Human bodies are home to a vast assortment of microbes, including bacteria, fungi, and viruses. These microbes live within their human hosts, interacting with each other and influencing states of health and disease. Despite their prevalence and importance, studying host-microbe interactions has been limited by the dearth of appropriate tools and approaches, and an underappreciation for the role of biophysics. \r\nThis thesis describes the development and application of novel tools and approaches for studying bacteria, fungi, and viruses to uncover their potential roles in human health and disease.
\r\n\r\nIn my first project, we investigated bacterial aggregation, a phenomenon related to important host-microbe interactions such as biofilm formation and the clearance of pathogens from the gastrointestinal tract. We found that bacteria aggregate in the presence of polymers (such as dietary fiber) via a mechanism that is qualitatively consistent with depletion-type forces under gut-like conditions. Surprisingly, motile bacteria aggregate more than nonmotile bacteria in viscous, high-polymer concentrations due to the higher effective diffusivity and inter-bacterial collisions enabled by motility. These two results give insight on how the foods (such as fiber) that we consume can physically affect the structure of microbes and other matter in the gut.
\r\n\r\nIn my next projects, we investigated viral-load kinetics to understand the best testing modality for early detection of SARS-CoV-2 via a large community-based household transmission study. By collecting longitudinal, paired saliva and nasal-swab specimens from SARS-CoV-2 patients starting from the incident of infection, we quantified the viral-load trajectories of COVID-19-positive participants in each specimen type over time. Our results revealed that viral loads increased quickly and reached a higher peak in nasal-swab specimens, whereas viral loads were detectable earlier but reached a lower maximum in saliva. Both specimen types exhibited a temporal trend whereby viral loads were higher in specimens collected in the morning compared with the evening. In samples where infectious viral titer was measured, we found that the ratio of N gene viral load and infectious viral titer did not remain consistent throughout the course of infection. These three results help us understand the heterogeneity of SARS-CoV-2 disease progression in different individuals, and how the analytical sensitivity of a diagnostic, the specimen type, and time of sampling can be crucial in conducting community surveillance programs during a pandemic. \r\nFinally, we extended and co-validated for fungi a novel sample-preparation method that enriches fungal cells in host-rich samples to enable the first demonstration of deep metagenomic sequencing of fungal communities directly from clinical samples (without a culture step). Our results show that this method depletes host DNA by over 1000-fold by mass, improving taxonomic classification and gene calling, as well as enabling de novo metagenome assembled genome (MAG) assembly in samples dominated by human biomass.
", "doi": "10.7907/7chb-wk98", "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:15248", "collection": "thesis", "collection_id": "15248", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312023-220301021", "primary_object_url": { "basename": "ling_bill_thesis_final3.pdf", "content": "final", "filesize": 20171760, "license": "other", "mime_type": "application/pdf", "url": "/15248/1/ling_bill_thesis_final3.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Acoustic Biomolecules for Diagnostic Ultrasound Imaging", "author": [ { "family_name": "Ling", "given_name": "Bill", "orcid": "0000-0002-1276-7204", "clpid": "Ling-Bill" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Chan", "given_name": "Warren C. W.", "orcid": "0000-0001-5435-4785", "clpid": "Chan-Warren-C-W" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nanotechnology has enabled significant breakthroughs in the early detection and treatment of disease, but many of these advances rely on expensive and less-accessible imaging modalities. Ultrasound, on the other hand, is a noninvasive imaging modality that stands out for its universal availability, cost-effectiveness, and safety. However, harnessing the benefits of nanomaterials for ultrasound has been challenging due to the size and stability constraints of typical ultrasound contrast agents. Recently, an innovative solution has emerged in the form of gas vesicles (GVs), a class of air-filled protein nanostructures found in certain aquatic microbes. These promising next-generation ultrasound contrast agents offer a crucial bridge between nanotechnology and ultrasonography.
\r\n\r\nIn this thesis, we investigate the in vivo behavior of GVs, explore their potential applications as nanodiagnostic agents, and consider key factors for their future clinical deployment. In Chapter 2, we examine the interactions of GVs with blood components, focusing on imaging performance and immunogenicity. In Chapter 3, we show that intravenously injected GVs are cleared by liver-resident macrophages and subsequently undergo lysosomal degradation. We leverage this finding to develop an ultrasound-based method for visualizing cellular degradative processes and demonstrate its potential as a liver disease diagnostic. In Chapter 4, we introduce bicone GVs, the smallest known ultrasound contrast agent. We show that these sub-80 nm particles can penetrate tumors, deliver potent ultrasound-induced mechanical effects, and are readily engineered for molecular targeting, extended circulation time, and payload conjugation.
\r\n\r\nTogether, these findings highlight the tremendous potential of GVs as injectable nanomaterials for ultrasound imaging, laying the foundation for future studies to further refine the design and application of these agents.
", "doi": "10.7907/va8g-tb47", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16062", "collection": "thesis", "collection_id": "16062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-194724728", "type": "thesis", "title": "Stochastic Foundations for Single-Cell RNA Sequencing", "author": [ { "family_name": "Gorin", "given_name": "Gennady", "orcid": "0000-0001-6097-2029", "clpid": "Gorin-Gennady" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Single-cell RNA sequencing, which quantifies cell transcriptomes, has seen widespread adoption, accompanied by proliferation of analysis methods. However, there has been relatively little systematic investigation of its best practices and their underlying assumptions, leading to challenges and discrepancies in interpretation. I present a set of generic, principled strategies for modeling the biological and technical components of sequencing experiments and use case studies to motivate their application to sequencing data.
", "doi": "10.7907/jn6n-x368", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15163", "collection": "thesis", "collection_id": "15163", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112023-130637882", "primary_object_url": { "basename": "Liaw_Eric_2023_Thesis.pdf", "content": "final", "filesize": 10234218, "license": "other", "mime_type": "application/pdf", "url": "/15163/2/Liaw_Eric_2023_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "A Novel, Rapid Phenotypic Assay for a Beta-Lactam Antibiotic Susceptibility and an Analysis of its Theoretical Limits", "author": [ { "family_name": "Liaw", "given_name": "Eric Jer-Jiun", "orcid": "0000-0003-2244-8335", "clpid": "Liaw-Eric-Jer-Jiun" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Current management of bacterial infections is limited by the slow turnaround time of culture-based antibiotic susceptibility testing (AST). Culture-free phenotypic AST methods, though faster, are limited not only by analytical sensitivity but also by the low number, density, and purity of live pathogens present in clinical specimens before culturing. Separating and concentrating pathogens from clinical specimen matrices and improving the analytic sensitivity of phenotypic measurement technologies remain active areas of research. However, to date, the literature lacks consensus over what is a reasonable goal for the minimum number of pathogens in a clinical specimen needed to accurately perform phenotypic AST.
\r\n\r\nI describe \"bulk filtration AST\" and \"digital filtration AST,\" two new filtration-based AST methods that improve an AST method previously published by others and myself. These methods use nucleic acid quantification to assess the activity of antibiotic classes (and only those classes) targeting peptidoglycan turnover, specifically the beta-lactams, which are the most frequently prescribed class of antibiotics. I use filtration AST to quantify the in vitro pharmacodynamics of beta-lactam antibiotics over time scales shorter than two hours, and I simultaneously validate the methods' accuracies on clinical isolates of Enterobacteriaceae. To analyze filtration AST results, either for fitting parameter values or for predicting susceptibility, I derive probabilistic models for the outcomes of each of the two filtration AST methods, then perform Bayesian parameter inference from my data.
\r\n\r\nI then propose a general mathematical framework for defining the concepts of the phenotypic assay and the ideal phenotypic assay. Within this framework, I calculate the ideal filtration AST performance as a function of the number of cells assayed, my fitted pharmacodynamic parameters, and other variables. Interestingly, the observed performance of my implementation of digital filtration AST is consistent with the implementation's approaching the ideal performance. I hope my demonstration of these new methods and my theoretical framework will help guide future research into rapid phenotypic AST.
", "doi": "10.7907/qhvg-7q92", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15009", "collection": "thesis", "collection_id": "15009", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08272022-063655097", "type": "thesis", "title": "Bioorthogonal Noncanonical Amino Acid Tagging for Understanding Bacterial Persistence", "author": [ { "family_name": "Liu", "given_name": "Xinyan", "orcid": "0000-0003-3258-5720", "clpid": "Liu-Xinyan" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenotypic heterogeneity in populations of isogenic bacterial cells includes variations in metabolic rates and responses to antibiotic treatment. In particular, sub-populations of \u201cpersister\u201d cells exhibit increased antibiotic tolerance. Understanding the mechanisms that underlie bacterial persistence would constitute an important step toward preventing and treating chronic infections. On the other hand, bacteria often have multiple molecular mechanisms to adapt to fluctuating environments. Understanding these mechanisms, and their redundancy, requires examinations in depth at the molecular level. This thesis describes a time- and cell state-selective proteome-labeling approach that enables researchers to investigate heterogeneous systems and molecular redundancy.
\r\n\r\nIn Chapter 1, we review the concept of bacterial persistence. The definition of bacterial persistence is introduced. Both the differences and connections between bacterial persistence and resistance are covered. In particular, we discuss research related to Pseudomonas aeruginosa (P. aeruginosa), an important opportunistic pathogen found in many cystic fibrosis patients. State-of-the-art technologies to investigate bacterial persistence are discussed, and we conclude that advanced tools are needed to advance research on bacterial persistence further.
\r\n\r\nIn Chapter 2, we highlight the concept of bioorthogonal noncanonical amino acid tagging (BONCAT). BONCAT is a powerful tool developed in the Tirrell and Schuman laboratories allowing the incorporation of noncanonical amino acids (ncAA) into newly-synthesized proteins. We review established strategies for proteomics, especially cell-selective proteomics. We introduce the concept and mechanism of BONCAT and address the advantages of BONCAT in the investigation of phenotypic heterogeneity and bacterial persistence.
