Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP\u2019s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP\u2019s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.
\r\n\r\nThe postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.
", "doi": "10.7907/Z9N877R8", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:6223", "collection": "thesis", "collection_id": "6223", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01132011-095304695", "primary_object_url": { "basename": "011611_Complete_Thesis.pdf", "content": "final", "filesize": 29546795, "license": "other", "mime_type": "application/pdf", "url": "/6223/55/011611_Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "New Tools for Studying O-G1cNAc Glycosylation and Chondroitin Sulfate Proteoglycans and Studies on the Roles of O-G1cNAc Glycosylation on the Transcription Factor CREB", "author": [ { "family_name": "Clark", "given_name": "Peter Michael", "clpid": "Clark-Peter-Michael" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The addition and removal of the monosaccharide N-acetyl-D-glucosamine (GlcNAc) to serine and threonine residues of proteins has emerged as a critical regulator of cellular processes. However, studies of O-GlcNAc in such complex systems as the brain have been limited, in part due to the lack of tools. Here we report the development of new tools for studying O-GlcNAc, and the application of these and other tools for studying the roles of O-GlcNAc in the brain.
\r\n\r\nWorking from a previously established chemoenzymatic method, we designed an isotopic labeling strategy for probing the dynamics of O-GlcNAc glycosylation using quantitative proteomics. With this tool, we show that O-GlcNAc is dynamically modulated on specific proteins by excitatory stimulation of the brain in vivo. Separately, we improved this chemoenzymatic strategy by integrating [3+2] azide-alkyne cycloaddition chemistry to attach biotin and fluorescent tags to O-GlcNAc residues. These tags allow for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc modified proteins. With this strategy, we identified over 146 novel glycoproteins from the mammalian brain.
\r\n\r\nThe transcription factor cAMP-response element binding protein (CREB) is critical for numerous functions in the brain, including neuronal survival, neuronal development, synaptic plasticity, and long-term memory. We show that CREB is highly glycosylated in the brain and discover new glycosylation sites on CREB in neurons. One of these sites is dynamically modulated and is important for regulating CREB. Removal of this glycosylation site alters CREB-mediated functions in vitro and in vivo. These studies are the first demonstration that O-glycosylation at a specific site on a specific protein is critical for neuronal function and behavior.
\r\n\r\nChondroitin sulfates (CS) are sulfated linear polysaccharides important in neuronal development and viral invasion. Depending on their sulfation patterns, CS molecules differ dramatically in their functions. We developed a computational method to model the structure and function of CS. Using this approach, we show that different CS tetrasaccharides have distinct solution structures. We also modeled the CS binding site on a variety of proteins and discovered that CS may be important in modulating protein-protein interactions.
", "doi": "10.7907/FTJS-8M82", "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:5903", "collection": "thesis", "collection_id": "5903", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012010-124413933", "type": "thesis", "title": "Signaling Proteins in the Post-Synaptic Density", "author": [ { "family_name": "Luong", "given_name": "Tinh Nghi", "clpid": "Luong-Tinh-Nghi" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The ability of an organism to respond to its environment stems from synapses and signaling in the post-synaptic density (PSD). Neurological disorders often occur at the level of faulty signal transduction in the PSD. Here we describe the behavioral characterization of Densin 180, a PSD-enriched scaffold protein. We also report on the regulation of Ras guanine release factor1 (RasGRF1), a guanine exchange factor that promotes activation of Ras and thus the ERK pathway as part of an NMDA (N-methyl-D-aspartate) receptor complex with CaMKII (Ca2+/calmodulin-dependent kinase). The Densin KO exhibits severe nest building deficits, elevated anxiety and aggressiveness, impaired sensorimotor gating, hyperlocomotion to novel objects, and short-term memory (hippocampal- and cortical- dependent) deficits. These behavioral abnormalities resemble schizophrenia and autism. Decreases in the schizophrenia susceptibility gene products, DISC1 and mGluR5, are observed in the KO relative to the WT and may be a result of a decrease in their common binding partner \u03b1-actinin. \u03b1-actinin is known to regulate mGluR5 surface levels. Cross-linking and stabilization of PSD protein architecture by scaffold proteins like Densin may contribute to some of the observed behavioral abnormalities. The Densin KO also has blunted activity-dependent gene expression. Steady-state levels of the immediate early genes Arc and c-fos are decreased in the hippocampus and cortex of brain sections. Levels of Arc induced in response to stimulation by the neurotrophin BDNF is significantly decreased in the Densin KO neurons after 8 hours of treatment. Impairments in BDNF signaling can lead to affective and cognitive disorder and has a role in cortical inhibition. Dysfunction in BDNF signaling and DISC1 signaling have been previously implicated in autism. The Densin KO also is susceptible to seizures, in particular when injected with Nembutal, a GABA(A) agonist. One point of intersection between signaling pathways that involve DISC1, mGluR, and BDNF is at the level of ERK signaling, which if impaired may establish a hypoglutamatergic state in the Densin KO.
\r\n\r\nIn addition to characterizing the Densin KO, we studied possible interactions of RasGRF1 and CaMKII with the NR2B subunit tail of the NMDA receptor. CaMKII phosphorylation sites on RasGRF1 were identified, including Ser916, by mass spectrometry. Immunoprecipitation from HEK cells revealed that RasGRF1 enhances CaMKII association with NR2B.
", "doi": "10.7907/GQ1D-CY75", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5903", "collection": "thesis", "collection_id": "5903", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012010-124413933", "type": "thesis", "title": "Signaling Proteins in the Post-Synaptic Density", "author": [ { "family_name": "Luong", "given_name": "Tinh Nghi", "clpid": "Luong-Tinh-Nghi" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The ability of an organism to respond to its environment stems from synapses and signaling in the post-synaptic density (PSD). Neurological disorders often occur at the level of faulty signal transduction in the PSD. Here we describe the behavioral characterization of Densin 180, a PSD-enriched scaffold protein. We also report on the regulation of Ras guanine release factor1 (RasGRF1), a guanine exchange factor that promotes activation of Ras and thus the ERK pathway as part of an NMDA (N-methyl-D-aspartate) receptor complex with CaMKII (Ca2+/calmodulin-dependent kinase). The Densin KO exhibits severe nest building deficits, elevated anxiety and aggressiveness, impaired sensorimotor gating, hyperlocomotion to novel objects, and short-term memory (hippocampal- and cortical- dependent) deficits. These behavioral abnormalities resemble schizophrenia and autism. Decreases in the schizophrenia susceptibility gene products, DISC1 and mGluR5, are observed in the KO relative to the WT and may be a result of a decrease in their common binding partner \u03b1-actinin. \u03b1-actinin is known to regulate mGluR5 surface levels. Cross-linking and stabilization of PSD protein architecture by scaffold proteins like Densin may contribute to some of the observed behavioral abnormalities. The Densin KO also has blunted activity-dependent gene expression. Steady-state levels of the immediate early genes Arc and c-fos are decreased in the hippocampus and cortex of brain sections. Levels of Arc induced in response to stimulation by the neurotrophin BDNF is significantly decreased in the Densin KO neurons after 8 hours of treatment. Impairments in BDNF signaling can lead to affective and cognitive disorder and has a role in cortical inhibition. Dysfunction in BDNF signaling and DISC1 signaling have been previously implicated in autism. The Densin KO also is susceptible to seizures, in particular when injected with Nembutal, a GABA(A) agonist. One point of intersection between signaling pathways that involve DISC1, mGluR, and BDNF is at the level of ERK signaling, which if impaired may establish a hypoglutamatergic state in the Densin KO.
\r\n\r\nIn addition to characterizing the Densin KO, we studied possible interactions of RasGRF1 and CaMKII with the NR2B subunit tail of the NMDA receptor. CaMKII phosphorylation sites on RasGRF1 were identified, including Ser916, by mass spectrometry. Immunoprecipitation from HEK cells revealed that RasGRF1 enhances CaMKII association with NR2B.
