Animal\u2013microbe symbioses are fundamental to the biology of nearly all multicellular organisms, yet many questions remain regarding how hosts recognize beneficial microbes and establish stable associations. The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri provides a powerful model for investigating these processes.
\r\n\r\nIn this dissertation, I examined host\u2013microbe interactions across multiple biological scales, from bacterial genetic variation and tissue-level responses to cellular mechanisms of symbiosis. I demonstrate that bacterial strain identity influences host transcriptional responses. I further identify the skin as an early site of host\u2013microbe interaction, revealing that hosts respond to symbiont exposure prior to stable colonization of the light organ. Finally, I establish methodologies for single-cell and single-nucleus transcriptomics in E. scolopes, providing a foundation for investigating symbiosis at cellular resolution.
\r\n\r\nTogether, these findings show that host responses to symbiotic bacteria emerge from interconnected processes spanning bacterial genetics, host tissues, and individual cell types. This work advances our understanding of how beneficial host\u2013microbe partnerships are established and maintained and provides new tools for studying symbiosis in cephalopods and other animal systems.
", "doi": "10.7907/q2b1-xn16", "publication_date": "2027", "thesis_type": "phd", "thesis_year": "2027" }, { "id": "thesis:18534", "collection": "thesis", "collection_id": "18534", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05012026-185528615", "type": "thesis", "title": "Smart Bandages for Chronic Wound Sampling, Monitoring, and Management", "author": [ { "family_name": "Wang", "given_name": "Canran", "orcid": "0000-0003-3297-9041", "clpid": "Wang-Canran" } ], "thesis_advisor": [ { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" } ], "thesis_committee": [ { "family_name": "Demirer", "given_name": "Gozde S.", "orcid": "0000-0002-3007-1489", "clpid": "Demirer-G\u00f6zde-S" }, { "family_name": "Gao", "given_name": "Wei", "orcid": "0000-0002-8503-4562", "clpid": "Gao-Wei" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Zhang", "given_name": "Anqi", "orcid": "0000-0001-6121-8095", "clpid": "Zhang-Anqi" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Chronic wounds are a major global health issue, incurring staggering economic costs and severely impacting patient well-being. Effective exudate management is crucial, yet current methods fail to balance moisture levels. Real-time analysis of biomarkers like reactive oxygen and nitrogen species could guide treatment, but existing systems lack the capacity required for continuous monitoring. Although wearable electronics have the potential to advance wound care, efficient management and analysis of wound exudate in real time remains challenging owing to its low secretion rate and complex composition. To address these issues, we introduce iCares, a wearable device for wound exudate management and continuous in situ analysis of crucial wound biomarkers. iCares contains a flexible nanoengineered sensor array that measures key reactive species such as NO, H\u2082O\u2082, and O\u2082, along with pH and temperature, providing multiparameter data to inform wound status. The device features a pump-free triad microfluidic modules with a superhydrophobic\u2013superhydrophilic Janus membrane, bioinspired wedge channels, and 3D graded micropillars for efficient unidirectional exudate collection, transport, and refreshing. The sensors demonstrate consistent response and analyte selectivity, validated in wound exudate. Rapidly manufacturable through advanced printing and laser-patterning techniques, iCares seamlessly integrates Bluetooth connectivity and enables scalable, wireless, long-term continuous reactive species monitoring without impeding daily activities. The iCares system was validated through in vivo testing in murine models of infection and fasting, where real-time monitoring was performed. In addition, clinical evaluation was conducted in 20 patients with chronic wounds, as well as in patients monitored before and after surgery, demonstrating the system\u2019s applicability across diverse wound conditions. iCares offers early infection detection and wound classification and outcome prediction using machine learning-enhanced data analysis.", "doi": "10.7907/cq9y-x940", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17749", "collection": "thesis", "collection_id": "17749", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11052025-233903791", "primary_object_url": { "basename": "AMoiseyenko_Thesis_2025.pdf", "content": "final", "filesize": 14928313, "license": "other", "mime_type": "application/pdf", "url": "/17749/1/AMoiseyenko_Thesis_2025.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Gut Microbiota as Modulators and Therapeutic Targets in Parkinson\u2019s Disease", "author": [ { "family_name": "Moiseyenko", "given_name": "Anastasiya O.", "orcid": "0000-0001-5379-7808", "clpid": "Moiseyenko-Anastasiya-O" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The gastrointestinal (GI) tract is a unique junction of the nervous system, immune system, and the gut microbiome. The gut microbiome, a complex community of bacteria, fungi, and viruses, is able to regulate host development, behavior, immunity, and disease. In Parkinson\u2019s disease (PD), a neurodegenerative disorder characterized by motor dysfunction, \u03b1-synuclein (\u03b1Syn) pathology, and common GI symptoms, the gut bacterial composition is significantly altered, with depletions in beneficial, anti-inflammatory taxa compared to healthy controls. This thesis explores whether specific gut bacteria may be disease-protective. We first assembled a consortium of taxa that are reduced in individuals with PD across multiple cohorts and geographies. We find that both therapeutic and prophylactic oral administration of this consortium to Thy-1-\u03b1Syn overexpressing (Thy1-ASO) mice, a preclinical model of PD, improves select motor and GI deficits and reduces \u03b1Syn pathology in the brain. We next identified three taxa that independently drive motor function improvements, with Faecalibacterium prausnitzii producing the most pronounced effects. Further characterization of treatment with F. prausnitzii revealed improvements in GI symptoms, reduced \u03b1Syn aggregates in the brain, remodeling of the gut microbiome, and induction of anti-inflammatory and tissue-regenerative pathways in the colon. Collectively, these findings provide a foundation for developing specific bacterial species as novel therapeutics for PD and highlight the broader potential of the gut microbiome to transform the way we understand and treat human health and disease.", "doi": "10.7907/3yxw-z226", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18770", "collection": "thesis", "collection_id": "18770", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022026-081334128", "primary_object_url": { "basename": "martinez_zachary_2026.pdf", "content": "final", "filesize": 14589104, "license": "other", "mime_type": "application/pdf", "url": "/18770/1/martinez_zachary_2026.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Tokens, Topologies, Taxa: Towards Declarative Biology and Bioengineering", "author": [ { "family_name": "Martinez", "given_name": "Zachary A.", "orcid": "0000-0002-7830-3162", "clpid": "Martinez-Zachary-A" } ], "thesis_advisor": [ { "family_name": "Thomson", "given_name": "Matthew W.", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Bois", "given_name": "Justin", "orcid": "0000-0001-7137-8746", "clpid": "Bois-Justin" }, { "family_name": "Thomson", "given_name": "Matthew W.", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Contemporary deep-learning models for the life-sciences have outpaced the tooling that lets experimentalists compose them. Three contributions are presented in response, a software platform, exemplary tasks built on it, and a predicted structural proteome of a defined gut microbiome. The underlying argument is that for experimentalists who use rather than build deep-learning methods, difficulties with composition and usability now outpace availability.
\r\n\r\nTRILL, a platform for AI-based protein engineering and analysis, is open-source, runs locally, and wraps models/methods behind a uniform vocabulary of thirteen top-level commands. Furthermore, TRILL is scalable, ranging from parallel fine-tuning of large models on a supercomputer to democratized, parameter off-loading in compute-limited scenarios. Models can be swapped with a one-argument change rather than a pipeline rewrite, and fast predictions can be paired with physics-based validation where overconfidence costs most.
\r\n\r\nProtein language models were fine-tuned using a homology-aware strategy, decreasing data leakage when evaluating generated proteins. Classifiers for cellulase, antimicrobial, and toxin activity were trained and applied to a scan of over two hundred million proteins from the NCBI non-redundant catalogue. An end-to-end pipeline carried seventeen predicted toxins of unknown function through structure prediction, binder design, and molecular dynamics on nearly nine hundred designed complexes.
\r\n\r\nThe third contribution targets hCom2, a defined synthetic gut consortium. We present a structural resource, where roughly four hundred thousand structures of its proteome were predicted using TRILL, segmented into eight hundred thousand domains, and assigned CATH designations. A case study demonstrating the utility of this structural database identifies nineteen carriers of the Helicobacter pylori virulence-factor TIPalpha fold across fourteen strains where sequence-only annotation fails.
", "doi": "10.7907/b5ye-jy33", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18682", "collection": "thesis", "collection_id": "18682", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292026-023647406", "primary_object_url": { "basename": "Horak_Richard_Thesis_PDF.pdf", "content": "final", "filesize": 37525468, "license": "other", "mime_type": "application/pdf", "url": "/18682/2/Horak_Richard_Thesis_PDF.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Metabolic Rewiring Promotes Bacterial Survival Under Oxidative and Reductive Stress", "author": [ { "family_name": "Horak", "given_name": "Richard Davis", "orcid": "0000-0003-0630-5481", "clpid": "Horak-Richard-Davis" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Ruby", "given_name": "Edward G.", "orcid": "0000-0002-4112-4830", "clpid": "Ruby-Edward" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Across the tree of life, all cells must follow unifying metabolic rules. Namely, organisms must balance electron flow to couple energy conservation with energy expenditure. Historically, studies in bacterial metabolism focused on exponential growth where cells are awash in nutrients and electron acceptors, exhibiting high bioenergetic levels. Therefore, from the perspective of both human biology and these fast-growing microbes, loss of redox balance is purely detrimental, leading to suppressed energetic states, growth arrest, and even death. Yet bacteria are commonly found under such conditions across diverse environments from industrial bioreactors to chronic infections to agricultural fields. This thesis was motivated by the remaining mystery behind how and why bacteria exist in such low energy survival states. Specifically, I focus on metabolic shifts during non-growth survival in the opportunistic pathogen Pseudomonas aeruginosa due to redox imbalance, as well as the potential benefits to such transitions. In the first section, I focus on oxidative stress, exploring bacterial survival during oxic nutrient starvation. I find that phenazines and toxoflavin \u2013 endogenous redox-active metabolites produced by P. aeruginosa and Burkholderia species respectively \u2013 lower the bioenergetic state of P. aeruginosa. Such bioenergetic self-poisoning would be traditionally deemed detrimental. Yet I find this phenomenon provides cells with increased tolerance to a variety of clinical antibiotics, suggesting cells might have agency over lowering their energetic state and that there is a benefit to doing so. In the following chapters, I turn my attention to reductive stress, examining the metabolic strategies P. aeruginosa uses to support anaerobic survival in the absence of terminal electron-acceptors. I discover that P. aeruginosa uses a phosphoketolase-mediated alternative glucose catabolic pathway under reductive stress, reminiscent of fermentative growth metabolisms in many obligate anaerobes. Moreover, this phosphoketolase plays a key role in mediating ribonucleotide homeostasis during survival-triggered macromolecule turnover. I find that many bacteria unable to grow in the absence of respiration contain phosphoketolases and show that at least two of these species, Dyella japonica and Paraburkholderia graminis, similarly rely on these enzymes for anaerobic survival. Finally, I speculate a generalizable role for phosphoketolases in supporting ribonucleotide turnover across bacterial taxa. These studies expose the large gaps remaining in our understanding of growth arrest metabolisms, even in well-studied model organisms. I hope this thesis motivates further exploration of these enigmatic yet important bacterial lifestyles.", "doi": "10.7907/240t-cx19", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18427", "collection": "thesis", "collection_id": "18427", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03162026-211941584", "type": "thesis", "title": "The Neural Basis of Brain-Body Communication", "author": [ { "family_name": "Wang", "given_name": "Tongtong", "orcid": "0000-0002-0408-2571", "clpid": "Wang-Tongtong" } ], "thesis_advisor": [ { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Understanding how the nervous system orchestrates physiology across the body has long been a central question in neuroscience. While neural mechanisms underlying behavior have been extensively characterized, the cellular and circuit principles that mediate brain-body communication remain underexplored. In this thesis, I investigate how internal physiological signals are detected and translated into coordinated regulation of organ functions through specialized sensory and autonomic pathways.
\r\n\r\nUsing molecular, behavioral, and genetic perturbation approaches, I first examine how changes in body fluid balance are detected by central sensory neurons. I identify distinct neuronal populations within forebrain circumventricular regions that detect hyperosmotic and hypovolemic challenges and drive modality-specific fluid consumption behaviors. Then I show how water signals in the gut are encoded by a dedicated vagal afferent population, providing feed-forward inputs that contribute to thirst satiation. These studies demonstrate that internal states are monitored through specialized channels spanning central and peripheral circuits.
\r\n \r\nNext, I investigate the circuit logic of sympathetic regulation in the abdomen, identifying molecularly defined neuronal populations that project selectively to visceral organs and differentially regulate gastrointestinal transit and digestive processes. These results demonstrate that sympathetic outputs are organized into discrete pathways that enable precise and independent control of physiology. I then synthesize current knowledge of autonomic organization, highlighting its molecular diversity and modular architecture as key features enabling selective regulation of organ function.
\r\n \r\nTogether, these findings reveal that brain-body communication is mediated by structured sensory pathways and modular autonomic circuits to achieve precise yet flexible control of physiology. This work provides a framework for understanding how neural systems coordinate internal stability and offers insight into how disruptions of these processes may contribute to diseases.
", "doi": "10.7907/a8mn-0e75", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:16285", "collection": "thesis", "collection_id": "16285", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02042024-025331192", "primary_object_url": { "basename": "Griffiths_Thesis_2_3_2024.pdf", "content": "final", "filesize": 28536604, "license": "other", "mime_type": "application/pdf", "url": "/16285/2/Griffiths_Thesis_2_3_2024.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Bidirectional Interactions Between the Gut Microbiome and Nervous System", "author": [ { "family_name": "Griffiths", "given_name": "Jessica Anne", "orcid": "0000-0002-5586-1567", "clpid": "Griffiths-Jessica-Anne" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "There is roughly one microbe for every human cell in your body. Though some are inconsequential hitchhikers, and some are potentially harmful, many perform beneficial roles. This thesis focuses on the function and interaction of resident microbes within laboratory mice, with the hope that it may translate to us as humans. Chapter (1) highlights recent findings of microbiome involvement in neurologic disorders. Each subsequent chapter presents a different interaction between the mammalian nervous system and gut microbiome. (2) Excitatory signaling in the brain is partially regulated by a genetic factor (Shank3), which is further modulated by environmental interactions through presence or absence of the gut microbiome. This genetic factor implicated in brain and behavior also affects gastrointestinal function and inflammation susceptibility. (3) Applying powerful genetic tools developed for the brain to the enteric nervous system reveals the impact of different enteric neuron populations on gut motility and fluid secretion as well as the immune system, pancreatic activity, and microbial populations. (4) Common opinion has shifted from the belief that microbes are primarily pathogens to viewing them as symbiotic organisms. With this paradigm shift, the artificially clean laboratory mouse microbiome has been found to stunt the immune system, and is being reevaluated. Male mice with natural \u201cwild\u201d microbiomes have altered behavioral and neurological profiles, which may reflect a more physiological state.", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16437", "collection": "thesis", "collection_id": "16437", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292024-221307093", "primary_object_url": { "basename": "BussMarjorie_thesis_v04.pdf", "content": "final", "filesize": 30293546, "license": "other", "mime_type": "application/pdf", "url": "/16437/1/BussMarjorie_thesis_v04.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Tools for Noninvasive Imaging and Control of Engineered Bacteria In Vivo", "author": [ { "family_name": "Buss", "given_name": "Marjorie Theresa", "orcid": "0000-0002-4266-9197", "clpid": "Buss-Marjorie-Theresa" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Genetically engineered bacteria are promising new cell-based diagnostic and therapeutic agents due to their ability to sense and respond to unique signals, access and interface with hard-to-reach areas of the body, and deliver therapeutics directly to these areas. However, currently tools to noninvasively monitor and control their activity in vivo are limited. Optical imaging methods, which are based on fluorescent and luminescent reporter genes, and optogenetics, which are based on light-activated proteins, are widely used in cell culture and rodent studies. However, these optical methods suffer from the poor penetration depth of light in tissue which limits their use in larger animals or humans. On the other hand, nuclear imaging methods such as PET and SPECT have good imaging depth but rely on radioactive tracers whose synthesis can be complex and exposes patients to radiation. Here I present tools for imaging and control of bacteria that based on non-ionizing forms of energy that easily penetrate tissue: sound waves and magnetic fields.