\r\n\r\nIn Chapter 3, we describe our work using BONCAT for understanding bacterial persistence. In particular, we investigated the process of persister resuscitation, as it is closely related to the reoccurrence of P. aeruginosa infections. The characteristics of the heterogeneity of persister cells during persister awakening were examined by survival assays and by ScanLag, an automated colony-based system allowing high-throughput acquisition of time-lapse images, quantification, and analysis of growth of bacterial colonies. Two BONCAT methods were developed in the P. aeruginosa strain PA14 by treating cells either with L-azidohomoalanine (Aha), which avoids extensive usage of antibiotic markers and allows direct integration with PA14 transposon insertion library, or with L-azidonorleucine (Anl), which has the advantage of specificity, as well as direct application in nutrition-rich medium. Through BONCAT enrichment experiments, we found proteins involved in the biosynthesis of pyochelin, a secondary siderophore involved in bacterial iron acquisition, were up-regulated in the regrowth phase. We further explored whether the up-regulation was a result of the modulation of HigB-HigA toxin-antitoxin system.
\r\n\r\nIn Chapter 4, we describe our work for understanding molecular redundancy. The chapter follows up on our observation of up-regulation of pyochelin-related proteins during persister regrowth. We discuss the hypothesis that pyochelin confers a growth advantage in persister cells subject to carbon-limited conditions. In addition, we discuss the potential role of Fur, a ferric uptake regulator, in bacterial persistence.
", "doi": "10.7907/q6bx-kt39", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15142", "collection": "thesis", "collection_id": "15142", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04242023-215416950", "primary_object_url": { "basename": "husic_thesis_submit.pdf", "content": "final", "filesize": 35655205, "license": "other", "mime_type": "application/pdf", "url": "/15142/1/husic_thesis_submit.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Strategies for the Mechanically Triggered Release of Small Molecules", "author": [ { "family_name": "Husic", "given_name": "Corey Christopher", "orcid": "0000-0003-0248-7484", "clpid": "Husic-Corey-Christopher" } ], "thesis_advisor": [ { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of force-responsive molecules called mechanophores is a central component of the field of polymer mechanochemistry. Mechanophores enable the design and fabrication of polymers for a variety of applications ranging from sensing to self-healing materials. Nevertheless, an insufficient understanding of structure\u2013activity relationships limits experimental development, and thus computation is necessary to guide structural design. Herein, we use the constrained geometries simulate external force (CoGEF) method to evaluate a library of covalent mechanophores using density functional theory (DFT). We use these results to identify key parameters that accurately predict experimentally determined mechanochemical reactivity.
\r\n \r\nPolymers that release small molecules upon external stimulation are promising for a wide range of applications, including sensing, catalysis, and drug delivery. Mechanophores are uniquely suited to enable molecular release with excellent selectivity and control. We have designed a general platform for mechanically gated small molecule release that leverages a latent 2-furylcarbinol species masked as a mechanically labile Diels\u2013Alder adduct. Here, we describe the computationally guided design of metastable 2-furylcarbinol derivatives through the prediction of activation energy values and construction of structure\u2013activity relationships. These results enable a molecular release platform suitable for a wide scope of cargo molecules across a broad range of chemical environments.
", "doi": "10.7907/0add-6n09", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14295", "collection": "thesis", "collection_id": "14295", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07062021-155047044", "type": "thesis", "title": "Engineering the Tryptophan Synthase \u03b2-Subunit for Synthesis of Noncanonical Amino Acids", "author": [ { "family_name": "Watkins-Dulaney", "given_name": "Ella Jenn\u00e1", "orcid": "0000-0002-0585-1598", "clpid": "Watkins-Dulaney-Ella-Jenn\u00e1" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Buller", "given_name": "Andrew R.", "orcid": "0000-0002-9635-4844", "clpid": "Buller-A-R" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The tryptophan synthase \u03b2-subunit (TrpB) naturally catalyzes a pyridoxal phosphate cofactor-mediated \u03b2-substitution reaction between indole and serine to form L-tryptophan. Almost half a century ago, it was realized that TrpB could accept nucleophiles other than indole to synthesize noncanonical amino acids (ncAAs), which are highly useful small-molecule building blocks that are found in many bioactive molecules. Since then, TrpB has been applied to synthesize a wide range of ncAAs. This thesis details the engineering of TrpB for synthesis of new and useful ncAAs and the application of TrpB as a model to study the principles that govern intra-protein interactions. Chapter I chronicles the history of tryptophan synthase, provides useful information about the enzyme\u2019s catalytic cycle, and describes how TrpB has been used to synthesize ncAAs in works preceding this thesis. Chapter II describes the evolution, application, and characterization of TrpB for the synthesis of a blue, fluorescent noncanonical amino acid \u03b2-(1-azulenyl)-L-alanine (AzAla). Chapter III details the engineering and mechanistic characterization of TrpB to asymmetrically catalyze C\u2013C bond formation with an entirely new class of nucleophile: ketones. Chapter IV describes the in vivo continuous evolution of TrpB which resulted in sequence-diverse TrpB orthologs that have been adapted to function at lower temperatures and display a range of substrate-selectivity profiles. Chapter V describes the development of a deep mutational scanning experiment of combinatorial site-saturation mutagenesis (SSM) libraries for generating a large dataset that maps enzyme sequence to function for the purpose of studying epistasis with machine learning. Overall, the work presented in this thesis expands the repertoire of ncAAs that can be synthesized by TrpB and demonstrates unique applications of TrpB as a model enzyme for continuous in vivo directed evolution and for generating a dataset that will be useful to the protein machine learning community.
", "doi": "10.7907/yekm-y267", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "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:14523", "collection": "thesis", "collection_id": "14523", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03222022-221436705", "type": "thesis", "title": "A Bubble Is Born: Nucleation and Early Growth of CO\u2082 Bubbles in Polymer Foams", "author": [ { "family_name": "Ylitalo", "given_name": "Andrew Samuel", "orcid": "0000-0003-4086-3508", "clpid": "Ylitalo-Andrew-Samuel" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "thesis_committee": [ { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Flagan", "given_name": "Richard C.", "orcid": "0000-0001-5690-770X", "clpid": "Flagan-R-C" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Gas bubble nucleation is a fundamental phenomenon both throughout the natural sciences and in the production of foams for lightweight, functional materials; it is also the basis for many a bubbly beverage. Enhancing bubble nucleation in polyurethane insulating foams used for refrigeration can further reduce their low thermal conductivity without resorting to hazardous blowing agents used in the past. Experimental challenges of measuring the kinetics of the rapid, multiscale process of bubble nucleation pose a roadblock to investigation of suitable processing conditions, as well as the development of theoretical models of bubbles and foams.
\r\n \r\nHere, using a microfluidic flow-focusing technique developed for measurement of protein and chemical kinetics, we built a microfluidic cell to probe gas bubble nucleation of CO\u2082 in polyol, a model system for polyurethane insulating foams, at controlled pressure with millisecond resolution over acquisition times sufficient for optical, IR, and X-ray measurements. This technique allows for repeated measurements of bubble nucleation at any degree of supersaturation without the interference of heterogeneous nucleation from surfaces. By extrapolating a model fit to high-speed optical microscopy measurements of bubble growth backward in time, we estimated the degree of supersaturation at nucleation for thousands of bubbles. Estimates of the nucleation rate based on Poisson statistics were consistent with predictions by a string method model based on density functional theory and G-ADSA measurements. This model predicted that the addition of cyclopentane (a common physical blowing agent in polyurethane foams) can dramatically reduce the nucleation energy barrier due to the formation of a liquid-like layer of cyclopentane and CO\u2082 along the surface of the bubble that reduces the interfacial tension, which previous models have only predicted at significantly higher saturation pressures. This prediction was supported by thermodynamic measurement of a three-phase coexistence under similar conditions, which is a known fingerprint for such nucleation pathways, and measurement of significantly higher bubble nucleation rates upon the addition of cyclopentane. These findings shed light on the possibility of a previously unappreciated role of physical blowing agents like cyclopentane in enhancing bubble nucleation by opening up a qualitatively distinct and more favorable nucleation pathway.
", "doi": "10.7907/cdgw-7c18", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14382", "collection": "thesis", "collection_id": "14382", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012021-223453672", "primary_object_url": { "basename": "20211001_PhD_Thesis_MA_Proofread_Final.pdf", "content": "final", "filesize": 2995353, "license": "other", "mime_type": "application/pdf", "url": "/14382/1/20211001_PhD_Thesis_MA_Proofread_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Development of Single-Cell SPRITE: a Tool for Measuring Heterogeneity of 3D DNA Organization", "author": [ { "family_name": "Arrastia", "given_name": "Mary Villanueva", "orcid": "0000-0002-0723-3574", "clpid": "Arrastia-Mary-Villanueva" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Across eukaryotic cells, DNA from each nucleus is organized in three dimensions in order to help regulate transcriptional activity. Decades of chromosome capture technologies have revealed fundamental chromatin structures, providing information about how DNA is assembled genome-wide. The majority of these methods utilize direct physical ligation of DNA molecules to generate pairwise interactions, which have provided information about short-range interactions and intra-chromosomal structures. Recent technologies have moved toward identifying multiple DNA interactions simultaneously without physical ligation of DNA molecules, revealing information about long-range interactions and inter-chromosomal structures. One of the biggest limitations of these methods is that they only study DNA organization in bulk, which misses the heterogeneity of chromosomal structures at the single-cell level. As a result, single-cell chromosome capture methods have been developed to begin probing into the cell-to-cell variability of DNA organization and answer long-standing questions regarding single-cell structure. However, single-cell methods are currently limited to identifying low-resolution, intra-chromosomal DNA interactions with few numbers of cells. This creates a need for an improved, high-throughput single-cell method that can capture high-resolution structures and simultaneous mapping of both intra- and inter-chromosomal interactions to better elucidate single-cell DNA organization. In this thesis, we describe the development of 'single-cell split-pool recognition of interactions by tag extension' (scSPRITE), a single-cell chromosome capture method that allows for mapping of high-resolution, intra- and inter-chromosomal structures across thousands of cells. Through scSPRITE, we were not only able to reveal fundamental information about single-cell DNA organizations, but we can also quantitatively measure the variability of DNA interactions from cell to cell.