", "doi": "10.7907/GQ1D-CY75", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5835", "collection": "thesis", "collection_id": "5835", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252010-112354381", "primary_object_url": { "basename": "AshleyWrightThesis_Final.pdf", "content": "final", "filesize": 102522512, "license": "other", "mime_type": "application/pdf", "url": "/5835/1/AshleyWrightThesis_Final.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Genetic Analysis of Axon Guidance in Drosophila melanogaster", "author": [ { "family_name": "Wright", "given_name": "Ashley Palani", "clpid": "Wright-AshleyPalani" } ], "thesis_advisor": [ { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Due to its genetic manipulability and relatively short reproductive cycle, genetic screens are often carried out in the fruit fly, Drosophila melanogaster. Deficiency \u201ckits\u201d that cover the Drosophila genome with a minimum number of lines have been established by other groups to facilitate gene mapping. These kits cannot be systematically analyzed for many phenotypes, however, because embryos homozygous for many deficiencies fail to develop due to the loss of key gene products. To create new kits that can be screened for more phenotypes, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ~400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. Screens of this kit can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16 (thus allowing for central nervous system development), covers ~50% of the genome. We have screened this smaller kit for motor axon guidance phenotypes, and we present examples of new axon guidance phenotypes in the central nervous system and neuromuscular system. Through screening of these kits, we also found deficiencies that fail to stain with monoclonal antibody BP102, which recognizes an unknown epitope on the proximal segments of central nervous system axons. In addition, we have found a deficiency that exhibits ectopic BP102 staining on peripheral sensory neurons. By defining the single genes under these deficiencies, we have obtained evidence that BP102 may recognize a chondroitin sulfate proteoglycan and that BP102 epitope expression is regulated by matrix metalloproteinase 1. Thus, in addition to this screen providing information about motor axon guidance in the embryo, we have also been able to further characterize an antibody that is frequently used by the Drosophila community.\r\n", "doi": "10.7907/FTNQ-S839", "publication_date": "2010-06-11", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:4333", "collection": "thesis", "collection_id": "4333", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10302008-123855", "primary_object_url": { "basename": "00fullthesis.pdf", "content": "final", "filesize": 23301278, "license": "other", "mime_type": "application/pdf", "url": "/4333/1/00fullthesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Understanding the Chemical Basis of Neuronal Development and Communication: I. The Role of Fucose \u03b1(1-2) Galactose Carbohydrates in Neuronal Growth. II. Structure-Function Analysis of Chondroitin Sulfate in the Brain", "author": [ { "family_name": "Gama", "given_name": "Cristal Ivette", "clpid": "Gama-Cristal-Ivette" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Although carbohydrates are known to participate in many processes including inflammation and cancer metastasis, their functional roles are only beginning to be understood on a molecular level. Unlike DNA and proteins, carbohydrate structures are not template-encoded and are challenging to detect in vivo and manipulate for structure-function analyses. New tools are needed to complement biochemical and genetic approaches to advance our understanding of carbohydrates and their physiological roles. We seek to understand the roles of carbohydrates in regulating the structure and function of proteins in the brain. Our focus is on two modifications that are important in neuronal communication and development: fucosylation (Part I) and chondroitin sulfate modifications (Part II).
\r\n\r\nIn Part I, we describe our progress in elucidating the molecular mechanisms by which fucosyl saccharides regulate neuronal communication. Previous studies have shown that preventing formation of fucose\u03b1(1-2)galactose saccharides causes reversible amnesia in animals, suggesting that these sugars play essential roles in learning and memory. However, proteins expressing the fucose\u03b1(1-2)galactose epitope or proteins binding this epitope have not been identified. Using chemical probes, we established that fucose\u03b1(1-2)galactose associated proteins participate in a novel carbohydrate-mediated pathway for regulating neuronal growth. Additionally, we found that fucose\u03b1(1-2)galactose glycoproteins are prevalent in developing brain and that synapsin Ia/Ib are the major fucose\u03b1(1-2)galactose glycoproteins in adult brain. In our attempts to identify Fuc\u03b1(1-2)Gal lectins, we have established that multivalent polymers enhance our ability to capture and characterize such proteins.
\r\n\r\nIn Part II, we describe our efforts toward understanding the role of chondroitin sulfate glycosaminoglycans in neuronal development. Chondroitin sulfate glycosaminoglycans are structurally complex and heterogeneous in nature, thus hampering efforts to understand their precise biological roles. It is thought that chondroitin sulfate activity is dictated by a sulfation code, where distinct sulfation sequences are spatially and temporally regulated. We have developed a chemical approach to evaluate the structure-activity relationship of chondroitin sulfate as it effects neuronal growth. We generated the first synthetic library of well-defined chondroitin sulfate oligosaccharides containing various sulfation sequences and have demonstrated that the chondroitin sulfate-E sequence is a stimulatory motif that promotes the growth of several neuron types. Moreover, we determined that chondroitin sulfate-E stimulation was facilitated through activation of the midkine/PTP\u03b6 and BDNF/TrkB pathways.
", "doi": "10.7907/CDGH-MJ49", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5222", "collection": "thesis", "collection_id": "5222", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06022009-194041", "primary_object_url": { "basename": "FinalThesis.pdf", "content": "final", "filesize": 82858582, "license": "other", "mime_type": "application/pdf", "url": "/5222/7/FinalThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Construction and Initial Characterization of the Densin Knockout Mouse", "author": [ { "family_name": "Medina-Marino", "given_name": "Andrew G. A.", "orcid": "0000-0002-2691-982X", "clpid": "Medina-Marino-Andrew-G-A" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Densin-180 is a core protein of postsynaptic densities (PSDs) in excitatory neurons. Densin is known to interact with Maguin-1 and PSD-95, suggesting that it plays a role in the NMDA receptor complex. Densin also interacts with \u03b4-Catenin and N-Cadherin, an adhesion complex known to play a role in spine morphology. A ternary complex of Densin, CaMKII, and alpha-actinin suggests that Densin plays a key role in cytoskeleton dynamics. Finally, Densin can directly bind to shank, a core scaffolding molecule of the postsynaptic density. The association of Densin with such diverse complexes of proteins suggests that it acts as an integrator of numerous signaling cascades. Here I describe the construction and initial characterization of a Densin knockout mouse. Mice homozygous for the Densin deletion are prone to seizures induced by barbiturates. Also, Densin^-/- animals have altered spine morphologies and show changes in the expression levels of other core PSD proteins. Densin^-/- neurons in culture exhibit an overall decrease in their dendritic complexity. Furthermore, we show that in the absence of the NMDA receptor, Densin can act to bind CaMKII in the PSD. A new high-throughput method for studying changes in gene transcription, RNA seq, was also used to study the effect of the Densin deletion on the forebrain and the hippocampus. This work represents the first time RNA seq has been used to study an animal with a knockout mutation. Two candidate genes that may mediate the seizure sensitivity, Npas4 and GABAA\u03b12, were identified by this method. Npas4 is known to directly affect the number of inhibitory synapses formed by neurons, and GABAA\u03b12 is a major GABA receptor subunit that mediates the effects of Nembutal. These results suggest that Densin may play a role in maintaining the balance between inhibitory and excitatory networks. Together, our results demonstrate that Densin is important for dendritic arbor formation, spine morphology, CaMKII localization in the PSD, and seizure susceptibility.\r\n", "doi": "10.7907/HYHX-A061", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1780", "collection": "thesis", "collection_id": "1780", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05132008-133518", "primary_object_url": { "basename": "SmithThesis2008.pdf", "content": "final", "filesize": 12412200, "license": "other", "mime_type": "application/pdf", "url": "/1780/1/SmithThesis2008.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Maternal Immune Activation and Abnormal Behavior in the Adult Offspring: Towards a Mechanism", "author": [ { "family_name": "Smith", "given_name": "Stephen Edward Paucha", "clpid": "Smith-Stephen-Edward-Paucha" } ], "thesis_advisor": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Maternal infection is a risk factor for both schizophrenia and autism. The offspring of women who develop an infection during pregnancy are several times more likely to develop these diseases compared to offspring from uncomplicated pregnancies. Modeling this risk factor in animals, when pregnant rodents are given an influenza infection during pregnancy, their offspring show several behavioral and histological abnormalities consistent with human mental illness. Maternal injection of non-infectious, immune-activating compounds, such as the dsRNA poly(I:C), yields similar results, suggesting that the maternal immune response causes deleterious changes in fetal brain development. The main focus of this thesis is establishing the importance of a single component of the maternal immune response, the cytokine interleukin-6 (IL-6), in mediating the observed changes in the brain development and behavior of the offspring. In addition, I report new observations on the offspring of poly(I:C)-activated pregnant mice that are consistent with findings in autism and schizophrenia: a localized deficit of Purkinje cells in the cerebellum, abnormal eye-blink conditioning, abnormal hippocampal-dependent behaviors and hyper-sensitivity to dopamine in the hippocampus. I also present data on the immune reaction to poly(I:C) in pregnant non-human primates. Finally, I describe preliminary findings on the identification of factors that act down-stream of IL-6. The mechanism through which maternal immune activation causes abnormal behavior in the offspring could illuminate important pathways that contribute to the pathogenesis of schizophrenia and autism.", "doi": "10.7907/CB5Y-P545", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4234", "collection": "thesis", "collection_id": "4234", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10242005-165226", "primary_object_url": { "basename": "HGdissertation.pdf", "content": "final", "filesize": 3024537, "license": "other", "mime_type": "application/pdf", "url": "/4234/1/HGdissertation.