\r\n\r\nThe first two parts of my thesis focuses on imaging bacteria in vivo with ultrasound, which is a widely available imaging modality that does not use ionizing radiation and has tissue penetration depth of several centimeters. Bacteria can be imaged with ultrasound by expressing acoustic reporter genes (ARGs) which result in the production of gas vesicles (GVs), air-filled protein nanostructures that aquatic microbes use to regulate their buoyancy. However, the first-generation acoustic reporter genes expressed too poorly under in vivo conditions to enable ultrasound imaging of bacteria in therapeutically relevant contexts. Here, we present a new and improved ARG construct that produces high levels of robust gas vesicle expression in the probiotic bacterium E. coli Nissle (EcN), enabling ultrasound imaging of these cells with high sensitivity. This second-generation ARG construct, bARGSer, uses genes derived from Serratia sp. ATCC 39006 and was optimized for plasmid-based expression in EcN. We demonstrate that with bARGSer, we can visualize the spatial distribution of engineered EcN after they home to and colonize tumors upon systemic administration. We also demonstrate that the engineered EcN can be imaged with ultrasound when colonizing the gastrointestinal tract of mice after sensing dietary sugars as well as biomarkers of inflammation. By enabling monitoring of the precise spatial location of engineered probiotic bacteria inside the body, this technology could greatly improve the development and eventual clinical use of this emerging class of microbial cell-based theranostics.
\r\n\r\nThe last part of my thesis focuses on control of bacteria in vivo with magnetic fields. Many bacteria have limited ability to selectively colonize specific targeted regions of the GI tract due to a lack of external control over their location and persistence. Magnetic fields are well suited to provide such control due to their ability to freely penetrate biological tissues, but they are difficult to apply with enough strength to directly manipulate magnetically labeled cells within deep tissue or viscous environments such as in the GI tract. Here, we show that ingestible micron-sized magnetic particles, combined with an externally applied magnetic field, act as in vivo magnetic field gradient amplifiers, enabling the trapping and retention of orally administered probiotic E. coli within the mouse GI tract. This technology improves the ability of these probiotic agents to accumulate at specific locations and stably colonize without antibiotic treatment. By enhancing the ability of GI-targeted cellular agents to be at the right place at the right time, cellular localization assisted by magnetic particles (CLAMP) adds external physical control to an important emerging class of biotherapeutics.
", "doi": "10.7907/mvgg-ch02", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16204", "collection": "thesis", "collection_id": "16204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10102023-024622119", "primary_object_url": { "basename": "Wang_Renee_Z_2023_THESIS.pdf", "content": "final", "filesize": 15451019, "license": "other", "mime_type": "application/pdf", "url": "/16204/2/Wang_Renee_Z_2023_THESIS.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "From Photosynthesis to Detoxification: Microbial Metabolisms Shape Earth\u2019s Surface Chemistry", "author": [ { "family_name": "Wang", "given_name": "Ren\u00e9e Zurui", "orcid": "0000-0003-3994-3244", "clpid": "Wang-Ren\u00e9e-Zurui" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Eiler", "given_name": "John M.", "orcid": "0000-0001-5768-7593", "clpid": "Eiler-J-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Earth\u2019s chemistry, through geologic time and in the present, is inextricably linked with biologically mediated reactions. All major elemental cycles on Earth\u2019s surface have arisen from two competing processes \u2013 life shaping its chemical environment through the evolution of key biochemical pathways, and the environment constraining metabolism by dictating which reactions will occur. Understanding this complicated interplay motivates the research presented in this thesis, which studies this phenomenon over two major elemental cycles \u2013 the modern Nitrogen (N) and ancient Carbon (C) cycle.
\r\n\r\nChapters One and Two focus on the evolution of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), the enzyme that catalyzes the key carbon fixation step in modern oxygenic photosynthesis. This reaction also imparts a large kinetic isotope effect (KIE) that causes the fixed carbon to be relatively depleted in natural abundance \u00b9\u00b3C compared to its substrate; this isotopic fingerprint can be seen in both the modern C cycle and in rock records recording the ancient C cycle. Therefore, this KIE has been used both in vitro (outside the cell) by biochemical models to rationalize rubisco\u2019s reaction mechanism, and in vivo (in the cell) as a proxy for environmental CO\u2082 concentrations in the past and present. However, both the in vitro and in vivo measurements are calibrated using modern organisms even though rubisco and oxygenic photosynthesis have undergone profound evolution over geologic time. Therefore, we measured the KIE in vitro and in vivo of a reconstructed ancestral Form IB rubisco dating to >> 1 Ga, and the KIE in vitro of a recently discovered Form I\u2019 rubisco that presents a modern analogue to ancestral Form I rubiscos prior to the evolution of the small subunit. Overall, we find that the KIEs of both rubiscos are smaller than their modern counterparts, which is surprising given that the rock record indicates overall carbon isotope fractionations in vivo are larger in the past. In addition, we find that models strictly based on modern organisms may not apply to the past, questioning the basic assumption that uniformitarianism can be readily applied to biological processes. However, these models can be rescued by accounting for other aspects of cell physiology.
\r\n\r\nChapter Three focuses on disentangling the source of key metabolites, like nitrous oxide (N\u2082O) in the modern N cycle. Like Chapters 1 and 2, an isotopic fingerprint that measures the \u2018preference\u2019 of \u00b9\u2075N for the central or outer nitrogen site in N\u2082O (\u201cSite Preference\u201d or \u201cSP\u201d) has primarily been calibrated using dissimilatory, or energy-generating, nitric oxide (NO) reductases (NORs). However, there exists a much larger and phylogenetically widespread class of NO-detoxifying enzymes; in particular, flavohemoglobin proteins (Fhp/Hmp) produce N\u2082O as a strategy to neutralize damaging NO-radicals in anoxic conditions. This enzyme, which generates N\u2082O in non-growing and anoxic conditions, may be more relevant to natural environments where N\u2082O production has been detected. Surprisingly, we found that Fhp imparts a distinct SP on N\u2082O that differs from both bacterial and eukaryotic NORs, and that this value better aligns with existing in situ measurements of N\u2082O from soils. In addition, we find that in strains with both Fhp and NOR, the Fhp signal dominates when cells are first exposed to high concentrations of NO in oxic conditions while growing before being shifted to an anoxic, non-growing state. Therefore, in addition to telling us \u2018Who\u2019s there,\u2019 the SP fingerprint may also be able to tell us something about cell physiology in vivo. We propose a new framework for interpreting the source of N\u2082O based on SP values.
", "doi": "10.7907/kf85-cq89", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:15138", "collection": "thesis", "collection_id": "15138", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04132023-174709152", "primary_object_url": { "basename": "Xinhong_thesis_4.13.2023.pdf", "content": "final", "filesize": 7942753, "license": "other", "mime_type": "application/pdf", "url": "/15138/1/Xinhong_thesis_4.13.2023.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Non-Invasive Functional Gene Delivery to the Central and Peripheral Nervous System Across Species", "author": [ { "family_name": "Chen", "given_name": "Xinhong", "orcid": "0000-0003-0408-0813", "clpid": "Chen-Xinhong" } ], "thesis_advisor": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The normal function of the central nervous system (CNS) and peripheral nervous system (PNS) relies on precise regulation. When this regulation breaks down in diseases, genetic access to the nervous system is critical for therapeutic intervention. However, access to the nervous system remains difficult, reflecting the critical need for development of effective and non-invasive gene delivery vectors across species. By applying directed evolution approach, we identified 2 capsids, AAV-MaCPNS1 and AAV-MaCPNS2, which efficiently transduced the PNS in rodents following intravenous administration. Combining with rational optimization, we also identified AAV-X1 capsid family, which transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds and rats. Some previously-engineered AAVs that target the nervous system fail to translate across non-human primate (NHP). We thus also further tested our novel vectors across species and showed that AAV-MaCPNS1/2 efficiently transduced both the PNS and CNS in NHPs. AAV-X1.1 also exhibit superior transduction of the CNS in rhesus macaques and ex vivo human brain slices although the endothelial tropism is not conserved across species.
\r\n\r\nWith these enhanced systemic AAVs, we wanted to explore whether they could enable neuronal recording and modulation which has been challenging with the nature AAV serotypes. We used AAV-MaCPNS1 to systemically deliver the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia. We observed specific nodose neuronal response to physiological modulation in the gut. Furthermore, we showed that the MaCPNS1-delivered neuronal actuator DREADD to dorsal root ganglia could enable non-invasive neuronal modulation and create a model of pain. The functional utility of the novel systemic vectors demonstrated here provide a non-invasive approach to better explore the nervous system, which would lead to better therapeutic intervention. To this end, we also demonstrated that the X1 capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory for production of therapeutic agents for CNS. We showed that vasculature-secreted Hevin (a synaptogenic protein), whose coding sequence is delivered by X1 vectors, rescued synaptic deficits in a mouse model.
\r\n\r\nAAV repeated dosing could be favorable for certain therapeutic applications, however, neutralizing antibody generated following the first injection creates major obstacle for second injection. We explored whether serotype switching with 2 AAV capsids that have a distinguished neutralizing antibody profile could be a potential solution. To this end, we firstly showed that the X1 capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ. We then combined the different engineered serotype to enable serotype switching for sequential AAV administration in mice, showing the first AAV-delivered receptor for the second AAV could boost its CNS targeting.
\r\n\r\nIn general, we developed strategies to enable non-invasive functional gene delivery to the central and peripheral nervous system across species, which would be incremental for both basic neuroscience research and gene therapies for neurological disorders.
", "doi": "10.7907/48a2-0y07", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15272", "collection": "thesis", "collection_id": "15272", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-003659646", "type": "thesis", "title": "Structural Insights into the Conformational Plasticity and Antibody Recognition of HIV-1 Env", "author": [ { "family_name": "Dam", "given_name": "Kim-Marie Anh", "orcid": "0000-0002-1416-4757", "clpid": "Dam-Kim-Marie-Anh" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Acquired immunodeficiency syndrome (AIDS) and its causal agent, the human immunodeficiency virus 1 (HIV-1), remain a global public health concern since they were first identified in the early 1980s. Diligent research and gradual scientific advances have led to innovative strategies in HIV-1/AIDS prevention and treatment, transforming an obscure and deadly disease into a manageable condition with a normal life expectancy. Despite this progress, researchers have yet to develop a safe and effective vaccine against HIV-1. The work presented here describes a structural perspective related to the HIV-1 Envelope (Env) glycoprotein, the sole viral target of vaccines that seek to elicit neutralizing antibodies.
\r\n\r\nEnv is the only viral protein on the surface of HIV-1 virions and is composed of a homotrimer of gp120/gp41 heterodimers. Env mediates entry into target cells by engaging the host receptor, CD4. CD4 triggers conformational changes in gp120, thereby enabling coreceptor recognition. Interactions with the host coreceptor trigger structural rearrangements in gp41 that facilitate fusion of host and viral membranes leading to infection. Our work builds upon our understanding of Env structural plasticity. First, we evaluated the conformational plasticity of soluble Env constructs using double electron-electron resonance (DEER) spectroscopy. This method measured distances between probes in Env subunits, allowing us to interpret the distribution in distances as Env flexibility. Our findings captured previously unseen nuances in static Env structures including gp41 elasticity and conformational heterogeneity associated with CD4-receptor binding. Although our work gave a new perspective on Env flexibility, it largely corroborated observations from static Env structures. Importantly, this suggested that soluble versions of Env, which serve as templates for immunogen design, retain favorable structural properties.
\r\n\r\nInformed with these insights in Env structure, we then sought to address a prevailing question related to receptor engagement: how many CD4 receptors are needed to induce gp120 conformation changes that lead to coreceptor binding followed by fusion? Prior work only characterized CD4-induced Env structural changes in Envs complexed with three soluble CD4 proteins. In our work, we designed and structurally characterized Envs bound to only one or two CD4 receptors. We found that Env engagement of one CD4 resulted in minor changes to the prefusion, closed Env conformation while Env bound to two CD4 molecules led to CD4-induced opening in the CD4-bound gp120s and a partially open conformation in the unliganded gp120.
\r\n\r\nStructural biology has also been leveraged to characterize the mechanism by which broadly neutralizing antibodies (bNAbs) recognize HIV-1 Env. We include an extensive review of how structural observations from antibodies bound to viral proteins contribute to our understanding of antibody-mediated viral neutralization. We also present a technical evaluation of bNAb binding assays that revealed how Env conformations can be unintentionally altered resulting in misleading antibody binding results and identified ideal methods to ensure reliable data.
\r\n\r\nAdditionally, we report on projects related to bNAbs that target the CD4 binding site (CD4bs) epitope of Env. In the first, we characterized the inferred germline (iGL) precursor of BG24, a VRC01-class bNAb with features that make it a promising target for vaccine design. We solved four cryo-EM structures of BG24iGL constructs complexed with different Envs and provided insight on the mode of iGL accommodation. The second project centers around the IOMA-class of CD4bs bNAbs. We characterized features of IOMA-class bNAbs and measured how different features contribute to neutralization breadth and potency. Taken together, the conclusions from our work provide guidance for the next generation of structure-based, CD4bs-targeting immunogen design.
", "doi": "10.7907/8pvb-dx31", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15062", "collection": "thesis", "collection_id": "15062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11112022-203906821", "primary_object_url": { "basename": "20221111_thesis.pdf", "content": "final", "filesize": 3261854, "license": "other", "mime_type": "application/pdf", "url": "/15062/1/20221111_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Gut Microbiota Modulation of Host Feeding Behavior", "author": [ { "family_name": "Ousey", "given_name": "James Anthony", "orcid": "0000-0003-4886-0053", "clpid": "Ousey-James-Anthony" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Prober", "given_name": "David A.", "orcid": "0000-0002-7371-4675", "clpid": "Prober-D-A" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-Carlos" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The rich, diverse community of microorganisms in the gastrointestinal tract of animals, or gut microbiota, regulates aspects of host metabolism, immunity, and neural function, with resulting effects on the expression of complex behaviors, including feeding.
\r\n\r\nIn this thesis, we sought to characterize gut microbiota influences on the behavioral response to palatable foods in mice. We discover that binge-like consumption of palatable foods, including high-sucrose pellets and a high-fat diet, is exacerbated in mice in the absence of a gut microbiota. Furthermore, using automated feeding dispensers and video analysis, we find that microbiota depletion with oral antibiotics results in elongated feeding bouts and conserved changes in the dynamics of palatable food intake. We show the hyperphagic phenotype of antibiotic-treated mice is reversible upon microbiota reconstitution via fecal microbiota transplant. Operant conditioning tests reveal that the motivation to pursue high-sucrose rewards is augmented in microbiota-depleted mice. The mesolimbic brain region activity induced upon high-sucrose pellet consumption is elevated in antibiotic-treated mice. Gut bacteria from the family S24-7 and genus Lactobacillus were identified by differential antibiotic treatment and fecal microbiota transplants as correlating with reduction of high-sucrose pellet consumption. Indeed, colonization of vancomycin-treated mice with a mixture of S24-7 and Lactobacillus johnsonii reduces overconsumption of high-sucrose pellets in a limited-access binge-eating model. The work in this thesis comprehensively demonstrates that the gut microbiota regulates feeding induced in response to palatable foods in mice.
