The size and shape of the spine are controlled by the actin cytoskeleton. I studied how stimulation of synaptic receptors drives changes in activation of proteins that regulate actin polymerization. We identified neuron-specific aspects of a canonical actin regulation pathway and characterized activity-regulated phosphatase activity.
\r\n\r\nChanges in spine size and other events associated with synaptic plasticity can begin within seconds of synaptic stimuli, but persistent changes require gene expression. For example, Arc, an immediate early gene required for changes in synaptic strength to persist, is the only transcript known to be both transcribed in response to synaptic stimulation and translocated specifically to the site of the stimulation. However, the role of Arc in promoting the plasticity of the synapse is still under investigation. We studied its binding partners and found that an interaction demonstrated in non-neuronal cells was not evident in neurons.
\r\n\r\nWe also studied changes in transcription over longer time periods. In order to identify pathways involving the postsynaptic protein densin, we assessed global changes in transcription with RNA-Seq, which uses ultra-high-throughput, short-read sequencing to measure transcript abundance. Compared to wild-type mice, densin knockout mice exhibit increased abundance of CaMKII\u03b1 (a densin binding partner), increased abundance of immediate early gene expression including Arc, and downregulated GABA_AR subunits.
\r\n\r\nIn summary, we investigated posttranslational modifications that take place within seconds of stimulation, binding interactions occurring in steady-state conditions in wild-type mice, and homeostatic adaptations to the chronic absence of a gene. These investigations into synaptic signaling illustrate not only the complexity of synapse-related regulatory networks but also the range of time scales they span.
", "doi": "10.7907/5NQ9-KX48", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5603", "collection": "thesis", "collection_id": "5603", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03142010-163350106", "primary_object_url": { "basename": "Kiowa_Bower_Thesis_2010.pdf", "content": "final", "filesize": 56594011, "license": "other", "mime_type": "application/pdf", "url": "/5603/1/Kiowa_Bower_Thesis_2010.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Chemical-Scale Studies of the 5-HT\u2083 and D2 Dopamine Receptors ", "author": [ { "family_name": "Bower", "given_name": "Kiowa San", "clpid": "Bower-Kiowa-San" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "During synaptic transmission in the central nervous system, neuroreceptors transduce a chemical signal into an electrical signal, a process that is mediated by both ligand-gated ion channels (LGICs) and G-protein coupled receptors (GPCRs). The work in this thesis examines structure-function relationships within these receptors, with a focus on elucidating the mechanism of molecular recognition during ligand binding. We utilize conventional and unnatural amino acid mutagenesis, structural derivatives of agonists, and homology models to identify specific interactions and the role of binding site residues in ligand binding and receptor activation. The technique of unnatural amino acid mutagenesis allows us to study these processes in greater detail than would otherwise be possible, even at the scale of a chemical bond.
\r\n\r\nChapter 2 covers structure-function investigations of a ligand-gated ion channel, the 5-HT\u2083 receptor, with a goal of understanding agonist binding and receptor activation. The project examines residues in close proximity to the ligand-binding site and focuses on polar interactions with hydrophilic residues. We identify 5-fluorotryptamine (5-FT) as a partial agonist of the 5-HT\u2083 receptors and show that size and electronegativity are important at the 5\u2019 position for efficient channel opening. Our investigation of the compound 1-OT revealed it to be an agonist of equal potency to the native agonist (5-HT), demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor. A study focusing on loop A residues led us to refine our homology model and propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Further studies of binding site residues identified an ionic interaction that likely participates in the conformational changes associated with receptor gating and characterized several other residues that play critical roles in receptor activation. Finally, we compare and contrast the behaviors of two structurally distinct agonist classes, 5-HT and its related structures, and m-chlorophenylbiguanide (mCPBG) and identify several residues that play critical roles in modulating agonist binding and gating in response to these agonists.
\r\n\r\nChapter 3 describes a study examining the binding site and the mechanism of agonist activation of a GPCR, the D2 dopamine receptor. A number of aromatic amino acids thought to be near the agonist binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at a conserved tryptophan of the D2 receptor establishes a cation-\u03c0 interaction between the agonist dopamine and this residue (W6.48), suggesting a reorientation of W6.48 on agonist binding, consistent with proposed \"rotamer switch\" models.
\r\n\r\nFinally, chapter 4 describes a project that seeks to extend the nonsense suppression methodology to include mammalian expression systems. Progress is made developing techniques for efficient transfection of cells in culture.
\r\n", "doi": "10.7907/17AK-6J11", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5290", "collection": "thesis", "collection_id": "5290", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012009-223603699", "type": "thesis", "title": "Elucidating the Hippocampal Dopaminergic Subproteome with Novel Bioorthogonal Techniques", "author": [ { "family_name": "Hodas", "given_name": "Jennifer Jin Lee", "clpid": "Hodas-Jennifer-Jin-Lee" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela Jane", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Both synaptic and behavioral plasticity require de novo protein synthesis. Dopamine is a critical neuromodulator, and abnormalities in dopaminergic regulation underlie disorders like Parkinson\u2019s disease, Alzheimer\u2019s disease, and schizophrenia\u2014 diseases that impair the ability of the brain to perform complex processes, including the formation and retrieval of memories. The stimulation of D1/D5 dopaminergic receptors in the hippocampus is critical for protein synthesis-dependent long-term potentiation (LTP), a process important for long-term synaptic plasticity and memory. The proteins synthesized upon activation of dopaminergic pathways, the dopaminergic subproteome, however, remain unknown.
\r\n\r\nHere, we describe the development of two sister technologies that employ bioorthogonal chemistry to effectively and specifically identify and visualize a proteome in an unbiased, nontoxic manner. In both bioorthogonal noncanonical amino acid tagging (BONCAT) and fluorescent noncanonical amino acid tagging (FUNCAT), we utilize methionine surrogates, either azidohomoalanine (AHA) or homopropargylglycine (HPG), which are conjugated via [3+2] copper (I)-catalyzed cycloaddition to either a biotin or fluorescent molecule-bearing probe. We demonstrate the utility of these methods by showing that both AHA and HPG can be used to examine two temporally distinct protein populations. Furthermore, we visualize the dendrite-specific contribution to the neuronal proteome by taking advantage of the spatial control achievable by FUNCAT. We then combine these techniques to address the question of the identity of the proteins in the specifically dendritic subproteome of the hippocampus. We confirm that upon stimulation with a D1/D5 dopamine receptor-specific agonist, there are significantly increased levels of protein synthesis in dendrites when compared to unstimulated dendrites. By utilizing a combination of these novel methods and more traditional techniques, we are able to provide the first comprehensive list of the dopaminergic dendritic subproteome of the hippocampus. These data suggest that the initial stages of D1/D5 receptor activation lead to the translation of proteins that may play a role in synaptic strengthening.
\r\n", "doi": "10.7907/DBGH-J143", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5298", "collection": "thesis", "collection_id": "5298", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10152009-180857846", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 21361360, "license": "other", "mime_type": "application/pdf", "url": "/5298/1/thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Neuromodulator-Mediated Control of Spatial and Nonspatial Information Processing in the Hippocampus", "author": [ { "family_name": "Ito", "given_name": "Hiroshi", "orcid": "0000-0001-7726-0781", "clpid": "Ito-Hiroshi" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Siapas", "given_name": "Athanassios G.", "orcid": "0000-0001-8837-678X", "clpid": "Siapas-A-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "How the brain implements learning is a long-standing question in neuroscience research. Many studies have indicated a critical role of the hippocampus in establishing memories of facts and episodes. As episodic memories require the association of many different sensory events in the environment, the hippocampus integrates multimodal information acquired from sensory systems. The brain area that sends major afferent inputs to the hippocampus, the entorhinal cortex, can be further divided into two subareas, the medial and lateral entorhinal cortex, each of which primarily transfers either spatial or nonspatial information to the hippocampus. The proper control of these two information streams is essential for constructing neuronal representations of the environment in hippocampus. To understand this process, my studies have focused primarily on the projection from the entorhinal cortex to area CA1, the temporoammonic pathway. Although this pathway has been relatively unexplored, recent studies have suggested that it plays a unique role in hippocampal function. I investigated how the temporoammonic synapses influence hippocampal function from three different perspectives; in single-neuron studies, local-circuit analyses, and behavioral manipulations. I propose that the temporoammonic pathway gives rise to a unique functional circuit in the hippocampus, which allows for the independent control of spatial and nonspatial information processing. Neuromodulators are a key component to this control as they differentially influence two streams of information from the entorhinal cortex. Finally, I describe my studies on the pathophysiology of schizophrenia-like behaviors at a neuronal circuit level. A mouse model of schizophrenia, generated by maternal immune activation, displays several behavioral abnormalities relevant to schizophrenia patients. We found that hippocampal slices prepared from these mice exhibit altered synaptic properties in the temporoammonic pathway. The mice also exhibit behavioral abnormality in novel object recognition. Taken together, my studies shed light on two information streams in hippocampal circuits. Anatomical or neuromodulatory-based disturbance of this control may underlie some of the behavioral abnormalities observed in several mental disorders.", "doi": "10.7907/3ZPG-0E52", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:2522", "collection": "thesis", "collection_id": "2522", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06082009-164212", "primary_object_url": { "basename": "ASThesis.pdf", "content": "final", "filesize": 2683675, "license": "other", "mime_type": "application/pdf", "url": "/2522/1/ASThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Intrabodies as Therapeutics for Huntington\u2019s Disease\r ", "author": [ { "family_name": "Southwell", "given_name": "Amber L.", "orcid": "0000-0002-6353-5008", "clpid": "Southwell-Amber-L" } ], "thesis_advisor": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Khoshnan", "given_name": "Ali", "clpid": "Khoshnan-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Huntington\u2019s disease (HD) is a devastating, genetic, neurodegenerative disease for which there is currently no effective therapy. The polyglutamine (polyQ) expansion that causes HD is in the first exon (HDx1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids (AAs) and two proline (polyP) repeat domains, modulate the toxicity of mutant Htt (mHtt). The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies; iAbs), we tested various epitopes for their roles in mHDx1 toxicity, aggregation, localization and turnover. Three domains in the P-rich region (PRR) of HDx1 are defined by iAbs: MW7 binds the two polyP domains, and Happs 1 and 3, two new iAbs, bind the unique, P-rich epitope located between the two polyP epitopes. In cultured cells, we find that the three PRR-binding iAbs, as well as VL12.3, which binds an epitope in the N-terminal 17 AA segment, decrease the toxicity and aggregation of mHDx-1, but they do so by different mechanisms. The PRR-binding iAbs have no effect on Htt localization, but they cause a significant increase in the turnover rate of mHtt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. These results suggest that the PRR domain regulates mHtt stability and toxicity. Thus, compromising this pathogenic epitope by iAb binding represents a novel therapeutic strategy for treating HD.
\r\n\r\nWe have tested this hypothesis by delivering both VL12.3 and Happ1 to the brains of HD model mice using an AAV2/1 viral vector with a modified CBA promoter. VL12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in assays of motor and cognitive deficits as well as in the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128 and BACH models of HD. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment possibility for HD.
\r\n", "doi": "10.7907/DDSD-7K09", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:42", "collection": "thesis", "collection_id": "42", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01062009-133603", "primary_object_url": { "basename": "ALE_Thesis.pdf", "content": "final", "filesize": 25714013, "license": "other", "mime_type": "application/pdf", "url": "/42/1/ALE_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigating Structure-Function Relationships in Ion Channels Using Unnatural Amino Acids", "author": [ { "family_name": "Eastwood", "given_name": "Amy Lynn", "clpid": "Eastwood-Amy-Lynn" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ion channels are proteins that traverse the cell membrane and form gated pores that open and close in response to various stimuli. In order to experimentally probe aspects of ion channel functionality, we performed subtle structure function studies using the in vivo nonsense suppression method, which allows for the incorporation of synthetically accessible unnatural amino acids and hydroxy acids into an ion channel at a site of interest.
\r\n\r\nFluorinated aromatic amino acids are good probes for a cation-\u03c0 interaction because fluorine substituents reduce the binding affinity of the aromatic for a cation in a linear, step-wise fashion. In collaboration with Professor Richard Horn at the Thomas Jefferson University, we substituted a series of fluorinated phenylalanines for important tyrosines in the Shaker B K\u207a channel and experimentally determined that TEA was binding to the residues through a cation-\u03c0 interaction. We also determined that Ca\u00b2\u207a binds to and blocks the NaV1.4 channel through a cation-\u03c0 interaction with a tyrosine at the top of the pore of this channel. We found that tetrodotoxin, another channel blocker, also binds to this same residue through a cation-\u03c0 interaction. Finally, we proved that lidocaine and other local anesthetics bind to a phenylalanine at the bottom of the pore of this channel through a cation-\u03c0 interaction.
\r\n\r\nAn important aspect of our work is the development of unnatural amino acids that can be used in the study of ion channels through the in vivo nonsense-suppression methodology. We determined that D-amino acids could not be incorporated into ion channels using this method. We synthesized several novel fluorescent-MTS reagents to be used in FRET studies. We probed the sterics around phenylalanines using the unnatural amino acid 3,5-dimethylphenylalanine. We also attempted to incorporate 4-amino-phenylalanine, but, unfortunately, we never saw the enhanced binding of a cationic ligand that was our expected phenotype.
\r\n\r\nFinally, we also designed and synthesized two \u03b1-hydroxy acids capable of site-specific proteolysis upon UV irradiation. We used a tripeptide model system to isolate and characterize the cleavage fragments, proving that these two residues are indeed capable of site-specific proteolysis through the predicted mechanism.
\r\n", "doi": "10.7907/86B5-SV57", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2522", "collection": "thesis", "collection_id": "2522", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06082009-164212", "primary_object_url": { "basename": "ASThesis.pdf", "content": "final", "filesize": 2683675, "license": "other", "mime_type": "application/pdf", "url": "/2522/1/ASThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Intrabodies as Therapeutics for Huntington\u2019s Disease\r ", "author": [ { "family_name": "Southwell", "given_name": "Amber L.", "orcid": "0000-0002-6353-5008", "clpid": "Southwell-Amber-L" } ], "thesis_advisor": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Khoshnan", "given_name": "Ali", "clpid": "Khoshnan-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Huntington\u2019s disease (HD) is a devastating, genetic, neurodegenerative disease for which there is currently no effective therapy. The polyglutamine (polyQ) expansion that causes HD is in the first exon (HDx1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids (AAs) and two proline (polyP) repeat domains, modulate the toxicity of mutant Htt (mHtt). The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies; iAbs), we tested various epitopes for their roles in mHDx1 toxicity, aggregation, localization and turnover. Three domains in the P-rich region (PRR) of HDx1 are defined by iAbs: MW7 binds the two polyP domains, and Happs 1 and 3, two new iAbs, bind the unique, P-rich epitope located between the two polyP epitopes. In cultured cells, we find that the three PRR-binding iAbs, as well as VL12.3, which binds an epitope in the N-terminal 17 AA segment, decrease the toxicity and aggregation of mHDx-1, but they do so by different mechanisms. The PRR-binding iAbs have no effect on Htt localization, but they cause a significant increase in the turnover rate of mHtt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. These results suggest that the PRR domain regulates mHtt stability and toxicity. Thus, compromising this pathogenic epitope by iAb binding represents a novel therapeutic strategy for treating HD.
\r\n\r\nWe have tested this hypothesis by delivering both VL12.3 and Happ1 to the brains of HD model mice using an AAV2/1 viral vector with a modified CBA promoter. VL12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in assays of motor and cognitive deficits as well as in the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128 and BACH models of HD. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment possibility for HD.
\r\n", "doi": "10.7907/DDSD-7K09", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:958", "collection": "thesis", "collection_id": "958", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03142008-155402", "primary_object_url": { "basename": "Princess_Imoukhuede.pdf", "content": "final", "filesize": 5887662, "license": "other", "mime_type": "application/pdf", "url": "/958/1/Princess_Imoukhuede.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Visualizing the Membrane Confinement, Trafficking and Structure of the GABA Transporter, GAT1", "author": [ { "family_name": "Imoukhuede", "given_name": "Princess Ikhianosen Uerenikhosen", "orcid": "0000-0002-4257-1085", "clpid": "Imoukhuede-Princess-Ikhianosen-Uerenikhosen" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Chow", "given_name": "Robert", "clpid": "Chow-Robert" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Transporter trafficking regulators can play an important role in maintaining the transporter density necessary for effective function. I determine interactions that confine GAT1 at the membrane by investigating GAT1 lateral mobility through fluorescence recovery after photobleaching (FRAP). I find that the mobility of GAT1 can be increased by depolymerizing actin or by blocking the GAT1 PDZ interacting domain. I also identify ezrin as the GAT1 adaptor to actin. Through fluorescence resonance energy transfer (FRET), the distance between GAT1-YFP and Ezrin-CFP is calculated as 64--68 \u00c5, and it can be significantly increased by disrupting the actin cytoskeleton. Altogether, my data reveals that actin confines GAT1 to the plasma membrane via ezrin, an interaction mediated through the GAT1-PDZ interaction domain.
\r\n\r\nDiscoveries in the field of vesicle fusion provide direct ties to translational research. While the study of vesicle fusion classically has been applied to neurotransmitter and neuropeptide containing vesicles; there is evidence that secretory vesicles physiologically differ from vesicles trafficking membrane protein. For instance, GAT1 resides on a vesicle lacking neurotransmitter but containing some v-SNARE proteins. These differences in the vesicle composition suggest inherent differences in trafficking mechanisms, which can only be confirmed through further study of membrane protein trafficking. To this end, I apply total internal reflection fluorescence microscopy (TIRFM) to quantify the number of GAT1 molecules on vesicles and to observe the movement of vesicles containing fluorescently tagged GAT1 into the plasma membrane. I determine that these vesicles contain 3--7 molecules of GAT1 and uncover a population of GAT1 vesicles with ATP-dependent lateral displacement.
\r\n\r\nThe protein-protein interactions, trafficking, and oligomerization of mouse GAT1 were studied using fourteen different fusions of mGAT1 with fluorescent protein. We determine that a natural PDZ-interacting motif is minimally required for wild-type GAT1 behavior. Fusions with wild-type function yielded up to 21% FRET efficiency, indicating efficient GAT1 oligomerization. Additionally, 45% FRET was observed between a GAT1 construct and YFP-syntaxin-1A. Inserting XFP between R565 and L566, resulted in 33% FRET but impaired function, which indicated the \"RL\" motif in the proximal C terminus governs export from the endoplasmic reticulum but not transporter oligomerization.
", "doi": "10.7907/3Q8S-CV89", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:190", "collection": "thesis", "collection_id": "190", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01152007-080704", "primary_object_url": { "basename": "CompleteThesis.pdf", "content": "final", "filesize": 4146525, "license": "other", "mime_type": "application/pdf", "url": "/190/5/CompleteThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Chemical Scale Investigations of the Gating Mechanism of Ion Channels", "author": [ { "family_name": "Lee", "given_name": "Lori Wai Hang", "clpid": "Lee-Lori-Wai-Hang" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The studies contained in this dissertation are aimed at utilizing chemistry to understand neurobiology and neuronal communication. Chapters 2 and 3 both address the gating of ion channels, describing structure-function studies to shed light on the gating mechanisms of two classes of ion channels. Chapter 2 studies the gating mechanism of the mechanosensitive channel of small conductance (MscS), which is voltage modulated. Elucidating the mechanism of voltage sensation in MscS may provide insight into how voltage-gated channels translate a change in membrane potential to channel gating. The research discussed in Chapter 2 is aimed at elucidating the role of two arginine residues, in the TM1 and TM2 of MscS, in voltage sensing. We generated two MscS mutants, Arg46Ala and Arg74Ala, to evaluate the effects of \"neutralizing\" the charged side chain on the voltage sensing ability of the channel. The mutants were evaluated using single channel analysis in E. coli spheroplasts. Our preliminary results indicated a potentially significant role for Arg46 in the voltage sensitivity of MscS, however this data set is not extensive due to inconsistency in the spheroplasts preparation.
\r\n\r\nIn Chapter 3, we utilized nonsense suppression to incorporate unnatural amino acids to study the gating of the cation-selective Cys-loop family of ion channel receptors. Specifically, it describes work aimed at elucidating the role of cis-trans isomerization of a proline residue in the gating mechanism of the serotonin-gated 5-hydroxy-tryptamine receptor 3A (5-HT3A) and the nicotinic acetylcholine receptor. A series of proline analgues, of varying cis preference were incorporated at proline 308 in the M2-M3 loop of the 5-HT3A receptor using in vivo nonsense suppression methodology in a Xenopus oocyte expression system. Electrophysiological analysis of the mutant channels revealed a linear relationship between the cis preference of the proline analog and the EC50 of the mutant channel\u2014suggesting that proline 308 may serve as a hinge during the gating 5-HT3A. From these data, we proposed a model of gating for the 5-HT3A receptor. Initial results from similar studies in nAChR suggests that the analogous proline does not play a role in its gating.
\r\n\r\nLastly, Chapter 4 addresses the role of fucose-galactose carbohydrates in learning and memory. It aims to identify lectins to fucose-alpha(1-2)-galactose as well as identify the corresponding glycoproteins bearing fucose-alpha(1-2)-galactose. Chemical probes were synthesized and used to study fucose-alpha(1-2)-galactose binding proteins. One of the probes was used to demonstrate the existence of fucose-alpha(1-2)-galactose binding proteins in hippocampal neurons. Furthermore, initial results from experiments with a photoreactive probe suggested that the design of our probe is sufficient to isolate fucose-alpha(1-2)-galactose binding proteins from the brain. Additionally, we were able to use antibodies specific to fucose-alpha(1-2)-galactose epitopes to examine fucose-alpha(1-2)-galactose bearing glycoproteins in the brain. Overall, results from both studies utilizing chemical probes and molecular probes strongly suggest that the modifications of proteins with fucose-alpha(1-2)-galactose epitopes and the expression of fucose-alpha(1-2)-galactose binding proteins are developmentally regulated.