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Novel Methods for Studying Ras/Erk MAP Kinase Signaling in Developing T Cells", "author": [ { "family_name": "Green", "given_name": "Harry Miguel", "clpid": "Green-Harry-Miguel" } ], "thesis_advisor": [ { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The Ras/Erk MAPK pathway has been shown to be important in multiple developmental contexts. The development of T cells in the thymus is one such developmental system. Thymocytes undergo positive and negative selection, processes by which they are \"chosen\" for their ability to recognize MHC molecules loaded with peptide on the surface of cells, but only to react when the peptide is foreign. The Ras/Erk cascade has been shown to be indispensable during the onset of positive selection, but the mechanism of Erk signaling in this process is unknown. In addition, it is unclear if the Ras/Erk cascade is involved in the differentiation phase of positive selection called CD4/CD8 lineage determination, where thymocytes either become CD4+ or CD8+ T cells. Furthermore, Erk signaling has been shown to be activated during negative selection, but seems dispensable. In this thesis, we describe novel methods for analyzing Erk signaling by applying new technologies to gain a different perspective on Erk signaling in thymocytes during selection. To this end, we have utilized a technique of intracellular staining to obtain data for single-cell Erk activation in the context of a population of fixed thymocytes. We also pursued the development of FRET-based, genetically-encoded intracellular sensors of Erk activity that could be applied to the analysis of Erk signaling in live thymocytes in vivo. To examine the involvement of Ras/Erk signaling during CD4/CD8 lineage determination, we applied a recently described method of lentiviral transgenesis to examine dose-dependent effects of a dominant negative form of Mek, the Erk MAPK kinase, in a single mouse generation. These studies have yielded insights into Erk signaling events and advanced the development of novel techniques to examine signaling during thymocyte selection.", "doi": "10.7907/8wa9-t690", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2462", "collection": "thesis", "collection_id": "2462", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06052006-142955", "primary_object_url": { "basename": "PhDthesis.pdf", "content": "final", "filesize": 4813951, "license": "other", "mime_type": "application/pdf", "url": "/2462/1/PhDthesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Quantitative Model of Calcium/Calmodulin- Dependent Protein Kinase II Activation", "author": [ { "family_name": "Mihalas", "given_name": "Stefan", "orcid": "0000-0002-2629-7100", "clpid": "Mihalas-Stefan" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" }, { "family_name": "Thorne", "given_name": "Kip S.", "clpid": "Thorne-K-S" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key element in the calcium second messenger cascades that lead to long term potentiation (LTP) of synaptic strength. In this thesis, I have constructed kinetic models of activation of CaMKII and measured some of the unknown parameters of the model. I used the models to elucidate mechanisms of activation of CaMKII and to study the kinetics of its activation under conditions similar to those in dendritic spines.
\r\n\r\nIn chapter 2, I developed a new experimental method to rapidly stop the autophosphorylation reaction. I used this method to measure the catalytic turnover number of CaMKII. To quantitatively characterize CaMKII atophosphorylation in nonsaturating calcium, I also measured the autophosphorylation turnover number when CaMKII is activated by calmodulin mutants that can bind calcium ions only in either the amino or the carboxyl lobes.
\r\n\r\nPrevious models of CaMKII activation assumed that binding of calmodulins to individual CaMKII subunits is independent and that autophosphorylation occurs within a ring of 6 subunits. However, a recent structure of CaMKII suggests that pairs of subunits cooperate in binding calmodulin and raises the possibility that the autophosphorylation occurs within pairs of subunits. In chapter 3, I constructed a model in which CaMKII subunits cooperate in binding calmodulin. This model reconciled previous experimental results from the literature that appeared contradictory. In chapter 4, I constructed two models for CaMKII autophosphorylation, in which autophosphorylation can occur either in rings or pairs, and used them to design experiments aimed at differentiating between these possibilities. Previously published measurements and the measurements that I performed are more consistent with autophosphorylation occurring within pairs.
\r\n\r\nIn chapter 5, I constructed a model for simultaneous interactions among calcium, calmodulin, and CaMKII, and I used an automatic parameter search algorithm to fit the parameters for this model. I used it to characterize which of the parameters of calcium transients are critical for CaMKII activation.
\r\n\r\nThis modeling work is part of a continuing effort to realistically model the spatial and temporal aspects of calcium second messenger signaling in dendritic spines.
", "doi": "10.7907/d0zv-g984", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2165", "collection": "thesis", "collection_id": "2165", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272006-184911", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 7841790, "license": "other", "mime_type": "application/pdf", "url": "/2165/1/THESIS.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Ubiquitin-Proteasome System at the Synapse", "author": [ { "family_name": "Bingol", "given_name": "Baris", "orcid": "0000-0002-8225-4367", "clpid": "Bingol-Baris" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "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" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Each neuron in the mammalian central nervous system makes up to ten thousand synaptic connections with other neurons yet is able to regulate the strength of individual connections locally. Synaptic enhancement or depression induced at one location on the dendritic arbor does not spread through out the entire neuron. This means neurons must be able to regulate the complement and concentration of the synaptic proteins locally, near synapses. The local concentration of synaptic proteins is influenced by many processes, including protein trafficking, buffering and sequestration, and most directly by protein synthesis and degradation. In recent years, it has been shown that neurons can synthesize proteins locally in their dendrites. These studies have suggested that any cellular process that regulates protein availability could be of importance in regulating synaptic function and plasticity. Indeed, the evidence for the contribution of local protein degradation to the regulation of synaptic function and plasticity has started to emerge in recent years.
\r\n\r\nHere, we show that synapses have the machinery required to degrade proteins and local protein degradation occurs in the dendrites. Furthermore, we demonstrate the requirement for protein degradation for one of the main cellular correlates of synaptic plasticity, namely the trafficking of glutamate receptors. In turn, we demonstrate how neuronal activity regulates protein degradation at synapses, specifically by mobilizing the enzymatic machinery for protein degradation. These data show that the interplay between protein degradation and synaptic activity functions to sculpt the protein composition of the synapses.
", "doi": "10.7907/qyq5-k147", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2327", "collection": "thesis", "collection_id": "2327", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05312005-114949", "primary_object_url": { "basename": "FinalThesisJoyce.pdf", "content": "final", "filesize": 22064932, "license": "other", "mime_type": "application/pdf", "url": "/2327/1/FinalThesisJoyce.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure and Function Prediction of Human Muscarinic Acetylcholine Receptor 1, Cation-\u03c0 Studies, and Protein Design", "author": [ { "family_name": "Peng", "given_name": "Joyce Yaochun", "clpid": "Peng-Joyce-Yaochun" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Vaidehi", "given_name": "Nagarajan", "orcid": "0000-0001-8100-8132", "clpid": "Vaidehi-N" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "(1) Muscarinic acetylcholine receptors, a G protein-coupled receptor, are responsible for a wide range of diseases. We predicted the 3D structure of the human M1 muscarinic receptor using the MembStruk method and validated its binding sites for 10 agonists and antagonists using the HierDock method. The predicted binding sites, the intramolecular contacts that stabilize the receptor conformation, and the in silico mutagenesis results, agree well with mutagenesis data. The calculated relative binding energies correlate well with measured binding affinities. In addition, the predicted binding sites provide a structural basis for the large reduction in ligand binding affinity and signaling efficacy by Trp 157 and Pro 159 mutations, which was not previously explained by homology models. The predicted binding sites illustrate the importance of aromatic residues in ligand binding through extensive cation-pi and aromatic-aromatic interactions, with new mutation candidates suggested. The predicted M1 structure improves our understanding of the muscarinic receptors, offers a basis for structure based drug design, and is a successful step toward applying these procedures in predicting the structures of other muscarinic receptor subtypes.