", "doi": "10.7907/8ghx-2b24", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16110", "collection": "thesis", "collection_id": "16110", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06122023-184806431", "primary_object_url": { "basename": "SWilbert_Thesis.pdf", "content": "final", "filesize": 26984088, "license": "other", "mime_type": "application/pdf", "url": "/16110/1/SWilbert_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Role of Context-Dependent Metabolic Interactions in Organizing Microbial Communities", "author": [ { "family_name": "Wilbert", "given_name": "Steven Alexander", "orcid": "0009-0008-4409-8974", "clpid": "Wilbert-Steven-Alexander" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "We can image the strikingly beautiful compositions of natural microbial communities, but we still lack an understanding of the factors that shape their organization. Understanding the drivers of these structures at the microscale may allow us to better predict and control large-scale community functions in dynamic environments. In this thesis, I developed quantitative image analysis pipelines for uncovering the spatiotemporal growth of aggregate biofilms within a developing oxygen gradient by expanding upon the Agar Block Biofilm Assay (ABBA). I then developed the Agar Disk Biofilm Assay (ADBA) for improved imaging resolution. These tools push the bounders of laboratory experiments to better capture the complexity of natural environments. Next, I built a synthetic microbial community reflecting a metabolic pathway often partitioned between members found in nature: Pseudomonas aeruginosa (PA) strains with a denitrification pathway genetically split at the nitric oxide (NO) node. I characterized the growth of a strict consumer and a strict producer of NO and found that PA metabolizes NO in a manner that supports growth, a previously underappreciated energy conservation strategy. Local oxygen flips this interaction from beneficial to detrimental by increasing toxicity. I found these principles drive context-dependent cellular organization. This work underscores the contributions of partitioned metabolic pathways, redox-active metabolites, and dynamic micro-niches to the organization of microbial communities. Finally, combining my efforts towards method development and an appreciation for how redox-active metabolites drive context-dependent microbial interactions, I show how phenazines promote a previously unrecognized form of slow growth under nutrient limited environments. Taken together, this thesis highlights the importance of understanding dynamic micron-scale microbial interactions and presents several methodological improvements to capture it.", "doi": "10.7907/7sv2-gj10", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14409", "collection": "thesis", "collection_id": "14409", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282021-191743624", "type": "thesis", "title": "Quantitative Sequencing and its Application to Studies of the Human Small-Intestine Microbiota", "author": [ { "family_name": "Barlow", "given_name": "Jacob T.", "orcid": "0000-0002-1842-4835", "clpid": "Barlow-Jacob-T" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Our understanding of the interplay between microbial species and the hosts they live on and in is continually expanding. New insights have focused not only microorganisms that drive specific disease states but also those that help maintain human health. As research drives towards mechanistic understanding of host-microbe relationships new quantitative tools are needed to help interrogate these complex interactions. Chapter I of this thesis discusses formulation of a method for rapid detection of antibiotic resistance in Neisseria gonorrhoeae. Our approach identified RNA signatures from transcriptional profiling of Neisseria gonorrhoeae after 10-minute antibiotic exposure. Utilization of these RNA markers allowed for rapid identification of antibiotic susceptibility or resistance to the antibiotic ciprofloxacin. Chapter II shifts focus to the development of a quantitative sequencing technique for the measurement of absolute taxon abundances in complex microbial communities. Combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing allowed for simultaneous quantitative profiling of all bacterial taxa in host-associated microbial communities. We extensively characterized our quantitative sequencing methodology in the presence of high host nucleic acid levels and low microbial loads to understand the limits of quantification and detection in complex sample types. Last, Chapter III applies the quantitative sequencing technology from Chapter II to investigate the microbial community of the human small intestine, specifically the duodenum. Data from the duodenum of 250 individuals revealed a wide range of total microbial loads and a distinct subset of microbes, termed disruptor taxa, that were associated with small intestinal bacterial overgrowth (SIBO) and GI symptom severity.
", "doi": "10.7907/ca28-fk21", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14413", "collection": "thesis", "collection_id": "14413", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302021-184745797", "primary_object_url": { "basename": "Magnus_Hoffmann_2021_Thesis_final version_No EBR.pdf", "content": "final", "filesize": 16843967, "license": "other", "mime_type": "application/pdf", "url": "/14413/4/Magnus_Hoffmann_2021_Thesis_final version_No EBR.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Nanoparticle Technologies to Cure and Prevent Infectious Diseases", "author": [ { "family_name": "Hoffmann", "given_name": "Magnus Adrian Gero", "orcid": "0000-0003-4923-9568", "clpid": "Hoffmann-Magnus-Adrian-Gero" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "abstract": "Despite almost 40 years of intensive research, there is still no curative treatment for HIV-1/AIDS. Anti-retroviral therapy (ART) prolongs the life expectancy of HIV-1-infected individuals but is associated with side effects, and multiple drugs need to be given in combination to prevent the development of viral resistance. In addition, treatment must continue for the lifetime of the individual due to the existence of a long-lived latent proviral reservoir. While a \"sterilizing\" cure remains difficult to achieve due to difficulties associated with identifying and clearing latently-infected cells, recent research has focused on designing a \"functional\" cure, i.e., a therapeutic strategy that enables long-term suppression of HIV-1 replication and remission of symptoms in the absence of ART. The work presented here describes a new therapeutic direction for the development of a functional cure against HIV-1. This approach is based on the hypothesis that HIV-1 is unable to escape from a nanoparticle (NP)-based decoy that presents clusters of the HIV-1 receptor CD4, because CD4-NPs mimic viral target cells more accurately than soluble CD4-based inhibitors and permit high-avidity interactions with trimeric HIV-1 Env proteins. We demonstrate that CD4-NPs are >10,000-fold more potent than soluble CD4 (sCD4) and prevent viral escape in vitro. AAV-mediated delivery of self-assembling CD4-NPs produced stable CD4-NP serum concentrations in mice that were almost 1,000-fold higher than concentrations required to neutralize HIV-1 in vitro, suggesting that these concentrations could be therapeutic. Viral challenge studies in non-human primates are underway to evaluate the potential of this therapeutic strategy.
\r\n\r\nAs an alternative approach to generate decoys against HIV-1, we generated engineered red blood cells (RBCs) that expressed viral receptors and potently inhibited HIV-1 infection of target cells in vitro. Because RBCs do not contain nuclei or functional organelles required for protein translation, infection of engineered RBCs represents a dead-end for a lentivirus such as HIV-1, which must integrate into the host cell genome as part of its lifecycle. We generated stable erythroid progenitor cell lines that continuously produced HIV-1 receptor-expressing RBCs that could be administered to HIV-1-infected individuals. As RBCs vastly outnumber CD4+ T-cells, HIV-1\u2019s main target cells, and have extended lifetimes, only a fraction of an individual\u2019s RBCs would need to be replaced with the engineered RBC viral traps in order to suppress HIV-1 infection in vivo.
\r\n\r\nMy work on CD4-NP therapeutics against HIV-1 also led to the invention and development of the EBR NP technology that is ideally suited for vaccine design applications. This technology can be used to modify any type of membrane protein to self-assemble into enveloped virus-like NPs without the need for additional proteins. EBR NP assembly is induced by inserting a short amino acid sequence into the cytoplasmic tail of the membrane protein, which was designed to recruit host proteins from the endosomal sorting complex required for transport (ESCRT) pathway. We applied this technology to design protein NP-based vaccines against Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which elicited potent serum neutralizing antibody responses in mice. The EBR NP technology is also ideally suited for the development of hybrid vaccine approaches that allow genetic encoding of protein-based NPs, thereby combining attributes of mRNA and protein-based NP vaccines. Pilot studies demonstrated that mRNA and DNA vaccines encoding the self-assembling SARS-CoV-2 spike-EBR construct elicited ~10-fold higher neutralizing antibody responses than mRNA and DNA vaccines encoding the unmodified spike protein. This hybrid approach has the potential to substantially enhance the potency of mRNA vaccines and could become a leading vaccine platform technology. Future applications for the EBR NP technology are discussed, including the development of a universal coronavirus vaccine to prevent future pandemics, and engineering EBR NPs to mRNA vaccines or therapeutic cargoes for efficient and targeted delivery.
", "doi": "10.7907/g0w1-rc77", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14517", "collection": "thesis", "collection_id": "14517", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03162022-173632582", "primary_object_url": { "basename": "Abdel-haq_Reem_2022_thesis.pdf", "content": "final", "filesize": 44751420, "license": "other", "mime_type": "application/pdf", "url": "/14517/1/Abdel-haq_Reem_2022_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Gut Microbiome Modulates Microglia Physiology in Homeostatic and Disease States", "author": [ { "family_name": "Abdel-Haq", "given_name": "Reem", "orcid": "0000-0002-7418-5736", "clpid": "Abdel-Haq-Reem" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The gastrointestinal tract (GI) harbors a complex community of ~100 trillion bacteria, fungi, and viruses collectively referred to as the gut microbiome. Through direct and indirect signaling mechanisms, the gut microbiome exerts its effects on almost every organ system, including the brain. Constant, bi-directional communication along the gut-brain axis is required for the normal and healthy development of the host Central Nervous System (CNS). One of the cells in the CNS shaped by microbial-derived cues is microglia, the resident immune cells in the brain. Aberrant microglia activity is a driving force of several neurological diseases in which the gut microbiome plays a role, including Parkinson\u2019s disease (PD). \r\n\r\nIn this thesis, we explore the interplay between gut microbiota signaling and microglia physiology during homeostatic and disease states. We first detail how microbial signaling along the gut-brain axis shapes microglial development and function. Next, we explore how the gut microbiome composition influences microglial activation states in the context of disease. Leveraging a preclinical mouse model of PD, we show that dietary-driven changes to the gut microbiome through the use of prebiotics attenuates motor deficits and \u03b1-synuclein aggregation. These effects result from changes in microglial gene expression and activation status. Collectively, these findings have broad implications for the gut microbiome research community and highlight potential for development of microbiome-based therapies for diseases of the brain.", "doi": "10.7907/ht1j-2461", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14262", "collection": "thesis", "collection_id": "14262", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082021-015935441", "primary_object_url": { "basename": "Alexander Cohen_Caltech-Thesis Final version .pdf", "content": "final", "filesize": 42649473, "license": "other", "mime_type": "application/pdf", "url": "/14262/1/Alexander Cohen_Caltech-Thesis Final version .pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Developing Multivalent Nanoparticle Vaccines Against Current and Future Viruses", "author": [ { "family_name": "Cohen", "given_name": "Alexander Armand", "orcid": "0000-0002-2818-656X", "clpid": "Cohen-Alexander-Armand" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Scott", "given_name": "David W.", "orcid": "0000-0003-2708-4251", "clpid": "Scott D-W" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The 1918-1919 flu pandemic resulted in an estimated 50 to 100 million deaths worldwide, making it the deadliest pandemic in modern history. It was caused by a new influenza virus that likely spilled over from birds and reassorted with a human influenza virus. Since the human population was immunologically na\u00efve to this virus, transmission and lethality was much higher than for seasonal influenza outbreaks. Numerous pandemic influenza viruses emerged within the next century, with none causing the same amount of carnage. There is likely to be future influenza pandemics, with wild migratory birds being carriers of a wide swath of different influenza A viruses. Zoonotic transmission of Avian influenza has taken place with limited human to human transmission. There is evidence showing that the barrier of human transmissibility by some of these avian viruses is not very high, and therefore emergence into humans is possible, with most if not all of the population immunologically na\u00efve. The humoral immune response to influenza is defined by the imprinting of the antibody response to immunodominant epitopes. Such responses can impair immunity, providing less adequate protection against seasonal and pandemic infections, as well as poorer immunity induced by seasonal vaccines. There are instances where imprinting can be advantageous and even offer protection against pandemic or avian viruses, particularly when conserved epitopes to the HA stalk are exploited. Manipulating the antibody response to recognizing conserved stalk epitopes on influenza HA is therefore a strategy being used for universal influenza vaccines. In the second Chapter of this thesis, a mosaic nanoparticle immunization strategy for inducing breadth of antibody responses against HA will be described. This strategy involves the co-display of HAs from up to eight different strains on a particle platform. Although the breadth of antibody responses elicited by immunization of these particles was limited, this work provides insight into the antigenicity of such particles, and a possible alternative to current influenza vaccines.
\r\n \r\nApproximately 100 years after the 1918-1919 flu pandemic, a deadly SARS-like coronavirus, known as SARS-CoV-2, emerged in the human population resulting in a currently ongoing pandemic. This came less than two decades after the small but deadly SARS outbreak, essentially a warning call for this class of coronaviruses. Other SARS-like coronavirus strains in bats have been identified and shown to be human tropic, though resulting in an attenuated infection. Some of these viruses can infect via hACE2 but there are others that may use an unknown receptor for entry into VERO cells as well as human cell lines. There is evidence that the major barrier to zoonosis is protease compatibility, which could be gained through recombination events or errors during replication. Therefore, future SARS-like coronaviruses (sarbecovirus) may emerge in humans, seeding future outbreaks. The antibody response to SARS-CoV-2 is robust and protective. Furthermore, there is the presence of conserved epitopes particularly on the RBD that can be targeted by antibodies that are cross-neutralizing against many SARS-like coronaviruses. Exploiting these cross-reactive epitopes is one strategy that can be used for developing a universal coronavirus vaccine. In Chapter 3 of this thesis, a similar mosaic nanoparticle immunization strategy will be described, that attempts to elicit cross-reactive antibodies against the SARS-like coronavirus family. The mosaic nanoparticles co-display the RBDs of eight different sarbecovirus strains including SARS-CoV-2. Immunization with these mosaic-RBD nanoparticles elicited polyclonal antibody responses that were cross-reactive as well as cross-neutralizing against sarbecoviruses strains both present and not present on the particles.
", "doi": "10.7907/r33z-kj46", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13861", "collection": "thesis", "collection_id": "13861", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09012020-121925511", "primary_object_url": { "basename": "Caltech-Thesis-Poceviciute-2020-v6.pdf", "content": "final", "filesize": 11133034, "license": "other", "mime_type": "application/pdf", "url": "/13861/30/Caltech-Thesis-Poceviciute-2020-v6.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Mucosal Landscape of the Gut: Development and Application of 3D Imaging Tools for Interrogation of Host-Microbe Mucosal Interface in Mice and Humans", "author": [ { "family_name": "Poceviciute", "given_name": "Roberta", "orcid": "0000-0002-6649-2170", "clpid": "Poceviciute-Roberta" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Mammalian gastrointestinal tract is inhabited by trillions of microbes that, in number, amount to the total number of cells in the human body. These microbes, collectively known as microbiota, are found on the skin and in body cavities, and come in close contact with the host on mucosal surfaces. Here, pivotal host-microbe interactions likely take place because close proximity to the host enhances the uptake of microbial metabolites by the host and enables direct contact. To aid the investigation of these interactions, we developed an imaging technology that preserves fragile mucosal structure, enables to explore large areas of mucosal surface, and image the structurally and biochemically complex host-microbe interface in 3D in a mouse. However, 3D imaging presents challenges, such as slow transport of large molecular weight reagents and low signal/background ratio at depth, and these challenges are further exacerbated in particularly thick samples, such as small intestinal samples with long finger-like villus protrusions and thick human gut samples. Therefore, we further advanced our technology to improve sensitivity and specificity at depth, and we have taken steps to translate our technology to precious resected human gut samples from inflammatory bowel disease patients. Finally, we applied these tools to interrogate Enterobacteriaceae \u2013 Bacteroidaceae interactions in the small intestine of a mouse weakened by malnutrition. Using complementary tools, we have first determined that Bacteroidaceae required malnutrition to increase in number in the jejunum digesta, whereas Enterobacteriaceae required both malnutrition and Bacteroidaceae. With imaging, we visualized that in malnourished mice not exposed to Enterobacteriaceae and Bacteroidaceae, bacteria were effectively cleared after digesta passage, whereas in exposed mice bacterial retention was detected, suggestive of bacterial adherence to and colonization of mucosa. Finally, we detected a rare event of abundant bacterial colonization of small intestinal mucosa and captured in 3D.
", "doi": "10.7907/83t8-mv42", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:11805", "collection": "thesis", "collection_id": "11805", "cite_using_url": "http://resolver.caltech.edu/CaltechTHESIS:10012019-095132591", "type": "thesis", "title": "Development of Analytical Tools and Animal Models for Studies of Small-Intestine Dysbiosis", "author": [ { "family_name": "Bogatyrev", "given_name": "Said R.", "orcid": "0000-0003-0486-9451", "clpid": "Bogatyrev-Said-R" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Our appreciation of the role of human-associated microbial communities in the context of human health and disease has grown dramatically in the past two decades, with modern research tools enabling deeper insights into the mechanisms of host-microbial interactions. The elusive notion of dysbiosis, a state of microbial imbalance related to a disease, has achieved widespread distribution across popular, scientific, and medical literature (on September 16, 2019 PubMed search yielded 6,064 records of scientific and medical publications containing this keyword). The conventional wisdom further narrows down the definition and understanding of dysbiosis towards a compositional \"imbalance\" of the microbiota (a community of all microorganisms inhabiting human body). There exists an additional and frequently overlooked aspect of microbial imbalance in the context of the human gastrointestinal system, something that we can define as a \"spatial imbalance\": a state of the microbial community in the host gastrointestinal system where even a \"healthy\" and \"balanced\" microbiota may be associated with or causative of a disease by being present in sections of the gastrointestinal tract where it is not \"supposed\" to be, with the most prominent example being small intestinal bacterial overgrowth (SIBO). This thesis describes the progress in the development of analytical tools (quantitative microbiome profiling described in Chapter I) and refinement of animal mouse models (non-coprophagic mouse model described in Chapter II) for exploring the normal function of small-intestine microbiota in health and for dissecting the mechanisms of emergence and the persistence of the small-intestine dysbiosis (SIBO) in the future.