", "doi": "10.7907/w70x-2294", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14467", "collection": "thesis", "collection_id": "14467", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01042022-184525578", "type": "thesis", "title": "Development and Scaling of Differentiation Circuit Architectures for Improving the Evolutionary Stability of Burdensome Functions in E. coli", "author": [ { "family_name": "Williams", "given_name": "Rory Logan", "orcid": "0000-0003-2605-5790", "clpid": "Williams-Rory-Logan" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Goentoro", "given_name": "Lea A.", "orcid": "0000-0002-3904-0195", "clpid": "Goentoro-L-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "With advances in the sequencing and synthesis of DNA, automation, and computation, we are increasingly able to rapidly and reliably program functions into cells. However, because the functions we engineer cells to perform are often both unnecessary for the cell\u2019s survival and burdensome to cell growth, mutation and natural selection can rapidly lead to loss of function. Though numerous strategies have made headway, improving the evolutionary stability of engineered functions remains a goal of the synthetic biology community. To address this problem generally, we developed a strategy relying on integrase-mediated recombination which allows non-producing progenitor cells to differentiate at a tunable rate, thereby continuously replenishing producer cells expressing the orthogonal T7 RNAP. While this strategy removes selective pressure for mutations inactivating the function of interest in the progenitor cell population, a strategy of terminal differentiation, in which the capacity of differentiated cells to grow is limited, was necessary to prevent the expansion of such mutations in the differentiated cell population. To experimentally implement terminal differentiation, we co-opted the R6K plasmid system, using differentiation to simultaneously activate expression of T7 RNAP, and inactivate expression of \u03c0 protein (an essential factor for R6K plasmid replication), thereby allowing limitation of differentiated cell growth through antibiotic selection. Critically, we demonstrated computationally that terminal differentiation endows the circuit with robustness to mutations which disrupt T7 RNAP driven expression, and to plasmid instability effects that result in decreased expression. Intuitively and computationally identifying the category of mutations which disrupt the differentiation process as the Achilles's heel of terminal differentiation, we developed a redundant architecture using a novel split-\u03c0 protein system which required 2 mutations to break the circuit. We experimentally demonstrated a trade-off between rate of production and duration of function as the differentiation rate is tuned, an increased benefit of terminal differentiation with higher-burden expression, and that redundancy improves the evolutionary stability of the terminal differentiation architecture. Specifically we achieve a maximum of ~2.8x (single-cassette terminal differentiation) and ~4.2x (redundant terminal differentiation) the total fluorescent protein production achieved from comparable high-burden naive expression in which all cells inducibly express T7 RNAP. We further demonstrate differentiation can enable the expression of even toxic functions, and develop a terminal differentiation circuit architecture which will allow the degree of redundancy and therefore the evolutionary stability of the architecture to be scaled to arbitrary degrees.
", "doi": "10.7907/5k67-b636", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14388", "collection": "thesis", "collection_id": "14388", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10072021-173853251", "type": "thesis", "title": "Nucleic Acid Measurements for Antibiotic Susceptibility Testing and Early Detection of SARS-CoV-2", "author": [ { "family_name": "Savela", "given_name": "Emily Sue", "orcid": "0000-0001-9614-4276", "clpid": "Savela-Emily-Sue" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_bbe" } ], "abstract": "Nucleic-acid-amplification tests (NAATs) are widely used in microbial detection both in environmental characterization and human diagnostics. NAATs offer highly sensitive and specific detection of target molecules among the noise of complex samples. This thesis covers two important applications of nucleic-acid quantification techniques in human clinical samples. First, I co-developed a new phenotypic antibiotic susceptibility test that uses species-specific DNA detection to detect bacterial cell-wall damage following incubation with beta-lactam antibiotics. Second, I helped compile a longitudinal dataset of SARS-CoV-2 viral loads during a community-based COVID-19 study run by the Ismagilov Lab through October 2020 \u2013 April 2021 in the greater Los Angeles County area, USA. Sensitive and specific nucleic-acid tests allowed for robust detection of pathogenic microbes in both these applications. Designing and implementing NAATs for these applications required consideration of biological constraints of the microorganisms, molecular stability over the time of quantification, and the practical constraints of acquiring and transporting samples. Continued innovation of NAAT technologies will be critical to contain present and future pandemics and empower medical professionals with data to inform treatment options.
", "doi": "10.7907/vp9a-n206", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "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:14204", "collection": "thesis", "collection_id": "14204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302021-051953086", "primary_object_url": { "basename": "CheeHuat(Linus)Eng_thesis.pdf", "content": "final", "filesize": 6063063, "license": "other", "mime_type": "application/pdf", "url": "/14204/1/CheeHuat(Linus)Eng_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Plus Ultra: Genome-Wide Spatial Transcriptomics with RNA seqFISH+", "author": [ { "family_name": "Eng", "given_name": "Chee Huat (Linus)", "orcid": "0000-0002-2521-9696", "clpid": "Eng-Chee-Huat-Linus" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Visualizing single cells and their organization in intact tissue is crucial to understanding their governing biological function. Even though single cell RNA sequencing has provided many insights into the heterogeneity and gene expression profiles across many tissue types, the dissociation process which loses the spatial information is hindering our deeper understanding of how these transcriptional distinct cell types are organized and interacting in their native tissue environment.
\r\n\r\nThe thesis begins by giving a background on how single cell RNA sequencing has transformed biology and the emergence of spatial technology such as sequential fluorescence in situ hybridization (seqFISH). While spatial methods are useful for mapping the cell types identified from single cell RNA sequencing, the need for turning spatial technology such as seqFISH, which has high detection efficiency of the transcriptome with spatial information, into an in situ discovery tool is discussed as the scientific community\u2019s goal heads towards building spatial atlases for every human tissues and organs such as the brain.
\r\n \r\nWhile seqFISH has high detection efficiency, it is still limited in the number of genes capable of profiling at once. The major obstacle is the optical crowding problems when more RNA species are targeted and imaged using a fluorescence microscope. In Chapter 2, we first investigated, if the RNA molecules are instead captured on a coverslip and profiled with sequential barcoding strategy, the FISH-based method will reliably characterize the transcriptome when molecular crowding is not an issue.
\r\n \r\nFinally, in Chapter 3, we demonstrate the barcoding strategy to break through the molecular crowding limit of multiplexed FISH. From being able to profile hundreds to a thousand genes by various multiplexed FISH methods at that time in the field, we succeeded in profiling 10,000 genes by RNA seqFISH+, an evolved version of seqFISH, in various intact tissue sections, turning seqFISH+ into a spatial discovery technology with its genome-wide coverage and high detection efficiency. The work described in this part of the thesis is highlighted in Nature Method\u2019s Method of The Year 2020- Spatially-resolved Transcriptomic article.
", "doi": "10.7907/nvfe-5j74", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14121", "collection": "thesis", "collection_id": "14121", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04152021-173245433", "type": "thesis", "title": "Mechanisms and Consequences of Bacterial Resistance to Natural Antibiotics", "author": [ { "family_name": "Perry", "given_name": "Elena Kim", "orcid": "0000-0002-7151-1479", "clpid": "Perry-Elena-Kim" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "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_bbe" } ], "abstract": "Many bacteria secrete natural antibiotics\u2014toxic small molecules that can kill or inhibit the growth of other microorganisms. Several of these compounds have been commercialized as antimicrobial drugs, and the mechanisms and public health consequences of bacterial resistance to clinically-used antibiotics are well understood. By contrast, the role of bacterially-produced antibiotics in natural environments, where they have existed for millions of years, remains an open question. Besides potentially serving as tools of warfare between competing microbes, natural antibiotics have been proposed to serve less antagonistic functions ranging from the acquisition of nutrients to the transmission of signals between cells. Indeed, despite evidence that natural antibiotics can suppress sensitive microbes in environments such as the soil surrounding plant roots, the ecological significance of the toxicity of these molecules has sometimes been questioned. At the same time, for most natural antibiotics, the mechanisms and prevalence of resistance are either poorly characterized or entirely unknown.
\r\n\r\nThis thesis addresses the molecular mechanisms and consequences of bacterial resistance to a particular class of redox-active natural antibiotics called phenazines. Phenazines are produced by a major opportunistic human pathogen, Pseudomonas aeruginosa, during infections, as well as by several bacterial species that associate with the roots of crops such as wheat, where they serve to protect their plant hosts against fungal pathogens. Resistance to this family of natural antibiotics is therefore potentially relevant to multiple sectors of human society. I begin by investigating the intrinsic phenazine resistance of a common soil bacterium, Agrobacterium tumefaciens, that does not itself produce phenazines. Using a functional genetics approach, I find that the composition of the respiratory electron transport chain plays a critical role in mitigating phenazine toxicity, one that cannot be compensated by increased expression of efflux pumps that transport phenazines out of the cell or oxidative stress responses that neutralize the toxic byproducts of phenazine redox-cycling. Subsequently, we turn to P. aeruginosa, the phenazine-producing opportunistic pathogen, and demonstrate that the defenses it activates against its own toxic phenazine, pyocyanin, collaterally accelerate the acquisition of resistance to certain clinical antibiotics. Other bacteria known to form multispecies infections with P. aeruginosa can also benefit from exposure to pyocyanin in the presence of these clinical antibiotics; we show that in at least one strain isolated from a patient, the effect of pyocyanin on the frequency of spontaneous antibiotic-resistant mutants rivals that of disruptions in DNA repair machinery. Importantly, a growing body of reports suggests that, besides pyocyanin, other metabolites produced by bacterial pathogens can also affect the efficacy of clinical antibiotics. We review the evidence for which types of bacterial metabolites alter susceptibility to antimicrobial drugs, as well as the mechanisms underlying this phenomenon. Finally, I examine the prevalence of bacterial resistance to an agriculturally-relevant phenazine in a wheat field where the use of native phenazine producers to control crop diseases has been studied for decades. I discover that while Gram-positive bacteria are generally more susceptible to this phenazine compared to Gram-negative bacteria, the sharpness of this distinction is pH-dependent; moreover, I uncover surprising heterogeneity in phenazine resistance within certain taxonomic groups. Taken together, these findings illuminate recurring themes in mechanisms of phenazine resistance and point to an underappreciated role for natural antibiotics in the resilience of opportunistic pathogens to clinical antibiotics. This thesis also lays the groundwork for developing a predictive model of phenazine resistance across diverse bacteria, with potential implications for optimizing the use of clinical antibiotics and improving agricultural sustainability.