\r\n\r\n(2) We used high-level quantum mechanics to quantify cation-pi interactions in the crystal structure of carbamylcholine binding to Acetylcholine-binding Protein, a nicotinic receptor homolog. The calculated effects of fluorinated unnatural amino acid substitutions also correlate excellently with experimental EC50 data, suggesting that quantum mechanics can accurately predict cation-pi binding in a protein environment and provides a good model system in developing force fields to better describe cation-pi interactions.
\r\n\r\n(3) Histidines are known to modulate pH responsive binding. We designed a series of histidine derivatives by substituting its imidazole ring with functional groups that are small in size and lack the ability to form hydrogen bonds. Quantum mechanical calculations of the acid dissociation constants (pKa) show that these substitutions shift the histidine pKa upward or downward. We report a list of histidine derivatives and their corresponding pKa values that can be used in designing tumor specific drugs (e.g. HER2-Herceptin antibody), drug delivery through pH sensitive hydrogels, drug recycling, catalysis, and biosensors development. An example of how these unnatural histidines can be used is illustrated with 2-methyl histidine incorporated in a c-Myc-Max heterodimer.
", "doi": "10.7907/XVJR-RN32", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "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: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:1038", "collection": "thesis", "collection_id": "1038", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03212003-142207", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 1337146, "license": "other", "mime_type": "application/pdf", "url": "/1038/1/thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Biochemical Characterization of Drosophila Receptor Tyrosine Phosphatases", "author": [ { "family_name": "Burkemper", "given_name": "Bruce Seymour", "orcid": "0000-0003-3689-7481", "clpid": "Burkemper-Bruce-Seymour" } ], "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": "Hay", "given_name": "Bruce A.", "clpid": "Hay-B-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Two classes of enzymes are responsible for modulation of intracellular phosphotyrosine levels, namely protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Together these enzymes maintain the appropriate balance of phosphoproteins required for a variety of developmental processes including axon pathfinding. In Drosophila, five receptor-like protein tyrosine phosphatases (RPTPs) regulate axon pathfinding, but little is known about their downstream signaling pathways or the means by which their enzymatic activity is regulated.
\r\n\r\nChapter 2 of this thesis deals with experiments to test whether dimerization regulates the activity of these enzymes. Crystallographic data indicates that some RPTPs form dimers in which each monomer is precluded from binding substrate due to the insertion of a helix-turn-helix segment of the opposing monomer into the active site. I introduced ?tagged? RPTP constructs into Drosophila S2 tissue culture cells and tested for dimer formation using immunoprecipitation and Western blotting. I did not detect stable dimers, however. This may suggest that dimer formation requires other protein components (such as the putative RPTP ligands) that are not expressed in S2 cells.
\r\n\r\nIn Chapter 3 I investigated the possibility that Roundabout (Robo), a receptor mediating axonal repulsion from the embryonic midline, is a substrate for RPTPs DPTP69D and/or DPTP10D. Previous genetic studies implicate these RPTPs in participating in the Robo signaling pathway. Experiments detailed here show that Robo can be phosphorylated on tyrosine residues in S2 cells, characteristic of an RPTP substrate. However, Robo did not co-immunoprecipitate with ?substrate trap? mutants of either of these RPTPs, possibly because their interaction is dependent on co-factors not present in the cell culture system.
\r\n\r\nChapter 4 is a characterization of DPTP69D-associated proteins purified from embryos expressing a substrate trap version of DPTP69D. We identified one of the associated proteins as non-muscle myosin II heavy chain (nmm II hc). Proper regulation of nmm II hc is essential for axon patterning in mushroom bodies (MBs). I found that expression of the DPTP69D trap in MBs results in an axon retraction phenotype similar to that seen when nmm II hc activity is elevated, suggesting that this protein may be a target for DPTP69D activity.
", "doi": "10.7907/P1HY-MF28", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "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: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:17734", "collection": "thesis", "collection_id": "17734", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282025-173845996", "primary_object_url": { "basename": "Kang_H_1997.pdf", "content": "final", "filesize": 59673872, "license": "other", "mime_type": "application/pdf", "url": "/17734/1/Kang_H_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Modulation of Synaptic Function by Neurotrophic Factors in the Adult Hippocampus", "author": [ { "family_name": "Kang", "given_name": "Hyejin", "clpid": "Kang-Hyejin" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "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": "Laurent", "given_name": "Gilles J.", "orcid": "0000-0002-2296-114X", "clpid": "Laurent-G-J" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The neurotrophins including nerve growth factor (NGF), brain-derived neurotrophic\r\nfactor (BDNF), and neurotrophin-3 (NT-3), are a group of signaling factors that are\r\ncrucial for neuronal survival and differentiation during development. Previous studies\r\nhave shown that the hippocampus is a prominent site of expression of the neurotrophins\r\nand their Trk receptors in the adult brain. Interestingly, the expression of BDNF, NT-3,\r\nand their receptors can be regulated by a variety of neuronal activity, which suggests that\r\nthe neurotrophins may also participate in adult synaptic plasticity.
\r\n\r\nThe possibility that the neurotrophins directly modulate synaptic strength in the\r\nmature brain was investigated at the Schaffer collateral-CAI synapses in the adult rat\r\nhippocampus. Transient application of BDNF or NT-3 but not NGF produced a dramatic\r\nand sustained (3 to 4 hours) enhancement of synaptic transmission. Both\r\nelectrophysiological and immunocytochemical evidence indicated that the penetration\r\nand the resulting synaptic potentiation by neurotrophins are influenced by the perfusion\r\nrate at which the neurotrophin is applied to hippocampal synapses.
\r\n\r\nThe potentiating effects ofBDNF and NT-3 could be completely blocked by\r\ninhibiting the function of Trk receptors using either a pharmacological inhibitor of\r\ntyrosine kinases or a function-blocking Trk antibody. In addition, blockade ofL-type\r\ncalcium channels or intracellular calcium stores significantly reduced the potentiation\r\ninduced by either neurotrophin. These data suggest that both the activation of Trk\r\nreceptors and the subsequent increase in intracellular calcium concentration are essential\r\nfor the initiation of neurotrophin-induced synaptic enhancement.
\r\n\r\nThe neurotrophin-induced plasticity exhibits an immediate requirement for protein\r\nsynthesis, which is not somatic in origin. The neurotrophins still produced synaptic\r\npotentiation at synapses isolated from their cell bodies; this plasticity displayed a\r\ndependence on protein synthesis, raising the possibility that these factors may stimulate\r\nlocal protein synthesis within dendrites and promote site-specific modification of\r\nsynaptic function.
\r\n\r\nFinally, I report that the neurotrophins play a functional role in certain forms of\r\nhippocampal long-term potentiation (LTP). The maintenance of LTP induced by thetaburst\r\nor pairing, but not strong tetanic stimulation, relied on intact neurotrophin signaling.\r\nIn long-term LTP, the neurotrophins were primarily involved in maintaining the late\r\nphases of synaptic enhancement, without significantly affecting an early phase of\r\npotentiation. Thus, during adult plasticity, synaptic activity-induced increases in\r\nneurotrophin synthesis and release contribute to the late phase of LTP and may eventually\r\nlead to structural changes at the synapse.
", "doi": "10.7907/08g9-gb22", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "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: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: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.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Laurent", "given_name": "Gilles J.", "clpid": "Laurent-G-J" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "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: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:7519", "collection": "thesis", "collection_id": "7519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03142013-100553837", "primary_object_url": { "basename": "Miller-sg-1988.pdf", "content": "final", "filesize": 11369163, "license": "other", "mime_type": "application/pdf", "url": "/7519/1/Miller-sg-1988.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Characterization of a Brain-Region Specific Isozyme and Regulation by Autophosphorylation", "author": [ { "family_name": "Miller", "given_name": "Stephen G.", "clpid": "Miller-Stephen-G" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Aswad", "given_name": "Dana W.", "clpid": "Aswad-Dana-W" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Davidson", "given_name": "Eric H.", "clpid": "Davidson-E-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A variety of molecular approaches have been used to investigate the structural and enzymatic properties of rat brain type ll Ca\u00b2\u207a and calmodulin-dependent protein kinase (type ll CaM kinase). This thesis describes the isolation and biochemical characterization of a brain-region specific isozyme of the kinase and also the regulation the kinase activity by autophosphorylation.