", "doi": "10.7907/bv54-5p15", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3671", "collection": "thesis", "collection_id": "3671", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09202008-110124", "primary_object_url": { "basename": "McGarvey_t_2006.pdf", "content": "final", "filesize": 8270442, "license": "other", "mime_type": "application/pdf", "url": "/3671/1/McGarvey_t_2006.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Ultra-Sensitive Absorption Measurements through Cavity-Enhanced Spectroscopy", "author": [ { "family_name": "McGarvey", "given_name": "Raymond Timothy James", "clpid": "McGarvey-Raymond-Timothy-James" } ], "thesis_advisor": [ { "family_name": "Mabuchi", "given_name": "Hideo", "clpid": "Mabuchi-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Mabuchi", "given_name": "Hideo", "clpid": "Mabuchi-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The desire to increase the sensitivity of solution-based absorption spectroscopy is motivated by the need for label-free biosensing (which provides a more authentic indication of the state of a biological system) and by the usefulness of characterizing the kinetics of biologically-relevant reactions (which may not be accurately characterizable at reagent concentrations required by standard methods. There are a number of techniques by which such increasingly sensitive measurements have been made, including cavity ringdown spectroscopy, incoherent cavity-enhanced spectroscopy, microsphere-based whispering-gallery mode sensing,and our cavity-enhanced measurements, which are the most sensitive to date and which can be conducted in real time with high bandwidth. Our current device has a demonstrated detection threshold of 1.7x 10^{-7}/sqrt{Hz} (4.36x10^{-6}cm^{-1}), which could with further technical work be improved to a shot-noise limited sensitivity of 1.93x 10^{-10}/sqrt{Hz} (1.06x10^{-8}cm^{-1}). The latter would correspond to an average of 700 strong absorbers (epsilon = 10^5 M^{-1}cm^{-1}) in the optical beam volume. The shot-noise limited detection threshold of our measurement method could potentially be improved by up to two orders of magnitude by incorporating state-of-the-art optical mirrors. With such mirrors, cavity-enhanced absorption experiments performed with gas-phase samples have previously demonstrated single molecule sensitivity. We have established that solution-based cavity-enhanced absorption measurements are more sensitive than standard single-pass measurements by the predicted enhancement factor for our present device (~ 20,000). These measurements provide the proof-of-principle for solution-based, cavity-enhanced spectroscopy and serve as the intermediate step towards the attainment of the theoretical sensitivity of this technique. We believe that this device will be of broad interest to the scientific community, because it is presently the most sensitive solution-based spectroscopic device. It can make real-time absorption measurements which would allow monitoring of the kinetics of chemical reactions in which the spectral properties of reactants change by even a small amount, and, near its theoretical limit of sensitivity (given currently available mirrors), such a device could potentially resolve single-molecule absorption events on the sub-millisecond timescale and below.\r\n", "doi": "10.7907/CGYD-6J27", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:1804", "collection": "thesis", "collection_id": "1804", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05142008-004518", "primary_object_url": { "basename": "Eric-Slimko-Thesis.pdf", "content": "final", "filesize": 2410055, "license": "other", "mime_type": "application/pdf", "url": "/1804/1/Eric-Slimko-Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Selective Silencing of Vertebrate Neurons: Strategies Using Invertebrate Ligand-Gated Ion Channels", "author": [ { "family_name": "Slimko", "given_name": "Eric Michael", "clpid": "Slimko-Eric-Michael" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of certain human neurological disorders and (2) provide insight into the roles of specific sets of neurons, both in the local circuit and in the behavior of the intact organism. This work focuses on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl \u03b1 and \u03b2) in vertebrate neurons first in vitro using viral and tranfection techniques, and then finally in vivo using genetic techniques, to produce inhibition via a Cl- conductance when activated with IVM. We have considerably engineered these two genes by (1) re-coding the genes such that vertebrate-preferred codons are used throughout the sequences, (2) incorporating fluorescent tags within the proteins, and (3) finding a mutation to remove the undesirable glutamate sensitivity of the channel while retaining IVM efficacy. Expression of this new channel does not affect the normal spike activity of the target cell, yet the experimentor can effectively \u201cshut-off\u201d the cell with concentrations of as low as 5 nM IVM. Chapter 1 provides a broad overview of the many \u201cselective silencing\u201d approaches that experimenters have tried. In Chapter 2, the author describes the basic \u201cGluCl/IVM\u201d technique and initial experiments in cultured hippocampal neurons. Chapter 3 refines the technique by describing the strategy and mutation that allowed great reduction in the native glutamate response while maintaining the IVM response. Chapter 4 develops the final engineering of the channel: recoding the sequence for optimal expression and the introduction of fluorescent tags for identification. Finally, Chapters 5 and 6 discuss the successes and failures of in vivo work with what we now call the \u201cGluCl/IVM method.\u201d ", "doi": "10.7907/RYJJ-JW20", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:4962", "collection": "thesis", "collection_id": "4962", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12122004-215151", "primary_object_url": { "basename": "Shapovalov_00_title.pdf", "content": "final", "filesize": 29183, "license": "other", "mime_type": "application/pdf", "url": "/4962/2/Shapovalov_00_title.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Mechanosensitive Channels of Bacteria: Structure and Function. Electrophysiology as a High Resolution Technique of Ion Channel Study", "author": [ { "family_name": "Shapovalov", "given_name": "George G.", "orcid": "0000-0001-9702-9317", "clpid": "Shapovalov-George-G" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mabuchi", "given_name": "Hideo", "orcid": "0000-0002-5156-7678", "clpid": "Mabuchi-H" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Mechanosensitive (MS) ion channels commonly play a role of transducers converting mechanical stimuli into electrical or chemical signaling, thus allowing the cell to regulate its behavior in response to changing environment conditions. MS channels participate in sensation of sound and orientation in inner ear (hair cells), in touch sensation and in osmoregulation of bacteria. Structure of bacterial MS channels of large (MscL) and small (MscS) conductance has been recently solved at atomic resolution, stimulating various structural and functional studies. In this work author presents series of experiments enhancing an understanding of mechanosensation in bacteria. In Chapter 2 author performs cysteine cross-linking experiments suggesting asymmetric gating pattern of Tb-MscL ion channel. Chapters 3 and 4 establish a possibility of successful synthesis of fully functional Tb- and Ec-MscL proteins displaying a phenotype identical to recombinant channels. Studies in Chapter 5 and Appendix 1 extend the resolution of single-channel patch clamping technique, and describe a fine structure of MS channel gating by collecting and characterizing intersubstate transitions.", "doi": "10.7907/XZ69-9A14", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2005", "collection": "thesis", "collection_id": "2005", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05242005-172213", "primary_object_url": { "basename": "00_Introduction.pdf", "content": "final", "filesize": 126053, "license": "other", "mime_type": "application/pdf", "url": "/2005/1/00_Introduction.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of Ion Channel Structure and Function. I. Studies of Nicotine Binding to the Acetylcholine Receptor. II. Development of Tools for Studying Learning and Memory with Unnatural Amino Acids", "author": [ { "family_name": "Petersson", "given_name": "Ernest James", "orcid": "0000-0003-3854-9210", "clpid": "Petersson-Ernest-James" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation can be divided into two main sections:
\r\n\r\nI. In previous studies, we have used fluorinated tryptophan derivatives to conclusively identify a cation-pi interaction with Trp 149 in the binding of acetylcholine (ACh) to the muscle-type nicotinic acetylcholine receptor (nAChR). We have incorporated mimics of ACh, termed tethered agonists, in the binding site to produce self-activating channels. Using tertiary tethered agonists that would only become cations and activate the channel when protonated, we identified a perturbed pKa for the binding pocket, which has implications for the binding of tertiary agonists like nicotine (Nic). It has been shown that Nic does not participate in a straight-forward cation-pi interaction as ACh does. We have examined a hydrogen bond between the Nic pyrrolidine N-H and the backbone carbonyl of Trp149 by introducing an ester linkage at this point, weakening the carbonyl H-bond accepting ability. Calculations performed on hydrogen bound complexes of ACh, Nic, and the Nic analog epibatidine (Epi) explain the trends observed for ligand activation of the nAChR. Expanding upon this study, we have performed molecular dynamics (MD) simulations of models of the ligand binding domain of the nAChR. Ligand-bound structures from these simulations have been taken on to quantum mechanical/molecular mechanical (QMMM) calculations to model the effects of unnatural amino acid mutations in an environment that simulates the full nAChR binding pocket.
\r\n\r\nII. The nAChR is essential to neurotransmission at the junction between nerve and muscle cells, and it plays an important role in many central nervous system processes. However, its role in learning and memory is limited, at least in our current molecular models of these events. In a sense, the formation of a memory consists of the strengthening of some synaptic connections and the weakening of others. These processes, termed long term potentiation (LTP) and depression (LTD) respectively, are primarily governed by modifications to glutamate receptors (GluRs). We have developed tools for studying the mechanism and timecourse of these modifications (of phosphorylation in particular), and we have demonstrated the first incorporation of unnatural amino acids into a GluR.
", "doi": "10.7907/yhty-re87", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1439", "collection": "thesis", "collection_id": "1439", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04212005-143332", "primary_object_url": { "basename": "SSF_thesis_TOC.PDF", "content": "final", "filesize": 94218, "license": "other", "mime_type": "application/pdf", "url": "/1439/8/SSF_thesis_TOC.PDF", "version": "v3.0.0" }, "type": "thesis", "title": "Cytoarchitecture of the Locust Olfactory System", "author": [ { "family_name": "Farivar", "given_name": "Shabnam Sarah", "clpid": "Farivar-Shabnam-Sarah" } ], "thesis_advisor": [ { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" } ], "thesis_committee": [ { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The insect mushroom body (MB) receives and processes olfactory information. The MB is a highly conserved structure found in all but a few insect species, and has been shown to be a relevant area in learning and memory of olfactory information. The functional properties of the intrinsic cells of the MB -- the Kenyon cells (KCs) -- have been extensively studied, and their integrative properties are starting to be understood, particularly in locust. To help decipher its role in odor processing, this thesis presents an in-depth study of the architecture of the locust MB, using a variety of anatomical techniques and original software. Four divisions in the MB's input area, the calyx, are defined and described, as well as a division in one of its output regions, the beta lobe. KCs are characterized based on their morphologies and extents within the calyx divisions and the beta lobe. MB input cells - the projection neurons - are described in relation to their own input area, the antennal lobe, as well as their output to MB calyx divisions. Two classes of cells downstream from the KCs are also defined anatomically and related to immunochemistry on neurotransmitters. A specific area within the brain - the lateral horn lobe - to which projection neurons and extrinsic cells project, is also defined. Similarities of these structures to other insect orders are discussed.", "doi": "10.7907/4Y60-KH68", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1912", "collection": "thesis", "collection_id": "1912", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05212004-144036", "primary_object_url": { "basename": "Elmore_Thesis.pdf", "content": "final", "filesize": 16831479, "license": "other", "mime_type": "application/pdf", "url": "/1912/8/Elmore_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Investigations of Ion Channel Structure-Function Relationships Using Molecular Modeling and Experimental Biochemistry", "author": [ { "family_name": "Elmore", "given_name": "Donald Eugene, Jr.", "orcid": "0000-0002-8723-8710", "clpid": "Elmore-Donald-Eugene-Jr" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ion channels are integral membrane proteins found in all cells that mediate the selective passage of specific ions or molecules across a cell membrane. These channels are important in a diverse range of physiological processes, including signal transmission in the nervous system, sensory perception, and regulation of vital systems, such as circulation. This thesis discusses the use of computational chemistry methods, such as molecular dynamics (MD) and ab initio calculations, and experimental biochemical techniques, such as site-directed mutagenesis, in vivo bacterial assays, chemical cross-linking, and circular dichroism spectroscopy, in tandem to elucidate ion channel structure-function relationships. This research was catalyzed by the solving of atomic resolution crystal structures of the mechanosensitive channels of large and small conductance (MscL and MscS) by the Rees group. Although interesting themselves, these bacterial channels also provide good model systems for considering more complex eukaryotic channels.
\r\n\r\nMscL is an ion channel gated only by membrane tension. Initial studies of MscL verified the relevance of the crystal structure conformation under physiological conditions and compared different MscL homologues. Other work began to elucidate potentially unique structural and functional roles of the M. tuberculosis MscL C-terminal helical bundle. As well, interactions between the MscL channel protein and surrounding lipid and the potential relevance of helical kinking in MscL gating pathways were investigated. MscS is also gated by membrane tension, but its gating can be modulated by changes in transmembrane potential. Thus, studies on MscS began to identify the specific amino acid residues that are responsible for giving the channel its voltage sensitivity. Finally, computations predicting the conformation of nicotine in different solvent environments are discussed. Nicotine is a small molecule ligand that binds to and gates nicotinic acetylcholine receptors, and a thorough understanding of nicotine structure could aid efforts to elucidate receptor structure-function relationships and design new pharmaceuticals.
", "doi": "10.7907/47GW-HT46", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2351", "collection": "thesis", "collection_id": "2351", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-144341", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 28014631, "license": "other", "mime_type": "application/pdf", "url": "/2351/2/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "SynGAP Controls Synapse Formation by Regulating Spine Development and Morphology", "author": [ { "family_name": "V\u00e1zquez", "given_name": "Luis Enrique", "orcid": "0000-0003-0197-2058", "clpid": "V\u00e1zquez-Luis-Enrique" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "SynGAP is a brain-specific Ras GTPase-activating protein that is an abundant component of the signaling complex associated with the NMDA-type glutamate receptor. We generated mutant mice lacking synGAP to study its physiological role. Homozygous mutant mice die in the first few days after birth; however, neurons from mutant embryos can be maintained in culture. Here we report that spine maturation and synapse formation are accelerated in cultured mutant neurons, and the spines of mature mutant neurons are significantly larger than those of wild type. Clusters of PSD-95, and subunits of AMPA-type and NMDA-type glutamate receptors are larger and brighter, and appear in spines of mutant neurons by day 10 in vitro; whereas in wild-type neurons they are still mostly located in dendritic shafts. The frequency and amplitude of miniature excitatory postsynaptic currents are larger in mutant neurons at day 10 in vitro, confirming that they have more functional synapses, with more AMPA receptors in them. At day 21 in vitro, the spines of mutant neurons remain significantly larger than those of wild type. The mutant phenotype at day 10 in vitro can be rescued by introduction of recombinant wild-type synGAP on day 9. In contrast, introduction of synGAP with a mutated GAP domain or a deletion of the terminal domain that binds to PSD-95 does not rescue the mutant phenotype, indicating that both domains play a role in control of spine maturation. Thus, the GAP activity of synGAP, as well as its association with PSD-95, is important for normal regulation of spine and synapse maturation in hippocampal neurons.", "doi": "10.7907/v7bp-jb59", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2081", "collection": "thesis", "collection_id": "2081", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262004-125208", "primary_object_url": { "basename": "01FullDissertation.pdf", "content": "final", "filesize": 29555061, "license": "other", "mime_type": "application/pdf", "url": "/2081/1/01FullDissertation.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Local Control of Synaptic Strength: Neurotrophic and Dopaminergic Modulation of Dendritic Protein Synthesis", "author": [ { "family_name": "Smith", "given_name": "W. Bryan", "clpid": "Smith-W-Bryan" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Understanding the cell biological mechanisms responsible for the plasticity of central neurons is key to our understanding of brain function. In an effort to better characterize the cell and molecular biology of individual neurons, we have studied the role of local, dendritic protein synthesis in hippocampal synaptic plasticity. Such a localized control of protein synthesis provides a means of achieving input-specificity, the observation that synapses on a given neuron are able to independently scale the strength of their connections. This property of synaptic enhancement is correlated with the encoding capacity of an individual cell: the greater the input specificity, the more information a cell can encode.
\r\n\r\nHere we show two pathways for inducing local protein synthesis, one mediated by the brain-derived neurotrophic factor, and another by the D1/D5 dopaminergic signaling system. Local protein synthesis stimulated by the dopaminergic pathway leads directly to an increase in synaptic strength by increasing production and synaptic localization of AMPA receptors, the glutamate-gated ion channels that mediate fast synaptic transmission at central synapses. We also present a set of software tools for quantitative analysis of 3-D colocalization and 2-D spatial correlation in immunofluorescence microscopy. These tools will greatly assist in exploratory data analysis of the dynamics of protein distributions in neurons and other cells.
", "doi": "10.7907/15vm-zk49", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:20", "collection": "thesis", "collection_id": "20", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01042004-210542", "primary_object_url": { "basename": "title&chpt1.pdf", "content": "final", "filesize": 4552547, "license": "other", "mime_type": "application/pdf", "url": "/20/5/title&chpt1.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Chemical-Scale Manipulation of Ion Channels: in vivo Nonsense Suppression and Targeted Disulfide Crosslinking", "author": [ { "family_name": "Zacharias", "given_name": "Niki Marie", "orcid": "0000-0002-9364-6016", "clpid": "Zacharias-Niki-Marie" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The study of the three-dimensional shape and structure-function relationships of ion channels is a very challenging field of research. Ion channels are integral-membrane proteins that when open allow ions to flux across the cell membrane. The structure and function of ion channels are dependent on the cell membrane that surrounds them. Because an ion channel must be embedded in a cell membrane, many techniques used to probe the structure of soluble proteins cannot be used in the study of ion channels.
\r\n\r\nOne versatile technique that has been shown to be quite valuable in the structure-function studies of ion channels is the in vivo nonsense suppression method for unnatural amino acid incorporation. This technique allows one to site-specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression. The method has been shown to accommodate a wide variety of unnatural amino acids and hydroxy acids. Chapter One will discusses the in vivo nonsense suppression method in greater detail.
\r\n\r\nA key component of this work is the design and synthesis of new unnatural amino acids that have novel properties. Chapter 2 discusses the synthesis and uses of 5-(o-nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH). NBSeOH is used to site-specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a selenium anion. The selenium anion then initiates an intramolecular SN2 displacement that cleaves the backbone of the protein. Preliminary data reveals that NBSeOH can be incorporated into a protein in vivo and in vitro, and photolysis of proteins and peptides containing NBSeOH does lead to protein backbone cleavage.
\r\n\r\nChapter 4 discusses how the in vivo nonsense suppression method was used to incorporate unnatural amino acids containing a quaternary ammonium moiety to mimic the quaternary ammonium on acetylcholine. These unnatural amino acids were used to probe the nicotinic acetylcholine receptor?s binding site. These unnatural amino acids are called tethered agonists because when they were incorporated into four different positions on the nicotinic acetylcholine receptor partial opening of the channel occurred even when agonist was not present. These tethered agonists were used to obtain distance information about where acetylcholine binds within the receptor.
\r\n\r\nAnother technique used to probe the structure of ion channels is targeted disulfide crosslinking. In the targeted disulfide crosslinking method, cysteine residues are introduced at various locations throughout a protein and oxidized to see whether disulfide bond formation can occur. Since only cysteine residues close in space will form a disulfide bond, this method can reveal fine structural aspects of a protein. The method was used to study the pore lining structure of the nicotinic acetylcholine receptor. Several cysteine mutants were made using mutagenesis and then studied in functional channels expressed in Xenopus oocytes. The channels were then exposed to oxidizing agents, and the ability of these mutant channels to form disulfide bonds was evaluated. Chapter 3 describes the work dealing with the targeted disulfide crosslinking experiments in the nicotinic acetylcholine receptor.
", "doi": "10.7907/y4ay-ph28", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2046", "collection": "thesis", "collection_id": "2046", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252004-153512", "primary_object_url": { "basename": "SLMthesis.pdf", "content": "final", "filesize": 57677639, "license": "other", "mime_type": "application/pdf", "url": "/2046/6/SLMthesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Site-Specific Incorporation of Unnatural Amino Acids into Receptors Expressed in Mammalian Cells", "author": [ { "family_name": "Monahan", "given_name": "Sarah Lynn", "clpid": "Monahan-Sarah-Lynn" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Unnatural amino acid incorporation into proteins by nonsense suppression has proven to be a valuable tool for structure-function studies. Using the in vivo nonsense suppression methodology, information on ligand binding and ion channel gating mechanisms has been obtained on a variety of ion channels. To date, such studies have been limited to the Xenopus oocyte heterologous expression system. There would be clear benefits to expanding the technology to a mammalian cell expression system. This would provide a more relevant environment for many proteins of mammalian origin and would allow for studies of cell-specific signal transduction pathways.
\r\n\r\nWe describe here unnatural amino acid incorporation into channels and receptors expressed in mammalian cells. Presented is a general method to deliver mRNA or DNA that codes for a protein of interest, amber suppressor tRNA, and a reporter gene to mammalian cells. Chapter 2 describes in detail the screening of several suppressor tRNAs, as well as various transfection methods tested for tRNA delivery including electroporation, lipofection, peptide-mediated transfection and biolistics. It was found that electroporation was the best method to deliver tRNA to adherent cells, and that THG73 was the most efficient suppressor tRNA. Chapter 3 describes studies, aimed at optimizing the protocol, that involved co-electroporation of a human serine amber suppressor tRNA with the DNA or mRNA corresponding to the protein of interest into adherent cells. This leads to highly efficient delivery of these components and efficient nonsense suppression. We demonstrate this for both enhanced green fluorescent protein and nicotinic acetylcholine receptor expression in CHO-K1 cells. We also show that the approach is successful in cultured hippocampal neurons. Finally, Chapter 4 demonstrates the application of the electroporation method to the delivery of aminoacyl-tRNA to cells for unnatural amino acid incorporation into the nicotinic acetylcholine receptor. When chemically aminoacylated with natural or unnatural amino acids, THG73 delivers the amino acid site-specifically into receptors expressed in CHO-K1 cells. Electrophysiology clearly reveals the expected shift in dose-response relations, establishing that the desired unnatural amino acid has been incorporated. In conclusion, we describe the first general method for unnatural amino acid incorporation in mammalian cells.
", "doi": "10.7907/B00K-QH65", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2990", "collection": "thesis", "collection_id": "2990", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07312002-120928", "primary_object_url": { "basename": "lintongli-thesis.pdf", "content": "final", "filesize": 1959356, "license": "other", "mime_type": "application/pdf", "url": "/2990/1/lintongli-thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Tethered Agonist Approach to Mapping Ion-Channel Proteins: Toward a Structural Model for the Agonist-Binding Site of the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Li", "given_name": "Lintong", "clpid": "Li-Lintong" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The integral membrane proteins of neurons and other excitable cells are generally resistant to high-resolution structural tools. In this thesis we present our efforts to probe the structure of the agonist-binding site of the nicotinic acetylcholine receptor (nAChR) using the tethered agonist approach, which combines chemical synthesis, the nonsense suppression methodology for unnatural amino acid incorporation and electrophysiology.
\r\n\r\nIn Chapter 2, we present the results of incorporating a series of tethered quaternary ammonium derivatives of tyrosine into the nAChR using the in vivo nonsense suppression methodology for incorporating unnatural amino acids site-specifically. At three sites, a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57, all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the binding site of the receptor can be developed by analogy to the acetylcholine esterase crystal structure.
\r\n\r\nIn Chapter 3, we report evidence that the N-terminal extracellular domain of nAChR is closely related to acetylcholine binding protein (AChBP), whose crystal structure was solved in May 2001. Based on the model obtained from docking acetylcholine into the structure of AChBP, we designed and incorporated a new tethered agonist, lysyl-carbamylcholine. Incorporation of this tethered agonist at several positions produced constitutively active receptors, with significant activity seen at alpha192, alpha193, and gamma119/delta121. These results demonstrated that the loop E residue gamma119/delta121 on the complementary subunit is very near the agonist-binding site. We also investigated the role of an intersubunit hydrogen bond, which was seen in the crystal structure of AChBP. Incorporation of tryptophan analogs that abolish the hydrogen bonding abilities slowed the desensitization of the receptor, which implied that this hydrogen bond might play a key role in the allosteric transitions of desensitization.