", "doi": "10.7907/tv8n-kr43", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:14138", "collection": "thesis", "collection_id": "14138", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05082021-185615529", "primary_object_url": { "basename": "McCardellReed2021thesis.pdf", "content": "final", "filesize": 7370942, "license": "other", "mime_type": "application/pdf", "url": "/14138/7/McCardellReed2021thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Genetic Circuits for the Control of Multi-Strain Bacterial Populations", "author": [ { "family_name": "McCardell", "given_name": "Reed Dillard", "orcid": "0000-0002-0955-3133", "clpid": "McCardell-Reed-Dillard" } ], "thesis_advisor": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Microbial species rarely exist alone. Nearly everywhere you could think to look, microorganisms of various species live together in harmony. Microbes together in their communities are incredibly powerful actors wherever they are found; they perform small miracles---the conversion of milk into yogurt---and large ones---production of most of the planet's oxygen and organic carbon.
\r\n\r\nOur burgeoning knowledge of microbial life combined with modern technologies to manipulate it create a critical, exciting opportunity to harness microbial power for the betterment of technology, people, and the planet. This thesis presents a body of work which explores the manipulation of microbial communities using the intersectional bio-engineering approach of synthetic biology. We demonstrate how molecular tools evolved by bacteria can be repurposed to create rationally designed systems for controlling features of bacterial populations.
\r\n\r\nWe begin by examining a genetic circuit that caps the size of a bacterial population by coordinating the deaths of population members -- the population capping or \"pop cap\" circuit. Briefly, E. coli cells in the pop cap circuit are engineered to synthesize a chemical -- a quorum sensing (QS) signal -- that reports the density of the population, sense this chemical, and produce the ccdB toxin to destroy themselves in response. The molecular tools that make up this circuit are drawn from organisms across the spectrum of bacterial diversity. Brought together, they create a feedback control circuit that controls population size by causing member cells to die when a target population size has been reached. To improve the performance of this population controller and reduce the influence of the environment on the circuit, we add the aiiA quorum sensing signal degradase to allow the experimenter control over the degradation rate of the QS density signal. Additionally, we explore RNA and protein mechanisms to sequester the death-causing toxin---inactivating it---allowing us to release a population cap. The resulting \"cap and release\" circuit is a flexible motif that can be scaled to control multi-strain populations, expanding the scope of control beyond the single-strain populations regulated by the base pop cap circuit.
\r\n\r\nUsing the scalable cap and release motif, we design a genetic circuit to regulate a multi-strain community. Two different cell strains expressing symmetric, interconnected cap and release systems form the \"A=B\" circuit, so named for its ability to control the composition of the community to a target ratio of A cells to B cells, or Apopulation = \u03b1Bpopulation. Through dynamical system models of the system, we explore the effects of active QS signal degradation on composition control performance and perform a parameter sensitivity analysis of the system to help determine the best method for building a functioning A=B system in the laboratory. We use a high throughput construction and screening protocol to create variants of the A=B system with identical architectures, but slightly differing component production rates. We crown the most successful variant with a series of experiments to determine if it indeed recapitulates our model's predictions for its performance. Our implementation of the A=B circuit can successfully regulate the composition of a community, with interesting additional effects on total population density.
\r\n\r\nThe cap and release and A=B circuits need parts that can do three things: 1) send a signal between cells to communicate information, 2) compare two signals, 3) regulate cell growth or death. We highlight bacteriocins, bacterial protein exotoxins that are released from a producer cell to kill other cells of similar species, as attractive tools for bacterial community engineering both for their multi-functionality and modular protein structure. By themselves, bacteriocins can perform all the functions needed for population control: they transmit themselves between cells, have unique high-affinity sequestering antitoxin proteins, and are toxins to receiver cells. We begin the process of their characterization and usage as synthetic biological \"parts\" by creating non-native expression systems that match native expression strengths. Using these experimenter-controlled systems we design preliminarily test a bacteriocin-based bacterial community control circuit. Additionally, given the E. coli colicin bacteriocins' unique, nearly plug-and-play modular domain structure, we explore possibilities for engineering colicin proteins themselves for increased functional diversity or uses outside of growth regulation.
", "doi": "10.7907/wgpp-vj97", "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:13684", "collection": "thesis", "collection_id": "13684", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04272020-152058259", "primary_object_url": { "basename": "Manuel_Bedrossian_Caltech_PhD_Thesis.pdf", "content": "final", "filesize": 121568711, "license": "other", "mime_type": "application/pdf", "url": "/13684/8/Manuel_Bedrossian_Caltech_PhD_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "A Novel Digital Holographic Microscope (DHM) to Investigate and Characterize Microbial Motility in Extreme Aquatic Environments", "author": [ { "family_name": "Bedrossian", "given_name": "Manuel M.", "orcid": "0000-0003-2524-3765", "clpid": "Bedrossian-Manuel-M" } ], "thesis_advisor": [ { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Nadeau", "given_name": "Jay L.", "orcid": "0000-0001-5258-0076", "clpid": "Nadeau-Jay-L" }, { "family_name": "Gharib", "given_name": "Morteza", "orcid": "0000-0003-0754-4193", "clpid": "Gharib-M" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Recent shifts in the astrobiological community have prompted the development of methods for the direct search for extant life within our solar system. In order to look for life elsewhere in our solar system, it is important to also investigate the broad spectrum of extant life on Earth. Over millions of years of evolution, life has continually adapted such that an 'extreme' environment has become a relative term. What is considered extreme for one type of organism is home to another and vice versa. Furthermore, very little is known about the organisms that inhabit these extreme environments, and even less in known about their in situ behavior. Investigating various extreme environments around Earth in order to understand the in situ behavior of organisms that inhabit it will better inform the astrobiological community when planning future space missions for the direct search for extant life within our solar system. However, no suitable instrument exists to conduct these in situ field campaigns, while also being physically robust enough to withstand the rugged terrains that can be expected from extreme environments.
\r\n\r\nThis thesis describes the development of a novel off-axis digital holographic microscope (DHM) for the direct in situ observation of microscale organisms in extreme aquatic environments. The hardware developments of this instrument are introduced and validated experimentally as well as software developments including autonomous particle detection and tracking algorithms. This instrument is then used in novel laboratory experiments involving the development of optical phase contrast agents, as well as deployed to multiple field campaigns where off-axis DHM is used to observe the in situ behavior of microorgansisms in various extreme aquatic environments around North America.
", "doi": "10.7907/m3a3-4610", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13728", "collection": "thesis", "collection_id": "13728", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282020-112303076", "primary_object_url": { "basename": "Xinran Liu Thesis 2020.pdf", "content": "final", "filesize": 12082731, "license": "other", "mime_type": "application/pdf", "url": "/13728/20/Xinran Liu Thesis 2020.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Cell-Selective Proteomic Profiling in Complex Biological Systems", "author": [ { "family_name": "Liu", "given_name": "Xinran", "orcid": "0000-0003-0045-8823", "clpid": "Liu-Xinran" } ], "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": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cells within biological systems are constantly adjusting their protein synthesis in response to various environmental changes. To study the rapid cellular regulations in complex biological systems, global proteomic profiling provides important information on system-level regulations, yet physiological properties characteristic of individual cellular subpopulations could be hidden under the characterization. Instead, cell-selective proteomic profiling allows researchers to reveal the heterogeneities in biological systems with phenotypically and even genetically distinct subpopulations under different microenvironments.
\r\n\r\nChapter 1 describes the development of bioorthogonal noncanonical amino acid tagging (BONCAT) for proteomic profiling with resolution in both space and time: its initial role is protein labeling with temporal resolution via pulse-addition of noncanonical amino acid, which could be recognized by endogenous aminoacyl tRNA-synthetase (aaRS), into systems of interest; later on, mutant aaRSs are identified through mutant synthetase library screening, which allows for efficient incorporation of various types of noncanonical amino acids that could hardly be activated by endogenous machineries. The identification and exploitation of mutant aaRSs allow sensitive cellular selectivity during protein labeling. With unprecedented spatiotemporal resolution of BONCAT, and the advancement in high-resolution mass spectrometry and computational algorithms, BONCAT is a powerful technique for selective proteomic profiling to study physiological regulations in a wide range of complex biological systems.\r\nChapter 2 describes the application of the BONCAT method in cell-selective proteomic profiling in Pseudomonas aeruginosa biofilms. In this work, we targeted an iron-starved subpopulation in biofilms and compared its proteomic profile with that of the entire system. Key gene and pathway regulations in the subpopulation are found through the analysis of the proteomic data, which suggest that iron-starved cells shift their priority towards housing keeping pathways, adapt an energy- and resources-saving mode to cope with their harsh local environmental conditions, and get prepared to disperse for better survival. Analysis of poorly studied proteins highly upregulated in the subpopulation led to the discovery of a previously uncharacterized protein (PA14_52000) that is potentially related to iron acquisition. The transposon insertion mutant PA14_52000::tn showed significantly enhanced pyoverdine production in rich medium and reduced biofilm formation.