\r\n\r\nThe cerebellar isozyme of the type ll CaM kinase was purified and its biochemical properties were compared to the forebrain isozyme. The cerebellar isozyme is a large (500-kDa) multimeric enzyme composed of multiple copies of 50-kDa \u03b1 subunits and 60/58-kDa \u03b2/\u03b2' subunits. The holoenzyme contains approximately 2 \u03b1 subunits and 8 \u03b2 subunits. This contrasts to the forebrain isozyme, which is also composed of \u03b1 and \u03b2/\u03b2' subunits, but they are assembled into a holoenzyme of approximately 9 \u03b1 subunits and 3 \u03b2/\u03b2' subunits. The biochemical and enzymatic properties of the two isozymes are similar. The two isozymes differ in their association with subcellular structures. Approximately 85% of the cerebellar isozyme, but only 50% of the forebrain isozyme, remains associated with the particulate fraction after homogenization under standard conditions. Postsynaptic densities purified from forebrain contain the forebrain isozyme, and the kinase subunits make up about 16% of their total protein. Postsynaptic densities purified from cerebellum contain the cerebellar isozyme, but the kinase subunits make up only 1-2% of their total protein.
\r\n\r\nThe enzymatic activity of both isozymes of the type II CaM kinase is regulated by autophosphorylation in a complex manner. The kinase is initially completely dependent on Ca\u00b2\u207a/calmodulin for phosphorylation of exogenous substrates as well as for autophosphorylation. Kinase activity becomes partially Ca\u00b2\u207a-independent after autophosphorylation in the presence of Ca\u00b2\u207a/calmodulin. Phosphorylation of only a few subunits in the dodecameric holoenzyme is sufficient to cause this change, suggesting an allosteric interaction between subunits. At the same time, autophosphorylation itself becomes independent of Ca\u00b2\u207a These observations suggest that the kinase may be able to exist in at least two stable states, which differ in their requirements for Ca\u00b2\u207a/calmodulin.
\r\n\r\nThe autophosphorylation sites that are involved in the regulation of kinase activity have been identified within the primary structure of the \u03b1 and \u03b2 subunits. We used the method of reverse phase-HPLC tryptic phosphopeptide mapping to isolate individual phosphorylation sites. The phosphopeptides were then sequenced by gas phase microsequencing. Phosphorylation of a single homologous threonine residue in the \u03b1 and \u03b2 subunits is correlated with the production of the Ca\u00b2\u207a-independent activity state of the kinase. In addition we have identified several sites that are phosphorylated only during autophosphorylation in the absence of Ca\u00b2\u207a/calmodulin.
", "doi": "10.7907/w18z-4r52", "publication_date": "1988", "thesis_type": "phd", "thesis_year": "1988" }, { "id": "thesis:11468", "collection": "thesis", "collection_id": "11468", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152019-165931960", "type": "thesis", "title": "Regional Distribution and Subcellular Associations of Type II Calcium and Calmodulin-Dependent Protein Kinase in Rat Brain", "author": [ { "family_name": "Erondu", "given_name": "Ngozi Emmanuel", "clpid": "Erondu-Ngozi-Emmanuel" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Four monoclonal antibodies generated against the Type II CaM kinase have been characterized. Two of these antibodies were used to confirm that both alpha and beta subunits were part of the holoenzyme complex. I also developed liquid phase and solid phase radioimmunoassays for the kinase.
\r\n\r\nWith the solid phase radioimmunoassay, the distribution of the kinase in rat brain was examined. This study revealed that the concentration of the kinase varies markedly in different brain regions. It is most highly concentrated in the telencephalon where it comprises approximately 2% of total hippocampal protein, 1.3% of cortical protein and 0.7% of striatal protein. It is less concentrated in lower brain regions ranging from 0.3% of hypothalamic protein to 0.1% of protein in the pons/medulla. The unusually high concentration of the kinase in telencephalic regions may confer upon their neurons specialized responses to calcium that are different from those of neurons in lower brain regions.
\r\n\r\nThe association of the kinase with elements of the cytoskeleton was also investigated. The results of this study showed that autophosphorylation causes an increase in the association of the enzyme with taxol-polymerized microtubules and F-actin. This increase in association was reversed by dephosphorylating phosphokinase with protein phosphatase. These results suggest that autophosphorylation could constitute a mechanism for the regulation of the subcellular associations of the Type II CaM kinase by neuronal activity.
", "doi": "10.7907/30tg-qa85", "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: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:11456", "collection": "thesis", "collection_id": "11456", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04122019-162032124", "type": "thesis", "title": "Brain Type II Calcium and Calmodulin-Dependent Protein Kinase: Purification, Characterization and Molecular Cloning", "author": [ { "family_name": "Bennett", "given_name": "Mark Knowles", "clpid": "Bennett-Mark-Knowles" } ], "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": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Tanouye", "given_name": "Mark", "clpid": "Tanouye-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A combination of biochemical, immunochemical, and molecular biological techniques have been employed to purify and characterize a rat brain Ca2+/calmodulin-dependent protein kinase. The enzyme, named type II Ca2+/calmodulin-dependent protein kinase (type II CaM kinase), was identified in rat brain homogenates by its ability to phosphorylate site II on the synaptic vesicle associated protein synapsin I.
\r\n\r\nType II CaM kinase has been purified 290 fold over crude homogenates and is found to be composed of multiple copies of two different subunits. Both subunits copurify with kinase activity and are coprecipitated with kinase activity by an anti-kinase monoclonal antibody. The two subunits have molecular weights of 50,000 (\u03b1) and 58,000/60,000 (\u03b2), and are present in a 3:1 \u03b1:\u03b2 ratio. The type II CaM kinase holoenzyme has a sedimentation coefficient of 16.4 S, a Stokes radius of 95 \u00c5, and a calculated molecular weight of 650,000. A dodecameric holoenzyme consisting of 9 \u03b1 subunits and 3 \u03b2 subunits has been proposed. The purified type II CaM kinase phosphorylates several substrates, in addition to synapsin I, at a significant rate, and may therefore be responsible for a number of neuronal responses to Ca2+.
\r\n\r\nThe \u03b1 subunit of type II CaM kinase has a number of biochemical characteristics which are similar to the major protein component of a subcellular fraction which is derived from brain postsynaptic densities (PSDs). A direct comparison between the a subunit of type II CaM kinase and the major PSD protein using immunochemical and biochemical techniques has revealed that they are in fact very similar or identical proteins.
\r\n\r\nTwo approaches have been taken to further characterize the subunits of type II CaM kinase at a molecular level. The first approach has been to isolate cDNA clones which code for the \u03b2 subunit. A number of clones have been isolated and sequenced. The ammo acid sequence for the \u03b2 subunit (predicted from the cDNA sequence) is homologous to several other protein kinases. Southern blot analysis with a \u03b2 subunit cDNA indicates the existence of a type II CaM kinase multigene family. The second approach to the molecular characterization of the type II CaM kinase subunits has been to determine the amino acid sequence of peptides derived from the \u03b1 subunit. Two regions of \u03b1 subunit sequence have been determined, and both are found to be homologous to regions of \u03b2 subunit amino acid sequence deduced from \u03b2 subunit cDNA clones.
\r\n\r\nThe molecular characterization of neuronal type II CaM kinase in vitro has both provided insight into the possible function of the enzyme in vivo and suggested experimental approaches which may eventually allow its in vivo function to be directly addressed.
", "doi": "10.7907/a6p9-vd59", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:11375", "collection": "thesis", "collection_id": "11375", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-172314851", "type": "thesis", "title": "The Genes for Myelin Basic Protein in Normal and Shiverer Mutant Mice", "author": [ { "family_name": "Roach", "given_name": "Arthur Henry", "clpid": "Roach-Arthur-Henry" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "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": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A cDNA library was constructed from the brains of 18 day old rats, and was screened with a synthetic DNA probe to yield clones representing myelin basic protein (MBP). One 1.9 kb clone was sequenced and found to encode the 14 kd MBP. Using this clone as a hybridization probe, cosmid clones from a library of wild type mouse DNA were selected and characterized. One clone was shown to carry five exons which encode 14 kd MBP, distributed over a 32 kb region. A sixth exon was detected with a synthetic DNA probe, and was found to encode the 41 amino acids which distinguish 18.5 kd from 14 kd MBP. The 5' end ot the gene was mapped with S1 nuclease protection and primer extension experiments to a position 47 bp 5' of the initator codon for MBP synthesis. It was shown that the gene cloned is probably the only MBP gene in the mouse genome.