", "doi": "10.7907/VJDZ-7B52", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13686", "collection": "thesis", "collection_id": "13686", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04282020-145500248", "primary_object_url": { "basename": "Thesis - Caltech - BBE - Bryan B. Yoo- FINAL - submit to library - compressed.pdf", "content": "final", "filesize": 9465428, "license": "other", "mime_type": "application/pdf", "url": "/13686/1/Thesis - Caltech - BBE - Bryan B. Yoo- FINAL - submit to library - compressed.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Host-Microbe Interactions Impacting and Mediated by Nervous Systems", "author": [ { "family_name": "Yoo", "given_name": "Bryan B.", "orcid": "0000-0003-1450-2696", "clpid": "Yoo-Bryan-B" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Animals and microbes coevolved, and thus it is not surprising that the trillions of microorganisms that harmoniously inhabit the mammalian gastrointestinal tract (GIT), collectively termed the gut microbiome, continue to be implicated in healthy and disease states. However, less is known about the mechanisms by which these states are maintained, and how deviations from homeostasis (i.e., dysbiosis) occurr. This thesis explores the relationship between host-microbe interactions and the central and peripheral nervous systems. Specifically, the first chapter of this thesis explores how the microbiome differs is patients with multiple sclerosis and how these differences alter diseases outcomes in a mouse model of the disease. Next, we introduce the enteric nervous system (ENS), the intrinsic nervous system of the GI tract which is supposed as a major conduit of the bidirectional communication between the gut and the brain. Lastly, by adopting biotechnologies in gene delivery and genetically encoded tools for neuroscience, we introduce a molecular toolkit to characterize the ENS in a robust and efficient manner and modulate the ENS to uncover novel mechanisms by which innervation of the GI mediates host-microbe interactions.", "doi": "10.7907/1zv5-ve82", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11687", "collection": "thesis", "collection_id": "11687", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052019-122355847", "primary_object_url": { "basename": "Frankiw_Luke_2019_Thesis_FINAL.pdf", "content": "final", "filesize": 6138653, "license": "other", "mime_type": "application/pdf", "url": "/11687/1/Frankiw_Luke_2019_Thesis_FINAL.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "mRNA Splicing-Mediated Gene Expression Regulation in Innate Immunity", "author": [ { "family_name": "Frankiw", "given_name": "Luke Steven", "clpid": "Frankiw-Luke-Steven" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "At the heart of an inflammatory response lies a tightly regulated gene expression program. Perturbations to this finely tuned response can result in unchecked or inappropriately scaled inflammation, shifting the balance from protective to destructive immunity. A variety of post-transcriptional mechanisms play a role in the fine-tuning of an inflammatory gene expression program. One such mechanism involves unproductive RNA splicing, whereby alternative splicing can frameshift the transcript or introduce a premature termination codon (PTC). These effects render the transcript nonfunctional and/or subject it to nonsense-mediated decay.
\r\n\r\nWe observed such an event in Irf7, the master regulator of the type I interferon response. We found a single intron was consistently retained at a level much greater than other introns in the Irf7 transcript. In an effort to understand trans-acting factors that regulate this retention, we used RNA-antisense purification followed by mass spectrometry (RAP-MS) to identify the factor BUD13 as a highly enriched protein on Irf7 transcripts. Deficiency in BUD13 was associated with increased retention, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection.
\r\n\r\nBeyond this intron retention event in Irf7, we identified a variety of other unproductive splicing events in a number of important genes involved with the innate immune response. This unproductive splicing was not restricted to intron retention events. For example, we identified a frequently used alternative splice site in the crucial murine antiviral response gene, oligoadenylate synthetase 1g (Oas1g) that led to both a frameshift and incorporation of a PTC. Genome editing was used to remove the alternative splice site in a macrophage cell line, which led to both increased Oas1g expression and improved viral clearance. We hypothesize these events exist as a means of mitigation for what might otherwise be an inappropriately scaled response. In doing so, they represent a previously underappreciated layer of gene expression regulation in innate immunity.
", "doi": "10.7907/NBQG-BS67", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11154", "collection": "thesis", "collection_id": "11154", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08222018-105935103", "primary_object_url": { "basename": "Catherine Schretter Thesis_8 22.pdf", "content": "final", "filesize": 3643354, "license": "other", "mime_type": "application/pdf", "url": "/11154/1/Catherine Schretter Thesis_8 22.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Microbial Modulation of Host Locomotion", "author": [ { "family_name": "Schretter", "given_name": "Catherine Elizabeth", "orcid": "0000-0002-3957-6838", "clpid": "Schretter-Catherine-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Coordinated locomotor behavior is critical for the survival and propagation of an individual and is modulated by internal and external sensory inputs. The microbiota regulates host metabolism, which is closely intertwined with motor behavior. However, little is known regarding influences by the gut microbiome on host locomotion, or the pathways involved. The work presented in this thesis examines microbial regulation of locomotor behavior from both bacterial and host perspectives. Removal of the microbiota results in hyperactivity in female D. melanogaster, which is reversible through colonization with specific bacteria or administration of bacterial-derived products, including xylose isomerase (Xi) from Lactobacillus brevis. We found that Xi modulates host speed via sugar metabolism and octopamine signaling in flies. Additionally, aspects of microbial regulation of host locomotion appear to be conserved in mice. This work suggests that microbial modulation of host physiology extends beyond local intestinal effects to locomotor behavior through alterations in energy-related pathways.", "doi": "10.7907/Z1DX-4J03", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11549", "collection": "thesis", "collection_id": "11549", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282019-090132145", "primary_object_url": { "basename": "BastaDavid2019thesis.pdf", "content": "final", "filesize": 17391825, "license": "other", "mime_type": "application/pdf", "url": "/11549/17/BastaDavid2019thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases", "author": [ { "family_name": "Basta", "given_name": "David Wagdi", "orcid": "0000-0003-4176-6566", "clpid": "Basta-David-Wagdi" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Growth arrest is the dominant mode of microbial existence on the planet, yet the molecular mechanisms that underpin survival during growth arrest remain far less studied than other growth states. A better understanding of these mechanisms would provide valuable insight into the activity of microbial communities in both biogeochemical and clinical contexts, including the treatment of chronic infections. This thesis investigates the genetic requirements for survival of the bacterium Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen that thrives in diverse environments in which growth arrest is often caused by energy limitation. After reviewing our current knowledge of the strategies used by growth-arrested bacteria to adjust metabolism, regulate transcription and translation, and maintain the chromosome, I perform a functional genomic screen to identify genes that promote fitness of P. aeruginosa during growth arrest caused by carbon or oxygen starvation. I find that P. aeruginosa can survive for days to weeks in these energy-starved conditions by maintaining a reduced steady-state level of ATP, and that many functional classes of genes are required for fitness. Intriguingly, a majority of genetic fitness determinants differ between carbon and oxygen starvation, despite the common endpoint of reduced ATP levels in these two conditions. Among the few genes generally required for fitness are the stress response sigma factor encoded by rpoS and the heat shock protease encoded by ftsH. Using independently-generated deletion strains, I show that mutants in distinct functional categories exhibit temporal fitness dynamics during oxygen starvation: regulatory genes generally manifest a phenotype early during growth arrest, whereas genes involved in cell wall metabolism are required later. Building on these findings, I investigate the functional role of FtsH during growth arrest more deeply and find a surprising negative genetic interaction between ftsH and rpoS, with mutations in rpoS alleviating the fitness defects of \u0394ftsH during growth arrest. I also find that FtsH functions coordinately with the other conserved heat shock proteases to maintain cellular integrity and delay aging of P. aeruginosa during growth arrest. Finally, I investigate the role of FtsH and the other heat shock proteases in a novel N-terminal protein degradation pathway and find that the molecular details of this pathway likely differ between E. coli and P. aeruginosa. Together, these findings uncover essential molecular processes that promote fitness of an important bacterial pathogen during growth and survival.
", "doi": "10.7907/K6X1-GS91", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11703", "collection": "thesis", "collection_id": "11703", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-035956740", "primary_object_url": { "basename": "WenChen_2019_Caltech-thesis.pdf", "content": "final", "filesize": 23199748, "license": "other", "mime_type": "application/pdf", "url": "/11703/1/WenChen_2019_Caltech-thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Proteomics Profiling and Functional Characterization of Caenorhabditis elegans Excreted/Secreted Proteins", "author": [ { "family_name": "Chen", "given_name": "Wen", "orcid": "0000-0001-8056-5711", "clpid": "Chen-Wen-Biochemistry-Molecular-Biophysics" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Excretory-secretory products (ESPs) are first characterized and defined in parasitic nematode proteomics studies as the combination of various biomolecules that are continuously excreted or secreted into the environment throughout the whole life cycle. ESPs are particularly interesting to many scientists as anti-parasitic vaccine candidates and\r\nas promising drug targets since large portions of ESPs are active enzymes that potentially function directly at the parasite-host or worm-environment interfaces. However, majority of the parasites lack whole genome sequence knowledge and genome-editing tools. Thus, the number of ESPs identified is limited and many functions of ES proteins cannot be elucidated. Therefore, we use the most studied nematode, Caenorhabditis elegans, as the model to characterize the composition of excreted/secreted proteins with the help of nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). We characterized more than 509 excreted/secreted proteins with mix-staged worms, including many metalloproteases, cysteine proteases, and lysozymes. Proteases and proteases inhibitors are a major group in C. elegans ESPs. We performed stable isotope dimethylvlabeling quantitative proteomics and compared C. elegans ESPs on different bacteria diets. Lysozymes are not only enriched in C. elegans ESPs but are also up-regulated in response to pathogen and bacteria.
\r\n\r\nComparative studies of expression profiles of developmental life stages and pathogen infections elucidate the dynamics in regulating ESP components. We successfully identified stage-specific ESP groups associated with L1, L3, adult, L2 dauer, and postdauer. We demonstrated that proteases activities are down regulated by increased protease inhibitor expressions, while during dauer exit proteases expressions are increased. The comparison between dauer excretome/secretome and RNA-seq dauer expression profiles revealed 91 ESP encoding genes that are highly expressed in dauers. We performed dauer formation assay to these dauer-associated gene mutants. The great prediction rate confirmed that our comparative method is the simplest way to quickly pick out candidates for functional assays. Similarly, we employed this comparative method to pathogeninduced transcriptomes. We reported a group of genes that are associated with Serratia marcescens infection and a group of bacterial pathogens responding genes. We confirmed the roles of C. elegans ESPs in immuoregulation by infection assays with various pathogens. Lysosomes and cysteine protease inhibitor are among the most important genes in innate immune response pathway of C. elegans defending pathogen infection.
\r\n\r\nThe recent discovery of a C. elegans sibling species, Caenorhabditis inopinata, allows the deeply comparative study for evolutional interpretation. The excretome/secretome of C. inopinata has not been characterized. We took advantage of the sensitive and highthroughput technique of nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) to directly characterize the protein components of C. inopinata excretome/secretome. Functional annotations reveal several protein families, including C-type lectins, Cathepsin Z, Cathepsin B family, transthyretin, and saposin-like families, suggesting ESPs play critical roles in regulating innate immune response. We compared C. inopinata excretome/secretome with C. elegans. The structures are highly conserved across species, suggesting the sibling species share common mechanism to respond to environmental stimuli.
", "doi": "10.7907/WTMK-7M75", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11033", "collection": "thesis", "collection_id": "11033", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062018-180959061", "type": "thesis", "title": "Targeted Nanoparticle Delivery of Therapeutics Across the Blood-Brain and Blood-Tumor Barriers to Breast Cancer Brain Metastases", "author": [ { "family_name": "Wyatt", "given_name": "Emily Ann", "orcid": "0000-0002-7534-0582", "clpid": "Wyatt-Emily-Ann" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Brain metastases of human epidermal growth factor receptor 2 (HER2)-positive breast cancer are presenting an increasing problem in the clinic. While HER2-targeted therapies effectively control systemic disease, their efficacy against brain metastases is hindered by their inability to penetrate the blood-brain and blood-tumor barriers (BBB and BTB). One promising strategy to increase brain penetration of systemic therapeutics is to exploit endogenous transport systems at the BBB to shuttle drugs into the brain. Previous studies showed that gold nanoparticles designed to shed transferrin receptor (TfR)-targeting ligands under acidic conditions encountered during transcytosis of the BBB demonstrated increased accumulation in the brain. The focus of this work was to determine whether therapeutic, TfR-targeted nanoparticles using an improved acid-cleavable chemistry could be used to deliver therapeutically useful amounts of drug to the brain.
\r\n\r\nTo accomplish this goal, a new animal model of HER2-positive breast cancer brain metastasis was developed in an attempt to create a clinically representative, impermeable barrier to standard therapeutics. This new model establishes brain metastases by methods that more closely resemble the human disease, forming whole-body tumors that eventually metastasize to the brain. Brain metastases formed by this new methodology show no response to standard HER2-targeted agents, mimicking the clinical situation.
\r\n\r\nNext, efficacy and brain uptake of TfR-targeted, single-agent therapeutic nanoparticles were investigated in the newly developed model, as well as two common models from the literature. These nanoparticles show significant tumor growth delay and increased accumulation in both brain metastases and healthy brain tissue in all three models, highlighting their therapeutic potential. Additionally, non-BBB-penetrant small molecule and non-targeted nanoparticle therapeutics elicit a substantial antitumor response as well as brain tumor accumulation in the most commonly used literature model. In contrast, the new model and one gaining popularity in the literature provide for a more clinically relevant, impermeable barrier to non-BBB-penetrant agents, indicating that the method used to establish brain metastases can affect efficacy and brain uptake of therapeutics.
", "doi": "10.7907/5qpd-0736", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10859", "collection": "thesis", "collection_id": "10859", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05082018-122340793", "primary_object_url": { "basename": "GPD thesis 2018.pdf", "content": "final", "filesize": 22341118, "license": "other", "mime_type": "application/pdf", "url": "/10859/1/GPD thesis 2018.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Colonization of the Intestinal Surface by Indigenous Microbiota", "author": [ { "family_name": "Donaldson", "given_name": "Gregory Paul", "orcid": "0000-0002-8551-374X", "clpid": "Donaldson-Gregory-Paul" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "3MT Competition (Caltech)" }, { "literal": "div_bbe" } ], "abstract": "The mammalian gut evolved to foster the development and maintenance of a community of specific bacterial symbionts that persist for years. Bacteroides fragilis is one of a number of species that are able to colonize the mucus of the large intestine in mice and humans. This thesis explores the mechanisms and functions of mucosal colonization, most notably by using reductionist approaches with gnotobiotic mice. Harnessing genetics on both the host and microbial side allowed the dissection of a pathway by which immunoglobulin A enhances mucosal colonization by B. fragilis. Novel colonization assays were developed to explore the importance of mucosal colonization to bacterial fitness. Finally, an enrichment method for host-associated bacterial transcriptomics was used to define the behavior of this symbiont within the mucus layer.
", "doi": "10.7907/6EZ0-3007", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10349", "collection": "thesis", "collection_id": "10349", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07222017-155020423", "primary_object_url": { "basename": "Stone_Thesis.pdf", "content": "final", "filesize": 28125680, "license": "other", "mime_type": "application/pdf", "url": "/10349/13/Stone_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Cell-Selective Chemoproteomics for Biological Discovery", "author": [ { "family_name": "Stone", "given_name": "Shannon Elizabeth", "orcid": "0000-0002-6617-3874", "clpid": "Stone-Shannon-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cellular protein synthesis changes rapidly in response to internal and external cues in ways that vary from cell to cell. Global proteomic analyses of microbial communities, tissues, and organisms have provided important insights into the behavior of such systems, but can obscure the diversity of responses characteristic of different cellular subpopulations. Recent advances in cell-specific proteomics\u2014fueled in part by the development of bioorthogonal chemistries, more sensitive mass spectrometers and more advanced mining algorithms\u2014have yielded unprecedented glimpses into how proteins are expressed in space and time. Whereas previous cell-specific proteomic analyses were confined to abundant cells in relatively simple systems, recent advances in chemoproteomics allow researchers to map the protein expression patterns of even rare cells in complex tissues and whole organisms.