\r\n\r\nChapter 3 describes the study of physiological regulations in Bacillus subtilis K-state subpopulation via BONCAT. A subset of B. subtilis cells, typically 10% - 20% of the entire population, enter K-state in a stochastic manner. With the low level of K-state entry rate and high randomness, we challenged BONCAT to specifically capture gene and pathway regulations in K-state cells and compared the proteomic profiling with that of the entire population. Regardless of the difficulties of selective protein labeling inherent in the system, our results indicate that BONCAT has high specificity and resolution in proteomic profiling for minor subpopulations and proteins with low overall absolute abundance. We found multiple pathways and genes characteristic of K-state regulated differentially from the entire population, either significantly up- or down-regulated. Proteins that are uncharacterized or previously known for functions irrelevant of K-state are highly abundant in the subpopulation, providing new insight toward their alternative functions critical for K-state cells and future investigation directions of K-state study.
", "doi": "10.7907/p18t-5j69", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13735", "collection": "thesis", "collection_id": "13735", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292020-131840076", "primary_object_url": { "basename": "200529_erik_jue_2020_thesis_final.pdf", "content": "final", "filesize": 13853909, "license": "other", "mime_type": "application/pdf", "url": "/13735/1/200529_erik_jue_2020_thesis_final.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Improved Tools for Point-of-Care Nucleic Acid Amplification Testing", "author": [ { "family_name": "Jue", "given_name": "Erik Bradley", "orcid": "0000-0001-7585-3794", "clpid": "Jue-Erik-Bradley" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "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" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "There is a critical need for improved diagnostic tools to detect infectious diseases, especially in low-resource regions. A sample-to-answer point-of-care nucleic acid amplification test (NAAT) would be incredibly valuable for many different applications (e.g. COVID-19, Chlamydia/Gonorrhoeae, Influenza, Ebola, Zika/Chikungunya/Dengue, etc.). However, sample preparation (purification of pure nucleic acids) is a challenging bottleneck. In Chapter 2, commercial NA extraction methods were studied and improved. In Chapter 3, commercial stocks of SARS-CoV-2 RNA used in FDA emergency-use authorizations were found to be inaccurate and were independently quantified using reverse transcription digital PCR. In Chapter 4, a 3D printed meter-mix device was developed for initial processing prior to the sample preparation device. In Chapter 5, a 3D printed sample-to-device interface was prototyped to facilitate loading multi-volume SlipChip devices with purified template mixed with LAMP reactants. In Chapters 6-7, advancements were made for image processing of commercial chips to study digital LAMP reactions. In Chapter 8, additional tools were developed towards sample-to-answer point-of-care NAAT including a sample preparation module, amplification module, cell-phone readout, and automated base station.", "doi": "10.7907/d6mf-5081", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13742", "collection": "thesis", "collection_id": "13742", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302020-111741817", "primary_object_url": { "basename": "Silverman_thesis_submission_200530.pdf", "content": "final", "filesize": 4442708, "license": "other", "mime_type": "application/pdf", "url": "/13742/3/Silverman_thesis_submission_200530.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Protein-Mediated Colloidal Assembly", "author": [ { "family_name": "Silverman", "given_name": "Bradley Ross", "orcid": "0000-0002-9256-8941", "clpid": "Silverman-Bradley-Ross" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Brady", "given_name": "John F.", "clpid": "Brady-J-F" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "Rosen Bioengineering Center" }, { "literal": "div_chem" } ], "abstract": "The assembly of colloidal-sized particles into larger structures by the manipulation of inter-particle forces has been a subject of significant research towards applications in materials science, soft matter physics, and synthetic biology. To date, much of this work has utilized manipulation of electrostatic or depletion interactions to drive the aggregation of the particles. More recently, specific (bio)-chemical interactions have been harnessed, particularly the use of deoxyribonucleic acid (DNA) linkers to program particle interactions by Watson-Crick base-pairing. In this thesis, we will demonstrate the use of an alternative set of biochemical interactions, protein-protein interactions, which have useful properties (in particular, their ability to be completely genetically-programmable).
\r\n\r\nIn Chapter 2, we discuss the development of a model system for the protein-mediated assembly of colloidal micro-particles. Associative proteins are grafted onto the surface of polystyrene micro-particles, enabling their assembly into aggregates either through reversible coiled-coil interactions or by irreversible isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of the immobilized associative proteins on their surface. Further, we show that particles grafted with different protein pairs show excellent self-sorting into separate aggregates. Finally, we demonstrate that these protein-protein interactions can be used to assemble complex core-shell aggregates. The principles of protein-mediated colloidal assembly learned in this chapter will be instructive as we attempt the more complex assembly of living microbial cells.
\r\n\r\nIn Chapter 3, we discuss the implementation of a protein-driven aggregation system in living bacterial cells. Similarly to Chapter 2, we demonstrate that we can drive the aggregation of bacteria by the surface display of proteins enabling reversible coiled-coil interactions or irreversible isopeptide bonds. The sizes of these aggregates are tunable by titration of surface expression levels by standard synthetic biology techniques. Finally, we show that this programmable aggregation of bacteria may have physiological consequences for the cells, in particular, the activation of a quorum sensing circuit due to a higher local concentration of bacteria.
\r\n\r\nIn Chapter 4, we further investigate how the properties of the aggregates described in Chapter 3 can be controlled and how these relate to the underlying properties of the associative proteins and shear field. we demonstrate control of the assembly kinetics and equilibrium sizes of the resulting flocs over several orders of magnitude using different associating proteins and expression levels. Finally, we show that a single point mutation in the associative protein leads to an unexpected ultra-sensitive pH-responsive coil, demonstrating the importance of molecular-scale interactions on the macro-scale properties of the aggregates.
\r\n\r\nIn Chapter 5, we discuss the ability of the bacterial aggregates described in Chapters 3 and 4 to enable substrate channeling between bacterial strains, leading to enhancement of titers in multi-step biosynthetic pathways. When biosynthetic pathways are split into separate bacterial strains, dilution of the intermediate compound into the bulk media may decrease reaction flux. By aggregating the bacteria, the intermediate compound is able to rapidly diffuse into the downstream cell without being diluted, enabling higher reaction fluxes. we demonstrate through the model flavonoid synthesis pathway that aggregation can lead to substantially higher titers of the desired compound without pathway re-engineering, and develop a mathematical model by which this result can be understood.
", "doi": "10.7907/x3ya-fq67", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11808", "collection": "thesis", "collection_id": "11808", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10062019-213347699", "primary_object_url": { "basename": "porubsky_nicholas_2019.pdf", "content": "final", "filesize": 28767176, "license": "other", "mime_type": "application/pdf", "url": "/11808/1/porubsky_nicholas_2019.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Enhanced Algorithms for Analysis and Design of Nucleic Acid Reaction Pathways", "author": [ { "family_name": "Porubsky", "given_name": "Nicholas James", "orcid": "0000-0001-6330-2645", "clpid": "Porubsky-Nicholas-James" } ], "thesis_advisor": [ { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "thesis_committee": [ { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nucleic acids provide a powerful platform for programming at the molecular level. This is possible because the free energy of nucleic acid structures is dominated by the local interactions of base pairing and base pair stacking. The nearest neighbor secondary structure model implied by these energetics has enabled development of a set of algorithms for calculating thermodynamic quantities of nucleic acid sequences. Molecular programmers and synthetic biologists continue to extend their reach to larger, more complicated nucleic acid complexes, reaction pathways, and systems. This necessitates a focus on new algorithm development and efficient implementations to enable analysis and design of such systems.
\r\n\r\nConcerning analysis of nucleic acids, we collect seemingly diverse algorithms under a unified three-component dynamic programming framework consisting of: 1) recursions that specify the dependencies between subproblems and incorporate the details of the structural ensemble and the free energy model, 2) evaluation algebras that define the mathematical form of each subproblem, 3) operation orders that specify the computational trajectory through the dependency graph of subproblems. Changes to the set of recursions allows operation over the complex ensemble including coaxial and dangle stacking states, affecting all thermodynamic quantities. An updated operation order for structure sampling allows simultaneous generation of a set of structures sampled from the Boltzmann distribution in time that scales empirically sublinearly in the number of samples and leads to an order of magnitude or more speedup over repeated single-structure sampling.
\r\n\r\nFor the problem of sequence design for reaction pathway engineering, we introduce an optimization algorithm to minimize the multitstate test tube ensemble defect, which simultaneously designs for reactant, intermediate, and product states along the reaction pathway (positive design) and against crosstalk interactions (negative design). Each of these on-pathway or crosstalk states is represented as a target test tube ensemble containing arbitrary numbers of on-target complexes, each with a target secondary structure and target concentration, and arbitrary numbers of off-target complexes, each with vanishing target concentration. Our test tube specification formalism enables conversion of a reaction pathway specification into a set of target test tubes. Sequences are designed subject to a set of hard constraints allowing specification of properties such as sequence composition, sequence complementarity, prevention of unwanted sequence patterns, and inclusion of biological sequences. We then extend this algorithm with soft constraints, enhancing flexibility through new constraint types and reducing design cost by up to two orders of magnitude in the most highly constrained cases. These soft constraints enable multiobjective design of the multitstate test tube ensemble defect simultaneously with heuristics for avoiding kinetic traps and equalizing reaction rates to further aid reaction pathway engineering.