\r\n\r\nCloned DNAs were used to analyze the MBP gene and its expression in the myelin deficient mutant mouse shiverer. It was shown that a deletion has removed five out of six MBP exons, leaving only the 5'-most exon and 13 kb of the first intervening sequence. The deletion completely prevents expression of normal 2.1 kb MBP mRNAs, but a 16-fold lower number of transcripts are observed which initiate correctly at the 5' end of the first exon, are not correctly spliced, and are rarely polyadenylated. If translated, they would direct synthesis of a 61 amino acid peptide containing the first 56 amino acids of MBP. The MBP gene was mapped to mouse chromosome 18 by hybridization of MBP probes with DNA from Chinese hamster-mouse hybrid cell lines, showing it to be linked to the shiverer mutation. It is proposed that the partial deletion of the MBP gene is the primary lesion of the shiverer mutation.
", "doi": "10.7907/4d7s-zs52", "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:11375", "collection": "thesis", "collection_id": "11375", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-172314851", "type": "thesis", "title": "The Genes for Myelin Basic Protein in Normal and Shiverer Mutant Mice", "author": [ { "family_name": "Roach", "given_name": "Arthur Henry", "clpid": "Roach-Arthur-Henry" } ], "thesis_advisor": [ { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "thesis_committee": [ { "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": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Strumwasser", "given_name": "Felix", "clpid": "Strumwasser-F" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A cDNA library was constructed from the brains of 18 day old rats, and was screened with a synthetic DNA probe to yield clones representing myelin basic protein (MBP). One 1.9 kb clone was sequenced and found to encode the 14 kd MBP. Using this clone as a hybridization probe, cosmid clones from a library of wild type mouse DNA were selected and characterized. One clone was shown to carry five exons which encode 14 kd MBP, distributed over a 32 kb region. A sixth exon was detected with a synthetic DNA probe, and was found to encode the 41 amino acids which distinguish 18.5 kd from 14 kd MBP. The 5' end ot the gene was mapped with S1 nuclease protection and primer extension experiments to a position 47 bp 5' of the initator codon for MBP synthesis. It was shown that the gene cloned is probably the only MBP gene in the mouse genome.
\r\n\r\nCloned DNAs were used to analyze the MBP gene and its expression in the myelin deficient mutant mouse shiverer. It was shown that a deletion has removed five out of six MBP exons, leaving only the 5'-most exon and 13 kb of the first intervening sequence. The deletion completely prevents expression of normal 2.1 kb MBP mRNAs, but a 16-fold lower number of transcripts are observed which initiate correctly at the 5' end of the first exon, are not correctly spliced, and are rarely polyadenylated. If translated, they would direct synthesis of a 61 amino acid peptide containing the first 56 amino acids of MBP. The MBP gene was mapped to mouse chromosome 18 by hybridization of MBP probes with DNA from Chinese hamster-mouse hybrid cell lines, showing it to be linked to the shiverer mutation. It is proposed that the partial deletion of the MBP gene is the primary lesion of the shiverer mutation.
", "doi": "10.7907/4d7s-zs52", "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:11295", "collection": "thesis", "collection_id": "11295", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12072018-092224939", "primary_object_url": { "basename": "Eatock_RA_1984.pdf", "content": "final", "filesize": 37219085, "license": "other", "mime_type": "application/pdf", "url": "/11295/1/Eatock_RA_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Sensory Adaptation in Hair Cells of the Bullfrog's Sacculus", "author": [ { "family_name": "Eatock", "given_name": "Ruth Anne", "orcid": "0000-0001-7547-2051", "clpid": "Eatock-Ruth-Anne" } ], "thesis_advisor": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "thesis_committee": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "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": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hair cells in the bullfrog's sacculus, a vestibular organ sensitive to linear acceleration, show sensory adaptation: the response to a constant stimulus peaks near the stimulus onset, then decays. This has been studied in two different preparations. In an excised in vitro preparation of the saccular sensory epithelium, intracellular responses of hair cells to step deflections of their hair bundles were recorded. The second set of experiments was conducted in vivo using steps of vertical linear acceleration as stimuli. The hair cell response was recorded extracellularly in the form of the saccular microphonic potential. Both the intracellular response to direct hair bundle deflection and the extracellular response to acceleration adapted to a steady-state value within the first 100 ms following the step onset. In both cases, the response decline was largely due to a shift in the operating range of the cells in the direction of the constant stimulus. This shift occurred without significant change in dynamic range or in sensitivity within the operating range. Thus the hair cells appear to respond to static stimuli by resetting the bias point of the operating range in the direction of the stimulus.
\r\n\r\nThe response of primary saccular neurons to acceleration steps also showed pronounced sensory adaptation. Comparison of the afferent activity and saccular microphonic potential suggests that adaptation of afferent responses to acceleration steps may be due largely to the adaptive operating range shift in the hair cell responses.
\r\n\r\nThe adaptation of saccular neurons to acceleration steps may be explained by the following simple model. The acceleration step causes displacement of the saccular otolith and deflection of the underlying hair bundles. The hair cells respond initially to the displacement, then adapt (as observed in vitro), and this information is faithfully translated postsynaptically into afferent spike rate. However, the possibility exists that the in vitro and in vivo adaptive shifts in operating range are not the same process. In vivo, one cannot distinguish an operating range shift within the hair cells from one due to mechanical adaptation of the stimulus to the hair bundles.
", "doi": "10.7907/6pam-hv78", "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:11295", "collection": "thesis", "collection_id": "11295", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12072018-092224939", "primary_object_url": { "basename": "Eatock_RA_1984.pdf", "content": "final", "filesize": 37219085, "license": "other", "mime_type": "application/pdf", "url": "/11295/1/Eatock_RA_1984.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Sensory Adaptation in Hair Cells of the Bullfrog's Sacculus", "author": [ { "family_name": "Eatock", "given_name": "Ruth Anne", "orcid": "0000-0001-7547-2051", "clpid": "Eatock-Ruth-Anne" } ], "thesis_advisor": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" } ], "thesis_committee": [ { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "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": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Pine", "given_name": "Jerome", "clpid": "Pine-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hair cells in the bullfrog's sacculus, a vestibular organ sensitive to linear acceleration, show sensory adaptation: the response to a constant stimulus peaks near the stimulus onset, then decays. This has been studied in two different preparations. In an excised in vitro preparation of the saccular sensory epithelium, intracellular responses of hair cells to step deflections of their hair bundles were recorded. The second set of experiments was conducted in vivo using steps of vertical linear acceleration as stimuli. The hair cell response was recorded extracellularly in the form of the saccular microphonic potential. Both the intracellular response to direct hair bundle deflection and the extracellular response to acceleration adapted to a steady-state value within the first 100 ms following the step onset. In both cases, the response decline was largely due to a shift in the operating range of the cells in the direction of the constant stimulus. This shift occurred without significant change in dynamic range or in sensitivity within the operating range. Thus the hair cells appear to respond to static stimuli by resetting the bias point of the operating range in the direction of the stimulus.
\r\n\r\nThe response of primary saccular neurons to acceleration steps also showed pronounced sensory adaptation. Comparison of the afferent activity and saccular microphonic potential suggests that adaptation of afferent responses to acceleration steps may be due largely to the adaptive operating range shift in the hair cell responses.
\r\n\r\nThe adaptation of saccular neurons to acceleration steps may be explained by the following simple model. The acceleration step causes displacement of the saccular otolith and deflection of the underlying hair bundles. The hair cells respond initially to the displacement, then adapt (as observed in vitro), and this information is faithfully translated postsynaptically into afferent spike rate. However, the possibility exists that the in vitro and in vivo adaptive shifts in operating range are not the same process. In vivo, one cannot distinguish an operating range shift within the hair cells from one due to mechanical adaptation of the stimulus to the hair bundles.
", "doi": "10.7907/6pam-hv78", "publication_date": "1984", "thesis_type": "phd", "thesis_year": "1984" }, { "id": "thesis:11830", "collection": "thesis", "collection_id": "11830", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-171906142", "type": "thesis", "title": "Identification and Characterization of Glial Growth Factor", "author": [ { "family_name": "Lemke", "given_name": "Greg Erwin", "clpid": "Lemke-Greg-Erwin" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" } ], "thesis_committee": [ { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts.