\r\n\r\nChapter 1 highlights recently developed strategies for cell-selective proteomics, including metabolic labeling strategies such as bioorthogonal noncanonical amino acid tagging (BONCAT). Bioorthogonal noncanonical amino acid tagging (BONCAT) is a chemoproteomic technique that enables temporal labeling of proteins. In cell-selective BONCAT, expressing a mutant aminoacyl-tRNA synthetase under the control of cell-specific genetic elements affords cellular resolution; only cells of interest can selectively incorporate a noncanonical amino acid into proteins for subsequent detection and identification. Chapter 2 details protocols to set up a cell-selective BONCAT system.
\r\n\r\nWhile BONCAT had previously been applied to studies of microbial pathogenesis in tissue culture-based models of infection, we sought to further develop the method to identify the proteome of methicillin-resistant Staphylococcus aureus (MRSA) within a mouse model of infection, as detailed in Chapter 3. We used this technique to enrich for staphylococcal proteins made within the host and in addition to finding many factors known to be important for infection, we also found many that had not previously been associated with infection. Screening several of these previously unknown factors in vivo led to the discovery of a novel protein important for MRSA infection. This unbiased approach to cell-selectively label pathogenic proteins during infection could be used as a global discovery tool for novel anti-infective strategies.
\r\n\r\nIn Chapter 4, we combine this cell-selective BONCAT strategy with microbial identification after passive clarity technique (MiPACT) to visualize both staphylococcal protein synthesis and ribosomal RNA within whole skin abscesses during infection. In Chapter 5, we continue developing cell-selective BONCAT to study microbial protein synthesis in the context of a living mouse by extending the system to Bacteroides fragilis, a common human gut commensal.
\r\n\r\nFinally, cell-selective BONCAT is wholly dependent on the bioorthogonal nature of the azide and its detection reagents. Fishing out an azide-tagged molecule from the rest of the cellular milieu requires optimization of enrichment-based strategies. In Chapter 6, we describe the development of a peptide to quantitate the gain of our enrichments.
\r\n\r\nWhile innovations in mass spectrometry and computational algorithms have facilitated the identification and quantification of thousands of proteins simultaneously from complex samples, this abundance of data does not necessarily lead to biological insight. Cell-specific proteomic techniques will play a key role in the identification of the mechanisms that govern cell specialization and that allow organisms to respond to changing environments. Overall, this work demonstrates the power of cell-selective chemoproteomics to ascertain biological insights in complex systems.
", "doi": "10.7907/Z9V122ZF", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10242", "collection": "thesis", "collection_id": "10242", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012017-074605131", "primary_object_url": { "basename": "170531 Rapp_Thesis_May2017_Final.pdf", "content": "final", "filesize": 5582593, "license": "other", "mime_type": "application/pdf", "url": "/10242/1/170531 Rapp_Thesis_May2017_Final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Diffusion and Molecular Association in Artificial Protein Hydrogels", "author": [ { "family_name": "Rapp", "given_name": "Peter Butterweck", "orcid": "0000-0002-9586-2126", "clpid": "Rapp-Peter-Butterweck" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Artificial proteins may be programmed to reversibly self-assemble into water-soluble networks, or \u201chydrogels\u201d, by encoding them with terminal coiled-coil forming domains. Such networks are model viscoelastic materials. The well-defined molecular structures adopted by proteins, combined with their facile preparation by recombinant synthesis, invite a careful exploration of the relationship between protein sequence and the resulting network properties.
\r\n\r\nThis work explores the relationship between network reorganization and diffusion from the perspective of single chains, using artificial elastin-like proteins as a model system. We make use of fluorescence recovery after photobleaching (FRAP), a classic biophysical technique, to measure chain mobilities as a function of network structure and probe architecture. Reversible network association is demonstrated to control the effective diffusivity of network-bound chains, and a novel mechanism of chain transport is proposed: the chains naturally partition into various bound states, and move by \u201chopping\u201d from site to site in between binding events.
\r\n\r\nA careful analysis of the equilibrium constants that control this partioning leads to the conclusion that the sequential binding of identical chain ends to the network is inherently asymmetric: the first association is always stronger than the second. This binding asymmetry is shown to arise from a strong entropic penalty for chain entry into the fully bound state due to local network structure. We derive a simple equation predicting the degree of binding asymmetry as a function of network geometry from equilibrium statistical mechanics. A large set of self-diffusivity measurements on a series of model telechelic proteins finds good agreement with this new theory. Generalized binding asymmetry for chains with many associative domains also holds.
\r\n\r\nFinally, the inherent viscoelasticity of the elastin-like network is found to couple with an entropically driven phase separation above a critical temperature set point. Relaxation of the viscoelastic stress throughout the process of phase domain segregation is found to induce highly dynamic phase patterns. The time evolution of these patterns illustrates that a delicate balance of surface tension and viscoelastic stress controls pattern formation in viscoelastic materials.
", "doi": "10.7907/Z9CV4FSF", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10230", "collection": "thesis", "collection_id": "10230", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312017-133325449", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 4208773, "license": "other", "mime_type": "application/pdf", "url": "/10230/1/THESIS.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Physiological and Biochemical Mechanisms of Phenazine-Mediated Survival in Pseudomonas aeruginosa", "author": [ { "family_name": "Glasser", "given_name": "Nathaniel Robert", "orcid": "0000-0002-2833-5166", "clpid": "Glasser-Nathaniel-Robert" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The opportunistic pathogen Pseudomonas aeruginosa secretes a class of colorful redox-active small molecules known as phenazines. Numerous functions have been proposed for phenazines, including antibiotic activity, virulence, cell-to-cell signaling, iron acquisition, and survival. This thesis delves into mechanisms of the latter role, that of long-term survival under oxidant-limiting conditions. Using a diverse array of methods, I investigated how phenazines support survival and how cells transfer electrons to phenazines, as well as the downstream effects that phenazines have on P. aeruginosa.
\r\n\r\nDirect measurements of NAD(H), ATP, the membrane potential, and fermentation products revealed that phenazines promote redox homeostasis and subsequently ATP synthesis. The ATP is used to maintain a membrane potential through the reverse action of the ATP synthase complex. Even though P. aeruginosa does not ferment on sugars, phenazines enable the anaerobic oxidation of glucose to acetate, suggesting P. aeruginosa may have previously under-appreciated metabolic flexibility in the absence of terminal electron acceptors. Activity assays with proteins purified natively from P. aeruginosa showed that glucose oxidation might be enabled in vivo by the pyruvate dehydrogenase complex, which can directly reduce phenazines using pyruvate as an electron donor. Liquid chromatography and mass spectrometry of culture supernatants showed that phenazines alter the chain length distribution of secreted quinolones, which may have indirect downstream signaling effects. Based on this result, combined with data from survival experiments, I hypothesize that phenazine-mediated redox homeostasis promotes \u03b2-oxidation and that fatty acid metabolism contributes to long-term survival. Further analysis also showed that P. aeruginosa cultures contain several previously-unreported sulfonated phenazines. In its natural environment, P. aeruginosa undoubtedly encounters other microbial species that consume or modify its phenazines. At least one of these, a Mycobacterium, contains a pyocyanin demethylating enzyme. The X-ray crystal structure of this protein revealed a novel reaction mechanism wherein the substrate is its own electron acceptor. Together, this work illuminates some of the many ways phenazines shape microbial communities in both clinical and environmental contexts.
\r\n", "doi": "10.7907/Z9SN070S", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9864", "collection": "thesis", "collection_id": "9864", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082016-141626444", "primary_object_url": { "basename": "Galimidi Thesis.pdf", "content": "final", "filesize": 70011604, "license": "other", "mime_type": "application/pdf", "url": "/9864/1/Galimidi Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Combating HIV with Novel Antibody Architectures ", "author": [ { "family_name": "Galimidi", "given_name": "Rachel P.", "clpid": "Galimidi-Rachel-P" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Zack", "given_name": "Jerome", "clpid": "Zack-J-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "More than 30 years has passed since the discovery of Human Immunodeficiency Virus (HIV) yet it remains one of the most important current threats to global public health. HIV is a T-lymphotrophic retrovirus that is the causative agent of Acquired Immune Deficiency Syndrome, and despite decades of research, there remains no cure. Vaccines are most effective when they are able to induce broadly neutralizing antibodies at concentrations capable of blocking viral infection. Notwithstanding all of the effort, a successful vaccine that is capable of inducing complete protection from the immune system has yet to be found. In this thesis, the first chapter provides a history of the discovery of HIV, the origins of the virus, description of the HIV genome, focusing primarily on the envelope glycoprotein, a trimeric spike on the surface of the HIV virion necessary for viral fusion and the sole epitope for broadly neutralizing antibodies. Lastly, the first chapter reviews an overview of the antiviral immune response specifically the role of humoral immune branch and broadly neutralizing antibodies, as well as their limitations in protection against HIV. Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. In addition to structural features of HIV\u2019s envelope spike that facilitate antibody evasion, we proposed that the low-density and limited lateral mobility of HIV spikes impedes bivalent binding by antibodies. The resulting predominantly monovalent binding minimizes avidity and thereby high affinity binding and potent neutralization, thus expanding the range of HIV mutations permitting antibody evasion. The work described in subsequent chapters attempts to overcome HIV\u2019s evasion strategy of low spike density through the design of novel antibody architectures.
\r\n\t\r\nWe postulated that anti-HIV-1 spike antibodies primarily bind monovalently because HIV\u2019s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike trimer structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and neutralization potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a \u201cmolecular ruler\u201d to measure intra-epitope distances on virion-bound spikes and to construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude of increased potency (>100-fold average increases across a virus panel) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection. These results shed light on dynamic spike conformations and are relevant to therapeutic interventions.
\r\n", "doi": "10.7907/Z9QC01FR", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9185", "collection": "thesis", "collection_id": "9185", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012015-132559997", "primary_object_url": { "basename": "2015 - Alexander M Sutherland Thesis - Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics.pdf", "content": "final", "filesize": 7315760, "license": "other", "mime_type": "application/pdf", "url": "/9185/1/2015 - Alexander M Sutherland Thesis - Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics", "author": [ { "family_name": "Sutherland", "given_name": "Alexander Muir", "clpid": "Sutherland-Alexander-Muir" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The first chapter of this thesis deals with automating data gathering for single cell microfluidic tests. The programs developed saved significant amounts of time with no loss in accuracy. The technology from this chapter was applied to experiments in both Chapters 4 and 5.
\r\n\r\nThe second chapter describes the use of statistical learning to prognose if an anti-angiogenic drug (Bevacizumab) would successfully treat a glioblastoma multiforme tumor. This was conducted by first measuring protein levels from 92 blood samples using the DNA-encoded antibody library platform. This allowed the measure of 35 different proteins per sample, with comparable sensitivity to ELISA. Two statistical learning models were developed in order to predict whether the treatment would succeed. The first, logistic regression, predicted with 85% accuracy and an AUC of 0.901 using a five protein panel. These five proteins were statistically significant predictors and gave insight into the mechanism behind anti-angiogenic success/failure. The second model, an ensemble model of logistic regression, kNN, and random forest, predicted with a slightly higher accuracy of 87%.
\r\n\r\nThe third chapter details the development of a photocleavable conjugate that multiplexed cell surface detection in microfluidic devices. The method successfully detected streptavidin on coated beads with 92% positive predictive rate. Furthermore, chambers with 0, 1, 2, and 3+ beads were statistically distinguishable. The method was then used to detect CD3 on Jurkat T cells, yielding a positive predictive rate of 49% and false positive rate of 0%.
\r\n\r\nThe fourth chapter talks about the use of measuring T cell polyfunctionality in order to predict whether a patient will succeed an adoptive T cells transfer therapy. In 15 patients, we measured 10 proteins from individual T cells (~300 cells per patient). The polyfunctional strength index was calculated, which was then correlated with the patient's progress free survival (PFS) time. 52 other parameters measured in the single cell test were correlated with the PFS. No statistical correlator has been determined, however, and more data is necessary to reach a conclusion.
\r\n\r\nFinally, the fifth chapter talks about the interactions between T cells and how that affects their protein secretion. It was observed that T cells in direct contact selectively enhance their protein secretion, in some cases by over 5 fold. This occurred for Granzyme B, Perforin, CCL4, TNFa, and IFNg. IL- 10 was shown to decrease slightly upon contact. This phenomenon held true for T cells from all patients tested (n=8). Using single cell data, the theoretical protein secretion frequency was calculated for two cells and then compared to the observed rate of secretion for both two cells not in contact, and two cells in contact. In over 90% of cases, the theoretical protein secretion rate matched that of two cells not in contact.
", "doi": "10.7907/Z9PK0D3K", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9835", "collection": "thesis", "collection_id": "9835", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032016-143422651", "primary_object_url": { "basename": "Thesis Final v2.pdf", "content": "final", "filesize": 75628401, "license": "other", "mime_type": "application/pdf", "url": "/9835/1/Thesis Final v2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Multiplexed Analysis of Diverse RNA Classes via Hybridization Chain Reaction", "author": [ { "family_name": "Acharya", "given_name": "Aneesh", "orcid": "0000-0002-4402-7147", "clpid": "Acharya-Aneesh" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Gene circuits are complex biological networks composed of numerous regulatory elements, including transcription factors, mRNAs, and microRNAs. Fluorescent in situ hybridization (FISH) is a powerful method for spatially mapping expression levels of RNA elements within an intact organism, but traditional methods exhibit at least one of the following drawbacks: low signal-to-background, arduous and/or destructive multiplexing, and non-quantitative signal. These issues are all overcome using in situ amplification based on the mechanism of hybridization chain reaction (HCR). With this approach, nucleic acid probes complementary to RNA targets trigger the self-assembly of fluorophore-labeled nucleic acid hairpins into tethered fluorescent amplification polymers. In situ HCR enables straightforward multiplexing, high signal-to-background, and quantitative signal. Here, we address three key scenarios in which HCR enables novel applications for in situ hybridization. First, we address the challenge of sorting cell subpopulations based on mRNA abundance using flow cytometry to enable high-throughput measurement of the signal intensity from individual cells. High signal is required to overcome the background autofluorescence integrated over the volume of each cell. Quantitative HCR signal amplification enables multi-dimensional sorting of mammalian cell lines based on expression levels of multiple target mRNAs. Second, we address the challenge of mapping multiple microRNA and mRNA targets simultaneously. Traditional methods enable mapping of single microRNA targets in isolation and use costly LNA probes with proprietary compositions that differ for each target. Here we develop in situ HCR for multiplexed mapping not only of microRNAs, but of microRNAs and mRNAs together, using non-proprietary 2'OMe-RNA probes for miRNA targets and DNA probes for mRNA targets. Third, to enable studies of gut flora, we address the challenge of mapping spatial relationships between different bacterial species within the intact mouse colon. In situ HCR enables multiplexed discrimination of multiple closely-related Bacteroides species with rRNAs that differ by only a few nucleotides. In summary, this thesis presents in situ HCR as a tool for multiplexed analysis of diverse RNA classes and expands the range of gene circuit regulatory elements that can be spatially and quantitatively mapped.", "doi": "10.7907/Z95M63N0", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9851", "collection": "thesis", "collection_id": "9851", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072016-121745268", "primary_object_url": { "basename": "Kim-Jocelyn-2016-thesis-FullVersion.pdf", "content": "final", "filesize": 9337223, "license": "other", "mime_type": "application/pdf", "url": "/9851/61/Kim-Jocelyn-2016-thesis-FullVersion.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Innate Immune System in Dendritic Cell-Targeted Lentiviral Vector Immunization and Cell-to-Cell Transmission of HIV-1", "author": [ { "family_name": "Kim", "given_name": "Jocelyn Tammy", "clpid": "Kim-Jocelyn-Tammy" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Yang", "given_name": "Otto", "clpid": "Yang-Otto" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Dendritic cells (DCs) are the sentinels of the immune system, and thus specialized in transporting foreign antigen to T cells and initiating activation of innate and adaptive immune responses. In this work, we first explore how DCs sense viral pathogens and stimulate antigen-specific T cell responses. In particular, we find the DC-targeting HIV-1 derived lentiviral vector (LV) is a potent T cell vaccine in vivo. However, the exact mechanism behind such efficient immunization is not clear. Interestingly, we find that DC activation is triggered by cellular DNA packaged in LVs and at least partially dependent on the STING protein. Innate immune activation is independent of MyD88, TRIF and IPS-1, ruling out an involvement of Toll-like receptors or RIG-I-like receptor signaling. Further, we find that antigenic protein delivered in viral particles via pseudotransduction is sufficient to stimulate an antigen-specific immune response. Delivery of the viral genome encoding the antigen increases the magnitude of this response in vivo, but is irrelevant in vitro. Thus, pseudotransduction, genomic transduction, and STING-mediated activation thus collaborate to make the DC-targeted LV a uniquely powerful immunogen. In addition, we explore how DCs mediate HIV-1 infection of T cells via cell-to-cell infection. In particular, we assess how DC-to-T cell transmission of HIV-1 allows for a concentrated amount of virus to be directed to an uninfected T cell. We report that DCs amplify the efficiency of T cell infection, resulting in anti-retroviral drug insensitivity compared to T cell infection in the absence of DCs. The DC-mediated amplification and drug-insensitivity of T cell infection are both entirely dependent on physical cellular interactions. Further, we find that the input of a virus is important to the drug insensitivity of DC-to-T cell infection, but not DC-free T cell infection. Thus, we have studied two separate roles of DCs: initiating immune responses to LVs and mediating transmission of infectious HIV-1. The study of these roles is important to discovering novel immune adjuvants and identifying targeted therapeutics to inhibit viral dissemination. ", "doi": "10.7907/Z93N21CS", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8737", "collection": "thesis", "collection_id": "8737", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12052014-154140797", "primary_object_url": { "basename": "AlborzThesisFinal.pdf", "content": "final", "filesize": 11354462, "license": "other", "mime_type": "application/pdf", "url": "/8737/1/AlborzThesisFinal.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Synthetic Biology Tools for Targeted Incorporation of Non-Canonical Amino Acids into Cellular Proteins", "author": [ { "family_name": "Mahdavi", "given_name": "Alborz", "orcid": "0000-0002-8790-8112", "clpid": "Mahdavi-Alborz" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Hajimiri", "given_name": "Ali", "orcid": "0000-0001-6736-8019", "clpid": "Hajimiri-A" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Proteins mediate many essential functions in cells, and methods to profile cellular proteins are of great interest for biological discovery. Whereas all of the cells in an organism share the same genome, the landscape of proteins (the proteome) varies between different cell types and over the lifetime of the organism. Rapid progress in mass spectrometers is enabling the detailed analysis of cellular proteomes. Whereas better instruments increase coverage, throughput, and measurement precision, new chemical reporters, metabolic tags, and synthetic biology techniques are required to enhance the specificity and spatiotemporal resolution of protein labeling and detection. This work introduces methods for cell-selective proteome analysis through the incorporation of non-canonical amino acids into newly synthesized proteins.