\r\n", "doi": "10.7907/M8CZ-MW98", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13636", "collection": "thesis", "collection_id": "13636", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02062020-145031701", "type": "thesis", "title": "Facilitating Miniaturized Bioanalytical Assays in Microfluidic Devices", "author": [ { "family_name": "Zhukov", "given_name": "Dmitriy Vladimirovich", "orcid": "0000-0002-4834-3147", "clpid": "Zhukov-Dmitriy-Vladimirovich" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Gao", "given_name": "Wei", "clpid": "Gao-Wei" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This work describes several efforts in making microfluidic lab-on-a-chip technology more convenient to use for bioanalysis in limited-resource settings (Chapters 2-3), and describes a device for miniaturized multistep process execution (Chapter 4). One underlying theme of these projects is the streamlining of the 'chip-to-world' interfacing to help bring this technology from specialized labs of its developers into more widespread utilization by potential users in other disciplines. Chapter 2 outlines a portable method for achieving stable fluid pumping for sample loading and flow control in microfluidic devices. Chapter 3 details a method for digital nucleic acid test readout with unmodified smartphone cameras. Chapter 4 demonstrates a lab-on-a-chip platform capable of carrying out complex multiplexed biochemical reactions requiring multiple sequential additions of reagents by performing RNA barcoding for multiplexed cDNA library generation.
", "doi": "10.7907/x9wq-h920", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11559", "collection": "thesis", "collection_id": "11559", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292019-112017023", "type": "thesis", "title": "Bioanalytical Tools to Develop Rapid Diagnostics and Study Physiology", "author": [ { "family_name": "Rolando", "given_name": "Justin Charles", "orcid": "0000-0001-8948-319X", "clpid": "Rolando-Justin-Charles" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This work details the development of bioanalytical tools for use in rapid diagnostics (Chapters 2-4) and in the study of physiology (Chapters 5-6). This research harness the power of real-time, singlemolecule microfluidics to study loop-mediated isothermal amplification in urinary tract infections (Chapter 2), chlamydia (Chapter 3), and gonorrhea (Chapter 4). In Chapter 5, non-reactive beads are designed and optimized to study the impact of polymers on murine gastrointestinal mucosa. Chapter 6 details the implementation of a mass spectrometry method to quantify bile acids and investigate their interaction with the microbiota in the murine gastro-intestinal tract.
", "doi": "10.7907/99NT-WP75", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11276", "collection": "thesis", "collection_id": "11276", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11162018-140640407", "type": "thesis", "title": "Bioresorbable Vascular Scaffolds Gain Ductility, Resistance to Hydrolysis, and Radial Strength via a Unique Poly L-lactide Microstructure", "author": [ { "family_name": "Ramachandran", "given_name": "Karthik", "orcid": "0000-0003-1820-7555", "clpid": "Ramachandran-Karthik" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Advances in tissue engineering over the past few decades are poised to revolutionize drug delivery and biomedical implants. Bioresorbable vascular scaffolds (BVS), which are made from the semicrystalline polymer poly (L-lactide), are an example of polymers saving and improving the quality of human life. BVSs are emerging as a promising alternative to metal stents for the treatment of coronary heart disease (CHD), one of the leading causes of death in the world. In contrast to permanent stents, BVSs are designed to have a limited lifespan in the body; they restore blood flow through the occluded artery by lending it support for 3-6 months, but are completely resorbed in 2-3 years, leaving behind a healthy artery. This transient character of BVS restores vasomotion in the treated artery and can eliminate the risk of thrombosis, a dreaded complication regarded as the bane of stenting.
\r\n\r\nThe promising success of the first and currently only clinically-approved BVS (FDA-approval in 2016) provides an impetus to continue its development. The struts of the BVS (~ 150\u03bcm) are nearly two times thicker than in metal stents (~ 80\u03bcm). A thicker device is challenging to implant and is unable to treat smaller and tortuous arteries. Furthermore, clinicians speculate that irregular blow flow over thicker struts may contribute towards thrombosis. An added complication of working with BVSs is that they are difficult to visualize with X-rays owing to the low atomic mass of polymers. The need for a BVS that is thinner, stronger, and radio-opaque is the motivation for this thesis, which aims to extend the benefits of transient implants to a broader patient population. Chapter I provides a brief chronological overview of the evolution of cardiovascular therapeutics to combat CHD. Chapters II and III elucidate micron-scale gradients in the PLLA microstructure of the clinically-approved BVS that overcome PLLA\u2019s inherent brittleness and provide lasting radial support to the artery. Chapter IV discusses the fabrication of novel instrumentation to establish structure-property relationships for scaffolds, and Chapter V explores polylactide nanocomposites that not only increase radial strength in a thinner profile but also provide radio-opacity.
", "doi": "10.7907/9146-5159", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11572", "collection": "thesis", "collection_id": "11572", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302019-162535966", "type": "thesis", "title": "Phenotypic Antimicrobial Susceptibility Testing Based on Nucleic Acid Analysis", "author": [ { "family_name": "Schoepp", "given_name": "Nathan Garrett", "orcid": "0000-0002-2406-3693", "clpid": "Schoepp-Nathan-Garrett" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Antimicrobial resistance (AMR) is one of the most widely recognized threats to global health, and one that continues to grow as new mechanisms of resistance evolve and resistant pathogens spread. Antibiotics are a cornerstone of modern medicine, but their misuse and overuse has constantly and consistently reduced their efficacy to the critically low levels we observe today. As a result, the rate of mortality as a direct result of AMR is approaching over a million deaths annually, with 20-year projections in the ten-millions. Rapid, phenotypic antimicrobial susceptibility testing (AST) that could be performed at the point of care (most notably in \u2264 30 min) would decrease the overuse of antimicrobials, allow physicians to make informed choices about which antimicrobials to prescribe, and improve patient outcomes. Today no such method exists. The ultimate goal of the below work is to allow physicians to choose, instead of guess, which antibiotics to use. We envision that development of these tests into distributable diagnostics will drastically improve patient outcomes, curb the spread of resistance, strengthen global antibiotic stewardship, and forestall the post-antibiotic era.
", "doi": "10.7907/5F6B-F452", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11586", "collection": "thesis", "collection_id": "11586", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312019-135625690", "primary_object_url": { "basename": "Khazaei Thesis - 5June2019.pdf", "content": "final", "filesize": 2655640, "license": "other", "mime_type": "application/pdf", "url": "/11586/1/Khazaei Thesis - 5June2019.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Metabolic Bi-Stability and Hysteresis in a Model Microbiome Community", "author": [ { "family_name": "Khazaei", "given_name": "Tahmineh", "orcid": "0000-0002-4743-2383", "clpid": "Khazaei-Tahmineh" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Doyle", "given_name": "John C.", "orcid": "0000-0002-1828-2486", "clpid": "Doyle-J-C" }, { "family_name": "Henry", "given_name": "Christopher S.", "clpid": "Henry-Christopher-S" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Changes in the species composition of the human microbiome are associated with a broad range of diseases, but elucidating causal mechanisms has been challenging. Some microbiome disease states persist in seemingly unfavorable conditions, e.g., the proliferation of aerobe\u2013anaerobe communities in oxygen-exposed environments in wounds or small intestinal bacterial overgrowth. In Chapter I, using two microbes relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and control theory to show that multi-stability and hysteresis (MSH) is a mechanism that can describe shifts to a resilient aerobe\u2013anaerobe community. We examine the impact of changing oxygen and nutrient regimes and identify factors, including changes in metabolism and gene expression, that lead to MSH. Where MSH explains microbiome shifts, it can profoundly improve our conceptual understanding of these paradoxically persistent disease states, and thereby facilitate effective interventions.
\r\n\r\nChapter II details a method for rapidly detecting the susceptibility and resistance of Neisseria gonorrhoeae to the antibiotic ciprofloxacin. Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1\u20132 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. In our approach, we explore the use of RNA signatures, which are among the first cellular responses to drug exposure, as an indicator of antibiotic susceptibility. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 minutes of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB), we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10-min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.
", "doi": "10.7907/Z588-5H60", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11586", "collection": "thesis", "collection_id": "11586", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312019-135625690", "primary_object_url": { "basename": "Khazaei Thesis - 5June2019.pdf", "content": "final", "filesize": 2655640, "license": "other", "mime_type": "application/pdf", "url": "/11586/1/Khazaei Thesis - 5June2019.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Metabolic Bi-Stability and Hysteresis in a Model Microbiome Community", "author": [ { "family_name": "Khazaei", "given_name": "Tahmineh", "orcid": "0000-0002-4743-2383", "clpid": "Khazaei-Tahmineh" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Doyle", "given_name": "John C.", "orcid": "0000-0002-1828-2486", "clpid": "Doyle-J-C" }, { "family_name": "Henry", "given_name": "Christopher S.", "clpid": "Henry-Christopher-S" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Changes in the species composition of the human microbiome are associated with a broad range of diseases, but elucidating causal mechanisms has been challenging. Some microbiome disease states persist in seemingly unfavorable conditions, e.g., the proliferation of aerobe\u2013anaerobe communities in oxygen-exposed environments in wounds or small intestinal bacterial overgrowth. In Chapter I, using two microbes relevant to the human microbiome, we combine genome-scale mathematical modeling, bioreactor experiments, transcriptomics, and control theory to show that multi-stability and hysteresis (MSH) is a mechanism that can describe shifts to a resilient aerobe\u2013anaerobe community. We examine the impact of changing oxygen and nutrient regimes and identify factors, including changes in metabolism and gene expression, that lead to MSH. Where MSH explains microbiome shifts, it can profoundly improve our conceptual understanding of these paradoxically persistent disease states, and thereby facilitate effective interventions.
\r\n\r\nChapter II details a method for rapidly detecting the susceptibility and resistance of Neisseria gonorrhoeae to the antibiotic ciprofloxacin. Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1\u20132 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. In our approach, we explore the use of RNA signatures, which are among the first cellular responses to drug exposure, as an indicator of antibiotic susceptibility. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 minutes of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB), we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10-min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.