\r\n\r\nThree independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel electrophoresis in SDS.
\r\n\r\nThree large-scale purifications of GGF, employing a combination of column chromatography steps and preparative electrophoreses, are described. The molecule has been purified to apparent homogeneity from anterior lobes of the bovine pituitary.
\r\n\r\nThrough the use of nucleic acid precursor incorporation assays, GGF has been shown to be markedly mitogenic for rat Schwann cells, astrocytes and fibroblasts, but inactive when assayed on oligodendrocytes or microglia. Electrophoretic analyses suggest that all responsive cell types are stimulated by a single (the same) molecular species. GGF is the only defined mitogen to which rat Schwann cells respond.
\r\n\r\nGlial growth factor from bovine brain has been found to be indistinguishable from bovine pituitary GGF, as determined by biochemical, immunological and bioactivity criteria. GGF is non-uniformly distributed among bovine brain regions. It is present in brain extracts prepared from a wide variety of vertebrate species.
\r\n\r\nPurified human platelet-derived growth factor (PDGF) shares many important properties with GGF. PDGF has been shown to be unable to significantly stimulate the division of rat Schwann cells, however, and therefore appears to be distinct.
\r\n\r\nObservations made in vitro suggest several possible biological roles for GGF in vivo. These are discussed.
\r\n", "doi": "10.7907/774r-7520", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11842", "collection": "thesis", "collection_id": "11842", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283", "type": "thesis", "title": "Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens", "author": [ { "family_name": "Nicholson", "given_name": "Bruce John", "orcid": "0000-0003-1649-7173", "clpid": "Nicholson-Bruce-John" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
\r\n\r\nComparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
\r\n\r\nThe extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
", "doi": "10.7907/bhjv-8053", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11842", "collection": "thesis", "collection_id": "11842", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10222019-124810283", "type": "thesis", "title": "Biochemistry and Diversity of the Gap Junction Protein: A Study of Liver, Heart and Lens", "author": [ { "family_name": "Nicholson", "given_name": "Bruce John", "orcid": "0000-0003-1649-7173", "clpid": "Nicholson-Bruce-John" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "thesis_committee": [ { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Fractions highly enriched for gap junctions by morphological criteria have been isolated from rat liver, heart and eye lens, although some question exists as to the nature of the structures from lens. The junctions from each tissue are comprised of a single major protein of Mr 28,000 in the liver, Mr 30,000 in the heart, and Mr 26,000 (MIP 26) in the lens. The polypeptide profile of the liver fraction is complicated by endogenous proteolysis and aggregation in SDS of the gap junction protein and the presence of about 20% non-junctional material. Heart and lens junction proteins are also found to aggregate in SDS, while endogenous proteolysis typically reduces the cardiac gap junction protein to Mr 28,000 during isolation.
\r\n\r\nComparisons of two-dimensional peptide maps of the junctional proteins from these tissues, and the use, where necessary, of a third dimension of resolution (HPLC), demonstrates the three proteins to be very different in terms of their primary structures. The protein of each tissue, however, seems well conserved between mammalian species. For liver and lens, this finding has been confirmed in amino acid analyses and partial NH2-terminal sequences (to 58 and 33 residues, respectively). Cleavage products of these two proteins have also been produced to allow further sequence analysis in the future. In spite of the differences in primary structure, some conservation of the tertiary structures of these proteins is suggested by proteolysis of intact junctions (likely restricted to the cytoplasmic surfaces). Liver and heart gap junction proteins are reduced by trypsin to two fragments of Mr 10,000, while a single Mr 21,000 fragment is produced from lens MIP 26. Sequence analysis (liver and lens only) indicates that most of the protein removed by tryptic hydrolysis is from the carboxy-terminus, although an additional loop of 4,000 daltons is excised from the center of the liver polypeptide and five residues are lost from the NH2-terminus of the lens protein.
\r\n\r\nThe extent and possible significance of this surprising tissue specificity of the gap junction protein are discussed in the light of these findings.
", "doi": "10.7907/bhjv-8053", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:11830", "collection": "thesis", "collection_id": "11830", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10182019-171906142", "type": "thesis", "title": "Identification and Characterization of Glial Growth Factor", "author": [ { "family_name": "Lemke", "given_name": "Greg Erwin", "clpid": "Lemke-Greg-Erwin" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" } ], "thesis_committee": [ { "family_name": "Brockes", "given_name": "Jeremy P.", "orcid": "0000-0002-3395-5159", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Hudspeth", "given_name": "A. James", "clpid": "Hudspeth-A-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "clpid": "Hood-L-E" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts.
\r\n\r\nThree independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel electrophoresis in SDS.
\r\n\r\nThree large-scale purifications of GGF, employing a combination of column chromatography steps and preparative electrophoreses, are described. The molecule has been purified to apparent homogeneity from anterior lobes of the bovine pituitary.
\r\n\r\nThrough the use of nucleic acid precursor incorporation assays, GGF has been shown to be markedly mitogenic for rat Schwann cells, astrocytes and fibroblasts, but inactive when assayed on oligodendrocytes or microglia. Electrophoretic analyses suggest that all responsive cell types are stimulated by a single (the same) molecular species. GGF is the only defined mitogen to which rat Schwann cells respond.
\r\n\r\nGlial growth factor from bovine brain has been found to be indistinguishable from bovine pituitary GGF, as determined by biochemical, immunological and bioactivity criteria. GGF is non-uniformly distributed among bovine brain regions. It is present in brain extracts prepared from a wide variety of vertebrate species.
\r\n\r\nPurified human platelet-derived growth factor (PDGF) shares many important properties with GGF. PDGF has been shown to be unable to significantly stimulate the division of rat Schwann cells, however, and therefore appears to be distinct.
\r\n\r\nObservations made in vitro suggest several possible biological roles for GGF in vivo. These are discussed.
\r\n", "doi": "10.7907/774r-7520", "publication_date": "1983", "thesis_type": "phd", "thesis_year": "1983" }, { "id": "thesis:10906", "collection": "thesis", "collection_id": "10906", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-173145200", "primary_object_url": { "basename": "Shotwell_SL_1982.pdf", "content": "final", "filesize": 41193889, "license": "other", "mime_type": "application/pdf", "url": "/10906/1/Shotwell_SL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "A Biochemical and Genetic Analysis of the Cyclic AMP Phosphodiesterase Defect in Dunce, a Memory Mutant of Drosophila", "author": [ { "family_name": "Shotwell", "given_name": "Sandra Lee", "clpid": "Shotwell-Sandra-Lee" } ], "thesis_advisor": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "thesis_committee": [ { "family_name": "Brockes", "given_name": "Jeremy P.", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Hudspeth", "given_name": "James", "clpid": "Hudspeth-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Drosophila can learn in several associative conditioning paradigms. Flies carrying the mutation dunce were selected for their poor performance in one such task, a negative reinforcement olfactory conditioning paradigm (Dudai et al., 1976). dunce flies express two other mutant phenotypes, female sterility, and reduced activity for one of the two cyclic AMP phosphodiesterases present in normal flies, PDE II (Byers et al., 1981) . . The experiments described below indicate that the normal dunce gene (dunce+) probably codes for PDE II itself, rather than for a regulator that affects PDE II and possibly other activities.
\r\n\r\nA micro-assay technique is described that allows the separate measurement of PDE I and PDE II when both are present in mixture. PDE II is shown to occur at high specific activity in the nervous system, which is consistent with a role for this enzyme in neuronal function. The phenotype of female sterility associated with dunce mutants can be suppressed by any of three suppressor mutations. These do not suppress the other two phenotypes of reduced PDE II activity and poor learning, indicating that these phenotypes are closer to the primary defect associated with dunce mutants. Reduced PDE II activity correlates with poor learning in dunce flies in all three developmental stages that were tested (first and third instar larvae, and adults), as well as in response to genetic modifications of dunce gene activity. The results of several biochemical and genetic experiments fail to reveal any abnormal regulation of PDE II activity in dunce flies. In Drosophila, as a rule, the activity level of an enzyme correlates linearly with the activity of the enzyme's structural gene. The specific activity of PDE II is shown to correlate in a one to one fashion with the level of normal dunce gene activity at five different doses of dunce+.