\r\n \r\nChapter I provides an overview of current technologies for translational profiling and proteomic analysis in cells. Strategies for the residue-specific incorporation of non-canonical amino acids and bioorthogonal non-canonical amino acid tagging are discussed. Chapter II introduces a new approach for the identification of secreted bacterial proteins from infected host cells using non-canonical amino acid labeling. This work demonstrates an application of cell-selective proteome labeling. Selectivity is achieved through controlled expression of a mutant aminoacyl tRNA synthetase (aaRS) enzyme that enables the metabolic incorporation of a non-canonical amino acid.
\r\n \r\nIdeally, the activity of multiple genes should be used to genetically control the extent of proteome labeling in cells. This is useful because many cell states are characterized by the activity of multiple genes and identified based on the expression of several proteins. Therefore chapter III introduces a novel approach to control proteome labeling as a function of multiple promoters using a genetically encoded AND gate based on a bisected methionyl-tRNA synthetase, a class I aaRS. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of this bisected aaRS. The utility of this tool is demonstrated by the selective labeling of proteins in subpopulations of bacterial cells in a laminar-flow microfluidic channel.
\r\n \r\nChapter IV extends the cell-selective incorporation of non-canonical amino acids from bacterial systems to mammalian cells by introducing a mutant mammalian methionyl-tRNA synthetase for cell-targeted proteome labeling. This enzyme is genetically encoded and can be conditionally activated for time-resolved and cell-targeted proteome analysis in a variety of different mammalian cell types. Chapter V uses this enzyme for lineage-specific proteomic analysis of mouse embryonic stem cells during differentiation to cardiac and mesoderm lineages. This approach for lineage-specific protein labeling enables the unbiased and comprehensive analysis of proteomic changes that occur during stem cell differentiation and cell-fate commitment.
\r\n \r\nAppendices A-G provide brief summaries of publications and research efforts during my PhD that are not directly related to this thesis. These publications are the result of a number of collaborations that I have been fortunate to be involved with during my graduate research.
\r\n\r\nThe technologies and methods introduced in this thesis provide versatile tools for the comprehensive and unbiased detection and identification of newly synthesized proteins in complex multicellular systems. Time-resolved, genetically encoded, and spatially defined non-canonical amino acid incorporation enables the identification of proteins involved in cell-cell interactions and the proteins made during specific cell states.
\r\n", "doi": "10.7907/Z9W66HS4", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8804", "collection": "thesis", "collection_id": "8804", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03252015-144135628", "primary_object_url": { "basename": "Mehta_Arnav_2015_Thesis_FINAL.pdf", "content": "final", "filesize": 35691475, "license": "other", "mime_type": "application/pdf", "url": "/8804/1/Mehta_Arnav_2015_Thesis_FINAL.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "MicroRNA-132 is a Physiological Regulator of Hematopoietic Stem Cell Function and B-cell Development", "author": [ { "family_name": "Mehta", "given_name": "Arnav", "clpid": "Mehta-Arnav" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression. Several microRNAs have been implicated in altering hematopoietic cell fate decisions. Importantly, deregulation of many microRNAs can lead to deleterious consequences in the hematopoietic system, including the onset of cancer, autoimmunity, or a failure to respond effectively to infection. As such, microRNAs fine-tune the balance between normal hematopoietic output and pathologic consequences. In this work, we explore the role of two microRNAs, miR-132 and miR-125b, in regulating hematopoietic stem cell (HSC) function and B cell development. In particular, we uncover the role of miR-132 in maintaining the appropriate balance between self-renewal, differentiation, and survival in aging HSCs by buffering the expression of a critical transcription factor, FOXO3. By maintain this balance, miR-132 may play a critical role in preventing aging-associated hematopoietic conditions such as autoimmune disease and cancer. We also find that miR-132 plays a critical role in B cell development by targeting a key transcription factor, Sox4, that is responsible for the differentiation of pro-B cells into pre-B cells. We find that miR-132 regulates B cell apoptosis, and by delivering miR-132 to mice that are predisposed to developing B cell cancers, we can inhibit the formation of these cancers and improve the survival of these mice. In addition to miR-132, we uncovered the role of another critical microRNA, miR-125b, that potentiates hematopoietic stem cell function. We found that enforced expression of miR-125b causes an aggressive myeloid leukemia by downregulation of its target Lin28a. Importantly, miR-125b also plays a critical role in inhibiting the formation of pro-B cells. Thus, we have discovered two microRNAs with important roles in regulating normal hematopoiesis, and whose dregulation can lead to deleterious consequences such as cancer in the aging hematopoietic system. Both miR-132 and miR-125b may therefore be targeted for therapeutics to inhibit age-related immune diseases associated with the loss of HSC function and cancer progression.", "doi": "10.7907/Z9XS5SBD", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8903", "collection": "thesis", "collection_id": "8903", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282015-153534197", "primary_object_url": { "basename": "Jessica Ricci Thesis 2015 FINAL.pdf", "content": "final", "filesize": 4770646, "license": "other", "mime_type": "application/pdf", "url": "/8903/1/Jessica Ricci Thesis 2015 FINAL.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Constraining the Interpretation of 2-Methylhopanoids through Genetic and Phylogenetic Methods", "author": [ { "family_name": "Ricci", "given_name": "Jessica Nicole", "clpid": "Ricci-Jessica-Nicole" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Hopanoids are a class of sterol-like lipids produced by select bacteria. Their preservation in the rock record for billions of years as fossilized hopanes lends them geological significance. Much of the structural diversity present in this class of molecules, which likely underpins important biological functions, is lost during fossilization. Yet, one type of modification that persists during preservation is methylation at C-2. The resulting 2-methylhopanoids are prominent molecular fossils and have an intriguing pattern over time, exhibiting increases in abundance associated with Ocean Anoxic Events during the Phanerozoic. This thesis uses diverse methods to address what the presence of 2-methylhopanes tells us about the microbial life and environmental conditions of their ancient depositional settings. Through an environmental survey of hpnP, the gene encoding the C-2 hopanoid methylase, we found that many different taxa are capable of producing 2-methylhopanoids in more diverse modern environments than expected. This study also revealed that hpnP is significantly overrepresented in organisms that are plant symbionts, in environments associated with plants, and with metabolisms that support plant-microbe interactions; collectively, these correlations provide a clue about the biological importance of 2-methylhopanoids. Phylogenetic reconstruction of the evolutionary history of hpnP revealed that 2-methylhopanoid production arose in the Alphaproteobacteria, indicating that the origin of these molecules is younger than originally thought. Additionally, we took genetic approach to understand the role of 2-methylhopanoids in Cyanobacteria using the filamentous symbiotic Nostoc punctiforme. We found that hopanoids likely aid in rigidifying the cell membrane but do not appear to provide resistance to osmotic or outer membrane stressors, as has been shown in other organisms. The work presented in this thesis supports previous findings that 2-methylhopanoids are not biomarkers for oxygenic photosynthesis and provides new insights by defining their distribution in modern environments, identifying their evolutionary origin, and investigating their role in Cyanobacteria. These efforts in modern settings aid the formation of a robust interpretation of 2-methylhopanes in the rock record. ", "doi": "10.7907/Z9MC8X0S", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8951", "collection": "thesis", "collection_id": "8951", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312015-231120184", "primary_object_url": { "basename": "06122015 Ruzbeh Mosadeghi Thesis.pdf", "content": "final", "filesize": 30704965, "license": "other", "mime_type": "application/pdf", "url": "/8951/1/06122015 Ruzbeh Mosadeghi Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanistic Dissection of the Cop9 Signalosome\u2019s Deneddylation Activity on Cullin-RING Ligases", "author": [ { "family_name": "Mosadeghi", "given_name": "Ruzbeh", "clpid": "Mosadeghi-Ruzbeh" } ], "thesis_advisor": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "We set out to understand the precise mechanisms that regulate the activation and deactivation of Cullin-RING Ligases (CRLs). While a great deal of work has already gone into identifying the players involved in these pathways and the cellular consequences associated with the loss of each, the biochemical mechanisms regulating these steps have remained elusive. In this work we sought to gain a better understanding of the mechanisms behind these steps by teasing apart specific their biochemical reactions. By measuring the individual microscopic rate constants of the reactions we have shed light on both the proper sequence of events in the regulation of CRLs as well as how they are in fact controlled.
\r\n\r\nPrior to this work, it was believed that CSN deactivated CRLs by binding them and enzymatically removing the activating post-translation modification Nedd8. It was believed that CSN could not bind to CRLs while they were active due to the steric hindrance by the CRL substrates, and that they would remain bound to deneddylated CRLs as a sequestering agent until a new substrate could displace it. We now have some insight that substrates themselves cannot inhibit CSN very well, but that the active ubiquitination by an E2 enzyme precludes CSN binding and activity. When the substrate for a CRL becomes depleted, CSN then binds to the CRL in a low affinity, low activity conformation. This triggers a conformational change that pulls the autoinhibitory Ins-1 loop away from the active site in the catalytic subunit Csn5, resulting in a large increase in affinity and cleavage of the isopeptide bond between CRLs and Nedd8. Upon dissociation of Nedd8, CSN rapidly returns to the low affinity state and dissociates from the CRL, allowing it reenter its activation cycle.
\r\n", "doi": "10.7907/Z9P26W3G", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8201", "collection": "thesis", "collection_id": "8201", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04242014-100521885", "type": "thesis", "title": "Gut Microbiota Promote Hematopoiesis to Control Bacterial Infection", "author": [ { "family_name": "Khosravi", "given_name": "Arya", "clpid": "Khosravi-Arya" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota. ", "doi": "10.7907/XM07-4X53", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8017", "collection": "thesis", "collection_id": "8017", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10262013-152651094", "primary_object_url": { "basename": "Lucey_KS_2014Thesis.pdf", "content": "final", "filesize": 9643634, "license": "other", "mime_type": "application/pdf", "url": "/8017/1/Lucey_KS_2014Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Catechol 2,3-Dioxygenase-Assisted Cleavage of Aromatics by \"Anaerobic\" Termite Gut Spirochetes and Genomic Evidence of a Complete Meta-Pathway", "author": [ { "family_name": "Lucey", "given_name": "Kaitlyn Shae", "clpid": "Lucey-Kaitlyn-Shae" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The termite hindgut microbial ecosystem functions like a miniature lignocellulose-metabolizing natural bioreactor, has significant implications to nutrient cycling in the terrestrial environment, and represents an array of microbial metabolic diversity. Deciphering the intricacies of this microbial community to obtain as complete a picture as possible of how it functions as a whole, requires a combination of various traditional and cutting-edge bioinformatic, molecular, physiological, and culturing approaches. Isolates from this ecosystem, including Treponema primitia str. ZAS-1 and ZAS-2 as well as T. azotonutricium str. ZAS-9, have been significant resources for better understanding the termite system. While not all functions predicted by the genomes of these three isolates are demonstrated in vitro, these isolates do have the capacity for several metabolisms unique to spirochetes and critical to the termite system\u2019s reliance upon lignocellulose. In this thesis, work culturing, enriching for, and isolating diverse microorganisms from the termite hindgut is discussed. Additionally, strategies of members of the termite hindgut microbial community to defend against O2-stress and to generate acetate, the \u201cbiofuel\u201d of the termite system, are proposed. In particular, catechol 2,3-dioxygenase and other meta-cleavage catabolic pathway genes are described in the \u201canaerobic\u201d termite hindgut spirochetes T. primitia str. ZAS-1 and ZAS-2, and the first evidence for aromatic ring cleavage in the phylum (division) Spirochetes is also presented. These results suggest that the potential for O2-dependent, yet nonrespiratory, metabolisms of plant-derived aromatics should be re-evaluated in termite hindgut communities. Potential future work is also illustrated.", "doi": "10.7907/KQB3-7010", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8403", "collection": "thesis", "collection_id": "8403", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272014-224131184", "primary_object_url": { "basename": "Sheng_Thesis_Final.pdf", "content": "final", "filesize": 36633278, "license": "other", "mime_type": "application/pdf", "url": "/8403/1/Sheng_Thesis_Final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Tunable Heparan Sulfate Glycomimetics for Modulating Chemokine Activity", "author": [ { "family_name": "Sheng", "given_name": "Gloria J.", "clpid": "Sheng-Gloria-J" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Heparan sulfate (HS) glycosaminoglycans participate in critical biological processes by modulating the activity of a diverse set of protein binding partners. Such proteins include all known members of the chemokine superfamily, which are thought to guide the migration of distinct subsets of immune cells through their interactions with HS proteoglycans on endothelial cell surfaces. Animal-derived heparin polysaccharides have been shown to reduce inflammation levels through the inhibition of HS-chemokine interactions; however, the clinical usage of heparin as an anti-inflammatory drug is hampered by its anticoagulant activity and potential risk for side effects, such as heparin-induced thrombocytopenia (HIT).
\r\n\r\nHere, we describe an expedient, divergent synthesis to prepare defined glycomimetics of HS that recapitulate the macromolecular structure and biological activity of natural HS glycosaminoglycans. Our synthetic approach uses a core disaccharide precursor to generate a library of four differentially sulfated polymers. We show that a trisulfated glycopolymer antagonizes the chemotactic activities of pro-inflammatory chemokine RANTES with similar potency as heparin polysaccharide, without potentiating the anticoagulant activities of antithrombin III. The same glycopolymer also inhibited the homeostatic chemokine SDF-1 with significantly more efficacy than heparin. Our work offers a general strategy for modulating chemokines and dissecting the pleiotropic functions of HS/heparin through the presentation of defined sulfation motifs within multivalent polymeric scaffolds.