", "doi": "10.7907/Z588-5H60", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11718", "collection": "thesis", "collection_id": "11718", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-153337463", "primary_object_url": { "basename": "Thesis-Yandong Zhang-Final.pdf", "content": "final", "filesize": 2182617, "license": "other", "mime_type": "application/pdf", "url": "/11718/1/Thesis-Yandong Zhang-Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Highly Multiplexed Imaging of E. Coli Chromosome and Sensitive Detection of Single-Cell Protein", "author": [ { "family_name": "Zhang", "given_name": "Yandong", "orcid": "0000-0003-3291-9209", "clpid": "Zhang-Yandong" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The driving force for biology research is the development of new techniques which allow high-sensitivity, high-throughput measurement in various contexts. Over the past decade, the emerging of a variety of single-cell techniques have greatly transformed our understanding of biological system. My thesis work was therefore focused on development of new single- cell techniques and use the techniques to generate new insights into biological system. Specifically, in the first part of my thesis work, we developed DNA seqFISH, a technique that allows us to image more than 100 different loci on the chromosome in single cells. We applied this technique to image E. coli chromosome with 50kb genomic resolution and 50nm spatial precision. Our data allows us to parse the E. coli chromosome structure according to their different spatial conformations and different cell-cycle stages. We identified two chromosome conformations with distinct domain structures, which is obscured from previous population-average research. We further characterized the domain structure dynamics during daughter chromosome segregation. Therefore, our data provides a high- resolution, dynamic view of E. coli chromosome structure.
\r\n\r\nIn the second part, we developed a novel method for sensitive detection of targeted protein and its post-translational modification (PTM) isoform in single cells. Instead of depending on antibodies to distinguish targeted protein and its PTM isoform, we developed an efficient covalent barcoding strategy to barcode targeted protein inside the cells. Thereafter, targeted protein and its PTM isoform are separated by conventional gel electrophoresis, while their single-cell identity is preserved in the covalently attached oligo. By counting the attached DNA oligos using next-generation sequencing, targeted protein, and its PTM isoform can be accurately measured. We demonstrated the utility of the technology by quantification of histone protein, H2B and its mono-ubiquitination isoform, H2Bub at single-cell level. Our method revealed the single-cell heterogeneities of H2Bub/H2B ratio and its cell-cycle dynamics. Our method therefore provides an antibody-free method for sensitive detection of proteins and its isoforms in single cells.
", "doi": "10.7907/CDSX-MR28", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11474", "collection": "thesis", "collection_id": "11474", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04162019-155405091", "type": "thesis", "title": "How Polymers Shape the Physicochemical Environment of the Gut", "author": [ { "family_name": "Preska Steinberg", "given_name": "Asher Raphael", "orcid": "0000-0002-8694-7224", "clpid": "Preska-Steinberg-Asher-Raphael" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The gut is where food is digested and nutrients are absorbed, therapeutics are often delivered, and many infections take hold. The gut microbiota is in symbiosis with its host, and can influence host health and behavior. Though the gut holds these central roles, little is understood about the physics of how soft materials interact with and shape the physicochemical environment of the gut. Soft materials abound in the gut in the form of particulates (e.g., microbes, viruses, therapeutic particles, food granules) and polymers (e.g., dietary fibers, therapeutics, food additives). This thesis explores the soft matter physics of the gut and how physicochemical interactions can influence gut structure and function. This is studied through a combination of mouse experiments and numerical calculations. In the first part of this thesis, we investigate how particulates interact with polymers in the small intestine. We find that polymers from dietary fiber can aggregate particulates by a mechanism that is qualitatively consistent with depletion interactions. This mechanism is distinct from agglutination via specific chemical interactions. In the second part of this thesis, we investigate how polymers interact with the colonic mucus hydrogel. Colonic mucus is the nexus of host-microbe interactions. It is a barrier which protects against microbial infiltration, and alterations to its physical structure have been linked to changes in host health. Here, we find that polymers compress the colonic mucus hydrogel. For uncharged polymers, this mechanism can be described by a simple, mean-field model based on Flory-Huggins solution theory. Further, we find that microbes can modulate the extent of mucus compression by degrading polymers in the gut. In the last part of this thesis, we find that charged polymers (polyelectrolytes) compress mucus by a Donnan mechanism.
", "doi": "10.7907/XPEZ-G864", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10399", "collection": "thesis", "collection_id": "10399", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08292017-182342238", "type": "thesis", "title": "Improving the Speed and Performance of Point-of-Care Diagnostics with Microfluidics", "author": [ { "family_name": "Schlappi", "given_name": "Travis Stratton", "orcid": "0000-0001-6132-6459", "clpid": "Schlappi-Travis-Stratton" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Microfluidic devices play an important role in improving global health because they reduce the study of biological phenomena into physiological scales and lay the foundation for point-of-care (POC) diagnostics. Health is improved and lives are saved because POC diagnostics can enable earlier diagnosis of diseases and therefore more effective treatment. Accurate and available diagnostics also prevent accelerated drug resistance that stems from overtreatment or mistreatment with antibiotics, which is projected to cause up to $100 trillion in lost economic output and 10 million deaths by 2050. This work details new diagnostic assays and theoretical analysis of microfluidic devices that can be implemented at the point-of-care to improve global health.", "doi": "10.7907/Z9K935Q6", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10267", "collection": "thesis", "collection_id": "10267", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042017-183300784", "type": "thesis", "title": "Discovering Biological Roles of Glycosaminoglycans and Protein O-GlcNAcylation Using Chemical Tools", "author": [ { "family_name": "Griffin", "given_name": "Matthew Everett", "orcid": "0000-0001-9549-4418", "clpid": "Griffin-Matthew-Everett" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Carbohydrates surround nearly every cell in the human body. Glycosaminoglycans like chondroitin sulfate and heparan sulfate on the cell surface regulate protein ligand engagement and receptor activation to control a variety of biological processes including development, angiogenesis, and neuronal growth. These polysaccharides exert activity through protein binding to their diverse chemical structures. Therefore, the development of methods to tailor glycosaminoglycan populations at the cell surface with defined structures could provide novel approaches to control biological activity. Herein, two new methods to engineer the cell surface glycocalyx with known glycosaminoglycans are reported. Together, these methods provide complementary short- and long-term approaches to change carbohydrate structures at the cell surface and guide neuronal growth and stem cell differentiation. It is also critical to identify unknown protein-carbohydrate interactions that underlie biological phenomena. Studies delineating novel GAG interactions with an orphan receptor and related soluble ligands are reported herein as well as work towards understanding the biological functions of these newly discovered interactions. These results showcase the utility of chemical biology and biochemical tools to discover and modulate various GAG-protein interactions in diverse biological systems.
\r\n\r\nWithin the cell, thousands of proteins are modified by O-GlcNAc glycosylation, a process that is uniquely catalyzed by a single transferase and hydrolase pair unlike many other post-translational modifications. O-GlcNAcylation functions in many biological contexts including transcription, translation, proteostasis, and metabolism. Key to understanding its effects on these physiological phenomena is the discovery of O-GlcNAc modification sites. However, due to a number of technical challenges, O-GlcNAc proteomics has not progressed nearly as quickly as phosphoproteomics. Thus, developing new methods to enrich O-GlcNAcylated substrates and map modification sites is critical to unravel the myriad functions of O-GlcNAc. Herein, a labeling approach using a chemically cleavable tag is reported as an improved method to capture and release O-GlcNAcylated substrates. Unlike other methods, the cleavable Dde tag is quantitatively removed under mild, neutral conditions and leaves a minimal residual tag on the O-GlcNAcylated peptide to be analyzed. Moreover, the Dde linker outcompetes a previously used UV-cleavable tag both at the protein and peptide enrichment levels. Together, these results highlight the potential usefulness of this method to illuminate novel roles of O-GlcNAcylation in diverse systems.