\r\n\r\nIn Chapter 4, we describe our efforts to prepare a short tethered agonist and the results of incorporating it into nAChR at alpha198 by chemical modification of cysteine mutants introduced by nonsense suppression methodology. Methanethiosulphonate ethyltrimethylammonium (MTSET) modification resulted in constitutive activity, which suggested the closeness of alpha198 to the agonist-binding site.
\r\n\r\nIn Chapter 5, methods in molecular biology, electrophysiology and molecular docking, and the synthesis of amino acids and dinucleotide dCA-amino acids are summarized.
", "doi": "10.7907/AMDZ-XE24", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:245", "collection": "thesis", "collection_id": "245", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01202003-221429", "primary_object_url": { "basename": "BrandtThesis.pdf", "content": "final", "filesize": 9256488, "license": "other", "mime_type": "application/pdf", "url": "/245/1/BrandtThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Site-Specific Incorporation of Synthetic Amino Acids into Functioning Ion Channels", "author": [ { "family_name": "Brandt", "given_name": "Gabriel Shaw", "orcid": "0000-0002-9148-8042", "clpid": "Brandt-Gabriel-Shaw" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic amino acids may be introduced into functioning proteins by means of nonsense suppression, using tRNA aminoacylated with unnatural amino acids. This technique can be extended to living cells through micro-injection of mRNA and tRNA into Xenopus laevis oocytes. Introduction of synthetic amino acids into proteins has been used, broadly, for three purposes. First, sensitive probes have been incorporated into proteins, using side chain chemistry unavailable to naturally encoded amino acids. Second, reactive side chains have been developed which can drive conformational rearrangements of the protein. Third, natural post-translational modifications of protein side chains have been mimicked. The work presented here applies all of these approaches to the study of ion channels. A series of fluorinated Trp residues was incorporated into the nicotinic acetylcholine receptor (nAChR) to probe electrostatic effects on cation-pi mediated binding of nicotine and other agonists. Site-specific protein backbone cleavage of the nicotinic acetylcholine and purinergic P2X\u2082 receptors was undertaken, along with intersubunit photo-crosslinking in the nAChR. Caged tyrosine was employed to study tyrosine phosphorylation of an important modulatory site in the potassium channel Kir2.1. Finally, caged phosphoamino acid analogs were synthesized for further characterization of the effects of phosphorylation on ion channels.", "doi": "10.7907/3PYX-4P72", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:2239", "collection": "thesis", "collection_id": "2239", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292003-182043", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 5936738, "license": "other", "mime_type": "application/pdf", "url": "/2239/1/thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Theory and Design of Relaxometric Probes", "author": [ { "family_name": "Qui\u00f1\u00f3nez", "given_name": "Carlo Joseph", "clpid": "Qui\u00f1\u00f3nez-Carlo-Joseph" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Peters", "given_name": "Jonas C.", "orcid": "0000-0002-6610-4414", "clpid": "Peters-J-C" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In an effort to rationally design an apoptosis-sensitive MRI contrast agent, two novel gadolinium complexes were designed, synthesized and evaluated as components of a relaxometric probe. The first, AEDO3A will prove a useful building block for in the area of the relaxometric probe design. AEDO3A-Gd has a relaxivity of 2.2 mM-1s-1 and 3.4 mM-1s-1 at 60Mhz and 500MHz, respectively If AEDO3A-Gd is the \"on\" state and assuming reasonable relaxivities for the \"off\" state, tissue contrast modeling of the smallest-detectable relaxivity change suggests AEDO3A is suitable for incorporation into relaxometric probes.
\r\n\r\n1-(2-Aspartryl-aminoethyl)-4,7,10-tri(carboxymethyl)-cyclen (Asp-AEDO3A) is evaluated as the second component of an apoptosis-sensitive relaxometric probe system. The synthesis and characterization of the ligand and its Gd and Tb complexes is described. Fluorescence lifetime data of the terbium complex indicate the presence of 0.6 water molecules in the inner coordination sphere. The gadolinium complex has a relaxivity of 1.4 mM-1s-1 and 1.7 mM-1s-1 at 60Mhz and 500MHz, respectively. Toxicity studies demonstrated Xenopus embryos tolerated Asp-AEDO3A-Gd at magnetically useful concentrations as predicted by tissue contrast modeling. X-ray crystallography data are presented for both the ligand and gadolinium complex.
\r\n\r\nUnfortunately, no enzymatic processing of Asp-AEDO3A-Gd was observed under any conditions. This phenomenon was attributed to heretofore-unknown coordination chemistry for gadolinium elucidated from the X-ray crystal structure. This novel N-carboxamido coordination, while problematic for the application of Asp-AEDO3A-Gd as a relaxometric probe, will be potentially useful in other areas of contrast agent design.
\r\n", "doi": "10.7907/X9G0-2M43", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:4188", "collection": "thesis", "collection_id": "4188", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10202002-002307", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 13085158, "license": "other", "mime_type": "application/pdf", "url": "/4188/11/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "I. Structure-Function Analysis of the Mechanosensitive Channel of Large Conductance. II. Design of Novel Magnetic Materials using Crystal Engineering", "author": [ { "family_name": "Maurer", "given_name": "Joshua Ahab", "orcid": "0000-0002-6663-0721", "clpid": "Maurer-Joshua-Ahab" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work presented here encompasses two distinct areas, with the first section addressing structure-function relationships in the mechanosensitive channel of large conductance (MscL) from bacteria. A high-throughput fluorescent screening technique has been developed for the E. coli homologue of MscL. This technique has been applied to a large library of random E. coli MscL mutations to provide insights into channel function. Additionally, attempts have been made to characterize the functionally important regions of MscL and comparisons have been made between the E. coli and M. tuberculosis homologues of MscL.
\r\n\r\nThe second section addresses the design of novel magnetic materials. The guanidinium sulfonate \"Ward lattice\" from crystal engineering has been used to develop a new family of frustrated magnetic materials.
", "doi": "10.7907/DNFY-8G73", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:1400", "collection": "thesis", "collection_id": "1400", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04172002-150548", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 7843633, "license": "other", "mime_type": "application/pdf", "url": "/1400/1/thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Neural Computations Leading to Space-specific Auditory Responses in the Barn Owl", "author": [ { "family_name": "Arthur", "given_name": "Benjamin Jacob", "clpid": "Arthur-Benjamin-Jacob" } ], "thesis_advisor": [ { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Sound localization is the ability to pinpoint the direction a sound is coming from based on auditory cues alone. Neurons in the brain which mediate this behavior are active only when sound comes from a particular direction. This thesis uses physiological and anatomical methods to investigate the computations which lead to such space-specific neural responses in the barn owl.
\r\n \r\nChapter 3 studies a behavioral and neural phenomenon called phase ambiguity, which arises from the way in which the auditory nerve and cochlear nuclei encode acoustic information. Phase ambiguity causes errors in sound localization to be made for tonal stimuli, and is resolved through the convergence of information across different frequencies in broadband noise stimuli. Data presented here show that a continuous band of noise is not necessary; a set of tones spaced at the critical bandwidth resolves phase ambiguity just as well as a noise stimulus. This is due to a sub-linear interaction for tones of nearby frequencies.
\r\n \r\nChapter 4 addresses the head-related transfer function (HRTF) model of sound localization. While traditional barn owl models use linear equations to relate interaural time differences (ITD) to azimuth and interaural intensity differences (IID) to elevation, the HRTF model purports that IID is dependent on frequency to such an extent that pattern recognition is used to match the spectral shape of IID in the stimulus to that characteristic of particular directions in space. Data presented here confirm predictions made by the HRTF model that IID tuning changes with frequency in space-mapped neurons, and that two-tone stimuli whose IIDs match these changes elicit better responses than those which do not.
\r\n \r\nChapter 5 investigates the computation of space-specificity in the forebrain. Previous anatomical studies have suggested that the space-specificity seen there is not merely inherited from the space map in the midbrain, but rather arises, at least in part, independently. The data presented here reconfirm that the forebrain pathway branches off from the midbrain pathway before the convergence across frequencies leads to space-specific neurons. All previous computations, however, including the formation of ITD-IID combination sensitivity, seem to be shared.
\r\n \r\nCollectively, these three studies expand our knowledge of the neurophysiology of sound localization in the barn owl by detailing specific mechanisms underlying the computation of space-specific neural responses.
", "doi": "10.7907/SY24-X458", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6772", "collection": "thesis", "collection_id": "6772", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01232012-132700275", "primary_object_url": { "basename": "Aakalu_g_2002.pdf", "content": "final", "filesize": 36543132, "license": "other", "mime_type": "application/pdf", "url": "/6772/1/Aakalu_g_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Building the Molecular Machinery of Memory: Local Protein Synthesis in Hippocampal Neurons", "author": [ { "family_name": "Aakalu", "given_name": "Girish Nanda", "clpid": "Aakalu-Girish-Nanda" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Synaptic plasticity is the most widely accepted cellular and molecular model for learning and memory. Although the idea that information is encoding through changes in\r\nsynaptic strength is simple, the requirement for new protein synthesis to maintain long-lasting forms of plasticity threatens to make this model untenably complex. This complexity arises from the fact that an individual neuron can have thousands of connections, small groups of which change strength independently of others. Since the\r\nnecessary proteins are likely the effectors of long-term plasticity, non-specific delivery would lead to loss of plasticity-encoded information. Thus it is critical that the effector proteins be faithfully delivered only to the correct sites.
\r\n\r\nOne way to accomplish this task would be to allow the synapses local control over the necessary protein synthesis and delivery. Previous studies have suggested this possibility of dendritic local protein synthesis (LPS). In this thesis we describe the visualization, in real time, of the synthesis of a GFP-based reporter in the dendrites of cultured rat hippocampal neurons. By utilizing a number of physical, optical and molecular manipulations we have insured that the observed synthesis was free of any\r\nsomatic contribution thereby providing the first definitive evidence for the existence of dendritic LPS in mature vertebrate neurons.
\r\n\r\nWe also describe the regulation of dendritic LPS by two forms of plasticity-inducing stimuli. First, we show that dendritic LPS can be stimulated by brain derived neurotrophic factor (BDNF), a molecule capable of causing persistent synaptic enhancement. Second, we show that a chronic blockade of synaptic activity, which results in a form of synaptic enhancement termed \"disuse hypersensitivity\", appears to enhance dendritic LPS.
\r\n\r\nFinally, we discuss a technique that can facilitate the study of the necessity of dendritic LPS for long-lasting plasticity. By using a \"caged\" protein synthesis inhibitor,\r\nwe are able to abolish protein synthesis in a spatially restricted manner. Thus it is now possible to conduct experiments where dendritic LPS is inhibited while somatic synthesis is permitted. If plasticity is not maintained under these conditions, we will have satisfying evidence of the necessity of dendritic LPS for long-lasting plasticity.
", "doi": "10.7907/CAY9-6643", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6968", "collection": "thesis", "collection_id": "6968", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04242012-143624169", "primary_object_url": { "basename": "Zirlinger_m_2002.pdf", "content": "final", "filesize": 9432064, "license": "other", "mime_type": "application/pdf", "url": "/6968/1/Zirlinger_m_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Application of Microarray, Laser Capture and Transgenic Technologies to the Study of Neural Diversity", "author": [ { "family_name": "Zirlinger", "given_name": "Mariela", "clpid": "Zirlinger-Mariela" } ], "thesis_advisor": [ { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" } ], "thesis_committee": [ { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A major quest in modem neurobiology is to understand how the brain controls behavior. To this end, the convergence of two traditionally separate fields, systems neuroscience and molecular neuroscience, is required. The delineation of brain regions responsible for different behaviors, and in particular, their underlying neural circuits should be accompanied by the appreciation of the molecules that compose such circuits.
\r\n\r\nI have taken two approaches toward unraveling the molecular signatures of specific neural structures.\r\nFirst, I conducted microarray-based RNA expression analyses to search, in a large scale and with no a priori constraints, for differentially expressed gene products in several brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb and periaqueductal gray. Interestingly, only 0.3% of the genes characterized to date showed restricted expression in distinct brain areas. Further characterization by in situ hybridization was performed for genes enriched in the amygdala, a structure that modulates emotional behavior. Remarkably, this revealed that most region-specific genes possessed expression domains whose limits respected subnuclear boundaries defined by classical cytoarchitectonic criteria.\r\nThese analyses were not only informative about the molecular composition of distinct brain areas, but also\r\nprovided tools to genetically dissect the role of different brain nuclei in specific behaviors.
\r\n\r\nSecond, I have used a genetic strategy to label all cellular derivatives of neural crest precursor cells\r\nexpressing a particular gene, Ngn2. Such lineage tracing study uncovered a segregated cellular subpopulation in the developing peripheral nervous system, which was strongly biased for the generation of sensory rather than autonomic neurons. Despite this fate bias, Ngn2-derived cells in the dorsal root ganglion were equally likely to give rise to neurons or glia. This suggests that some neural crest cells\r\nbecome restricted to sensory or autonomic sub lineages before becoming committed to neuronal or glial\r\nfates. In general, visualization of the behavior of neural progenitors during the formation of the nervous\r\nsystem may further our understanding of the generation of specific neuronal subtypes and, eventually,\r\nneuronal connections that shape the functioning brain.
\r\n\r\nThe combination of strategies here described will enable the characterization of brain regions at the molecular level on a broad, systems-based approach.
\r\n", "doi": "10.7907/c701-pk41", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:14292", "collection": "thesis", "collection_id": "14292", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07022021-160304976", "type": "thesis", "title": "Negative Regulation of Cell Fate Specification by the lin-15 Locus During Vulval Induction in Caenorhabditis elegans", "author": [ { "family_name": "Gonzalez-Serricchio", "given_name": "Aidyl Sofia", "clpid": "Gonzalez-Serricchio-Aidyl-Sofia" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "We have visualized extrachromosomal arrays by targeting the green fluorescent protein (GFP) to a specific DNA sequence (lac operator) incorporated into Caenorhabditis elegans' transgenes. This system can be used to determine polyploidy and to investigate chromosome segregation. This technique also allows rapid, accurate determination of spontaneous loss of an array, thereby allowing high-resolution mosaic analysis. We carried out genetic mosaic analysis on lin-3 (epidermal growth factor) using the GFP-Lacl + lacO method. This methodology confirmed lin-3's site of action for vulval induction is at the anchor cell. This result also proved this technique works.
\r\n\r\nWe used both the GFP-Lacl + lacO256 system as well as the ncl-1 gene as genetic mosaic markers to determine the site of action of lin-15A and lin-15B. Both markers indicate that lin-15A gene function is required within the vulval precursor cells (VPCs) to prevent an excessive number of VPCs from generating vulval progeny. The mosaic expression pattern for lin-15B is broad therefore, proven difficult to pinpoint a site of action.
\r\n\r\nThe products of the lin-15 gene were first defined genetically as negative regulators of the vulval induction pathway. It encodes two novel hydrophilic proteins, LIN-15A and LIN-15B. According to antibody stainings and GFP expression patterns, both proteins are nuclear and present in almost all the cells. lin-15 is part of the synthetic multivulva (synMuv) set of genes which are comprised of two classes, A and B. Mutation of both an A and a B gene is required to obtain a multivulva (Muv) phenotype. Further characterization of the lin-15 locus reveals an effect on fertility.
", "doi": "10.7907/ppq9-ps50", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:7588", "collection": "thesis", "collection_id": "7588", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04082013-160433360", "primary_object_url": { "basename": "Siegel_ms_2000.pdf", "content": "final", "filesize": 21296002, "license": "other", "mime_type": "application/pdf", "url": "/7588/1/Siegel_ms_2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Genetically Engineered Sensors of Cell Signaling", "author": [ { "family_name": "Siegel", "given_name": "Micah Seth", "clpid": "Siegel-Micah-Seth" } ], "thesis_advisor": [ { "family_name": "Mead", "given_name": "Carver", "orcid": "0000-0003-4051-0462", "clpid": "Mead-C-A" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Isacoff", "given_name": "Ehud Y.", "orcid": "0000-0003-4775-9359", "clpid": "Isacoff-Ehud-Y" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Mead", "given_name": "Carver", "orcid": "0000-0003-4051-0462", "clpid": "Mead-C-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is\r\na fundamental problem for the study of neural development and information processing. To address this\r\nproblem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane\r\nvoltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive\r\npotassium channel so that voltage dependent rearrangements in the potassium channel induce\r\nchanges in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be\r\nintroduced into an organism non-invasively and targeted to specific developmental stages, brain regions,\r\ncell types, and sub-cellular compartments.
\r\n\r\nWe also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used\r\nmultiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal\r\noutput via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh\r\nvariants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to\r\ndesign functional assays for receptor activation in living cells.
", "doi": "10.7907/92c5-r851", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:13852", "collection": "thesis", "collection_id": "13852", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08112020-100312577", "primary_object_url": { "basename": "Chen_WJ_2000.pdf", "content": "final", "filesize": 44757842, "license": "other", "mime_type": "application/pdf", "url": "/13852/1/Chen_WJ_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Extragenic Suppressors of Heat Shock Activated GO\u03b1. Topic I: Cyclin in Heat Shock Response. Topic II: Signaling by Go and Gq in C. elegans", "author": [ { "family_name": "Chen", "given_name": "Wen J.", "clpid": "Chen-Wen-J" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Dunphy", "given_name": "William G.", "clpid": "Dunphy-W-G" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "clpid": "Meyerowitz-E-M" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In the nematode C. elegans, heterotrimeric G proteins have been shown to regulate the behavior of locomotion, feeding and egg-laying. Go belongs to Gi family and only exists in organisms with a nervous system. The signaling downstream of Go has been a mystery since its discovery, and it is what we are determined to find out.
\r\n\r\nLoss of Go\u03b1 causes animals to be hyperactive and lay eggs constitutively. Overexpressing Go\u03b1 causes opposite phenotypes. Another \u03b1 protein a subunit, Gq, causes phenotypes opposite to Go. To identify G protein effectors in C. elegans, we performed a forward genetic screen for suppressors of activated Go\u03b1 under the control of the heat-shock promoter hsp16-2. Because of the nature of the screen design, we identified two categories of genes. One category acts on heat shock response and the other category acts on G protein pathways. We characterized and positional cloned genes from both categories.
\r\n\r\nThe second chapter of the thesis described sag-4, a cyclin L homologue that specifically affects heat shock promoters and decreases heat-shock induced protein expression. We propose that cyclin Lis likely to be involved in heat shock induced transcription. Other genes in this category, sag-3, sag-5 and sag-8, may also function in similar mechanisms.
\r\n\r\nThe third chapter of the thesis focused on G protein signaling. eat-16 was identified in the screen for suppressors of activated Go\u03b1. We positional cloned it and found it encodes a RGS7 homologue. RGS proteins have been studied as GTPase Activating Protein for the \u03b1 subunits of heterotrimeric G proteins. Although eat-16 was identified in a suppressor screen for activated Go (goa-1), both genetic and biochemical evidence showed that eat-16 is a GAP for Gq (egl-30). We propose that Go and Gq antagonize each other, thereby regulating behaviors. Go might negatively regulate Gq signaling, possibly through eat-16 or other unrevealed genes.
\r\n\r\nChapter four describes our reconstituted system in mammalian cell culture. EAT-16 decreases Gq/G11 mediated PLC activity. GOA-1 and GPB-2 (C. elegans G\u03b25 homologue) also decrease PLC activity induced by Gq/G11. These results are consistent with the hypothesis that Go negatively regulate Gq signaling, and the interaction between G\u03b25 and RGS7 can be one of the steps between Go and Gq.
", "doi": "10.7907/2sv5-sd03", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:8871", "collection": "thesis", "collection_id": "8871", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202015-142019062", "primary_object_url": { "basename": "Dvorak-Carbone, Hannah..pdf", "content": "final", "filesize": 41704677, "license": "other", "mime_type": "application/pdf", "url": "/8871/1/Dvorak-Carbone, Hannah..pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Contribution of the Temporoammonic Pathway to Hippocampal Processing", "author": [ { "family_name": "Dvorak-Carbone", "given_name": "Hannah", "clpid": "Dvorak-Carbone-Hannah" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The temporoammonic (TA) pathway is the direct, monosynaptic projection from layer III of entorhinal cortex to the distal dendritic region of area CA1 of the hippo\u00ad campus. Although this pathway has been implicated in various functions, such as memory encoding and retrieval, spatial navigation, generation of oscillatory activity, and control of hippocampal excitability, the details of its physiology are not well understood. In this thesis, I examine the contribution of the TA pathway to hippocampal processing. I find that, as has been previously reported, the TA pathway includes both excitatory, glutamatergic components and inhibitory, GABAergic components. Several new discoveries are reported in this thesis. I show that the TA pathway is subject to forms of short-term activity-dependent regulation, including paired-pulse and frequency\u00ad dependent plasticity, similar to other hippocampal pathways such as the Schaffer collateral (SC) input from CA3 to CA1. The TA pathway provides a strongly excitatory input to stratum radiatum giant cells of CA1. The excitatory component of the TA pathway undergoes a long-lasting decrease in synaptic strength following low-frequency stimulation in a manner partially dependent on the activation of NMDA receptors. High\u00ad frequency activation of the TA pathway recruits a feedforward inhibition that can prevent CA1 pyramidal cells from spiking in response to SC input; this spike-blocking effect shows that the TA pathway can act to regulate information flow through the hippocampal trisynaptic pathway.", "doi": "10.7907/AYRT-5Y12", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:17657", "collection": "thesis", "collection_id": "17657", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08282025-214605106", "primary_object_url": { "basename": "Tang_L_1999.pdf", "content": "final", "filesize": 64643673, "license": "other", "mime_type": "application/pdf", "url": "/17657/1/Tang_L_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Role for the Cadherin Family of Cell Adhesion Molecules in Synaptic Function in the Adult Hippocampus", "author": [ { "family_name": "Tang", "given_name": "Lixin", "clpid": "Tang-Lixin" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The cadherins are a family of type I single-pass integral membrane glycoproteins that\r\nspan intercellular junctions and mediate Ca2+-dependent homophilic intercellular interactions.\r\nThe highly conserved cytoplasmic C termini of cadherins interact with the catenins\r\nand the cytoskeleton. The extracellular domain is composed of five repeats with the most\r\ndistal repeat, especially a region containing the highly conserved His-Ala-Val (HA V)\r\nsequence, being critical for homophilic binding.