", "doi": "10.7907/JQ2Z-EN67", "publication_date": "2014-06-12", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7333", "collection": "thesis", "collection_id": "7333", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12122012-183251952", "primary_object_url": { "basename": "Lee_S-EM_Thesis.pdf", "content": "final", "filesize": 14747110, "license": "other", "mime_type": "application/pdf", "url": "/7333/1/Lee_S-EM_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanism of Intestinal Colonization by Symbiotic Bacteria", "author": [ { "family_name": "Lee", "given_name": "Sung-Eun Melanie", "clpid": "Lee-Sung-Eun-Melanie" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "All animals live in symbiosis. Shaped by eons of co-evolution, host-bacterial associations have developed into prosperous relationships creating mechanisms for mutual benefits to both microbe and host. No better example exists in biology than the astounding numbers of bacteria harbored by the lower gastrointestinal tract of mammals. This community of symbionts establishes a life-long habitat within the distal gut and profoundly impacts host health. Although many recent investigations have led to determination of the microbiota composition, molecular mechanisms mediating establishment and maintenance of the microbial community within the gut is poorly described.
\r\n\r\nWe use gnotobiotic mice to elucidate mechanisms of colonization by Bacteroides, one of the most numerically prominent genera in the intestine. We generate mutant strains of Bacteroides fragilis that lack the ability to express multiple capsular polysaccharides and demonstrate defect in colonization in competition with wild-type strain, suggesting a role for surface sugar architecture during host-symbiont mutualism. Through a functional in vivo genetic screen of colonization, we identify a novel operon from the genome of B. fragilis that is highly conserved among many sequenced intestinal Bacteroides and that mediate a species-specific colonization profile. We have named this genetic locus the commensal colonization factor (ccf). B. fragilis deleted in the ccf genes exhibit colonization defects in both germ-free and complex microbiota harboring mice. The ccf genes of B. fragilis are up-regulated during gut colonization, preferentially at the mucosal surface, supporting an in vivo function. Indeed, deletion of ccf genes leads to reduced mucosal association and a defect in bacterial occupation of colonic crypts of mice. The ability of B. fragilis to repopulate the gut after antibiotic perturbation or gastroenteritis requires expression of ccf, suggesting the niche within colonic crypts represents a colonization reservoir for the gut microbiota following environmental stress. These findings suggest a novel and evolutionarily conserved mechanism for persistent gut colonization by the Bacteroides species.
\r\n", "doi": "10.7907/24H5-8V73", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7337", "collection": "thesis", "collection_id": "7337", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12172012-110932095", "primary_object_url": { "basename": "EYH_thesis_final.pdf", "content": "final", "filesize": 34264054, "license": "other", "mime_type": "application/pdf", "url": "/7337/1/EYH_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Brain, Gut and Immune Interactions in Autism Spectrum Disorder", "author": [ { "family_name": "Hsiao", "given_name": "Elaine Yih-Nien", "clpid": "Hsiao-Elaine-Yih-Nien" } ], "thesis_advisor": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Anderson", "given_name": "David J.", "clpid": "Anderson-D-J" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Autism spectrum disorder (ASD) is a class of complex neurodevelopmental disabilities that are characterized by the presence and severity of stereotyped behaviors, impaired communication, and abnormal social interactions. The incidence of autism has rapidly increased to 1 in 88 children in the United States, making ASD one of the most significant medical and social burdens of our time. However, drugs are often used to treat autism-related conditions, including anxiety, hyperactivity, epilepsy, and obsessive-compulsive behaviors, and therapies for treating the core symptoms of autism are limited. Moreover, molecular diagnostics are not available for the reproducible identification of ASD; as yet, the disorder is diagnosed based on standardized behavioral assessments. Much research into ASD has focused on genetic, behavioral, and neurological aspects of the illness. However, primary roles for environmental risk factors and peripheral disruptions, such as immune dysregulation and gastrointestinal distress, have gained significant attention.
\r\n\r\nThe work described in this thesis uncovers molecular mechanisms involved in the pathogenesis of autism-related endophenotypes in a mouse model of a primary autism risk factor, maternal immune activation (MIA). MIA is founded upon the strong epidemiological link between maternal infection and increased autism risk in the offspring. This risk factor can be translated to a mouse model with face and construct validity for autism, wherein pregnant mice injected with the immunogenic, double-stranded RNA poly(I:C) yield offspring with the core behavioral and neuropathological features of autism. Specifically, we report that MIA critically alters placental immune status and endocrine function, reflecting a key pathway by which fetal development may be disrupted to manifest in ASD-related phenotypes. We identify signature changes to the fetal brain transcriptome in response to multiple modes of MIA, highlighting a converging pathway involved in the development of autism-related behaviors and neuropathologies. We characterize peripheral, neural, and enteric immune alterations in MIA offspring and uncover an immune contribution to autism-related behavioral abnormalities. Finally we demonstrate that a microbe-based therapeutic can ameliorate intestinal pathology, metabolic function, and autism-related behaviors in MIA mice, which supports a role for the gut-immune-brain axis in ASD.
\r\n", "doi": "10.7907/DEVQ-1P16", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7613", "collection": "thesis", "collection_id": "7613", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04122013-165433477", "primary_object_url": { "basename": "Thesis by Jimmy Zhao_submission.pdf", "content": "final", "filesize": 7263144, "license": "other", "mime_type": "application/pdf", "url": "/7613/1/Thesis by Jimmy Zhao_submission.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Function of MicroRNA-146a and NF-\u03baB in Physiologic and Pathologic Hematopoiesis", "author": [ { "family_name": "Zhao", "given_name": "Jimmy Liu", "clpid": "Zhao-Jimmy-Liu" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Rao", "given_name": "Dinesh S.", "clpid": "Rao-Dinesh-S" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a\u2014expressed in all blood cell types\u2014produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-\u03baB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression. ", "doi": "10.7907/99A5-DR49", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7658", "collection": "thesis", "collection_id": "7658", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05022013-181603910", "primary_object_url": { "basename": "Thesis_MMDR_final.pdf", "content": "final", "filesize": 3415310, "license": "other", "mime_type": "application/pdf", "url": "/7658/1/Thesis_MMDR_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Analysis of a Transcriptional Network Involving PU.1, Notch, and Gata3 in the Lymphomyeloid Lineage Decision during Early T-cell Development", "author": [ { "family_name": "Del Real", "given_name": "Marissa Morales", "clpid": "Del-Real-Marissa-Morales" } ], "thesis_advisor": [ { "family_name": "Rothenberg", "given_name": "Ellen V." } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway. ", "doi": "10.7907/PEWS-KM18", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7755", "collection": "thesis", "collection_id": "7755", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282013-062935856", "primary_object_url": { "basename": "SaxenaAbigail2013_thesis.pdf", "content": "final", "filesize": 13911949, "license": "other", "mime_type": "application/pdf", "url": "/7755/31/SaxenaAbigail2013_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Sulfur-Cycling in Methane-Rich Ecosystems: Uncovering Microbial Processes and Novel Niches", "author": [ { "family_name": "Saxena", "given_name": "Abigail Green", "orcid": "0000-0002-8502-6589", "clpid": "Saxena-Abigail-Green" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Microbial sulfur cycling communities were investigated in two methane-rich ecosystems, terrestrial mud volcanoes (TMVs) and marine methane seeps, in order to investigate niches and processes that would likely be central to the functioning of these crucial ecosystems. Terrestrial mud volcanoes represent geochemically diverse habitats with varying sulfur sources and yet sulfur-cycling in these environments remains largely unexplored. Here we characterized the sulfur-metabolizing microorganisms and activity in 4 TMVs in Azerbaijan, supporting the presence of active sulfur-oxidizing and sulfate-reducing guilds in all 4 TMVs across a range of physiochemical conditions, with diversity of these guilds being unique to each TMV. We also found evidence for the anaerobic oxidation of methane coupled to sulfate reduction, a process which we explored further in the more tractable marine methane seeps. Diverse associations between methanotrophic archaea (ANME) and sulfate-reducing bacterial groups (SRB) often co-occur in marine methane seeps, however the ecophysiology of these different symbiotic associations has not been examined. Using a combination of molecular, geochemical and fluorescence in situ hybridization coupled to nano-scale secondary ion mass spectrometry (FISH-NanoSIMS) analyses of in situ seep sediments and methane-amended sediment incubations from diverse locations, we show that the unexplained diversity in SRB associated with ANME cells can be at least partially explained by preferential nitrate utilization by one particular partner, the seepDBB. This discovery reveals that nitrate is likely an important factor in community structuring and diversity in marine methane seep ecosystems. The thesis concludes with a study of the dynamics between ANME and their associated SRB partners. We inhibited sulfate reduction and followed the metabolic processes of the community as well as the effect of ANME/SRB aggregate composition and growth on a cellular level by tracking 15N substrate incorporation into biomass using FISH-NanoSIMS. We revealed that while sulfate-reducing bacteria gradually disappeared over time in incubations with an SRB inhibitor, the ANME archaea persisted in the form of ANME-only aggregates, which are capable of little to no growth when sulfate reduction is inhibited. These data suggest ANME are not able to synthesize new proteins when sulfate reduction is inhibited.
", "doi": "10.7907/Z9125QKD", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6536", "collection": "thesis", "collection_id": "6536", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07042011-125318322", "type": "thesis", "title": "Pheromones in Free-Living and Parasitic Nematodes", "author": [ { "family_name": "Choe", "given_name": "Andrea", "clpid": "Choe-Andrea" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Wold", "given_name": "Barbara J.", "clpid": "Wold-B-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Platzer", "given_name": "Edward G.", "clpid": "Platzer-E-G" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Nematodes are among the most diverse phyla of animals, occupying almost every ecological niche available. Their ubiquity has led to a number of problems for civilization, including the loss of crops and the spread of neglected tropical diseases. Because they are responsible for a broad range of agricultural and human diseases, many pheromone-mediated nematode behaviors have been described but very few pheromones have been identified.
\r\n\r\nWe report, via high-performance liquid chromatography electrospray ionization mass spectrometry, the discovery that many free-living and parasitic nematodes secrete small-molecule pheromones called ascarosides. These pheromones, called ascarosides, were first found to play a role in sex attraction and induction into a stress-resistant diapausal life stage in the free-living organism, Caenorhabditis elegans. We have performed a double-blind purification of the female sex pheromone in the sour paste nematode Panagrellus redivivus and report that the female sex pheromone is composed of at least two ascarosides. We have also found that both free-living and parasitic nematodes respond to different concentrations of ascarosides through attraction or repulsion, demonstrating cross-species communication. These results suggest that ascarosides could be a universal nematode cue, similar to the role of N-Acyl homoserine lactones in bacteria quorum sensing.
\r\n\r\nBecause ascarosides are nonvolatile, they can only mediate close-range communication. Nematodes have a well-characterized capacity for long-range chemoattraction to a range of volatile cues. However, no studies have been done towards characterizing natural volatile cues derived from nematodes. Here I describe the discovery of volatile cues are produced by male-female species in the genus Caenorhabditis, but are lacking in the hermaphroditic species C. elegans, C. briggsae, and C. sp11. These volatile cues attract males (and sometimes females) from other Caenorhabditis species, demonstrating a cross-species gonochoristic cue.
\r\n", "doi": "10.7907/DX46-C462", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6649", "collection": "thesis", "collection_id": "6649", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09042011-230002591", "type": "thesis", "title": "MicroRNAs 155 and 125b Physiologically and Pathologically Regulate Hematopoiesis and Immunity", "author": [ { "family_name": "Chaudhuri", "given_name": "Aadel Ahmed", "clpid": "Chaudhuri-Aadel-Ahmed" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "MicroRNAs are a class of ~22 nucleotide RNA molecules with roles in diverse biological processes. Here I focus on two microRNAs, miR-155 and miR-125b, and reveal pathways by which their dysregulation leads to myeloproliferative disorder (MPD) and leukemia, respectively. I begin by searching for miR-155 target genes relevant to MPD. By writing an algorithm to search microarray data for predicted microRNA target genes, I identified 89 candidate target genes for miR-155 in myeloid cells. Literature search whittled this list down to 11, and one gene among them, SHIP1, turned out to be largely responsible for miR-155\u2019s ability to cause MPD. My focus shifted to miR-125b when I noticed that miR-125b was enriched in macrophages and thus might play important roles in that cell type. Indeed, gain- and loss-of-function experiments indicated that miR-125b is a potent activator of macrophage activation, and I identified IRF4 as the primary target gene in this process. Finally I asked whether miR-125b plays pathophysiological roles in the development of the hematopoietic system. Thus I overexpressed miR-125b in the hematopoietic system and, to my surprise, observed a very aggressive myeloid leukemia capable of infiltrating peripheral organs including the lungs, liver, kidneys and brain. To determine whether miR-125b is physiologically necessary for normal hematopoietic development, I designed a loss-of-function sponge vector that acts as a decoy, attracting the microRNA away from its normal targets. Use of the sponge in the mouse hematopoietic system led to significantly decreased overall hematopoietic ouput, indicating that miR-125b is physiologically required for normal hematopoiesis. Next, I assayed in vitro a panel of miR-125b target genes and saw that one, Lin28, was superior to the rest. Indeed, Lin28 gain- and loss-of-function in vivo recapitulated major aspects of miR-125b loss- and gain-of-function, respectively. Thus I identified Lin28 as a primary target of miR-125b in the hematopoietic system. In summary, my work shows that two microRNAs, miR-155 and miR-125b, physiologically and pathologically control hematopoietic development. I also identify important target genes for each of these microRNAs in their respective disease processes. Indeed, therapeutic targeting of these pathways may prove useful in the treatment of cancer.", "doi": "10.7907/HFSQ-8278", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6936", "collection": "thesis", "collection_id": "6936", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162012-151100376", "primary_object_url": { "basename": "Thesis-Ken_Yu-Final-Embedded.pdf", "content": "final", "filesize": 31495047, "license": "other", "mime_type": "application/pdf", "url": "/6936/1/Thesis-Ken_Yu-Final-Embedded.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineering Immunity Against HIV\r ", "author": [ { "family_name": "Yu", "given_name": "Kenneth Kwok-Chang", "clpid": "Yu-Kenneth-Kwok-Chang" } ], "thesis_advisor": [ { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An effective vaccine against the human immunodeficiency virus (HIV)-1 has so far been elusive. Anti-viral vaccines against other viruses work by stimulating the production of neutralizing antibodies that block infection. To be useful, an anti-HIV vaccine preparation needs to elicit potent neutralizing antibody response with sufficient breadth to cover the diversity of HIV variants. Despite sustained research efforts, such an immunogen has been difficult to develop. We could overcome this difficulty by using gene therapy to directly instruct the body to produce anti-HIV broadly neutralizing antibodies (bNAbs). In this thesis, I describe a technology I developed termed the \u201cMolecular Rheostat\u201d for directing the simultaneous expression of anti-HIV surface and secreted immunoglobulins using mutant 2A \u201cself-cleaving\u201d peptides. I describe the application of this system to the programming of hematopoeitic stem cells to generate anti-HIV B cells as a strategy to \u201cvaccinate\u201d against HIV infection. I then pivot to consider alternatives to B-cell programming to produce antibodies against HIV. I investigate the modification of non-lymphoid hematopoietic cells to produce antibodies using retroviral vectors and describe the use of lentiviral vectors to program muscle to produce anti-HIV broadly neutralizing antibodies. In addition to presenting a novel tool for controlling the simultaneous expression of full-length and truncated proteins, the work described here furnishes a foundation for future development into potential gene-therapeutic prophylaxis against HIV.", "doi": "10.7907/GFQ8-M763", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6766", "collection": "thesis", "collection_id": "6766", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01122012-225941177", "primary_object_url": { "basename": "jtngo_thesis_final.pdf", "content": "final", "filesize": 22194602, "license": "other", "mime_type": "application/pdf", "url": "/6766/1/jtngo_thesis_final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis", "author": [ { "family_name": "Ngo", "given_name": "John Tuan", "clpid": "Ngo-John-Tuan" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein\u2019s gene (or genes) of interest. This approach, often termed \u201cresidue-specific incorporation,\u201d allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. Chapter I of this thesis describes how this strategy has been used to track cellular protein synthesis with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a \"pulse\" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins.