\r\n", "doi": "10.7907/Z9610XCH", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9898", "collection": "thesis", "collection_id": "9898", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07292016-195927111", "primary_object_url": { "basename": "David Selck Thesis.pdf", "content": "final", "filesize": 11243250, "license": "other", "mime_type": "application/pdf", "url": "/9898/73/David Selck Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Microfluidics for Molecular Measurements and Quantitative Distributable Diagnostics", "author": [ { "family_name": "Selck", "given_name": "David Anthony", "orcid": "0000-0002-0591-4165", "clpid": "Selck-David-Anthony" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Guttman", "given_name": "Mitchell", "clpid": "Guttman-M" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A major challenge in global health care is a lack of portable and affordable quantitative diagnostic devices. This is because classic quantification of biomolecules is typically performed using kinetic assays that require strict control only found in controlled laboratory environments. By using the power of microfluidics, quantitative assays can be performed robustly in a \"digital\" format that is decoupled from precise kinetics through highly parallelized qualitative reactions. The benefits of performing quantitative assays in a digital format extend beyond just assay robustness to reduction of instrumental complexity, increase in quantitative precision, and an increase in the amount of information that can be gained from a single experiment. These microfluidic architectures, however, are not limited to usage in scenarios of quantification of biomolecules. These architectures can also potentially be extended to answering complex biological questions in single cells, such as determining the 3-dimensional organization of nuclear DNA and RNA.", "doi": "10.7907/Z9ZC80XT", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9704", "collection": "thesis", "collection_id": "9704", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05062016-131543245", "primary_object_url": { "basename": "babin_thesis_final.pdf", "content": "final", "filesize": 10129687, "license": "other", "mime_type": "application/pdf", "url": "/9704/39/babin_thesis_final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Bioorthogonal Noncanonical Amino Acid Tagging for Selective Analysis of the Pseudomonas aeruginosa Proteome", "author": [ { "family_name": "Babin", "given_name": "Brett Michael", "orcid": "0000-0002-4133-6665", "clpid": "Babin-Brett-Michael" } ], "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": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA\u2019s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.", "doi": "10.7907/Z94X55S1", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9728", "collection": "thesis", "collection_id": "9728", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05182016-140524270", "primary_object_url": { "basename": "DAT_Thesis_Final.pdf", "content": "final", "filesize": 16203253, "license": "other", "mime_type": "application/pdf", "url": "/9728/79/DAT_Thesis_Final.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Chemical Reaction Dynamics of the Liquid/Vapor Interface Studied by Mass Spectrometry", "author": [ { "family_name": "Thomas", "given_name": "Daniel Aaron", "orcid": "0000-0001-9415-5991", "clpid": "Thomas-Daniel-Aaron" } ], "thesis_advisor": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Flagan", "given_name": "Richard C.", "clpid": "Flagan-R-C" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis presents investigations of chemical reactions occurring at the liquid/vapor interface studied using novel sampling methodologies coupled with detection by mass spectrometry. Chapters 2 and 3 utilize the recently developed technique of field-induced droplet ionization mass spectrometry (FIDI-MS), in which the application of a strong electric field to a pendant microliter droplet results in the ejection of highly charged progeny droplets from the liquid surface. In Chapter 2, this method is employed to study the base-catalyzed dissociation of a surfactant molecule at the liquid/vapor interface upon uptake of ammonia from the gas phase. This process is observed to occur without significant modulation of the bulk solution pH, suggesting a transient increase in surface pH following the uptake of gaseous ammonia. Chapter 3 presents real-time studies of the oxidation of the model tropospheric organic compound glycolaldehyde by photodissociation of iron (III) oxalate complexes. The oxidation products of glycolaldehyde formed in this process are identified, and experiments in a deoxygenated environment identify the role of oxygen in the oxidation pathway and in the regeneration of iron (III) following photo-initiated reduction. Chapter 4 explores alternative methods for the study of heterogeneous reaction processes by mass spectrometric sampling from liquid surfaces. Bursting bubble ionization (BBI) and interfacial sampling with an acoustic transducer (ISAT) generate nanoliter droplets from a liquid surface that can be sampled via the atmospheric pressure interface of a mass spectrometer. Experiments on the oxidation of oleic acid by ozone using ISAT are also presented. Chapters 5 and 6 detail mechanistic studies and applications of free-radical-initiated peptide sequencing (FRIPS), a technique employing gas-phase free radical chemistry to the sequencing of peptides and proteins by mass spectrometry. Chapter 5 presents experimental and theoretical studies on the anomalous mechanism of dissociation observed in the presence of serine and threonine residues in peptides. Chapter 6 demonstrates the combination of FRIPS with ion mobility-mass spectrometry (IM-MS) for the separation of isomeric peptides.", "doi": "10.7907/Z9SN06XG", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8763", "collection": "thesis", "collection_id": "8763", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01302015-101318815", "primary_object_url": { "basename": "Han_Chao_2015_Thesis.pdf", "content": "final", "filesize": 5996704, "license": "other", "mime_type": "application/pdf", "url": "/8763/1/Han_Chao_2015_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Wide Field-of-View Microscopes and Endoscopes for Time-Lapse Imaging and High-Throughput Screening", "author": [ { "family_name": "Han", "given_name": "Chao", "clpid": "Han-Chao" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Choo", "given_name": "Hyuck", "clpid": "Choo-Hyuck" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Lai", "given_name": "Lily L.", "clpid": "Lai-Lily-L" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.
\r\n\r\nThe ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm \u00d7 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.
\r\n\r\nWe developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 \u03bcm and a FOV of ~13 mm2. We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells\u2019 dynamic response to an anticancer drug.
\r\n \r\nWe present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 \u03bcm, a maximum FOV of 100 mm \u00d7 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm \u00d7 120 mm in a single shot with a resolution of 100\u2500140 \u03bcm. In this work we demonstrate the PanCam\u2019s performance in imaging a stained tissue sample.
", "doi": "10.7907/Z9SF2T49", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8095", "collection": "thesis", "collection_id": "8095", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02212014-174719213", "primary_object_url": { "basename": "Thesis_final_SALee.pdf", "content": "final", "filesize": 3968641, "license": "other", "mime_type": "application/pdf", "url": "/8095/1/Thesis_final_SALee.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Bright-Field and Fluorescence Chip-Scale Microscopy for Biological Imaging", "author": [ { "family_name": "Lee", "given_name": "Seung Ah", "clpid": "Lee-Seung-Ah" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Tai", "given_name": "Yu-Chong", "clpid": "Tai-Yu-Chong" }, { "family_name": "Vaidyanathan", "given_name": "P. P.", "clpid": "Vaidyanathan-P-P" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Choo", "given_name": "Hyuck", "clpid": "Choo-Hyuck" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_eng" } ], "abstract": "Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term \u201cchip-scale microscopy\u201d refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.
\r\n\r\nOur strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 \u00b5m at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.
\r\n\r\nNext, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 \u00b5m) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm \u00d7 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.
\r\n\r\nThen, we demonstrate the adaptation of a smartphone\u2019s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.
\r\n\r\nFinally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-\u00b5m resolution for fluorescence imaging over a wide field-of-view (4.8 mm \u00d7 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.
\r\n", "doi": "10.7907/HNWJ-J182", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8218", "collection": "thesis", "collection_id": "8218", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052014-142031176", "primary_object_url": { "basename": "Bing_Sun_2014_thesis_complete.pdf", "content": "final", "filesize": 3582945, "license": "other", "mime_type": "application/pdf", "url": "/8218/67/Bing_Sun_2014_thesis_complete.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Mechanistic Studies of Reactions at the Single-Molecule Level using Microfluidics with Applications in Molecular Diagnostics", "author": [ { "family_name": "Sun", "given_name": "Bing", "clpid": "Sun-Bing" } ], "thesis_advisor": [ { "family_name": "Ismagilov", "given_name": "Rustem", "clpid": "Ismagilov-R-F" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Pierce", "given_name": "Niles A.", "clpid": "Pierce-N-A" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Motivated by needs in molecular diagnostics and advances in microfabrication, researchers started to seek help from microfluidic technology, as it provides approaches to achieve high throughput, high sensitivity, and high resolution. One strategy applied in microfluidics to fulfill such requirements is to convert continuous analog signal into digitalized signal. One most commonly used example for this conversion is digital PCR, where by counting the number of reacted compartments (triggered by the presence of the target entity) out of the total number of compartments, one could use Poisson statistics to calculate the amount of input target.
\r\n\r\nHowever, there are still problems to be solved and assumptions to be validated before the technology is widely employed. In this dissertation, the digital quantification strategy has been examined from two angles: efficiency and robustness. The former is a critical factor for ensuring the accuracy of absolute quantification methods, and the latter is the premise for such technology to be practically implemented in diagnosis beyond the laboratory. The two angles are further framed into a \u201cfate\u201d and \u201crate\u201d determination scheme, where the influence of different parameters is attributed to fate determination step or rate determination step. In this discussion, microfluidic platforms have been used to understand reaction mechanism at single molecule level. Although the discussion raises more challenges for digital assay development, it brings the problem to the attention of the scientific community for the first time.
\r\n\r\nThis dissertation also contributes towards developing POC test in limited resource settings. On one hand, it adds ease of access to the tests by incorporating massively producible, low cost plastic material and by integrating new features that allow instant result acquisition and result feedback. On the other hand, it explores new isothermal chemistry and new strategies to address important global health concerns such as cyctatin C quantification, HIV/HCV detection and treatment monitoring as well as HCV genotyping.
\r\n", "doi": "10.7907/BT81-YX06", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7483", "collection": "thesis", "collection_id": "7483", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02182013-221231959", "primary_object_url": { "basename": "truong_frank_2012_thesis final.pdf", "content": "final", "filesize": 2431877, "license": "other", "mime_type": "application/pdf", "url": "/7483/1/truong_frank_2012_thesis final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Expanding Protein Sequence Space through Incorporation of Non-Canonical Amino Acids", "author": [ { "family_name": "Truong", "given_name": "Frank", "clpid": "Truong-Frank" } ], "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": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "orcid": "0000-0002-3680-4399", "clpid": "Ismagilov-R-F" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein sequence space has been augmented by researchers wanting to expand the diversity of chemical functionalities that can exist within proteins. Artificial amino acids can range from simple atom substitutions such as fluorination, to installation of reactive handles like azides and alkynes. Researchers build upon the framework of natural proteins and have developed methods of installing a wide variety of artificial amino acids into proteins. Chapter 1 discusses in detail the two methods for metabolic incorporation of unnatural amino acids: site-specific incorporation and residue-specific incorporation. Advantages and disadvantages to each method are detailed as well as applications of these methods to the examination of problems in chemical biology.
\r\n\r\nNon-canonical amino acids analogues that are structurally similar to their canonical counterparts can be recognized by the endogenous translational machinery for residue-specific incorporation. Chapter 2 describes the directed evolution of the methionyl-tRNA synthetase (MetRS) to incorporate propargylglycine, an alkyne analogue that is not recognized by the wild-type MetRS. A new MetRS variant active towards propargylglycine was identified after screening libraries of both active site mutations and error-prone PCR mutations. PraRS is capable of producing proteins where methionine is quantitatively replaced by propargylglycine. PraRS also does not recognize azidonorleucine, an azide methionine analogue for which the NLL-MetRS was evolved in order to enable cell-specific protein labeling. A method to identify cellular origins of proteins from two different bacterial strains in co-culture was developed using the NLL-MetRS and PraRS.
\r\n\r\nhapter 3 illustrates the effects of global incorporation of non-canonical amino acids into globular proteins. Although trifluoroleucine and homoisoleucine have shown to increase the thermostability of model proteins, incorporation into more chloramphenicol acetyltransferase (CAT) does not yield the same benefits. We find that mutations that stabilize CAT for fluorinated amino acid incorporation do not protect against homoisoleucine incorporation.
\r\n\r\nLastly, access to new chemical reactions for protein modification requires synthesis and incorporation of new non-canonical amino acid analogues. Chapter 4 describes the design of two new artificial amino acids, S-allyl-homocysteine and 3-furanylalanine, for residue specific incorporation without expression of mutant synthetases. Also, a third amino acid, azidomethylphenylalanine, was designed for activation by a previously discovered phenylalanine-tRNA synthetase mutant. Incorporation of these three analogues provides chemical handles that are potential reagents for cross metathesis, Diels-Alder cycloaddition, and generation of a molecular epitope for binding to synthetic receptors.
\r\n", "doi": "10.7907/6CXB-2092", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" } ]