\r\n\r\nI first examined the expression of cadherins, especially the neural- (N-) and epithelial-\r\n(E-) subtypes, in the adult hippocampus. In situ hybridization experiments indicated\r\nthe presence of mRNAs for both N- and E- cadherins in the adult hippocampus. Immunoblot\r\nanalysis revealed the expression of cadherin proteins in the hippocampal synaptosome\r\nfraction. Immunofluorescent staining indicated that cadherins and catenins are\r\nexpressed at synaptic sites.
\r\n\r\nI investigated the possible role of cadherins in synaptic plasticity at the CAI synapses\r\nin the adult hippocampus. Preincubation of hippocampal slices with function-blocking\r\ncadherin antibodies or HA V-containing antagonistic peptides greatly reduced long-term\r\npotentiation (LTP) whereas basal synaptic properties including input-output relations, and\r\npaired-pulse facilitation were normal. The HAV peptides inhibited LTP in a concentration-\r\ndependent and LTP induction protocol-independent manner.
\r\n\r\nA decrease in the extracellular Ca2+ associated with LTP induction may increase the\r\nvulnerability of Ca2+-sensitive cadherin bonds to cadherin inhibitory reagents. In support\r\nof this hypothesis, I found that doubling of the extracellular Ca2+ abolished the inhibition\r\nof LTP by HAV peptides. Moreover, HAV peptides delivered in a lower Ca2+ solution\r\nreduced previously potentiated responses, suggesting a role for cadherins in both the\r\ninduction and expression of LTP.
\r\n\r\nA recombinant adenovirus containing a dominant-inhibitory cadherin cDNA was\r\nconstructed. I found that hippocampal slices infected with this virus exhibited normal\r\nsynaptic properties but less LTP than adjacent slices infected with an adenovirus containing\r\na reporter gene.
\r\n\r\nI also examined the effect of HAV peptides on presynaptic vesicle exocytosis in\r\nhippocampal cultures using the fluorescent membrane dye FM 1-43. HAV peptides do\r\nnot affect the dye release following stimulation, suggesting cadherin function is not\r\nrequired for normal exocytosis.
\r\n\r\nTaken together, these data suggest cadherins make important contributions to synaptic\r\nplasticity in the adult hippocampus.
", "doi": "10.7907/qh82-x455", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:8871", "collection": "thesis", "collection_id": "8871", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202015-142019062", "primary_object_url": { "basename": "Dvorak-Carbone, Hannah..pdf", "content": "final", "filesize": 41704677, "license": "other", "mime_type": "application/pdf", "url": "/8871/1/Dvorak-Carbone, Hannah..pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Contribution of the Temporoammonic Pathway to Hippocampal Processing", "author": [ { "family_name": "Dvorak-Carbone", "given_name": "Hannah", "clpid": "Dvorak-Carbone-Hannah" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The temporoammonic (TA) pathway is the direct, monosynaptic projection from layer III of entorhinal cortex to the distal dendritic region of area CA1 of the hippo\u00ad campus. Although this pathway has been implicated in various functions, such as memory encoding and retrieval, spatial navigation, generation of oscillatory activity, and control of hippocampal excitability, the details of its physiology are not well understood. In this thesis, I examine the contribution of the TA pathway to hippocampal processing. I find that, as has been previously reported, the TA pathway includes both excitatory, glutamatergic components and inhibitory, GABAergic components. Several new discoveries are reported in this thesis. I show that the TA pathway is subject to forms of short-term activity-dependent regulation, including paired-pulse and frequency\u00ad dependent plasticity, similar to other hippocampal pathways such as the Schaffer collateral (SC) input from CA3 to CA1. The TA pathway provides a strongly excitatory input to stratum radiatum giant cells of CA1. The excitatory component of the TA pathway undergoes a long-lasting decrease in synaptic strength following low-frequency stimulation in a manner partially dependent on the activation of NMDA receptors. High\u00ad frequency activation of the TA pathway recruits a feedforward inhibition that can prevent CA1 pyramidal cells from spiking in response to SC input; this spike-blocking effect shows that the TA pathway can act to regulate information flow through the hippocampal trisynaptic pathway.", "doi": "10.7907/AYRT-5Y12", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:6397", "collection": "thesis", "collection_id": "6397", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162011-132252955", "primary_object_url": { "basename": "Su_a_1998.pdf", "content": "final", "filesize": 26600640, "license": "other", "mime_type": "application/pdf", "url": "/6397/1/Su_a_1998.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Backbone Flexibility in Protein Design Theory and Experiment", "author": [ { "family_name": "Su", "given_name": "Yao-ying Alyce", "clpid": "Su-Yao-ying-Alyce" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" }, { "family_name": "Frautschi", "given_name": "Steven C.", "clpid": "Frautschi-S-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Tombrello", "given_name": "Thomas A.", "clpid": "Tombrello-T-A" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The role of backbone flexibility in protein design was studied. First, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G\u03b21 domain (G\u03b21). Concerted backbone motion was introduced by varying G\u03b21's supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally. Core variants containing as many as six of ten possible mutations retained native-like properties. This result demonstrates that backbone flexibility can be combined with amino acid side-chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of the native parameter values.\r\n\r\nSecond, a general, quantitative design method for computing de novo backbone templates was developed. The method had to compute atomic resolution backbones compatible with the atomistic sequence selection algorithm we were using and it had to be applicable to all protein motifs. We again developed a method that uses super-secondary structure parameters to determine the orientation among secondary structural elements, given a target protein fold. Possible backbone arrangements were screened using a cost function which evaluates core packing, hydrogen bonding, loop closure, and backbone torsional geometry. Given a specified number of residues for each secondary structural element, a family of optimal configurations was found. We chose three motifs to test our method (\u03b2\u03b2\u03b1, \u03b2\u03b1\u03b2, and \u03b1\u03b1) since their combination could be used to approximate most possible backbone fold. The best structure found for the \u03b2\u03b2\u03b1 motif is similar to a zinc finger, and the best structure for the \u03b2\u03b2\u03b1 motif is similar to a segment of a \u03b2-barrel. The backbone obtained for the \u03b1\u03b1 motif resembles minimized protein A.\r\n\r\nLast, our backbone design method was evaluated by testing the thermal stability and structural properties of the designed peptides using circular dichroism and 1D nuclear magnetic resonance. From these results, a set of heuristic rules was derived. Taken together, these studies suggest that de novo backbones assembled using our backbone design method may serve as adequate input templates for atomistic sequence selection algorithms.", "doi": "10.7907/MY6J-1191", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:7182", "collection": "thesis", "collection_id": "7182", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08062012-111300229", "primary_object_url": { "basename": "Vaughn_de_1998.pdf", "content": "final", "filesize": 21770345, "license": "other", "mime_type": "application/pdf", "url": "/7182/1/Vaughn_de_1998.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular Mechanism of pH Dependent Antibody Binding: Structure/Function Studies on the Neonatal Fc Receptor", "author": [ { "family_name": "Vaughn", "given_name": "Daniel E.", "clpid": "Vaughn-Daniel-E" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The work described here is an investigation of the molecular mechanism of pH\r\ndependent immunoglobulin G (IgG) binding by the neonatal Fc receptor (FcRn). FcRn\r\nbinds IgG at acidic, but not alkaline pHs, in two important physiological processes. These\r\nprocesses are the acquisition of passive immunity by the fetus or newborn and protecting\r\nIgG from a default degradative pathway.
\r\n\r\nA biosensor assay is used to characterize the interaction of a soluble form of FcRn\r\nwith IgG. Immobilization of FcRn on the biosensor surface reproduces the high affinity\r\nIgG binding observed for membrane bound FcRn, whereas immobilization of IgG results\r\nin lower affinity binding similar to that of the FcRn/IgG interaction in solution. The\r\nstatistical method of cross-validation is used to show that there are two classes of noninteracting binding sites. The IgG binding interaction is characterized for several mutant FcRns with designed amino acid substitutions. These mutations map the functional IgG\r\nbinding site on FcRn.
\r\n\r\nThe structure of FcRn at an alkaline pH is described. This structure determination\r\nreveals an extensive carbohydrate mediated interaction between the dimer related FeRn\r\nmolecules. The physiological relevance of this interaction is discussed in the context of the FcRn dimerization literature. A further refined structure of FcRn at an acidic pH is\r\ndescribed that includes additional carbohydrate structure. These structures are compared\r\nwith specific attention to the pH dependence of FcRn stability and IgG affinity. Finally, a\r\nmechanism for pH dependent antibody binding to FcRn is proposed based on these structures and the body of structure/function literature concerning this interaction.
", "doi": "10.7907/bzyh-hn75", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:6397", "collection": "thesis", "collection_id": "6397", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162011-132252955", "primary_object_url": { "basename": "Su_a_1998.pdf", "content": "final", "filesize": 26600640, "license": "other", "mime_type": "application/pdf", "url": "/6397/1/Su_a_1998.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Backbone Flexibility in Protein Design Theory and Experiment", "author": [ { "family_name": "Su", "given_name": "Yao-ying Alyce", "clpid": "Su-Yao-ying-Alyce" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" }, { "family_name": "Frautschi", "given_name": "Steven C.", "clpid": "Frautschi-S-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Tombrello", "given_name": "Thomas A.", "clpid": "Tombrello-T-A" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The role of backbone flexibility in protein design was studied. First, the effect of explicit backbone motion on the selection of amino acids in protein design was assessed in the core of the streptococcal protein G\u03b21 domain (G\u03b21). Concerted backbone motion was introduced by varying G\u03b21's supersecondary structure parameter values. The stability and structural flexibility of seven of the redesigned proteins were determined experimentally. Core variants containing as many as six of ten possible mutations retained native-like properties. This result demonstrates that backbone flexibility can be combined with amino acid side-chain selection and that the selection algorithm is sufficiently robust to tolerate perturbations as large as 15% of the native parameter values.\r\n\r\nSecond, a general, quantitative design method for computing de novo backbone templates was developed. The method had to compute atomic resolution backbones compatible with the atomistic sequence selection algorithm we were using and it had to be applicable to all protein motifs. We again developed a method that uses super-secondary structure parameters to determine the orientation among secondary structural elements, given a target protein fold. Possible backbone arrangements were screened using a cost function which evaluates core packing, hydrogen bonding, loop closure, and backbone torsional geometry. Given a specified number of residues for each secondary structural element, a family of optimal configurations was found. We chose three motifs to test our method (\u03b2\u03b2\u03b1, \u03b2\u03b1\u03b2, and \u03b1\u03b1) since their combination could be used to approximate most possible backbone fold. The best structure found for the \u03b2\u03b2\u03b1 motif is similar to a zinc finger, and the best structure for the \u03b2\u03b2\u03b1 motif is similar to a segment of a \u03b2-barrel. The backbone obtained for the \u03b1\u03b1 motif resembles minimized protein A.\r\n\r\nLast, our backbone design method was evaluated by testing the thermal stability and structural properties of the designed peptides using circular dichroism and 1D nuclear magnetic resonance. From these results, a set of heuristic rules was derived. Taken together, these studies suggest that de novo backbones assembled using our backbone design method may serve as adequate input templates for atomistic sequence selection algorithms.", "doi": "10.7907/MY6J-1191", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:14037", "collection": "thesis", "collection_id": "14037", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12212020-221414771", "primary_object_url": { "basename": "bradley-jcr_1996.pdf", "content": "final", "filesize": 74031718, "license": "other", "mime_type": "application/pdf", "url": "/14037/1/bradley-jcr_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Molecular Analysis of Olfactory Signal Transduction", "author": [ { "family_name": "Bradley", "given_name": "Jonathan Christopher Robert", "clpid": "Bradley-Jonathan-Christopher-Robert" } ], "thesis_advisor": [ { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" } ], "thesis_committee": [ { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Laurent", "given_name": "Gilles J.", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Olfactory receptor neurons respond to odorant stimulation with a rapid and transient increase in intracellular cAMP that opens cyclic nucleotide-gated (cng) cation channels. Cng channels in rat olfactory neurons are activated by cAMP in the low micromolar range and are outwardly rectifying. The cloned rat olfactory cng channel, (rOCNC1), however, is much less sensitive to cAMP and exhibits very weak rectification. We have investigated this discrepancy between native and cloned channels, and have cloned a new rat cng channel subunit, denoted rOCNC2. rOCNC2 does not form functional channels when expressed alone in HEK 293 cells. When rOCNC1 and rOCNC2 are coexpressed, however, an outwardly rectifying cation conductance with cAMP sensitivity near that of the native channel is observed. In situ hybridization with probes specific for the two subunits shows they are coexpressed in olfactory receptor neurons. Further, subunit specific antibodies coimmunoprecipitate the other subunit from olfactory cilia membrane extracts. These data indicate that the native olfactory cng channel is likely to be a hetero-oligomer of the rOCNC1 and rOCNC2 subunits (Bradley et al. Proc. Natl. Acad. Sci. USA 91, 8890-8894 1994).
\r\n\r\nThe olfactory cng channels are also expressed in non sensory neurons in the brain. We have determined by in situ hybridization, immunocytochemistry, and Western blot that the olfactory cng channels are expressed in the hippocampus, cerebellum, and cortex of adult rats. Cultured hippocampal neurons from embryonic day 17 rats also express the olfactory cng channels as detected by immunofluorescence. Whole cell and excised inside-out patch recordings indicate that these cells have cng channels sensitive to 10\u03bcMcAMP, are outwardly rectifying, and insensitive to block by nickel ions. Consistent with the identification of these channels as the two subunits of the olfactory cng channel.
\r\n\r\nIn order to identify and characterize olfactory receptors for specific odorant chemicals, we have developed a method for generating an electrophysiological signal in response to cAMP elevation in Xenopus oocytes. To do this, we expressed the cystic fibrosis transmembrane regulator (CFTR), a chloride channel that is controlled via phosphorylation by cAMP-dependent protein kinase A (Uezono et al. Receptors and Channels 1:223-241 1993). Pools of synthetic mRNAs from dories of putative olfactory receptor genes were coinjected into oocytes together with CFTR mRNA and tested with odorant mixtures. We have preliminary data indicating that single clones can mediate odorant responses. These responses are quite variable and we have determined using immunofluorescence that this is likely due to a trafficking problem of the expressed receptor protein inside the cell. We have observed this trafficking problem in both the Xenopus oocytes and HEK293 cells. To circumvent this problem we isolated the small fraction (1%) of transfected HEK293 cells that express receptor protein on their surface by fluorescence activated cell sorting (FACS). These cells can then be assayed functionally for odorant interaction using a fura based Ca\u00b2\u207a imaging set-up. Here, the reporter is the cng channels which conduct Ca\u00b2\u207a into the cell in response the receptor mediated rise in intracellular cAMP.
", "doi": "10.7907/9h9a-zx14", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:11901", "collection": "thesis", "collection_id": "11901", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11072019-170728389", "primary_object_url": { "basename": "figl-a-1996.pdf", "content": "final", "filesize": 4412867, "license": "other", "mime_type": "application/pdf", "url": "/11901/1/figl-a-1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Analysis of the \u03b2 Subunit of Neuronal Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Figl", "given_name": "Antonio", "clpid": "Figl-Antonio" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nicotinic receptors belong to the superfamily of ligand-gated ion channels. Since evidence was rapidly accumulating implicating the non-\u03b1 subunits in ligand-binding events, we decided to investigate eventual contributions of the neuronal \u03b2 subunit to these events by performing a series of increasingly detailed experiments on a series of chimeric \u03b2 subunits. In the first set of experiments, we constructed a variety of chimeric \u03b2 subunits consisting of NH2-terminal neuronal \u03b24 sequences and COOH-terminal \u03b22 sequences and expressed them with the \u03b13 subunit in Xenopus oocytes. The results showed that (a) two residues in the extracellular domain of chimeric \u03b24\u2022\u03b22 subunits (108\u03b22Phe\u2194\u03b24Val, 110\u03b22Ser\u2194\u03b24Thr) account for much of the relative cytisine sensitivity; and (b) four extracellular residues of chimeric \u03b24\u2022\u03b22 subunits (112\u03b22Ala\u2194\u03b24Val, 113\u03b22Val\u2194\u03b24Ile and 115\u03b22Ser\u2194\u03b24Arg, 116\u03b22Tyr\u2194\u03b24Ser) account for most of the relative tetramethylammonium sensitivity.
\r\n\r\nEncouraged by the above results, we continued our experiments with additional chimeras of the \u03b22 and \u03b24 neuronal nicotinic subunits to locate regions that contribute to differences between the acetylcholine dose-response relationships of \u03b13\u03b22 and \u03b13\u03b24 receptors. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other, suggesting that amino acids within the first 120 residues of \u03b22 and the corresponding region of \u03b24 contribute to an agonist binding site that bridges the \u03b1 and \u03b2 subunits in neuronal nicotinic receptors.
\r\n\r\nSince the EC50 phenotypes caused by the \u03b22 and \u03b24 subunits could be due to a difference in gating or binding properties, we attempted to unravel this question by performing voltage-jump relaxations for the series of neuronal nicotinic acetylcholine receptors we constructed previously. The chimeric \u03b24/\u03b22 subunits showed a transition in the concentration dependence of the relaxation rate constants in the region between residues 94 and 109, analogous to our previous observation with steady-state dose-response relationships. The data reinforce previous conclusions that the region between residues 94 and 109 on the \u03b2 subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.
", "doi": "10.7907/e390-k051", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:35", "collection": "thesis", "collection_id": "35", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01052007-083947", "primary_object_url": { "basename": "Apperson_ml_1996.pdf", "content": "final", "filesize": 43077789, "license": "other", "mime_type": "application/pdf", "url": "/35/1/Apperson_ml_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular Analysis of the Postsynaptic Density: Cloning and Characterization of Densin-lSO, a Novel Postsynaptic Density-Associated Adhesion Molecule", "author": [ { "family_name": "Apperson", "given_name": "Michelle Louise", "clpid": "Apperson-Michelle-Louise" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Dunphy", "given_name": "William G.", "clpid": "Dunphy-W-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The postsynaptic density (PSD) is an electron dense structure just beneath the postsynaptic membrane. Several functions have been proposed for the PSD including regulating receptor number and clustering, anchoring signal transduction molecules at the synapse and mediating adhesion between the presynaptic and postsynaptic membranes. However, little was known about the proteins that make up the PSD until the biochemical purification of a PSD fraction from brain was established in 1974. Since then, several interesting proteins have been localized to the PSD fraction. The most abundant PSD protein is the [alpha] subunit of the type II calcium/calmodulin dependent protein kinase ([alpha]CaMKII). This protein is likely to play a role in the calcium-mediated signal transduction at the synapse that mediates certain forms of synaptic plasticity. Another major PSD protein is PSD-95, a member of the guanylate kinase family (GUK) of proteins.\r\n\r\nHere, I describe the purification and identification of three additional PSD proteins that comigrate at a molecular weight of 180 kDa on SDS-polyacrylamide gels. First, PSDgp180 is identified as the 2B subunit of the N-methyl-D-aspartate receptor (NR2B). NR2B is a major component of the PSD fraction and binds to PSD-95 in vitro. This interaction may anchor NMDA receptors at the synapse.\r\n\r\nNext, I report the cloning and characterization of densin-180, a 180 kDa PSD protein with a novel adhesion molecule-like sequence. Densin-180 is a brain-specific sialomucin that is enriched in the PSD fraction and localized to the synapse by immunocytochemistry.\r\n\r\nIn order to study the assembly of PSD proteins at the synapse, I use antibodies against [alpha]CaMKII, PSD-95 and densin-180 for double labeling cultured hippocampal neurons. In these cultures, densin-180 protein is the first marker to be expressed and this early densin-180 expression is in a diffuse membrane pattern along dendrites. When synapse formation begins at about 5 days after plating, the densin-180 protein is clustered at synapses and PSD-95 expression is induced. PSD-95 colocalizes with densin-180 clusters. The [alpha]CaMKII protein is expressed later in synapse formation (7 to 9 days in vitro) and may be a marker of mature excitatory neurons.\r\n\r\nIn the brain, densin-180 is localized to the neuropil regions in a punctate pattern likely to represent synaptic staining. In addition, anti-densin-180 is localized to a specific set of cells and that may represent undifferentiated neurons and small processes that may represent dendritic filopodia.\r\n\r\nThe third 180 kDa PSD protein is citron, a recently identified Rho/Rac binding protein. The citron sequence contains numerous motifs found in signal transduction proteins and a myosin-like coiled coil domain. Citron may be a target for Rho/Racdependent signal transduction at the synapse and may mediate physical stabilization of the postsynaptic density.", "doi": "10.7907/ze5z-f533", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:7609", "collection": "thesis", "collection_id": "7609", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04112013-092807415", "primary_object_url": { "basename": "Ryckebusch-sa-1994.pdf", "content": "final", "filesize": 38103119, "license": "other", "mime_type": "application/pdf", "url": "/7609/1/Ryckebusch-sa-1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Central Nervous Control of Walking in the Locust Schistocerca americana", "author": [ { "family_name": "Ryckebusch", "given_name": "Sylvie Adrienne", "clpid": "Ryckebusch-Sylvie-Adrienne" } ], "thesis_advisor": [ { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Mead", "given_name": "Carver", "orcid": "0000-0003-4051-0462", "clpid": "Mead-C-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Barr", "given_name": "Alan H.", "clpid": "Barr-A-H" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Mead", "given_name": "Carver", "orcid": "0000-0003-4051-0462", "clpid": "Mead-C-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Rhythmic motor behaviors in all animals appear to be under the control of \"central pattern generator\" circuits, neural circuits which can produce output patterns appropriate for behavior even when isolated from their normal peripheral inputs. Insects have been a useful model system in which to study the control of legged terrestrial locomotion. Much is known about walking in insects at the behavioral level, but to date there has been no clear demonstration that a central pattern generator for walking exists. The focus of this thesis is to explore the central neural basis for locomotion in the locust, Schistocerca americana.
\r\n\r\nRhythmic motor patterns could be evoked in leg motor neurons of isolated thoracic ganglia of locusts by the muscarinic agonist pilocarpine. These motor patterns would be appropriate for the movement of single legs during walking. Rhythmic patterns could be evoked in all three thoracic ganglia, but the segmental rhythms differed in their sensitivities to pilocarpine, their frequencies, and the phase relationships of motor neuron antagonists. These different patterns could be generated by a simple adaptable model circuit, which was both simulated and implemented in VLSI hardware. The intersegmental coordination of leg motor rhythms was then examined in preparations of isolated chains of thoracic ganglia. Correlations between motor patterns in different thoracic ganglia indicated that central coupling between segmental pattern generators is likely to contribute to the coordination of the legs during walking.