\r\n \r\nChapter II of this thesis describes how the selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. The work in Chapter III illustrates how the extent of temporal and spatial resolution of protein labeling can be enhanced through controlled expression of mutant synthetases. Use of characterized promoters to direct transcription of mutant synthetase genes can limit labeling to relevant cells and physiological states in settings of increased complexity. Chapter IV presents a novel strategy with which ncAAs can be uniquely incorporated at the N-terminal positions of nascent proteins while excluded from insertion at internal positions. This approach permits \"site-selective\" tagging of cellular proteins, and its use in tagging and visualization of cell-cycle dependent protein synthesis is described.
\r\n\r\nThe work described throughout this thesis was designed with the objective of providing powerful and versatile methods for the study of protein synthesis in complex multicellular systems, including live animals. Thus, Chapter V considers how these strategies might be used to dissect protein synthesis in living animals.
", "doi": "10.7907/XNEA-GM53", "publication_date": "2012-12-15", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6781", "collection": "thesis", "collection_id": "6781", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01262012-055600494", "type": "thesis", "title": "Global Analysis of Dynamic Epigenetic Marking and Transcriptional Regulation Underlying T-Cell Lineage Commitment\r ", "author": [ { "family_name": "Zhang", "given_name": "Jingli A.", "clpid": "Zhang-Jingli-A" } ], "thesis_advisor": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Bronner", "given_name": "Marianne E.", "clpid": "Bronner-M-E" }, { "family_name": "Stathopoulos", "given_name": "Angelike", "clpid": "Stathopoulos-A" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "T-cell lineage specification and commitment success depends on precise temporal induction of T-lineage specific genes, as well as repression of lineage-inappropriate programs. After entry into the thymus, T-cell progenitors still retain inherited lineage plasticity, reflected by the mixed-lineage pattern of gene expression and the abilities to give rise to alternative lineages. Although Notch-Delta signaling is an essential force to trigger and sustain T-lineage differentiation, it does not appear to be the only requirement for this process. Successful commitment also depends on additional transcription factors, which often cooperatively interact with Notch-Delta signaling. However, the molecular mechanism by which pro-T cells are advanced to become committed T cells, in particular how the alternative lineage potentials are eliminated, is not fully understood. Using the genome-wide high-throughput sequencing, we track global shifts in gene expression pattern and transcriptional activity associated histone modifications in five successive stages of T-cell differentiation that span the commitment process. Our results show that T-lineage commitment is defined by the surprisingly complex downregulation of progenitor- and/or alternative lineage-associated programs, with relatively few regulatory genes are substantially upregulated. Rather than being silenced by a single global repression event, progenitor- and/or alternative lineage-associated genes are regulated by individual gene-specific mechanisms, indicated by the unsynchronized epigenetic transformations at discrete cis-elements of genes loci linked to progenitor and/or alternative lineage programs. We also investigate the genome-wide occupancies of PU.1 and GATA-3, two regulatory factors that have critical but complementary roles in early T-cell development. Binding sites choices of these two factors imply that transcriptional regulation by one particular factor is developmental context as well as dosage dependent. Furthermore, We combine this genome-wide approach with gene perturbation to study the function of Bcl11b, a transcription factor required for the completion of T-cell lineage commitment. Our analyses reveal that, in part through directly or indirectly regulation of Notch1 and GATA-3, Bcl11b mediates the modulation of T-cell lineage specification and commitment.", "doi": "10.7907/9Q4G-E674", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7140", "collection": "thesis", "collection_id": "7140", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062012-145546209", "primary_object_url": { "basename": "Yue_Shen_Thesis.pdf", "content": "final", "filesize": 6255334, "license": "other", "mime_type": "application/pdf", "url": "/7140/1/Yue_Shen_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Interkingdom Communication of a Bacterial Mutualist and its Mammalian Host", "author": [ { "family_name": "Shen", "given_name": "Yue", "clpid": "Shen-Yue" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Grant", "given_name": "Jensen", "clpid": "Jensen-G-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Microbial molecules have evolved to promote transient and/or permanent associations with mammals. Although numerous examples of secretion systems employed by pathogens during infection have been described, mechanisms by which commensal bacteria export molecules during symbiosis remain unknown. The human gut mutualist Bacteroides fragilis produces a capsular polysaccharide (PSA) that directs host immune development. We reveal herein that outer membrane vesicles (OMVs) deliver PSA to dendritic cells (DCs), promoting development of regulatory T cells and inducing anti-inflammatory cytokines during in vivo protection of intestinal disease. OMV mediated regulatory responses required the Growth Arrest and DNA-Damage-Inducible protein (Gadd45\u03b1) in DCs. DCs treated with OMVs containing PSA protect mice from experimental colitis, whereas Gadd45\u03b1-/- DCs are unable to support T cell regulatory response and are defective in suppressing proinflammatory cytokine production and host pathology. Our findings demonstrate DC-induced protection from disease via interaction with a beneficial microbial molecule delivered by OMVs, uncovering a novel paradigm for interkingdom communication between the microbiota and mammals. \r\nIn another effort to test the immunomodulatory activity of PSA outside of the gut, we found systemic treatment with PSA protects animals from experimental sepsis, a model for systemic inflammatory disease. More interestingly, this protection is mediated by B cells but not T cells because Rag-/- mice reconstituted with B cells gained the protection by PSA while those reconstituted with T cells were not protected. We further showed that a subset of B cells, marginal B cells, which are known to produce natural antibodies against bacterial antigens, were sufficient in mediating this protection. Preliminary data also suggested that secretion of IgM and/ or expression of type II Interleukin 1 receptor (IL-1R2) from marginal zone B cells might be critical for the suppression of the excessive inflammation during disease. This study will help to uncover the systemic effect of PSA, a microbial molecule from a gut commensal, and its potential as a novel therapy for human sepsis.", "doi": "10.7907/56YV-8J33", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6368", "collection": "thesis", "collection_id": "6368", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05022011-211415511", "primary_object_url": { "basename": "Chow,_Janet_Thesis.pdf", "content": "final", "filesize": 2002472, "license": "other", "mime_type": "application/pdf", "url": "/6368/1/Chow,_Janet_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "A Pathobiont of the Mammalian Microbiota Balances Intestinal Inflammation and Colonization", "author": [ { "family_name": "Chow", "given_name": "Janet", "clpid": "Chow-Janet" } ], "thesis_advisor": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Humans and mammals are colonized by a multitude of microbial organisms that have co\u2010evolved with their hosts for millions of years. The majority of these microbes reside in the gastrointestinal (GI) tract as a complex and dynamic consortium. Though most associations with the host are symbiotic or commensal, some resident bacteria have the potential to cause disease under certain conditions. We refer to these bacteria as \u2018pathobionts.\u2019 Pathobionts are distinct from opportunistic pathogens, which are often acquired from the environment and cause acute infections. Bacterial type VI secretion systems (T6SSs) are one mechanism for mediating close host\u2010microbial interactions. Herein we report that the T6SS of H. hepaticus, a pathobiont of the murine intestinal microbiota, mediates critical protective functions during association with its mammalian host. In cell cultures, infection of intestinal epithelial cells (IECs) with H. hepaticus T6SS mutants results in increased bacterial association compared to wild\u2010type bacteria. In animals, T6SS mutants colonize the lower GI tract to a higher degree. Most importantly, H. hepaticus defective in type VI secretion is unable to restrain potent innate and adaptive immune responses in an animal model of experimental colitis. In addition, the H. hepaticus T6SS directs an anti\u2010inflammatory gene expression profile in IECs, and CD4+ T cells from mice colonized with T6SS mutants produce increased proinflammatory interleukin\u201017 cytokine in response to IECs presenting H. hepaticus antigens. Thus, our findings reveal that H. hepaticus has evolved a T6SS as a mechanism to actively maintain a non\u2010pathogenic, symbiotic relationship in the GI tract by regulating bacterial colonization and host inflammation. Disturbances in the dynamic interaction between gut bacteria and the intestinal immune system may lead to exacerbated host inflammation. As intestinal bacteria profoundly influence host biology, our findings support an emerging hypothesis that alterations in the composition of the microbiota, known as dysbiosis, is a critical factor in various human disorders such as inflammatory bowel disease and colon cancer.", "doi": "10.7907/4FZ1-A113", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5997", "collection": "thesis", "collection_id": "5997", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182010-104705359", "primary_object_url": { "basename": "Full_Dissertation_(N._Ballor).pdf", "content": "final", "filesize": 13949251, "license": "other", "mime_type": "application/pdf", "url": "/5997/1/Full_Dissertation_(N._Ballor).pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Hydrogenases and Hydrogen Sensors in the Symbiotic Microbial Communities of Wood-Feeding Termites", "author": [ { "family_name": "Ballor", "given_name": "Nicholas R.", "clpid": "Ballor-Nicholas-R" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Mazmanian", "given_name": "Sarkis", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite\u2019s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities. ", "doi": "10.7907/621E-9221", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5786", "collection": "thesis", "collection_id": "5786", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072010-142709280", "primary_object_url": { "basename": "XZhang_Complete_Thesis.pdf", "content": "final", "filesize": 24930564, "license": "other", "mime_type": "application/pdf", "url": "/5786/12/XZhang_Complete_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "I. Formate Dehydrogenase Gene Diversity in Lignocellulose-Feeding Insect Gut Microbial Communities. II. Metabolic Impacts on the Hydrogen Isotope Content of Bacterial Lipids ", "author": [ { "family_name": "Zhang", "given_name": "Xinning", "orcid": "0000-0003-2763-1526", "clpid": "Zhang-Xinning" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Sessions", "given_name": "Alex L.", "clpid": "Sessions-A-L" }, { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "I.) Symbiotic CO\u2082-reducing acetogens are important bacterial members of lignocellulose-feeding termite and roach gut communities. Acetogens are the major consumers of H\u2082 derived from lignocellulose fermentation and can contribute up to 1/3 of the acetate that serves as fuel for the insect host. Many acetogens in wood-feeding termites belong to a diverse group of relatively unstudied, uncultured spirochetes within the genus Treponema. Here, I use the gene sequence for hydrogenase-linked formate dehydrogenase, an enzyme utilized in sugar fermentation and the acetogenic metabolism of the spirochete isolate Treponema primitia, to investigate the diversity, evolution, and activity of uncultured acetogenic spirochetes in lignocellulose-feeding insect guts. The results suggest that (a) the trace element selenium has shaped the gene content of acetogenic spirochetes in gut communities over evolutionary time scales, (b) acetogenic spirochete populations have undergone extinctions and radiations associated with an evolutionary bottleneck, convergent evolutions, and possibly even invasion during termite evolution, and (c) termite gut acetogenesis is largely mediated by only a few spirochete species, which represent a small portion of total acetogenic spirochete diversity.
\r\n\r\nII.) The hydrogen-stable isotope compositions (D/H) of lipids in the environment vary greatly. All variations have been assumed to result from changes in the D/H of water, a source of lipid hydrogen. However, several studies suggest that water D/H may not be the only influential factor. In this study, I report that lipid D/H values can vary by 500\u2030 in bacterial cultures despite constant water D/H. This indicates variations in lipid/water fractionation need to be considered when interpreting environmental data. More significantly, I demonstrate that lipid D/H values are systematically related to the utilization of different central metabolic pathways in bacteria. The results suggest that different cellular mechanisms for NADPH synthesis result in lipids with characteristic D/H. Implications for the use of lipid D/H as an isotopic marker of energy metabolism are discussed.
", "doi": "10.7907/ETXQ-D671", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:2413", "collection": "thesis", "collection_id": "2413", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06022009-172223", "primary_object_url": { "basename": "06FullThesis.pdf", "content": "final", "filesize": 5764113, "license": "other", "mime_type": "application/pdf", "url": "/2413/6/06FullThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA Encoded Biotechnologies for Informative Cancer Diagnostics", "author": [ { "family_name": "Kwong", "given_name": "Gabriel Abner", "orcid": "0000-0002-6255-6755", "clpid": "Kwong-Gabriel-Abner" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Guo", "given_name": "Chin-Lin", "clpid": "Guo-Chin-Lin" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "This thesis describes the development of DNA-encoded, multi-parametric, sensing platforms for informative cancer diagnostics. In the first part of this thesis, I will present a technology called \u201cDNA-encoded antibody library (DEAL).\u201d In this approach, computationally derived, orthogonal ssDNA sequences are conjugated to antibodies specific for protein targets and cell surface markers. The resulting collection of conjugates is applied to a biological sample of interest, binds to their cognate antigens, and is detected after the complexes are hybridized to a glass substrate printed with spatially distinct complementary DNA sequences. By using DNA assembly, the DEAL platform enables the simultaneous detection of the major classes of biological molecules, namely nucleic acids, proteins and cells.
\r\n\r\nThe second part of this thesis focuses on the development of a cell sorting platform that can detect antigen-specific T cells called \u201cNucleic Acid Cell Sorting (NACS).\u201d In NACS, ssDNA encoding is used to assemble peptide major histocompatability complexes (p/MHC) on glass substrates by hybridization to cDNA microarrays. These assembled peptide/MHC microarrays are then used to sort mixed populations of antigen-specific T cells. This spatially encoded scheme addresses the widespread desire for methods that allow the multiplexed detection of antigen-specific T cells. The sensitivity and selectivity of NACS is similar to flow cytometry, demonstrated in key experiments with T cells derived from multiple sources, including endogenous and TCR-engineered T cells collected from cancer patients. Finally, this platform is used to monitor the persistence of cancer-specific T cells in peripheral blood collected from a patient undergoing T cellular immunotherapy.
\r\n\r\nLastly, a scheme for the detection of cell surface markers is presented. In this approach, DEAL and NACS conjugates prepared with UV labile ssDNA oligonucleotides are allowed to bind to target cell samples in solution. The ssDNA tags are released in solution by UV-induced photocleavage. The presence and expression of the cognate antigen is determined by collecting the pool of reporter ssDNA tags followed by exponential amplification by PCR. A DEAL conjugate specific for the oncogene EGFR was used to determine the expression level of EGFR in a low-passage brain tumor primary cell line. The feasibility of using ssDNA-p/MHC complexes for detecting unique TCRs was also demonstrated. Finally an experimental flow is described for integration with second generational high-throughput sequencing platforms for global and quantitative surface-ome profiling.
\r\n", "doi": "10.7907/AXMP-AM12", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:3888", "collection": "thesis", "collection_id": "3888", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10032008-170935", "primary_object_url": { "basename": "00_CompleteThesis.pdf", "content": "final", "filesize": 9019615, "license": "other", "mime_type": "application/pdf", "url": "/3888/1/00_CompleteThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Biology and Community Structure of CO\u2082-Reducing Acetogens in the Termite Hindgut", "author": [ { "family_name": "Ottesen", "given_name": "Elizabeth Ann", "orcid": "0000-0002-7898-2425", "clpid": "Ottesen-Elizabeth-Ann" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "clpid": "Mazmanian-S-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "In the guts of wood-feeding termites, CO\u2082-reductive acetogenesis serves as the dominant sink for H\u2082 generated during the fermentation of wood polysaccharides. This activity can generate up to 1/3 of the acetate that powers the energy metabolism of the host insect. The gene for formyl-tetrahydrofolate synthetase (FTHFS), a key gene in the acetyl-CoA pathway, can be used as a genetic marker of acetogenic capability. The dominant FTHFS types in the guts of wood-feeding termites are known to cluster phylogenetically with those from acetogenic Treponemes. In this work, we present the discovery that the guts of wood-feeding roaches are also dominated by Treponeme-like sequences. Phylogenetic analysis of roach-derived FTHFS sequences reveals a cluster that forms a basal radiation of the termite Treponeme cluster. This suggests that the Treponemes found in roach guts represent an ancient divergence, present in the last common ancestor of these insects, rather than a modern lineage acquired by cross-species symbiont transfer. The FTHFS sequences present in the guts of higher termites were also examined. Wood-, palm-, and litter-feeding termites were found to be dominated by acetogenic Treponemes, while subterranean soil/grass feeders were found to be dominated by a novel cluster of Firmicute-like FTHFS types. Also presented herein is the development of microfluidic digital PCR for molecular characterization of individual bacteria from environmental samples. We used this technique to retrieve FTHFS and 16S rRNA gene sequences from single bacterial cells, thereby discovering the 16S rRNA sequences of uncultured acetogens in the termite gut. This technique should provide a valuable tool for molecular analyses of termite gut acetogens, and can potentially be adapted for the characterization of uncultured bacteria that carry any metabolic gene of interest.\r\n", "doi": "10.7907/V57J-FB39", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" } ]