\r\n\r\nTaken as a whole, these experiments provide strong support for the hypothesis that PDE II represents the primary product of the dunce gene, indicating a role for this enzyme in the learning of Drosophila.
", "doi": "10.7907/67ye-6j88", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:10906", "collection": "thesis", "collection_id": "10906", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162018-173145200", "primary_object_url": { "basename": "Shotwell_SL_1982.pdf", "content": "final", "filesize": 41193889, "license": "other", "mime_type": "application/pdf", "url": "/10906/1/Shotwell_SL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "A Biochemical and Genetic Analysis of the Cyclic AMP Phosphodiesterase Defect in Dunce, a Memory Mutant of Drosophila", "author": [ { "family_name": "Shotwell", "given_name": "Sandra Lee", "clpid": "Shotwell-Sandra-Lee" } ], "thesis_advisor": [ { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" } ], "thesis_committee": [ { "family_name": "Brockes", "given_name": "Jeremy P.", "clpid": "Brockes-Jeremy-P" }, { "family_name": "Benzer", "given_name": "Seymour", "clpid": "Benzer-S" }, { "family_name": "Hudspeth", "given_name": "James", "clpid": "Hudspeth-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Owen", "given_name": "Ray David", "clpid": "Owen-R-D" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Drosophila can learn in several associative conditioning paradigms. Flies carrying the mutation dunce were selected for their poor performance in one such task, a negative reinforcement olfactory conditioning paradigm (Dudai et al., 1976). dunce flies express two other mutant phenotypes, female sterility, and reduced activity for one of the two cyclic AMP phosphodiesterases present in normal flies, PDE II (Byers et al., 1981) . . The experiments described below indicate that the normal dunce gene (dunce+) probably codes for PDE II itself, rather than for a regulator that affects PDE II and possibly other activities.
\r\n\r\nA micro-assay technique is described that allows the separate measurement of PDE I and PDE II when both are present in mixture. PDE II is shown to occur at high specific activity in the nervous system, which is consistent with a role for this enzyme in neuronal function. The phenotype of female sterility associated with dunce mutants can be suppressed by any of three suppressor mutations. These do not suppress the other two phenotypes of reduced PDE II activity and poor learning, indicating that these phenotypes are closer to the primary defect associated with dunce mutants. Reduced PDE II activity correlates with poor learning in dunce flies in all three developmental stages that were tested (first and third instar larvae, and adults), as well as in response to genetic modifications of dunce gene activity. The results of several biochemical and genetic experiments fail to reveal any abnormal regulation of PDE II activity in dunce flies. In Drosophila, as a rule, the activity level of an enzyme correlates linearly with the activity of the enzyme's structural gene. The specific activity of PDE II is shown to correlate in a one to one fashion with the level of normal dunce gene activity at five different doses of dunce+.
\r\n\r\nTaken as a whole, these experiments provide strong support for the hypothesis that PDE II represents the primary product of the dunce gene, indicating a role for this enzyme in the learning of Drosophila.
", "doi": "10.7907/67ye-6j88", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:10886", "collection": "thesis", "collection_id": "10886", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594", "primary_object_url": { "basename": "Gard_DL_1982.pdf", "content": "final", "filesize": 118360636, "license": "other", "mime_type": "application/pdf", "url": "/10886/1/Gard_DL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Intermediate Filaments and Myogenesis in vitro", "author": [ { "family_name": "Gard", "given_name": "David Lynn", "clpid": "Gard-David-Lynn" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis describes my investigations into the composition and function of intermediate filaments (IF) during myogenesis in vitro. I have found that avian embryonic myotubes cultured in vitro contain two intermediate filament subunits, desmin and vimentin. Prior to myoblast fusion vimentin is the sole IF subunit protein detectable by electrophoretic and immunological techniques. The onset of desmin synthesis and its cytoplasmic accumulation appear to coincide with fusion of myoblasts into multinucleate myotubes. Immunofluorescence reveals dense networks of desmin- and vimentin-containing filaments in the sarcoplasm of immature myotubes; however, late in myogenesis antisera to both desmin and vimentin are observed to stain the Z-lines of myofibrils. Double immunofluorescence microscopy using antisera to \u03b1-actinin and desmin revealed that this association occurs after the assembly of \u03b1-actinin into Z-lines, at a time when individual myofibrils are being organized into bundles. Phosphorylation of intermediate filament proteins in muscle has been previously reported (O'Connor et al., Proc. Natl. Acad. Sci. U.S.A. 76: 819-823, 1979). Using two-dimensional tryptic analysis I have found that desmin from embryonic myotubes is phosphorylated at multiple sites which correspond to sites phosphorylated by cAMP-dependent protein kinase in vitro. I have observed phosphorylation of desmin and vimentin in intact myotubes at all stages of myogenesis. However, treatment of mature (7 day and older) myotubes with 8-BrcAMP or isoproterenol results in a specific 2-3 fold increase in phosphorylation of these proteins, with a corresponding increase in 32PO4 incorporation into one major phosphopeptide of desmin. Preliminary evidence indicates that treatment of 6-8 day myotubes with 8-BrcAMP or isoproterenol significantly inhibits the transition of intermediate filaments to the Z-line which occurs during normal myogenesis. These observations suggest that intermediate filaments containing desmin and vimentin are responsible for the organization of skeletal muscle myofibrils into an integral contractile machinery, and that cAMP-dependent phosphorylation of desmin and vimentin plays an important role in the regulation of this function.
", "doi": "10.7907/e5gk-4c77", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" }, { "id": "thesis:10886", "collection": "thesis", "collection_id": "10886", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142018-174410594", "primary_object_url": { "basename": "Gard_DL_1982.pdf", "content": "final", "filesize": 118360636, "license": "other", "mime_type": "application/pdf", "url": "/10886/1/Gard_DL_1982.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Intermediate Filaments and Myogenesis in vitro", "author": [ { "family_name": "Gard", "given_name": "David Lynn", "clpid": "Gard-David-Lynn" } ], "thesis_advisor": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" } ], "thesis_committee": [ { "family_name": "Lazarides", "given_name": "Elias", "clpid": "Lazarides-E" }, { "family_name": "Brokaw", "given_name": "Charles J.", "clpid": "Brokaw-C-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mitchell", "given_name": "Herschel K.", "clpid": "Mitchell-H-K" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis describes my investigations into the composition and function of intermediate filaments (IF) during myogenesis in vitro. I have found that avian embryonic myotubes cultured in vitro contain two intermediate filament subunits, desmin and vimentin. Prior to myoblast fusion vimentin is the sole IF subunit protein detectable by electrophoretic and immunological techniques. The onset of desmin synthesis and its cytoplasmic accumulation appear to coincide with fusion of myoblasts into multinucleate myotubes. Immunofluorescence reveals dense networks of desmin- and vimentin-containing filaments in the sarcoplasm of immature myotubes; however, late in myogenesis antisera to both desmin and vimentin are observed to stain the Z-lines of myofibrils. Double immunofluorescence microscopy using antisera to \u03b1-actinin and desmin revealed that this association occurs after the assembly of \u03b1-actinin into Z-lines, at a time when individual myofibrils are being organized into bundles. Phosphorylation of intermediate filament proteins in muscle has been previously reported (O'Connor et al., Proc. Natl. Acad. Sci. U.S.A. 76: 819-823, 1979). Using two-dimensional tryptic analysis I have found that desmin from embryonic myotubes is phosphorylated at multiple sites which correspond to sites phosphorylated by cAMP-dependent protein kinase in vitro. I have observed phosphorylation of desmin and vimentin in intact myotubes at all stages of myogenesis. However, treatment of mature (7 day and older) myotubes with 8-BrcAMP or isoproterenol results in a specific 2-3 fold increase in phosphorylation of these proteins, with a corresponding increase in 32PO4 incorporation into one major phosphopeptide of desmin. Preliminary evidence indicates that treatment of 6-8 day myotubes with 8-BrcAMP or isoproterenol significantly inhibits the transition of intermediate filaments to the Z-line which occurs during normal myogenesis. These observations suggest that intermediate filaments containing desmin and vimentin are responsible for the organization of skeletal muscle myofibrils into an integral contractile machinery, and that cAMP-dependent phosphorylation of desmin and vimentin plays an important role in the regulation of this function.
", "doi": "10.7907/e5gk-4c77", "publication_date": "1982", "thesis_type": "phd", "thesis_year": "1982" } ]