\r\n\r\nThe work described here clearly demonstrates that segmental pattern generators for walking exist in insects. The pattern generators produce motor outputs which are likely to contribute to the coordination of the joints of a limb, as well as the coordination of different limbs. These studies lay the groundwork for further studies to determine the relative contributions of central and sensory neural mechanisms to terrestrial walking.
\r\n", "doi": "10.7907/1aey-8096", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7707", "collection": "thesis", "collection_id": "7707", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-132218781", "primary_object_url": { "basename": "McDonough 1994.pdf", "content": "final", "filesize": 30805977, "license": "other", "mime_type": "application/pdf", "url": "/7707/1/McDonough 1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Pharmacology and pore-forming domains of the cystic fibrosis transmembrane conductance regulator", "author": [ { "family_name": "McDonough", "given_name": "Stefan I.", "clpid": "McDonough-S-I" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The cystic fibrosis transmembrane conductance regulator (CFTR) is a\r\nchloride channel member of the ATP-binding cassette (ABC) superfamily of\r\nmembrane proteins. CFTR has two homologous halves, each consisting of six\r\ntransmembrane spanning domains (TM) followed by a nucleotide binding fold,\r\nconnected by a regulatory (R) domain. This thesis addresses the question of\r\nwhich domains are responsible for Cl^- selectivity, i.e., which domains line the\r\nchannel pore.
\r\n\r\nTo address this question, novel blockers of CFTR were characterized.\r\nCFTR was heterologously expressed in Xenopus oocytes to study the\r\nmechanism of block by two closely related arylaminobenzoates,\r\ndiphenylamine-2-carboxylic acid (DPC) and flufenamic acid (FFA). Block by\r\nboth is voltage-dependent, with a binding site \u2248 40% through the electric field\r\nof the membrane. DPC and FFA can both reach their binding site from either\r\nside of the membrane to produce a flickering block of CFTR single channels.\r\nIn addition, DPC block is influenced by Cl^- concentration, and DPC blocks with\r\na bimolecular forward binding rate and a unimolecular dissociation rate.\r\nTherefore, DPC and FFA are open-channel blockers of CFTR, and a residue of\r\nCFTR whose mutation affects their binding must line the pore.
\r\n\r\nScreening of site-directed mutants for altered DPC binding affinity\r\nreveals that TM-6 and TM-12 line the pore. Mutation of residue 5341 in TM-6\r\nabolishes most DPC block, greatly reduces single-channel conductance, and\r\nalters the direction of current rectification. Additional residues are found in\r\nTM-6 (K335) and TM-12 (T1134) whose mutations weaken or strengthen DPC\r\nblock; other mutations move the DPC binding site from TM-6 to TM-12. The\r\nstrengthened block and lower conductance due to mutation T1134F is\r\nquantitated at the single-channel level. The geometry of DPC and of the\r\nresidues mutated suggest \u03b1-helical structures for TM-6 and TM-12. Evidence is\r\npresented that the effects of the mutations are due to direct side-chain\r\ninteraction, and not to allosteric effects propagated through the protein.\r\nMutations are also made in TM-11, including mutation S1118F, which gives\r\nvoltage-dependent current relaxations. The results may guide future studies on\r\npermeation through ABC transporters and through other Cl^- channels.
\r\n", "doi": "10.7907/eyre-8424", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:2628", "collection": "thesis", "collection_id": "2628", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06172004-110344", "primary_object_url": { "basename": "Adolphs_r_1993.pdf", "content": "final", "filesize": 12552657, "license": "other", "mime_type": "application/pdf", "url": "/2628/1/Adolphs_r_1993.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Processing of Interaural Level Differences in the Auditory Brainstem of the Barn Owl", "author": [ { "family_name": "Adolphs", "given_name": "Ralph", "orcid": "0000-0002-8053-9692", "clpid": "Adolphs-Ralph" } ], "thesis_advisor": [ { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" } ], "thesis_committee": [ { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Nervous systems process information about the environment in order to generate adaptive behavior. Sensory information that is obtained through different modalities, gleaned from different interactions of the animal with its surroundings, generated by different neural algorithms, or used to infer different distal stimulus properties, is often processed by distinct pathways in the brain. The auditory system compares sounds at the two ears in order to derive the location of the source. In the barn owl, Tyto alba, this is accomplished by interaural comparisons of the time that a sound reaches each ear, and of the level (intensity) at each ear. Interaural time differences code for horizontal positions of sound sources while interaural level differences, due to a vertical asymmetry in the owl's ears, can encode the vertical position of a sound. These two cues together can assign unique locations to sound sources in space. The barn owl processes time- and level differences in separate neural channels that converge in the inferior colliculus. This structure is the first site of neurons with spatially restricted auditory receptive fields, and with a neural map of auditory space. Downstream projections from here provide the sensory input for accurate sound localization by saccadic head movements.
\r\n\r\nI report that the owl's two auditory processing streams are also segregated histochemically. The pathway that computes level differences stains especially strongly for the enzyme acetylcholinesterase, which may underlay processing of scalar (intensity) information over large dynamic ranges. This staining is complementary to immunohistochemical staining for calbindin, which has been shown previously to stain the pathway that processes interaural time differences.
\r\n\r\nIn further hodological and physiological experiments, I describe the algorithms that generate tuned responses in the inferior colliculus that encode vertical sound source position. This study shows that a lemniscal nucleus, nucleus ventralis lemnisci lateralis pars posterior (VLVp), projects bilaterally to a subdivision of the inferior colliculus (the shell of ICc). This projection appears to preserve tonotopy, and to provide inhibition by sounds of large interaural level difference. This probably GABAergic mechanism leads to the synthesis of neuronal responses in the inferior colliculus that are narrowly tuned to interaural level difference. My methodological strategy was to increase or decrease activity in VLVp by injection of blockers or agonists of GABA-A receptors, and then to record downstream in the inferior colliculus any changes in response tuning that resulted. The study suggests that the bilateral inhibition by VLVp is sufficient to explain the peaked responses to level differences of collicular neurons. Excitatory input to the inferior colliculus is conveyed by fibers of the lateral lemniscus, and may arise from a number of stations, including lemniscal and cochlear nuclei. The circuits I describe determine the tuning of cells to interaural level differences, but are independent of and have no effect on the tuning to interaural time differences, and further support that time and level are processed separately in the owl's brainstem.
\r\n", "doi": "10.7907/QN2E-YR81", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:2628", "collection": "thesis", "collection_id": "2628", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06172004-110344", "primary_object_url": { "basename": "Adolphs_r_1993.pdf", "content": "final", "filesize": 12552657, "license": "other", "mime_type": "application/pdf", "url": "/2628/1/Adolphs_r_1993.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Processing of Interaural Level Differences in the Auditory Brainstem of the Barn Owl", "author": [ { "family_name": "Adolphs", "given_name": "Ralph", "orcid": "0000-0002-8053-9692", "clpid": "Adolphs-Ralph" } ], "thesis_advisor": [ { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" } ], "thesis_committee": [ { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Nervous systems process information about the environment in order to generate adaptive behavior. Sensory information that is obtained through different modalities, gleaned from different interactions of the animal with its surroundings, generated by different neural algorithms, or used to infer different distal stimulus properties, is often processed by distinct pathways in the brain. The auditory system compares sounds at the two ears in order to derive the location of the source. In the barn owl, Tyto alba, this is accomplished by interaural comparisons of the time that a sound reaches each ear, and of the level (intensity) at each ear. Interaural time differences code for horizontal positions of sound sources while interaural level differences, due to a vertical asymmetry in the owl's ears, can encode the vertical position of a sound. These two cues together can assign unique locations to sound sources in space. The barn owl processes time- and level differences in separate neural channels that converge in the inferior colliculus. This structure is the first site of neurons with spatially restricted auditory receptive fields, and with a neural map of auditory space. Downstream projections from here provide the sensory input for accurate sound localization by saccadic head movements.
\r\n\r\nI report that the owl's two auditory processing streams are also segregated histochemically. The pathway that computes level differences stains especially strongly for the enzyme acetylcholinesterase, which may underlay processing of scalar (intensity) information over large dynamic ranges. This staining is complementary to immunohistochemical staining for calbindin, which has been shown previously to stain the pathway that processes interaural time differences.
\r\n\r\nIn further hodological and physiological experiments, I describe the algorithms that generate tuned responses in the inferior colliculus that encode vertical sound source position. This study shows that a lemniscal nucleus, nucleus ventralis lemnisci lateralis pars posterior (VLVp), projects bilaterally to a subdivision of the inferior colliculus (the shell of ICc). This projection appears to preserve tonotopy, and to provide inhibition by sounds of large interaural level difference. This probably GABAergic mechanism leads to the synthesis of neuronal responses in the inferior colliculus that are narrowly tuned to interaural level difference. My methodological strategy was to increase or decrease activity in VLVp by injection of blockers or agonists of GABA-A receptors, and then to record downstream in the inferior colliculus any changes in response tuning that resulted. The study suggests that the bilateral inhibition by VLVp is sufficient to explain the peaked responses to level differences of collicular neurons. Excitatory input to the inferior colliculus is conveyed by fibers of the lateral lemniscus, and may arise from a number of stations, including lemniscal and cochlear nuclei. The circuits I describe determine the tuning of cells to interaural level differences, but are independent of and have no effect on the tuning to interaural time differences, and further support that time and level are processed separately in the owl's brainstem.
\r\n", "doi": "10.7907/QN2E-YR81", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7289", "collection": "thesis", "collection_id": "7289", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11282012-105047936", "primary_object_url": { "basename": "Bernander_jo_1993.pdf", "content": "final", "filesize": 46272689, "license": "other", "mime_type": "application/pdf", "url": "/7289/1/Bernander_jo_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Synaptic Integration and its Control in Neocortical Pyramidal Cells", "author": [ { "family_name": "Bernander", "given_name": "Jan \u04e6jvind", "clpid": "Bernander-Jan-\u04e6jvind" } ], "thesis_advisor": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "thesis_committee": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Barr", "given_name": "Alan H.", "clpid": "Barr-A-H" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The main goal of this thesis is to investigate the input/output relationship of single regular-firing neocortical pyramidal neurons and how this relationship can be controlled by external inputs. The thesis can be divided into three main parts. First, a detailed single cell model was developed, based on the morphology of reconstructed cells and experimental values for membrane conductances and synaptic input distributions. This model was used for the following investigations.
\r\n\r\nSecond, the spatio-temporal integration of single and multiple inputs was studied. Several measures for the efficacy and time delay of single synapses were defined and shown to vary dramatically. For example, a somatic synapse was only 2.2 times stronger than a very distal synapse using the charge attenuation measure, but more than 450 times stronger in the voltage attenuation measure. The effect that temporal synchronicity of multiple inputs had on firing rate was shown to vary with the number of inputs: for just-threshold input rates, synchronicity increased firing rate; for large inputs, high synchronicity strongly reduced firing rate, due to inputs being \"wasted\" during the refractory period.
\r\n\r\nThird, a subset of the inputs were considered to constitute a control signal, and their effect on other inputs was studied for three cases. The first case considers the level of synaptic background activity to be a control signal; since each synapse is a small conductance change, and not a voltage-independent current source, the sum total of all \"background\" synapses will constitute the lion's share of the membrane conductance. The background firing rate, f_b, will therefore determine the electrotonic structure of the cell. For f_b in the range of 0-10 H z, a more than 10-fold decrease was seen in both input resistance (50.4-5.1 M\u03a9) and membrane time constant (33.7-1.6 msec). Electrotonic length and resting potential were similarly affected. The second case treats input to the apical trunk as the control signal; when this input was weak and excitatory, the more distal input to the apical tuft could be facilitated, but when this input was strong or combined with inhibition, more distal input were reduced. The third case involves distributing two types of active conductances throughout the apical dendrites. The activation curves of these conductances were \"designed\" to ensure that the current delivered to the soma was linear in the input rate and amplified, since a passive tree strongly attenuates large apical inputs. The linearization was implemented with a persistent potassium conductance in the superficial layer I- III and the amplification with a persistent calcium conductance in the apical trunk (layer IV). The amplification gain could be set arbitrarily by modulating the channel density of either the potassium or calcium conductance.
", "doi": "10.7907/rz58-rh86", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7289", "collection": "thesis", "collection_id": "7289", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11282012-105047936", "primary_object_url": { "basename": "Bernander_jo_1993.pdf", "content": "final", "filesize": 46272689, "license": "other", "mime_type": "application/pdf", "url": "/7289/1/Bernander_jo_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Synaptic Integration and its Control in Neocortical Pyramidal Cells", "author": [ { "family_name": "Bernander", "given_name": "Jan \u04e6jvind", "clpid": "Bernander-Jan-\u04e6jvind" } ], "thesis_advisor": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" } ], "thesis_committee": [ { "family_name": "Koch", "given_name": "Christof", "orcid": "0000-0001-6482-8067", "clpid": "Koch-C" }, { "family_name": "Allman", "given_name": "John Morgan", "clpid": "Allman-J-M" }, { "family_name": "Barr", "given_name": "Alan H.", "clpid": "Barr-A-H" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The main goal of this thesis is to investigate the input/output relationship of single regular-firing neocortical pyramidal neurons and how this relationship can be controlled by external inputs. The thesis can be divided into three main parts. First, a detailed single cell model was developed, based on the morphology of reconstructed cells and experimental values for membrane conductances and synaptic input distributions. This model was used for the following investigations.
\r\n\r\nSecond, the spatio-temporal integration of single and multiple inputs was studied. Several measures for the efficacy and time delay of single synapses were defined and shown to vary dramatically. For example, a somatic synapse was only 2.2 times stronger than a very distal synapse using the charge attenuation measure, but more than 450 times stronger in the voltage attenuation measure. The effect that temporal synchronicity of multiple inputs had on firing rate was shown to vary with the number of inputs: for just-threshold input rates, synchronicity increased firing rate; for large inputs, high synchronicity strongly reduced firing rate, due to inputs being \"wasted\" during the refractory period.
\r\n\r\nThird, a subset of the inputs were considered to constitute a control signal, and their effect on other inputs was studied for three cases. The first case considers the level of synaptic background activity to be a control signal; since each synapse is a small conductance change, and not a voltage-independent current source, the sum total of all \"background\" synapses will constitute the lion's share of the membrane conductance. The background firing rate, f_b, will therefore determine the electrotonic structure of the cell. For f_b in the range of 0-10 H z, a more than 10-fold decrease was seen in both input resistance (50.4-5.1 M\u03a9) and membrane time constant (33.7-1.6 msec). Electrotonic length and resting potential were similarly affected. The second case treats input to the apical trunk as the control signal; when this input was weak and excitatory, the more distal input to the apical tuft could be facilitated, but when this input was strong or combined with inhibition, more distal input were reduced. The third case involves distributing two types of active conductances throughout the apical dendrites. The activation curves of these conductances were \"designed\" to ensure that the current delivered to the soma was linear in the input rate and amplified, since a passive tree strongly attenuates large apical inputs. The linearization was implemented with a persistent potassium conductance in the superficial layer I- III and the amplification with a persistent calcium conductance in the apical trunk (layer IV). The amplification gain could be set arbitrarily by modulating the channel density of either the potassium or calcium conductance.
", "doi": "10.7907/rz58-rh86", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:2922", "collection": "thesis", "collection_id": "2922", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07182007-074159", "primary_object_url": { "basename": "McCormack_k_1991.pdf", "content": "final", "filesize": 4889181, "license": "other", "mime_type": "application/pdf", "url": "/2922/1/McCormack_k_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-function studies of Drosophila shaker potassium channels", "author": [ { "family_name": "McCormack", "given_name": "Ken", "clpid": "McCormack-K" } ], "thesis_advisor": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "thesis_committee": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Voltage-dependent ion channels mediate electrical signals in the nervous system; many sodium (Na+), calcium (Ca++) and potassium (K+) selective channels are structurally related, and thus represent a family. These proteins undergo interesting conformational changes in response to alterations in transmembrane potential. However, the functional determinants involved in these transitions are not well understood. Chapters 2A and 2B describe the identification and characterization of an amino acid sequence motif (a leucine-heptad repeat) that is evolutionarily conserved among this family of voltage-dependent ion channels. Conservative, single amino-acid substitutions within this region of Drosophila Shaker (Sh) proteins have substantial effects on the voltage-dependence of activation. The observed alterations suggest that the heptad-repeat region is an important determinant in the conformational transitions leading to channel opening.\n\nNa+ and Ca++ channels are composed of four homologous domains, each of which is equivalent to a single K+ channel subunit. Thus, K+ channels are thought to be functional multimers. Furthermore, there are a large number of different voltage-dependent K+ genes and alternatively spliced products that potentially can be expressed in the same cell. Therefore, the potential number of different K+ channel multimers could be quite extensive. Chapter 3 describes the physiological characteristics of combinations of K+ channels belonging to the Sh family that have been coexpressed in Xenopus oocytes. Members of the same molecular class of Sh channel form heteromultimers with novel functional properties, adding to the diversity of K+ channel function. Members of different molecular classes do not form heteromultimeric channels, suggesting that there are distinct K+ channel systems. The Appendix describes an alternative exon in the \"constant\" region of the Drosophila Sh gene, the existence of which suggests, that the molecular diversity of this gene is greater than previously determined.", "doi": "10.7907/63q7-hw24", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2842", "collection": "thesis", "collection_id": "2842", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07092007-132214", "primary_object_url": { "basename": "Lochrie_ma_1991.pdf", "content": "final", "filesize": 10397132, "license": "other", "mime_type": "application/pdf", "url": "/2842/1/Lochrie_ma_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans", "author": [ { "family_name": "Lochrie", "given_name": "Michael Alan", "clpid": "Lochrie-M-A" } ], "thesis_advisor": [ { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "thesis_committee": [ { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis examines the molecular biology of G protein alpha subunits from bovine photoreceptors and the nematode Caenorhabditis elegans.\n\nChapter one describes the nucleotide sequence of the bovine cone photoreceptor transducin alpha subunit (Tc[alpha]). Analysis of the sequence defined regions homologous to other GTP binding proteins which may be involved in guanine nucleotide binding, allowed the positions of amino acids which are ADP-ribosylated by pertussis toxin and cholera toxin to be determined, and led to the prediction that G proteins are posttranslationally modified with lipids, which serve to anchor G protein alpha subunits to membranes. Comparison of the Tc[alpha] amino acid sequence with other alpha subunit sequences as they became available provided the first indications that G proteins would be more numerous and diverse than previously thought. The diversity observed among G protein subunits and its structural and functional implications are reviewed in the introduction.\n\nIn Chapter 2 the characterization of G protein alpha subunits in the nematode C. elegans is described. Two genes were isolated and their DNA sequences were determined. The protein products of these genes appear to be unique to C. elegans. A cDNA encoding a homolog of Go[alpha] was also isolated and sequenced. Thus, C. elegans has identifiable homologs of mammalian G proteins as well as G proteins that may be unique to it. The chromosomal positions of the genes were determined. Each maps to a unique location near mutations that could be in G protein alpha subunits.\n\nIn Appendix 1 the characterization of photoreceptor specific gene expression in human retinoblastoma cultures is described. These cells express cone photoreceptor-specific genes, but not rod photoreceptor specific genes. Therefore they may provide a system for studying the DNA elements required for the expression of genes specifically in cone cells.\n\nAppendix 2 surveys systems for the heterologous expression of transducin alpha subunits in E. coli, yeast, and insect cells. The E. coli expression system offers the most promise for obtaining adequate amounts of active, pure protein.", "doi": "10.7907/zqph-5443", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:2970", "collection": "thesis", "collection_id": "2970", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07232007-144526", "primary_object_url": { "basename": "Patton_bl_1991.pdf", "content": "final", "filesize": 15997429, "license": "other", "mime_type": "application/pdf", "url": "/2970/1/Patton_bl_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Autophosphorylation Sites of the Type II Ca\u00b2\u207a/Calmodulin-Dependent Protein Kinase: Identification, Regulation of Kinase Activity, and Site-Specific Antibodies", "author": [ { "family_name": "Patton", "given_name": "Bruce Lowell", "clpid": "Patton-Bruce-Lowell" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Biochemical and immunological approaches have been developed to study the regulation of the rat neuronal type II Ca\u00b2\u207a/calmodulin-dependent protein kinase (type II CaM kinase) by autophosphorylation. This thesis describes the identification of in vitro autophosphorylation sites on the CaM kinase and their role in regulating the catalytic activity of the CaM kinase. In addition, this thesis describes the development of antibodies against the type II CaM kinase that specifically recognize either the autophosphorylated kinase or the nonphosphorylated kinase.
\r\n\r\nThe autophosphorylation sites on in vitro autophosphorylated type II CaM kinase were identified by tryptic phosphopeptide mapping using reverse phase HPLC to isolate individual autophosphorylation sites. The sequence of the purified phosphopeptides was determined by gas phase microsequencing and compared to the known sequences of the kinase subunits, deduced from the cDNAs encoding them. The rates of site-specific autophosphorylation, or dephosphorylation by protein phosphatases, was compared with the rate of change in the Ca\u00b2\u207a/calmodulin-dependence of kinase catalytic activity. In the presence of Ca\u00b2\u207a and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the \u03b1 and \u03b2 subunits of the type II CaM kinase, Thr\u00b2\u2078\u2076 and Thr\u00b2\u2078\u2077, respectively. Phosphorylation of this site correlated with the generation of Ca\u00b2\u207a-independent catalytic activity. Removal of free Ca\u00b2\u207a ion from the autophosphorylation reaction resulted in the autophosphorylation of two pairs of homologous residues, Thr\u00b3\u2070\u2075 and Ser\u00b3\u00b9\u2074 in the a subunit a and Thr\u00b3\u2070\u2076 and Ser\u00b3\u00b9\u2075 in the kinase catalytic activity. In the presence of Ca\u00b2\u207a and calmodulin, type II CaM kinase autophosphorylated an homologous residue in the \u03b2 subunit. Ser\u00b3\u00b9\u2074/\u00b3\u00b9\u2075 is resistant to dephosphorylation by purified protein phosphatases 1 and 2A. Selective dephosphorylation of the Thr\u00b3\u2070\u2075/\u00b3\u2070\u2076 autophosphorylation site demonstrated that the presence of phosphate on Thr\u00b3\u2070\u2075/\u00b3\u2070\u2076 inhibits Ca\u00b2\u207a/calmodulin-stimulated catalytic activity. The presence of phosphate on Ser\u00b3\u00b9\u2074/\u00b3\u00b9\u2075 slightly decreases the sensitivity of the kinase to Ca\u00b2\u207a/calmodulin.
