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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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" } ]