\r\n\r\nAntibodies that bind to the type II CaM kinase at the Thr\u00b2\u2078\u2076/\u00b2\u2078\u2077 autophosphorylation site were produced in note and rabbits by immunization with thiophosphorylated and nonphosphorylated peptide haptens. A monoclonal antibody was obtained that specifically recognized the autophosphorylated type IICaM kinase. The monoclonal antibody recognized the Thr\u00b2\u2078\u2076/\u00b2\u2078\u2077 autophosphorylation site. A polyclonal antisera was obtained that, when affinity purified, specifically recognized the nonphosphorylated type II CaM kinase. Autophosphorylation of type II CaM kinase on Thr\u00b2\u2078\u2077 potently inhibited binding of the polyclonal antibodies. The monoclonal antibody and polyclonal antisera recognized type II CaM kinase in immunocytochemical sections and were used to assess the extent and distribution type II CaM kinase autophosphorylation in organotypic cultures of rat brain hippocampal slices. Double immunofluorescence immunocytochemistry with the antibodies specific for phosphorylated and nonphosphorylated type II CaM kinase indicated that most neurons and dendrites contain a mixture of phosphorylated and nonphosphorylated kinase, in varying proportions. Removal of extracellular Ca\u00b2\u207a greatly reduced the immunoreactivity specific for the phosphorylated kinase, implying that the type II CaM kinase phosphorylation state is in dynamic equilibrium in neurons.
", "doi": "10.7907/ete7-xp75", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:7829", "collection": "thesis", "collection_id": "7829", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042013-091916969", "type": "thesis", "title": "Electron Transfer in Cytochrome c Oxidase: The Rate of Electron Equillibration Between Cytochrome a and Copper A", "author": [ { "family_name": "Morgan", "given_name": "Joel Edgar", "clpid": "Morgan-Joel-Edgar" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by means of perturbed equilibrium techniques. We have prepared a three electron reduced, CO inhibited form of the enzyme in which cytochrome a and copper A are partially reduced an in intramolecular redox equilibrium. When these samples were photolyzed using a nitrogen laser (0.6 \u00b5s, 1.0 mJ pulses) changes in absorbance at 598 nm and 830 nm were observed which are consistent with a fast electron from cytochrome a to copper A. The absorbance changes at 598 nm have an apparent rate of 17,200 \u00b1 1,700 s-1 (1\u03c3), at pH 7.0 and 25.5 \u00b0C. These changes were not observed in either the CO mixed valence or CO inhibited fully reduced forms of the enzyme. The rate is fastest at about pH 8.0, and falls off in either direction, and there is a small, but clear temperature dependence. The process was also observed in the cytochrome c -- cytochrome c oxidase high affinity complex.
\r\n\r\nThis rate is far faster than any rate measured or inferred previously for the cytochrome a -- copper A electron equilibration, but the interpretation of these results is hampered by the fact that the relaxation could only be followed during the time before CO became rebound to the oxygen binding site. The meaning of our our measured rate is discussed, along with other reported rates for this process. In addition, a temperature-jump experiment on the same system is discussed.
\r\n\r\nWe have also prepared a partially reduced, cyanide inhibited form of the enzyme in which cytochrome a, copper A and copper B are partially reduced and in redox equilibrium. Warming these samples produced absorbance changes at 605 nm which indicate that cytochrome a was becoming more oxidized, but there were no parallel changes in absorbance at 830 nm as would be expected if copper A was becoming reduced. We concluded that electrons were being redistributed from cytochrome a to copper B. The kinetics of the absorbance changes at 605 nm were investigated by temperature-jump methods. Although a rate could not be resolved, we concluded that the process must occur with an (apparent) rate larger than 10,000 s-1.
\r\n\r\nDuring the course of the temperature-jump experiments, we also found that non-redox related, temperature dependent absorbance changes in fully reduced CO inhibited cytochrome c oxidase, and in the cyanide mixed valence enzyme, took place with an (apparent) rate faster that 30,000 s-1.
", "doi": "10.7907/88fd-2939", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:7829", "collection": "thesis", "collection_id": "7829", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042013-091916969", "type": "thesis", "title": "Electron Transfer in Cytochrome c Oxidase: The Rate of Electron Equillibration Between Cytochrome a and Copper A", "author": [ { "family_name": "Morgan", "given_name": "Joel Edgar", "clpid": "Morgan-Joel-Edgar" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by means of perturbed equilibrium techniques. We have prepared a three electron reduced, CO inhibited form of the enzyme in which cytochrome a and copper A are partially reduced an in intramolecular redox equilibrium. When these samples were photolyzed using a nitrogen laser (0.6 \u00b5s, 1.0 mJ pulses) changes in absorbance at 598 nm and 830 nm were observed which are consistent with a fast electron from cytochrome a to copper A. The absorbance changes at 598 nm have an apparent rate of 17,200 \u00b1 1,700 s-1 (1\u03c3), at pH 7.0 and 25.5 \u00b0C. These changes were not observed in either the CO mixed valence or CO inhibited fully reduced forms of the enzyme. The rate is fastest at about pH 8.0, and falls off in either direction, and there is a small, but clear temperature dependence. The process was also observed in the cytochrome c -- cytochrome c oxidase high affinity complex.
\r\n\r\nThis rate is far faster than any rate measured or inferred previously for the cytochrome a -- copper A electron equilibration, but the interpretation of these results is hampered by the fact that the relaxation could only be followed during the time before CO became rebound to the oxygen binding site. The meaning of our our measured rate is discussed, along with other reported rates for this process. In addition, a temperature-jump experiment on the same system is discussed.
\r\n\r\nWe have also prepared a partially reduced, cyanide inhibited form of the enzyme in which cytochrome a, copper A and copper B are partially reduced and in redox equilibrium. Warming these samples produced absorbance changes at 605 nm which indicate that cytochrome a was becoming more oxidized, but there were no parallel changes in absorbance at 830 nm as would be expected if copper A was becoming reduced. We concluded that electrons were being redistributed from cytochrome a to copper B. The kinetics of the absorbance changes at 605 nm were investigated by temperature-jump methods. Although a rate could not be resolved, we concluded that the process must occur with an (apparent) rate larger than 10,000 s-1.
\r\n\r\nDuring the course of the temperature-jump experiments, we also found that non-redox related, temperature dependent absorbance changes in fully reduced CO inhibited cytochrome c oxidase, and in the cyanide mixed valence enzyme, took place with an (apparent) rate faster that 30,000 s-1.
", "doi": "10.7907/88fd-2939", "publication_date": "1989", "thesis_type": "phd", "thesis_year": "1989" }, { "id": "thesis:4478", "collection": "thesis", "collection_id": "4478", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11092007-084226", "type": "thesis", "title": "Neuron-Microdevice Connections", "author": [ { "family_name": "Regehr", "given_name": "Wade Gordon", "clpid": "Regehr-Wade-Gordon" } ], "thesis_advisor": [ { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" } ], "thesis_committee": [ { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" }, { "family_name": "Bellan", "given_name": "Paul Murray", "clpid": "Bellan-P-M" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "A new method for long-term recording and stimulation applicable to cultured neurons has been developed. Silicon-based microelectrodes have been fabricated using integrated-circuit technology and micromachining. The chronic connection is made by positioning the electrode tip into contact with the cell body, and gluing the device to the bottom of the culture dish. These \"diving-board electrodes\" consist of an insulated lead exposed only at the tip sealed to the cell body of a cultured neuron. A two-way electrical connection to Helisoma B19 neurons has been established for up to four days. Preliminary experiments with cultured superior cervical ganglion neurons indicate diving-board electrodes can be used with cultured neurons larger than 20 \u00b5m in diameter.
\r\n\r\nIn a related technique Helisoma neurons grown on special dish containing a multielectrode array were found to seal to the dish electrodes, establishing similar long-term connections. This capability will make it possible to conduct experiments with either diving-board electrodes or dishes that cannot be performed using conventional techniques.
", "doi": "10.7907/nshf-ww49", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7493", "collection": "thesis", "collection_id": "7493", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02222013-111607349", "type": "thesis", "title": "Models for the Avoidance Response in Phycomyces", "author": [ { "family_name": "Matus Bloch", "given_name": "Ivan J.", "clpid": "Matus-Bloch-Ivan-J" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The sporangiophore of the fungus Phycomyces is able to avoid obstacles placed a few millimeters from its growing zone. The work described in this thesis presents evidence that the avoidance response is mediated by gases. Avoidance occurs in still air in the diffusion limit, even at relative humidities close to 100%. It is shown that the effect of the surfaces of the obstacles cannot only be to reflect these gases: the surfaces must play a more active role. Models in which the surfaces adsorb growth-inhibitors or adsorb an inert precursor that is re-emitted as a growth-promoter that decays are in agreement with our experimental observations.
", "doi": "10.7907/c5e3-v765", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7493", "collection": "thesis", "collection_id": "7493", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02222013-111607349", "type": "thesis", "title": "Models for the Avoidance Response in Phycomyces", "author": [ { "family_name": "Matus Bloch", "given_name": "Ivan J.", "clpid": "Matus-Bloch-Ivan-J" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The sporangiophore of the fungus Phycomyces is able to avoid obstacles placed a few millimeters from its growing zone. The work described in this thesis presents evidence that the avoidance response is mediated by gases. Avoidance occurs in still air in the diffusion limit, even at relative humidities close to 100%. It is shown that the effect of the surfaces of the obstacles cannot only be to reflect these gases: the surfaces must play a more active role. Models in which the surfaces adsorb growth-inhibitors or adsorb an inert precursor that is re-emitted as a growth-promoter that decays are in agreement with our experimental observations.
", "doi": "10.7907/c5e3-v765", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7512", "collection": "thesis", "collection_id": "7512", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03122013-111504785", "primary_object_url": { "basename": "Soha_jm_1988.pdf", "content": "final", "filesize": 53092840, "license": "other", "mime_type": "application/pdf", "url": "/7512/1/Soha_jm_1988.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Specificity and Competition During Maturation of Neuromuscular Synapses", "author": [ { "family_name": "Soha", "given_name": "James Martin", "clpid": "Soha-James-Martin" } ], "thesis_advisor": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "thesis_committee": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread. Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established. I have investigated this possibility by studying the degree of specificity during reinnervation of neonatal rabbit soleus muscle. Fiber type composition was assayed by measuring the twitch rise times of motor units within two days of the onset of functional reinnervation. In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times. Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting muscle fibers. An alternative possibility, that specific reinnervation was masked by contractile de-differentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval. Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation. Computer simulations were employed to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation. Based on this analysis, I conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied.
\r\n\r\nIn a second experiment, I compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. No significant difference in the time course of synapse elimination was observed for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low.
\r\n\r\nIn a third experiment, I examined a phenomenon in which compound endplate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. Several lines of evidence indicate that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact, and that genuinely polyinnervated fibers are very rare at this stage, if present at all.
\r\n\r\nA computer model of neuromuscular synapse elimination was developed to serve as an analytical tool in exploring the potential roles of candidate mechanisms in regulating the normal process and in shaping its response to experimental perturbations. Synapse elimination is a complex process likely to involve the dynamic interaction of several specific mechanisms. This situation limits the reliability of a strictly inductive theoretical investigation into how these mechanisms might act. Three mechanisms which have been previously proposed and discussed in the literature are simulated, including a synaptic stabilization molecule, a muscle derived trophic factor, and a hypothesized intrinsic tendency of motor neurons to limit their arbor. The model is stochastic rather than deterministic in character, and is also dynamic, tracing the growth and retraction of individual presynaptic terminals at each iteration as they compete for limited synaptic space.
\r\n\r\nNine experimental observations were selected to guide development of the model and evaluate its performance. All but one of the experimental observations canbe simulated by at least one of the mechanisms studied. No single mechanism, however, is adequate to duplicate the entire body of experimental evidence. A relative advantage for larger terminals appears critical for convergence in both the scaffolding and trophic factor mechanisms. Several alternative roles for activity are compared.
", "doi": "10.7907/3ebg-mv36", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7512", "collection": "thesis", "collection_id": "7512", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03122013-111504785", "primary_object_url": { "basename": "Soha_jm_1988.pdf", "content": "final", "filesize": 53092840, "license": "other", "mime_type": "application/pdf", "url": "/7512/1/Soha_jm_1988.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Specificity and Competition During Maturation of Neuromuscular Synapses", "author": [ { "family_name": "Soha", "given_name": "James Martin", "clpid": "Soha-James-Martin" } ], "thesis_advisor": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "thesis_committee": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Konishi", "given_name": "Masakazu", "clpid": "Konishi-M" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Bower", "given_name": "James M.", "clpid": "Bower-J-M" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread. Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established. I have investigated this possibility by studying the degree of specificity during reinnervation of neonatal rabbit soleus muscle. Fiber type composition was assayed by measuring the twitch rise times of motor units within two days of the onset of functional reinnervation. In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times. Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting muscle fibers. An alternative possibility, that specific reinnervation was masked by contractile de-differentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval. Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation. Computer simulations were employed to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation. Based on this analysis, I conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied.
\r\n\r\nIn a second experiment, I compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. No significant difference in the time course of synapse elimination was observed for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low.
\r\n\r\nIn a third experiment, I examined a phenomenon in which compound endplate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. Several lines of evidence indicate that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact, and that genuinely polyinnervated fibers are very rare at this stage, if present at all.
\r\n\r\nA computer model of neuromuscular synapse elimination was developed to serve as an analytical tool in exploring the potential roles of candidate mechanisms in regulating the normal process and in shaping its response to experimental perturbations. Synapse elimination is a complex process likely to involve the dynamic interaction of several specific mechanisms. This situation limits the reliability of a strictly inductive theoretical investigation into how these mechanisms might act. Three mechanisms which have been previously proposed and discussed in the literature are simulated, including a synaptic stabilization molecule, a muscle derived trophic factor, and a hypothesized intrinsic tendency of motor neurons to limit their arbor. The model is stochastic rather than deterministic in character, and is also dynamic, tracing the growth and retraction of individual presynaptic terminals at each iteration as they compete for limited synaptic space.
\r\n\r\nNine experimental observations were selected to guide development of the model and evaluate its performance. All but one of the experimental observations canbe simulated by at least one of the mechanisms studied. No single mechanism, however, is adequate to duplicate the entire body of experimental evidence. A relative advantage for larger terminals appears critical for convergence in both the scaffolding and trophic factor mechanisms. Several alternative roles for activity are compared.
", "doi": "10.7907/3ebg-mv36", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:7416", "collection": "thesis", "collection_id": "7416", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222013-094834563", "type": "thesis", "title": "Molecular Biology of Shaker, a Drosophila Gene that Encodes Multiple Potassium Channel Components", "author": [ { "family_name": "Kamb", "given_name": "Alexander", "clpid": "Kamb-Alexander" } ], "thesis_advisor": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "thesis_committee": [ { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Lewis", "given_name": "Edward B.", "clpid": "Lewis-E-B" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Shaker (Sh) mutants of Drosophila suffer from a characteristic leg-shaking behavioral defect. Previous genetic and physiological experiments suggested that Sh encodes at least one component of a fast, transient, or A-type K\u207a channel. To address questions pertaining to the structure, function and heterogeneity of K\u207a channels, we have undertaken a molecular analysis of Sh. We have isolated molecular clones for the genomic region encompassing Sh as part of a 350 kb chromosomal walk. Using a combination of classical and molecular genetics, we have mapped several Sh mutations within this region, and localized the Sh gene. Sh mutations scatter over at least 65 kb of genomic DNA, and the Sh gene itself is large, spanning at least 95 kb. Comparative studies on a collection of Sh cDNA clones show that Sh encodes a diverse array of gene products. The basis for this diversity is a mechanism that generates a limited number of different 5' and 3' end segments, and splices these segments onto a central constant region. This differential splicing mechanism produces at least 10, and possibly 28 or more, predicted Sh proteins that differ at the carboxyl and/or amino terminus. The primary structures of Sh proteins deduced from the cDNAs reveal two general types of polypeptide: a protein that contains seven potential membrane-spanning domains, including a positively charged segment that is similar to a sequence called S4 in Na\u207a channels, and a smaller protein that lacks S4 and contains only three potential membrane-spanning regions. Variants of the flrst protein type range in size from 493 a.a. to 656 a.a., whereas variants of the second type range from 303 a.a. to 337 a.a. These polypeptides may assemble as homomultimers and/or as heteromultimers to produce K\u207a channels with different features.
", "doi": "10.7907/a32z-ye28", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:17519", "collection": "thesis", "collection_id": "17519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07112025-215055238", "primary_object_url": { "basename": "Lowe_G_1987.pdf", "content": "final", "filesize": 51427723, "license": "other", "mime_type": "application/pdf", "url": "/17519/1/Lowe_G_1987.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Rotation of Bacterial Flagella at High Frequency", "author": [ { "family_name": "Lowe", "given_name": "Graeme", "clpid": "Lowe-Graeme" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Goodstein", "given_name": "David L.", "clpid": "Goodstein-D-L" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Flagellated bacteria propel themselves through their fluid\r\nenvironment by rotating one or more helical filaments which are driven\r\nat their base by a reversible motor powered by a protonmotive force. In\r\nsome species many filaments join. together to form a single flagellar\r\nbundle during swimming. Past work has characterized the functional\r\nproperties of the motor at low speed and high torque from studies of\r\ncells tethered by a single filament. This thesis describes work in which\r\nproperties of the motor at high speed and low torque were investigated\r\nby studying free swimming cells.
\r\n\r\nA method was developed for measuring the rotation rates of filaments\r\nin bundles of swimming cells. Images of cell bodies were projected onto\r\nthe photocathode of a photomultiplier tube whose sensitivity is\r\nspatially inhomogeneous, and the power spectral density of the output\r\nwas computed using the Fast Fourier Transform. Averages of many spectra\r\nrevealed a peak at high frequency due to the vibration of the cell body\r\nby the rotating filaments. This method was analysed in detail by\r\nmechanical simulations and computer models. Techniques were also\r\ndeveloped for following the rotation of single motors by attaching\r\nmarkers to sheared flagella. Most experiments were done with a motile\r\nstrain of Streptococcus. At 22\u00b0C, filaments of this organism rotate at\r\nca. 100 Hz relative to the cell body. Higher frequencies were seen in\r\nEscherichia coli at the same temperature, ca. 180 Hz.
\r\n\r\nThe relation between torque and speed of the flagellar motor at\r\nfixed protonmotive force was determined by varying the viscosity of the\r\nmedium. Torque was found to drop linearly with increasing speed over the\r\nupper half of the speed range. A comparison with the torque generated by\r\ntethered cells suggests that linearity may hold over the entire speed\r\nrange. This behavior is consistent with a scheme whereby the free energy\r\navailable per proton is dissipated in a series of small steps.
\r\n\r\nThe bundle frequency of glycolysing Streptococcus was found to\r\nincrease linearly with temperature, by a factor of 7 from 10\u00b0C to 42\u00b0C,\r\ncorresponding to an increase in torque by a factor of 3. The\r\nprotonmotive force did not vary by more than 10% from 16\u00b0C to 32\u00b0C.\r\nConditions were found under which cells swam when artificially energized\r\nby a combination of transmembrane pH gradient and potassium diffusion\r\npotential. These cells exhibited a large deuterium isotope effect, their\r\nspeed dropping by 30 - 50% in D20. Thus, proton transfer reactions\r\nappear to be limiting the rate of motor rotation in swimming cells.
", "doi": "10.7907/yh7f-q174", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:17519", "collection": "thesis", "collection_id": "17519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07112025-215055238", "primary_object_url": { "basename": "Lowe_G_1987.pdf", "content": "final", "filesize": 51427723, "license": "other", "mime_type": "application/pdf", "url": "/17519/1/Lowe_G_1987.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Rotation of Bacterial Flagella at High Frequency", "author": [ { "family_name": "Lowe", "given_name": "Graeme", "clpid": "Lowe-Graeme" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Goodstein", "given_name": "David L.", "clpid": "Goodstein-D-L" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Flagellated bacteria propel themselves through their fluid\r\nenvironment by rotating one or more helical filaments which are driven\r\nat their base by a reversible motor powered by a protonmotive force. In\r\nsome species many filaments join. together to form a single flagellar\r\nbundle during swimming. Past work has characterized the functional\r\nproperties of the motor at low speed and high torque from studies of\r\ncells tethered by a single filament. This thesis describes work in which\r\nproperties of the motor at high speed and low torque were investigated\r\nby studying free swimming cells.
\r\n\r\nA method was developed for measuring the rotation rates of filaments\r\nin bundles of swimming cells. Images of cell bodies were projected onto\r\nthe photocathode of a photomultiplier tube whose sensitivity is\r\nspatially inhomogeneous, and the power spectral density of the output\r\nwas computed using the Fast Fourier Transform. Averages of many spectra\r\nrevealed a peak at high frequency due to the vibration of the cell body\r\nby the rotating filaments. This method was analysed in detail by\r\nmechanical simulations and computer models. Techniques were also\r\ndeveloped for following the rotation of single motors by attaching\r\nmarkers to sheared flagella. Most experiments were done with a motile\r\nstrain of Streptococcus. At 22\u00b0C, filaments of this organism rotate at\r\nca. 100 Hz relative to the cell body. Higher frequencies were seen in\r\nEscherichia coli at the same temperature, ca. 180 Hz.
\r\n\r\nThe relation between torque and speed of the flagellar motor at\r\nfixed protonmotive force was determined by varying the viscosity of the\r\nmedium. Torque was found to drop linearly with increasing speed over the\r\nupper half of the speed range. A comparison with the torque generated by\r\ntethered cells suggests that linearity may hold over the entire speed\r\nrange. This behavior is consistent with a scheme whereby the free energy\r\navailable per proton is dissipated in a series of small steps.
\r\n\r\nThe bundle frequency of glycolysing Streptococcus was found to\r\nincrease linearly with temperature, by a factor of 7 from 10\u00b0C to 42\u00b0C,\r\ncorresponding to an increase in torque by a factor of 3. The\r\nprotonmotive force did not vary by more than 10% from 16\u00b0C to 32\u00b0C.\r\nConditions were found under which cells swam when artificially energized\r\nby a combination of transmembrane pH gradient and potassium diffusion\r\npotential. These cells exhibited a large deuterium isotope effect, their\r\nspeed dropping by 30 - 50% in D20. Thus, proton transfer reactions\r\nappear to be limiting the rate of motor rotation in swimming cells.
", "doi": "10.7907/yh7f-q174", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11877", "collection": "thesis", "collection_id": "11877", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10312019-114338923", "primary_object_url": { "basename": "Yu_L_1987.pdf", "content": "final", "filesize": 51911087, "license": "other", "mime_type": "application/pdf", "url": "/11877/1/Yu_L_1987.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Nicotinic Acetylcholine Receptor: Gene Expression and Ion Channel Function", "author": [ { "family_name": "Yu", "given_name": "Lei", "clpid": "Yu-Lei" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The nicotinic acetylcholine receptor (AChR) is a complex protein, which functions as a ligand-gated ion channel on the postsynaptic membrane at the neuromuscular junction and mediates signal transmission from neuron to muscle. Research on the AChR has had a long history and has benefited from the endeavors of scientists from many disciplines. The intensive, multidisciplinary studies have yielded valuable knowledge about this molecule, which serves as a model for the understanding of many fundamental questions in biological sciences. Chapter 1 presents a review of the AChR.
\r\n\r\nAs a tissue-specific and developmental stage-specific molecule, AChR is under temporal and spatial control for its synthesis. Chapter 2 reports a qualitative and quantitative study of AChR gene activity during muscle cell differentiation, using a cDNA clone isolated from a murine muscle cell line, which codes for the \u03b3 subunit of the mouse AChR. The results indicate that the regulation of mRNA accumulation levels is a major mechanism in the differential synthesis of the AChR.
\r\n\r\nThe marriage between AChR and molecular biology resulted in many cDNA clones which, after being introduced to African frogs, produced the next generation \u2014 Xenopus oocytes with exotic AChRs on them. Chapter 3 describes the attempt to localize \"determinants\" that specify species subunit identity in the AChR by constructing chimeric cDNA clones composed of fragments from different origins, taking advantage of the Xenopus oocyte expression system. The results from surface toxin-binding assay and two-electrode voltage-clamp recording suggested that while the species specificity can be dictated by certain subunits, the determination of subunit identity does not reside at a defined locus in the fragments tested.
\r\n\r\nDoes the complex composition of multisubunits in the AChR bear any functional significance? Chapter 4 addresses this question through the study of mouse-Torpedo AChR hybrids. The complete substitution of AChR subunits between mouse and Torpedo receptors generated all 16 combinations, and a systematic analysis of these hybrids revealed an interesting pattern with respect to the voltage sensitivity in the ACh-induced response: The identity of the \u03b2 subunit determines, while the interaction between the \u03b2 and \u03b4 subunits modulates, the AChR voltage sensitivity. The results, therefore, suggest that different subunits of AChR may play a central role in different functional properties.
\r\n\r\nPatch-clamp technique has offered an opportunity for analyzing transmembrane current flow with the high resolution of single-channel recording. Chapter 5 describes such a study on homologous and hybrid AChRs. Voltage influence on the three parameters were evaluated, and the results indicate that the single channel conductance is independent of membrane potential and that the channel closing and opening rates together constitute the basis for the voltage sensitivity in whole-cell recording with the closing rate making the major contribution. Also investigated were the subunit roles in species specificity of channel-open duration and voltage dependence. The results are in agreement with those reported on channel duration and support the conclusions of our previous work on the subunit involvement in determining the voltage sensitivity of the AChR response.
", "doi": "10.7907/fj5c-4h83", "publication_date": "1987", "thesis_type": "phd", "thesis_year": "1987" }, { "id": "thesis:11465", "collection": "thesis", "collection_id": "11465", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-153632089", "type": "thesis", "title": "Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Mayne", "given_name": "Katherine Mixter", "clpid": "Mayne-Katherine-Mixter" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An introduction to the work in the study of the nicotinic acetylcholine receptor is presented. The author reviews the field to place the work of the present volume in its proper context The major developments in studying the protein biochemistry of the receptor are reviewed, including the subunit makeup, ligand binding, and protein sequences. These studies led to the cloning and sequencing of many of the subunits as cDNA or genomic DNA constructions. This wealth of sequence information has allowed the formulation of detailed models of receptor structure. Current work centers on testing various aspects of these models and expanding the scope of the field into different species and tissues that utilize this receptor.
\r\n\r\nPartial cDNA clones specific for the \u03b2 and \u03b4 subunits of the acetylcholine receptor of Torpedo californica were isolated by the following method. A cDNA library was constructed from electric organ poly( A)+ RNA and enriched by screening for clones more abundantly represented in electric organ than in brain or-liver mRNA preparations. These clones were tested by hybridization selection of clone specific mRNA which was then translated in vitro. Protein products were immunoprecipitated and analyzed by gel electrophoresis. The isolated clones were used to screen a library of Torpedo genomic DNA which resulted in the isolation of the gene for the Torpedo \u03b4 subunit. The \u03b4 gene was found to be single copy in Torpedo, and it contains at least four introns.
\r\n\r\nA cDNA library was constructed in \u03bbgt10 from membrane bound poly(A)+ RNA from mouse BC3H-1 cells. This library was screened with cDNA encoding the complete protein region of the Torpedo \u03b3 and \u03b4 subunits. Positively hybridizing clones isolated with the Torpedo \u03b3 subunit were sequenced and compared with published data. The deduced amino acid sequence was more highly homologous to the Torpedo \u03b4 than to the Torpedo \u03b3 and on this basis the mouse clone was tentatively identified as a \u03b4 subunit of the acetylcholine receptor. The mouse nucleotide sequence has several stretches of strong homology with the Torpedo \u03b3 subunit cDNA, but no such homology with the Torpedo \u03b4 subunit . A genomic blotting experiment indicated that there is probably one, but at most two chromosomal genes encoding this or closely related sequences.
\r\n\r\nIn order to test the assignment of the mouse \u03b4 cDNA by a more functional criterion than simple amino acid homology, the following experiment was done. The phage SP6 transcription system was used to transcribe mRNA from the four individual Torpedo subunits and from the mouse \u03b4. When the four Torpedo subunit specific mRNAs were injected into Xenopus oocytes, functional receptors appeared in the oocyte membrane. If the \u03b2 or \u03b3 subunit RNA was omitted, no response to acetylcholine was detected, while a small response was detected if the \u03b4 subunit RNA was omitted. When mouse \u03b4 specific RNA was injected in place of the Torpedo \u03b4, a 3-4 fold larger response was measured in response to acetylcholine under voltage clamp conditions. The replacement of Torpedo \u03b3 RNA with mouse \u03b4 RNA gave no detectable response. Surface binding of \u03b1-bungarotoxin was not significantly altered by exchanging the \u03b4 subunits, which indicates that the difference is intrinsic to the channel rather than a matter of stability or synthesis rates. Examination of the amino acid sequences of the two \u03b4 subunits and the Torpedo \u03b3 did not identify an obvious region of subunit specific homology. The amino acid features necessary to determine a specific subunit are not obvious from simple homology comparisons.
\r\n\r\nWe have constructed a series of chimeric subunits to try to localize subunit determining regions of the acetylcholine receptor polypeptides. Each chimera was tested in the oocyte system by replacing its RNA for each of the parent RNAs in turn. None of the chimeras we have constructed retained enough of either parental subunit characteristics to function fully in place of that parent subunit to form an acetylcholine receptor that is responsive to acetylcholine. We conclude that a minimum of two subunit-specific regions are widely dispersed over the subunit length. These data are also consistent with the conclusion that there are no discrete regions that determine subunit identity, but instead that this information is rather evenly distributed along subunit length. In some combinations, the chimeras were incorporated into surface AchRs, although these complexes were only weakly responsive to Ach. We further conclude that there are regions needed for efficient function of these subunits that are not necessary for the formation of surface complexes. We have demonstrated that the \u03b1 subunits of both mouse and chick form functional receptors in the Xenopus oocyte system in combination with the \u03b2 and \u03b3 subunits from Torpedo and a \u03b4 from either Torpedo or mouse. The responses of these hybrid AchRs are smaller than the response from the Torpedo AchR. In contrast, the mouse \u03b3 subunit did not form functional AchRs in any combination of the subunits mentioned above.
\r\n\r\nThe present author spent the early part of her career studying the molecular biology of the actin genes of Drosophila melanogaster. Portions of each of the six actin genes were sequenced. These sequences revealed that the amino acid sequence of actin is highly conserved but that the positions of introns in these genes are strikingly nonconserved. Further, each of the Drosophila actins resembles the cytoplasmic isoforms from vertebrates, while none resemble the muscle isoforms.
", "doi": "10.7907/nvv4-qt08", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11472", "collection": "thesis", "collection_id": "11472", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-154502622", "type": "thesis", "title": "The Avoidance Response of Phycomyces in a Controlled Environment", "author": [ { "family_name": "Meyer", "given_name": "Paul Wells", "clpid": "Meyer-Paul-Wells" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "If an object is placed 1 mm away from the growing zone of a Phycomyces sporangiophore growing in air, then after 2 to 6 min the sporangiophore bends away from the object, without ever touching it, at a rate of about 1\u00b0/min. The sporangiophore stops bending after about 30 min. This is called the avoidance response of Phycomyces.
\r\n\r\nHow does the sporangiophore detect the object? It seems likely that a chemical mechanism is involved, since other physical stimuli (light, electric and magnetic fields) have already been ruled out.
\r\n\r\nA simple mechanism was proposed 10 years ago, in which the ambient air currents near the surface of an object modify the distribution of a hypothetical, short-lived effector gas emitted by the sporangiophore. However, the avoidance response occurs at its usual rate in the complete absence of ambient air currents. Thus, the suppression of air currents near the surface of a solid object cannot provide the signal for the response.
\r\n\r\nThe avoidance rate depends significantly on the recent history of the experimental chamber, on the length of time the sporangiophore has spent inside the experimental chamber, and on other factors. By carefully controlling environmental variables, the variation in avoidance rate of different sporangiophores in successive experiments can be held to less than \u00b110%. This allows accurate determination of the distance dependence of the response, and accurate comparison of different types of barriers.
\r\n\r\nThe rate of the response falls off above 90 % relative humidity - but does not fall to zero. Surprisingly, the sporangiophore avoids a thin, 120 \u00b5m diameter parallel wire placed 0.5 mm away at about the same rate as it avoids another sporangiophore placed at the same distance. Also, the distance dependence of the avoidance response appears to be much weaker than previously reported, and the response may depend on the chemical composition of the object, in contrast to previous reports. These findings, combined with the results of calculations presented in Appendix 3, argue strongly against the hypothesis that the barrier acts merely by reflecting a diffusible substance emitted by the sporangiophore.
\r\n\r\nThe only remaining viable chemical mechanism for the avoidance response requires that the signal molecule emitted by the sporangiophore be adsorbed by the surface of the avoided object for a nonzero length of time, and not just be reflected by it. Three new versions of this hypothesis are presented which are consistent with the experimental results.
", "doi": "10.7907/s6zv-r568", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11472", "collection": "thesis", "collection_id": "11472", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-154502622", "type": "thesis", "title": "The Avoidance Response of Phycomyces in a Controlled Environment", "author": [ { "family_name": "Meyer", "given_name": "Paul Wells", "clpid": "Meyer-Paul-Wells" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "If an object is placed 1 mm away from the growing zone of a Phycomyces sporangiophore growing in air, then after 2 to 6 min the sporangiophore bends away from the object, without ever touching it, at a rate of about 1\u00b0/min. The sporangiophore stops bending after about 30 min. This is called the avoidance response of Phycomyces.
\r\n\r\nHow does the sporangiophore detect the object? It seems likely that a chemical mechanism is involved, since other physical stimuli (light, electric and magnetic fields) have already been ruled out.
\r\n\r\nA simple mechanism was proposed 10 years ago, in which the ambient air currents near the surface of an object modify the distribution of a hypothetical, short-lived effector gas emitted by the sporangiophore. However, the avoidance response occurs at its usual rate in the complete absence of ambient air currents. Thus, the suppression of air currents near the surface of a solid object cannot provide the signal for the response.
\r\n\r\nThe avoidance rate depends significantly on the recent history of the experimental chamber, on the length of time the sporangiophore has spent inside the experimental chamber, and on other factors. By carefully controlling environmental variables, the variation in avoidance rate of different sporangiophores in successive experiments can be held to less than \u00b110%. This allows accurate determination of the distance dependence of the response, and accurate comparison of different types of barriers.
\r\n\r\nThe rate of the response falls off above 90 % relative humidity - but does not fall to zero. Surprisingly, the sporangiophore avoids a thin, 120 \u00b5m diameter parallel wire placed 0.5 mm away at about the same rate as it avoids another sporangiophore placed at the same distance. Also, the distance dependence of the avoidance response appears to be much weaker than previously reported, and the response may depend on the chemical composition of the object, in contrast to previous reports. These findings, combined with the results of calculations presented in Appendix 3, argue strongly against the hypothesis that the barrier acts merely by reflecting a diffusible substance emitted by the sporangiophore.
\r\n\r\nThe only remaining viable chemical mechanism for the avoidance response requires that the signal molecule emitted by the sporangiophore be adsorbed by the surface of the avoided object for a nonzero length of time, and not just be reflected by it. Three new versions of this hypothesis are presented which are consistent with the experimental results.
", "doi": "10.7907/s6zv-r568", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11465", "collection": "thesis", "collection_id": "11465", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-153632089", "type": "thesis", "title": "Structural and Functional Studies on the Subunits of the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Mayne", "given_name": "Katherine Mixter", "clpid": "Mayne-Katherine-Mixter" } ], "thesis_advisor": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An introduction to the work in the study of the nicotinic acetylcholine receptor is presented. The author reviews the field to place the work of the present volume in its proper context The major developments in studying the protein biochemistry of the receptor are reviewed, including the subunit makeup, ligand binding, and protein sequences. These studies led to the cloning and sequencing of many of the subunits as cDNA or genomic DNA constructions. This wealth of sequence information has allowed the formulation of detailed models of receptor structure. Current work centers on testing various aspects of these models and expanding the scope of the field into different species and tissues that utilize this receptor.
\r\n\r\nPartial cDNA clones specific for the \u03b2 and \u03b4 subunits of the acetylcholine receptor of Torpedo californica were isolated by the following method. A cDNA library was constructed from electric organ poly( A)+ RNA and enriched by screening for clones more abundantly represented in electric organ than in brain or-liver mRNA preparations. These clones were tested by hybridization selection of clone specific mRNA which was then translated in vitro. Protein products were immunoprecipitated and analyzed by gel electrophoresis. The isolated clones were used to screen a library of Torpedo genomic DNA which resulted in the isolation of the gene for the Torpedo \u03b4 subunit. The \u03b4 gene was found to be single copy in Torpedo, and it contains at least four introns.
\r\n\r\nA cDNA library was constructed in \u03bbgt10 from membrane bound poly(A)+ RNA from mouse BC3H-1 cells. This library was screened with cDNA encoding the complete protein region of the Torpedo \u03b3 and \u03b4 subunits. Positively hybridizing clones isolated with the Torpedo \u03b3 subunit were sequenced and compared with published data. The deduced amino acid sequence was more highly homologous to the Torpedo \u03b4 than to the Torpedo \u03b3 and on this basis the mouse clone was tentatively identified as a \u03b4 subunit of the acetylcholine receptor. The mouse nucleotide sequence has several stretches of strong homology with the Torpedo \u03b3 subunit cDNA, but no such homology with the Torpedo \u03b4 subunit . A genomic blotting experiment indicated that there is probably one, but at most two chromosomal genes encoding this or closely related sequences.
\r\n\r\nIn order to test the assignment of the mouse \u03b4 cDNA by a more functional criterion than simple amino acid homology, the following experiment was done. The phage SP6 transcription system was used to transcribe mRNA from the four individual Torpedo subunits and from the mouse \u03b4. When the four Torpedo subunit specific mRNAs were injected into Xenopus oocytes, functional receptors appeared in the oocyte membrane. If the \u03b2 or \u03b3 subunit RNA was omitted, no response to acetylcholine was detected, while a small response was detected if the \u03b4 subunit RNA was omitted. When mouse \u03b4 specific RNA was injected in place of the Torpedo \u03b4, a 3-4 fold larger response was measured in response to acetylcholine under voltage clamp conditions. The replacement of Torpedo \u03b3 RNA with mouse \u03b4 RNA gave no detectable response. Surface binding of \u03b1-bungarotoxin was not significantly altered by exchanging the \u03b4 subunits, which indicates that the difference is intrinsic to the channel rather than a matter of stability or synthesis rates. Examination of the amino acid sequences of the two \u03b4 subunits and the Torpedo \u03b3 did not identify an obvious region of subunit specific homology. The amino acid features necessary to determine a specific subunit are not obvious from simple homology comparisons.
\r\n\r\nWe have constructed a series of chimeric subunits to try to localize subunit determining regions of the acetylcholine receptor polypeptides. Each chimera was tested in the oocyte system by replacing its RNA for each of the parent RNAs in turn. None of the chimeras we have constructed retained enough of either parental subunit characteristics to function fully in place of that parent subunit to form an acetylcholine receptor that is responsive to acetylcholine. We conclude that a minimum of two subunit-specific regions are widely dispersed over the subunit length. These data are also consistent with the conclusion that there are no discrete regions that determine subunit identity, but instead that this information is rather evenly distributed along subunit length. In some combinations, the chimeras were incorporated into surface AchRs, although these complexes were only weakly responsive to Ach. We further conclude that there are regions needed for efficient function of these subunits that are not necessary for the formation of surface complexes. We have demonstrated that the \u03b1 subunits of both mouse and chick form functional receptors in the Xenopus oocyte system in combination with the \u03b2 and \u03b3 subunits from Torpedo and a \u03b4 from either Torpedo or mouse. The responses of these hybrid AchRs are smaller than the response from the Torpedo AchR. In contrast, the mouse \u03b3 subunit did not form functional AchRs in any combination of the subunits mentioned above.
\r\n\r\nThe present author spent the early part of her career studying the molecular biology of the actin genes of Drosophila melanogaster. Portions of each of the six actin genes were sequenced. These sequences revealed that the amino acid sequence of actin is highly conserved but that the positions of introns in these genes are strikingly nonconserved. Further, each of the Drosophila actins resembles the cytoplasmic isoforms from vertebrates, while none resemble the muscle isoforms.
", "doi": "10.7907/nvv4-qt08", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:9686", "collection": "thesis", "collection_id": "9686", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04262016-091328237", "primary_object_url": { "basename": "Lewis_rs_1985.pdf", "content": "final", "filesize": 56597337, "license": "other", "mime_type": "application/pdf", "url": "/9686/1/Lewis_rs_1985.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Ionic Basis of Frequency Selectivity in Hair Cells of the Bullfrog's Sacculus", "author": [ { "family_name": "Lewis", "given_name": "Richard Sheridan", "orcid": "0000-0002-6010-7403", "clpid": "Lewis-Richard-Sheridan" } ], "thesis_advisor": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hair cells from the bull frog's sacculus, a vestibular organ responding to substrate-borne vibration, possess electrically resonant membrane properties which maximize the sensitivity of each cell to a particular frequency of mechanical input. The electrical resonance of these cells and its underlying ionic basis were studied by applying gigohm-seal recording techniques to solitary hair cells enzymatically dissociated from the sacculus. The contribution of electrical resonance to frequency selectivity was assessed from microelectrode recordings from hair cells in an excised preparation of the sacculus.
\r\n\r\nElectrical resonance in the hair cell is demonstrated by damped membrane-potential oscillations in response to extrinsic current pulses applied through the recording pipette. This response is analyzed as that of a damped harmonic oscillator. Oscillation frequency rises with membrane depolarization, from 80-160 Hz at resting potential to asymptotic values of 200-250 Hz. The sharpness of electrical tuning, denoted by the electrical quality factor, Qe, is a bell-shaped function of membrane voltage, reaching a maximum value around eight at a membrane potential slightly positive to the resting potential.
\r\n\r\nIn whole cells, three time-variant ionic currents are activated at voltages more positive than -60 to -50 mV; these are identified as a voltage-dependent, non-inactivating Ca current (Ica), a voltage-dependent, transient K current (IA), and a Ca-dependent K current (Ic). The C channel is identified in excised, inside-out membrane patches on the basis of its large conductance (130-200 pS), its selective permeability to Kover Na or Cl, and its activation by internal Ca ions and membrane depolarization. Analysis of open- and closed-lifetime distributions suggests that the C channel can assume at least two open and three closed kinetic states.
\r\n\r\nExposing hair cells to external solutions that inhibit the Ca or C conductances degrades the electrical resonance properties measured under current-clamp conditions, while blocking the A conductance has no significant effect, providing evidence that only the Ca and C conductances participate in the resonance mechanism. To test the sufficiency of these two conductances to account for electrical resonance, a mathematical model is developed that describes Ica, Ic, and intracellular Ca concentration during voltage-clamp steps. Ica activation is approximated by a third-order Hodgkin-Huxley kinetic scheme. Ca entering the cell is assumed to be confined to a small submembrane compartment which contains an excess of Ca buffer; Ca leaves this space with first-order kinetics. The Ca- and voltage-dependent activation of C channels is described by a five-state kinetic scheme suggested by the results of single-channel observations. Parameter values in the model are adjusted to fit the waveforms of Ica and Ic evoked by a series of voltage-clamp steps in a single cell. Having been thus constrained, the model correctly predicts the character of voltage oscillations produced by current-clamp steps, including the dependencies of oscillation frequency and Qe on membrane voltage. The model shows quantitatively how the Ca and C conductances interact, via changes in intracellular Ca concentration, to produce electrical resonance in a vertebrate hair cell.
", "doi": "10.7907/MZGR-KW63", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:9686", "collection": "thesis", "collection_id": "9686", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04262016-091328237", "primary_object_url": { "basename": "Lewis_rs_1985.pdf", "content": "final", "filesize": 56597337, "license": "other", "mime_type": "application/pdf", "url": "/9686/1/Lewis_rs_1985.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Ionic Basis of Frequency Selectivity in Hair Cells of the Bullfrog's Sacculus", "author": [ { "family_name": "Lewis", "given_name": "Richard Sheridan", "orcid": "0000-0002-6010-7403", "clpid": "Lewis-Richard-Sheridan" } ], "thesis_advisor": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hair cells from the bull frog's sacculus, a vestibular organ responding to substrate-borne vibration, possess electrically resonant membrane properties which maximize the sensitivity of each cell to a particular frequency of mechanical input. The electrical resonance of these cells and its underlying ionic basis were studied by applying gigohm-seal recording techniques to solitary hair cells enzymatically dissociated from the sacculus. The contribution of electrical resonance to frequency selectivity was assessed from microelectrode recordings from hair cells in an excised preparation of the sacculus.
\r\n\r\nElectrical resonance in the hair cell is demonstrated by damped membrane-potential oscillations in response to extrinsic current pulses applied through the recording pipette. This response is analyzed as that of a damped harmonic oscillator. Oscillation frequency rises with membrane depolarization, from 80-160 Hz at resting potential to asymptotic values of 200-250 Hz. The sharpness of electrical tuning, denoted by the electrical quality factor, Qe, is a bell-shaped function of membrane voltage, reaching a maximum value around eight at a membrane potential slightly positive to the resting potential.
\r\n\r\nIn whole cells, three time-variant ionic currents are activated at voltages more positive than -60 to -50 mV; these are identified as a voltage-dependent, non-inactivating Ca current (Ica), a voltage-dependent, transient K current (IA), and a Ca-dependent K current (Ic). The C channel is identified in excised, inside-out membrane patches on the basis of its large conductance (130-200 pS), its selective permeability to Kover Na or Cl, and its activation by internal Ca ions and membrane depolarization. Analysis of open- and closed-lifetime distributions suggests that the C channel can assume at least two open and three closed kinetic states.
\r\n\r\nExposing hair cells to external solutions that inhibit the Ca or C conductances degrades the electrical resonance properties measured under current-clamp conditions, while blocking the A conductance has no significant effect, providing evidence that only the Ca and C conductances participate in the resonance mechanism. To test the sufficiency of these two conductances to account for electrical resonance, a mathematical model is developed that describes Ica, Ic, and intracellular Ca concentration during voltage-clamp steps. Ica activation is approximated by a third-order Hodgkin-Huxley kinetic scheme. Ca entering the cell is assumed to be confined to a small submembrane compartment which contains an excess of Ca buffer; Ca leaves this space with first-order kinetics. The Ca- and voltage-dependent activation of C channels is described by a five-state kinetic scheme suggested by the results of single-channel observations. Parameter values in the model are adjusted to fit the waveforms of Ica and Ic evoked by a series of voltage-clamp steps in a single cell. Having been thus constrained, the model correctly predicts the character of voltage oscillations produced by current-clamp steps, including the dependencies of oscillation frequency and Qe on membrane voltage. The model shows quantitatively how the Ca and C conductances interact, via changes in intracellular Ca concentration, to produce electrical resonance in a vertebrate hair cell.
", "doi": "10.7907/MZGR-KW63", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11393", "collection": "thesis", "collection_id": "11393", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02132019-181420941", "type": "thesis", "title": "Chemotaxis of Escherichia coli Studied Using Ionophoretic Stimulation", "author": [ { "family_name": "Segall", "given_name": "Jeffrey Edward", "clpid": "Segall-Jeffrey-Edward" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Fender", "given_name": "Derek H.", "clpid": "Fender-D-H" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Chemotactic responses of the bacterium Escherichia coli were elicited by iontophoretic ejection of charged compounds from micropipettes. Responses were measured using cells tethered by a single flagellum and following the direction of rotation of the cell body (with cells viewed from above the surface to which the flagellum is attached) . Mean response latencies to addition of attractant or repellent were 0.1 to 0.2 seconds. Brief pulses of attractants and repellents were used to determine the impulse response for chemotaxis. In response to a brief pulse of attractant the counterclockwise (CCW) bias (fraction of time that the flagellar motor spent rotating CCW) increased rapidly to a peak, then fell below the prestimulus value, returning to baseline within 5 seconds -- the response was biphasic. Repellent impulse responses were similar but inverted. The attractant impulse response accurately predicted responses to step changes, linear ramp changes and sine wave changes in receptor occupancy. Mutants defective in the enzymes responsible for methylation (cheR) and demethylation (cheB), which do not adapt to attractant stimuli, gave monophasic impulse responses--only the initial peak was evident. Mutants defective in the cheZ gene product had responses that were slower than the wild-type by a factor of about 10. The responses of flagellar motors on filamentous cells also were studied. The reversals of two motors on the same cell were not correlated, but fluctuations in bias were correlated for motors less than l 0 microns apart. Responses of motors of filamentous cells to iontophoretic application of aspartate indicated that the internal chemotaxis signal travels about 2 microns in cells lacking the cheR and cheB gene products and about 6 microns in cells with a defective cheZ gene product. These data are consistent with a simple model involving destruction of a cellular chemotaxis signal by the cheZ gene product.
", "doi": "10.7907/6r2y-ba72", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:11393", "collection": "thesis", "collection_id": "11393", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02132019-181420941", "type": "thesis", "title": "Chemotaxis of Escherichia coli Studied Using Ionophoretic Stimulation", "author": [ { "family_name": "Segall", "given_name": "Jeffrey Edward", "clpid": "Segall-Jeffrey-Edward" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Fender", "given_name": "Derek H.", "clpid": "Fender-D-H" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Chemotactic responses of the bacterium Escherichia coli were elicited by iontophoretic ejection of charged compounds from micropipettes. Responses were measured using cells tethered by a single flagellum and following the direction of rotation of the cell body (with cells viewed from above the surface to which the flagellum is attached) . Mean response latencies to addition of attractant or repellent were 0.1 to 0.2 seconds. Brief pulses of attractants and repellents were used to determine the impulse response for chemotaxis. In response to a brief pulse of attractant the counterclockwise (CCW) bias (fraction of time that the flagellar motor spent rotating CCW) increased rapidly to a peak, then fell below the prestimulus value, returning to baseline within 5 seconds -- the response was biphasic. Repellent impulse responses were similar but inverted. The attractant impulse response accurately predicted responses to step changes, linear ramp changes and sine wave changes in receptor occupancy. Mutants defective in the enzymes responsible for methylation (cheR) and demethylation (cheB), which do not adapt to attractant stimuli, gave monophasic impulse responses--only the initial peak was evident. Mutants defective in the cheZ gene product had responses that were slower than the wild-type by a factor of about 10. The responses of flagellar motors on filamentous cells also were studied. The reversals of two motors on the same cell were not correlated, but fluctuations in bias were correlated for motors less than l 0 microns apart. Responses of motors of filamentous cells to iontophoretic application of aspartate indicated that the internal chemotaxis signal travels about 2 microns in cells lacking the cheR and cheB gene products and about 6 microns in cells with a defective cheZ gene product. These data are consistent with a simple model involving destruction of a cellular chemotaxis signal by the cheZ gene product.
", "doi": "10.7907/6r2y-ba72", "publication_date": "1985", "thesis_type": "phd", "thesis_year": "1985" }, { "id": "thesis:8905", "collection": "thesis", "collection_id": "8905", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-161004818", "primary_object_url": { "basename": "Krouse_me_1984.pdf", "content": "final", "filesize": 14875600, "license": "other", "mime_type": "application/pdf", "url": "/8905/1/Krouse_me_1984.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigation of Competitive Antagonist Binding to the Nicotinic Acetylcholine Receptor Using Voltage-Jump and Light-Flash Techniques", "author": [ { "family_name": "Krouse", "given_name": "Mauri Eugene", "clpid": "Krouse-Mauri-Eugene" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "1. The effect of 2,2\u2019-bis-[\u03b1-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.
\r\n\r\n2. 2BQ, at concentrations below 3 \u03bc\u039c, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.
\r\n\r\n3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (Ki) of .33 and 1.0 \u03bc\u039c, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.
\r\n\r\n4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis\u2192trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.
\r\n\r\n5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans\u2192cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20\u00b0C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 108/M/s. This agrees with the association rate constant of 1.8 x 108/M/s estimated from the binding constant (Ki). The Q10 of the dissociation rate constant of cis-2BQ was 3.3 between 6\u00b0 and 20\u00b0C. The rate constants for association and dissociation of cis-28Q at receptors are\r\nindependent of voltage, agonist concentration, and the nature of the agonist.
\r\n\r\n6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same Ki as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 109/M/s or roughly as fast as possible for an encounter-limited reaction.
\r\n", "doi": "10.7907/wb48-ph26", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11220", "collection": "thesis", "collection_id": "11220", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10032018-123356944", "primary_object_url": { "basename": "Gordon_H_1984.pdf", "content": "final", "filesize": 43234524, "license": "other", "mime_type": "application/pdf", "url": "/11220/1/Gordon_H_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Postnatal Development of Motor Units in Rabbit and Rat Soleus Muscles", "author": [ { "family_name": "Gordon", "given_name": "Herman J.", "clpid": "Gordon-Herman-J" } ], "thesis_advisor": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" } ], "thesis_committee": [ { "family_name": "Van Essen", "given_name": "David C.", "orcid": "0000-0001-7044-4721", "clpid": "Van-Essen-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Grinnell", "given_name": "Alan D.", "clpid": "Grinnell-Alan-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The development of motor unit properties in the soleus muscle of rabbits and rats was used to study the control of innervation and elimination of synapses from mammalian skeletal muscle fibers and the differentiation of those muscle fibers into various twitch types.
\r\n\r\n1. Motor unit twitches with distinctly different time courses were found in rabbit soleus muscle at a stage in development when all muscle fibers were polyinnervated. This observation implies that (1) muscle fibers have already begun their physiological differentiation into twitch types while still polyinnervated and (2) motor neurons of a specific type preferentially polyinnervate muscle fibers of a corresponding type.
\r\n\r\n2. It has recently been claimed that synapse elimination occurs preferentially among motor neurons from the more rostral of the two spinal roots contributing to the soleus muscle of the rat. Using an assay based on measurements of motor unit twitch tens ions, it was found, contrary to the previous claim, that synapses were lost to the same extent by motor neurons passing through all contributing spinal roots to both the rabbit and rat soleus muscles.
\r\n\r\n3. Two lines of evidence indicate that rabbit soleus motor neurons redistribute their terminals at a time after wholesale polyinnervation has been lost from the muscle. (1) Between 11 and 18 days and 5 weeks of age, the frequency of histochemically defined type I fibers increases from 30% to 65% while the incidence of physiologically defined slow motor units does not obviously change. (2) Over the same time period, the ratio of average slow twitch tension to average fast twitch tension quadruples after correction for changes 1n muscle fiber cross sectional area. I hypothesize that during this 3 week time window, slow twitch motor neurons take over end plates previously occupied by fast twitch motor terminals.
\r\n\r\n4. Activity has previously been shown to play a role in the overall rate of synapse elimination. I have conducted preliminary experiments to address whether a competition on active muscle fibers between terminals of active and tetrodotoxin-inactivated motor axons results in a preferential retention of active connections. With the paradigm used, there was at most a small bias favoring the survival of active synapses.
\r\n", "doi": "10.7907/3ecg-0z58", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11202", "collection": "thesis", "collection_id": "11202", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09262018-173300466", "type": "thesis", "title": "Chemotactic Responses of Tethered Bacteria", "author": [ { "family_name": "Block", "given_name": "Steven Michael", "clpid": "Block-Steven-Michael" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Fender", "given_name": "Derek H.", "clpid": "Fender-D-H" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli swim in a three-dimensional random walk of alternating runs and tumbles, using their flagella for propulsion. When moving in a gradient of an attractant or repellent, they bias the walk in such a way as to migrate into a favorable region; this is a basis for chemotaxis. Bacteria may be tethered to a glass surface by means of a single flagellum. When tethered, cell bodies spin alternately clockwise (CW) and counterclockwise (CCW) under the influence of the rotary motor that drives the flagellum. The CCW state corresponds to the run mode and the CW state to the tumble mode. Tethered bacteria remain fixed in place, thereby providing an opportunity to study chemotactic behavior by direct manipulation of attractant or repellent concentration near the cells. Two experimental approaches have been used to exploit this opportunity. In the first, a mixing device that provides programmable concentration changes was used to stimulate tethered cells with exponential temporal gradients or exponentiated sine waves of the attractant \u03b1-methyl-D,L-aspartate. Such changes cause chemoreceptor occupancy to be changed linearly or sinusoidally, respectively. Exponential temporal gradients (both positive and negative) were found to shift the rotational bias (defined as the fraction of time spent spinning CCW) by a fixed amount related to the steepness of the gradient. The bias shifts produced indicate that cells are exquisitely sensitive to small changes in chemoreceptor occupancy. Distributions of CW and CCW intervals remained exponential during such gradients. This result is inconsistent with a response regulator model in which rotational transitions are associated with level-crossings of a fluctuating, hypothetical intermediate. It is consistent with a model in which transitions occur at random between rotational states, the transition probabilities being governed by chemotactic signals. In the second approach, short bursts of an attractant or repellent were delivered iontophoretically, producing an impulse response in the tethered bacteria. Properties of the impulse response show both adaptive and integrative behavior, and imply that cells respond maximally to changes in concentration which occur over times comparable with the length of a run. The impulse response can be used to predict the behavior of cells towards an arbitrary stimulus in the linear domain. Impulse responses from a series of chemotaxis mutants showed that some were defective in adaptation but not excitation; others were defective in both. Taken together, the experiments provide information about the spectral response of bacteria to concentration changes with frequencies ranging from 10-3 Hz up to almost 10 Hz. Both sets of data are consistent with the notion of a cellular \"bias regulator\" signal that sets the transition probabilities between two states; one representing CCW and the other representing CW rotation.
", "doi": "10.7907/2fzz-tb74", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11202", "collection": "thesis", "collection_id": "11202", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09262018-173300466", "type": "thesis", "title": "Chemotactic Responses of Tethered Bacteria", "author": [ { "family_name": "Block", "given_name": "Steven Michael", "clpid": "Block-Steven-Michael" } ], "thesis_advisor": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "thesis_committee": [ { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" }, { "family_name": "Hopfield", "given_name": "John J.", "clpid": "Hopfield-J-J" }, { "family_name": "Fender", "given_name": "Derek H.", "clpid": "Fender-D-H" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli swim in a three-dimensional random walk of alternating runs and tumbles, using their flagella for propulsion. When moving in a gradient of an attractant or repellent, they bias the walk in such a way as to migrate into a favorable region; this is a basis for chemotaxis. Bacteria may be tethered to a glass surface by means of a single flagellum. When tethered, cell bodies spin alternately clockwise (CW) and counterclockwise (CCW) under the influence of the rotary motor that drives the flagellum. The CCW state corresponds to the run mode and the CW state to the tumble mode. Tethered bacteria remain fixed in place, thereby providing an opportunity to study chemotactic behavior by direct manipulation of attractant or repellent concentration near the cells. Two experimental approaches have been used to exploit this opportunity. In the first, a mixing device that provides programmable concentration changes was used to stimulate tethered cells with exponential temporal gradients or exponentiated sine waves of the attractant \u03b1-methyl-D,L-aspartate. Such changes cause chemoreceptor occupancy to be changed linearly or sinusoidally, respectively. Exponential temporal gradients (both positive and negative) were found to shift the rotational bias (defined as the fraction of time spent spinning CCW) by a fixed amount related to the steepness of the gradient. The bias shifts produced indicate that cells are exquisitely sensitive to small changes in chemoreceptor occupancy. Distributions of CW and CCW intervals remained exponential during such gradients. This result is inconsistent with a response regulator model in which rotational transitions are associated with level-crossings of a fluctuating, hypothetical intermediate. It is consistent with a model in which transitions occur at random between rotational states, the transition probabilities being governed by chemotactic signals. In the second approach, short bursts of an attractant or repellent were delivered iontophoretically, producing an impulse response in the tethered bacteria. Properties of the impulse response show both adaptive and integrative behavior, and imply that cells respond maximally to changes in concentration which occur over times comparable with the length of a run. The impulse response can be used to predict the behavior of cells towards an arbitrary stimulus in the linear domain. Impulse responses from a series of chemotaxis mutants showed that some were defective in adaptation but not excitation; others were defective in both. Taken together, the experiments provide information about the spectral response of bacteria to concentration changes with frequencies ranging from 10-3 Hz up to almost 10 Hz. Both sets of data are consistent with the notion of a cellular \"bias regulator\" signal that sets the transition probabilities between two states; one representing CCW and the other representing CW rotation.
", "doi": "10.7907/2fzz-tb74", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:10584", "collection": "thesis", "collection_id": "10584", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052017-094450464", "type": "thesis", "title": "Part I. Studies on the Organization of the Eye of Aplysia californica. Part II. Studies on the Interrelationship between Two Neuronal Circadian Oscillators in Aplysia californica", "author": [ { "family_name": "Audesirk", "given_name": "Gerald Joseph", "clpid": "Audesirk-Gerald-Joseph" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Wiersma", "given_name": "Cornelis A. G.", "clpid": "Wiersma-C-A-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pettigrew", "given_name": "John D.", "clpid": "Pettigrew-J-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Part I
\r\n\r\n\r\nThe isolated eye of Aplysia californica produces a bursting\r\npattern of spontaneous compound action potentials (CAPs) when recordings \r\nare made from the optic nerve in darkness. The CAP frequency varies\r\nwith a circadian rhythm. The light response, also composed of CAPs,\r\nmay be separated into an initial phasic response and a late tonic \r\nresponse similar in form to the dark discharge. Solutions containing\r\nLa+++ or high Mg++ with low Ca++, which are expected to block chemical\r\nsynapses, stop the dark discharge and tonic light response but not the \r\nphasic light response. The suppression of dark discharge by high Mg++\r\nwith low Ca++ is usually temporary, lasting about 0.5 to 4 hours.\r\nSynchrony of the CAPs is not affected by either La+++ or high Mg++, low \r\nCa++. These results indicate that the dark discharge is driven through \r\nchemical synapses, but the light response is not. Replacement of \r\nchloride in the bathing medium by propionate, which uncouples electrical \r\njunctions in the crayfish septate axon, abolishes all CAPs for varying \r\nperiods of time, usually several hours. Propionate leaves the ERG\r\nintact and the optic nerve electrically excitable. A model for inter-neuronal \r\nconnections in the Aplysia eye is constructed from these data. \r\nIt is postulated that the light response is initiated in the photoreceptors, \r\nwith the receptor depolarization passing through electrical \r\nsynapses to higher order cells. Spikes are produced in these cells and \r\npass down their axons in the optic nerve. Spontaneous dark activity\r\nalso represents spiking in these higher order cells, but is initiated\r\nthrough chemical synapses by pacemaker cell(s). Synchrony of the CAPs \r\nis facilitated by electrical synapses between higher order cells. In\r\nlow Ca++ media, these higher order cells may become hyperexcitable to\r\nthe point of autoactivity.
\r\n\r\n\r\n\r\nPart II
\r\n\r\n\r\nThe circadian rhythm of spike output of the single neuron R15 \r\nin the isolated PVG of Aplysia californica can be entrained in vivo \r\nby light. The timing of the rhythm depends not only on the lighting\r\nschedule to which the animal was exposed prior to dissection, but also \r\non the time of dissection relative to that light schedule. Entrainment \r\nof the rhythm by light proceeds very slowly, if at all, in Aplysia \r\nwith their eyes removed. An indirect inhibitory neural pathway is\r\nshown to exist between the eyes and R15, but cutting nervous \r\nconnections containing this and any other neural paths from the eyes \r\nto R15 does not prevent entrainment by light in a majority of animals. \r\nIn vitro experiments show that the eyes can influence the activity \r\nof R15 even when the eyes and the PVG are not neurally connected.\t\r\nThe eyes therefore must release a water soluble factor which can affect\r\nR15, either directly or through some other neurons in the PVG. If the \r\neyes and PVGs from different animals are incubated together for\r\nseveral days and then separated, the subsequent spiking behavior of R15 \r\nis similar to that observed after in vivo entrainment to a light \r\nschedule equivalent in phase to the circadian rhythm of the eyes in \r\nvitro. It is a strong possibility that the factor released by the\r\neyes can entrain the circadian rhythm of R15.
\r\n\r\n \r\n\r\n", "doi": "10.7907/BT9J-NK43", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" }, { "id": "thesis:10584", "collection": "thesis", "collection_id": "10584", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052017-094450464", "type": "thesis", "title": "Part I. Studies on the Organization of the Eye of Aplysia californica. Part II. Studies on the Interrelationship between Two Neuronal Circadian Oscillators in Aplysia californica", "author": [ { "family_name": "Audesirk", "given_name": "Gerald Joseph", "clpid": "Audesirk-Gerald-Joseph" } ], "thesis_advisor": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" }, { "family_name": "Wiersma", "given_name": "Cornelis A. G.", "clpid": "Wiersma-C-A-G" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Pettigrew", "given_name": "John D.", "clpid": "Pettigrew-J-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Part I
\r\n\r\n\r\nThe isolated eye of Aplysia californica produces a bursting\r\npattern of spontaneous compound action potentials (CAPs) when recordings \r\nare made from the optic nerve in darkness. The CAP frequency varies\r\nwith a circadian rhythm. The light response, also composed of CAPs,\r\nmay be separated into an initial phasic response and a late tonic \r\nresponse similar in form to the dark discharge. Solutions containing\r\nLa+++ or high Mg++ with low Ca++, which are expected to block chemical\r\nsynapses, stop the dark discharge and tonic light response but not the \r\nphasic light response. The suppression of dark discharge by high Mg++\r\nwith low Ca++ is usually temporary, lasting about 0.5 to 4 hours.\r\nSynchrony of the CAPs is not affected by either La+++ or high Mg++, low \r\nCa++. These results indicate that the dark discharge is driven through \r\nchemical synapses, but the light response is not. Replacement of \r\nchloride in the bathing medium by propionate, which uncouples electrical \r\njunctions in the crayfish septate axon, abolishes all CAPs for varying \r\nperiods of time, usually several hours. Propionate leaves the ERG\r\nintact and the optic nerve electrically excitable. A model for inter-neuronal \r\nconnections in the Aplysia eye is constructed from these data. \r\nIt is postulated that the light response is initiated in the photoreceptors, \r\nwith the receptor depolarization passing through electrical \r\nsynapses to higher order cells. Spikes are produced in these cells and \r\npass down their axons in the optic nerve. Spontaneous dark activity\r\nalso represents spiking in these higher order cells, but is initiated\r\nthrough chemical synapses by pacemaker cell(s). Synchrony of the CAPs \r\nis facilitated by electrical synapses between higher order cells. In\r\nlow Ca++ media, these higher order cells may become hyperexcitable to\r\nthe point of autoactivity.
\r\n\r\n\r\n\r\nPart II
\r\n\r\n\r\nThe circadian rhythm of spike output of the single neuron R15 \r\nin the isolated PVG of Aplysia californica can be entrained in vivo \r\nby light. The timing of the rhythm depends not only on the lighting\r\nschedule to which the animal was exposed prior to dissection, but also \r\non the time of dissection relative to that light schedule. Entrainment \r\nof the rhythm by light proceeds very slowly, if at all, in Aplysia \r\nwith their eyes removed. An indirect inhibitory neural pathway is\r\nshown to exist between the eyes and R15, but cutting nervous \r\nconnections containing this and any other neural paths from the eyes \r\nto R15 does not prevent entrainment by light in a majority of animals. \r\nIn vitro experiments show that the eyes can influence the activity \r\nof R15 even when the eyes and the PVG are not neurally connected.\t\r\nThe eyes therefore must release a water soluble factor which can affect\r\nR15, either directly or through some other neurons in the PVG. If the \r\neyes and PVGs from different animals are incubated together for\r\nseveral days and then separated, the subsequent spiking behavior of R15 \r\nis similar to that observed after in vivo entrainment to a light \r\nschedule equivalent in phase to the circadian rhythm of the eyes in \r\nvitro. It is a strong possibility that the factor released by the\r\neyes can entrain the circadian rhythm of R15.
\r\n\r\n \r\n\r\n", "doi": "10.7907/BT9J-NK43", "publication_date": "1975", "thesis_type": "phd", "thesis_year": "1975" } ]