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Since the school's inception in 1979, students in the Morningside Graduate School of Biomedical Sciences (GSBS) at UMass Chan Medical School have contributed more than a thousand doctoral dissertations and masters theses to the field of biomedical sciences. This collection makes this body of work accessible to our students, faculty, potential recruits, the citizens of Massachusetts, and the world.

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Recently Published

  • A Pilot Clinical Trial of an Informatics-Informed Mobile Transitional Care Program: the Paramedic Assisted Community Evaluation after Discharge (PACED) Intervention

    O'Connor, Laurel (2024-04-03)
    Introduction: Early rehospitalization of frail older adults after hospital discharge is detrimental to patients and hospital systems. Implementing effective strategies to execute a feasible and effective transitional care plan is challenging. Mobile integrated health (MIH) programs, which deploy mobile assets into the community to care for patients, may present a possible solution to facilitating effective transitional care back to home environments after hospitalization. However, there have been few previous studies investigating MIH models for transitional care delivery. The objective of this project is to assess the implementation and effectiveness of an informatics-supported paramedic-led MIH transitional care program for frail older adults. Methods: Patients 65 and older preparing for discharge from the hospital with an eFrailty index of 0.24 or greater were enrolled to participate in a structured post-discharge transitional home visit conducted by community paramedics within 72 hours of discharge. Demographic and clinical information, as well as healthcare utilization patterns, were recorded at enrollment and 30 days after the index hospitalization. Additionally, a separate control group of patients that were screened for the intervention but excluded due to geographical location of residence were aggregated and their electronic health record data including demographical and outcomes data was abstracted. Categorical group comparisons were conducted using chi-square tests and continuous variables group comparisons were conducted using the Kruskal–Wallis equality-of-populations rank test. Crude and adjusted binomial regressions were used for comparative outcomes. Results: In total 100 subjects were enrolled in the intervention (median age 81, 64% female) and 47 were included in the control group (median age 80, 55.2% female). The recruitment rate was 18.0%. The complete intervention protocol was completed and documented by paramedics for 90 (90.0%) patients. The crude and relative risk of 30-day rehospitalization was decreased in the PACED group compared to the control (RR=0.40, CI 0.19-0.84, p=0.03). There was a non-significant trend toward decreased risk of 30-day ED visits (RR=0.61, CI=0.37-1.37, p=0.23). Paramedics identified medication errors in 34 (34.0%) of the participants; the errors were remediated during the visit in 31 (91.2%). Additionally, 67 (67.0%) of subjects screened positive for high fall risk and 7 subjects (7.0%) screened positive for delirium. Conclusions: This pilot study of MIH intervention transition care program programs was feasible with high protocol fidelity and yields preliminary evidence that the intervention results in a decreased risk of rehospitalization in frail older adults.
  • Introducing Telomere-Dysfunction Induced Cellular Senescence in iPSC-Based Modeling Of Neurodegenerative Diseases

    Neherin, Kashfia (2024-03-06)
    Investigation of disease-associated cellular changes in vitro has leaped forward significantly with the innovation of induced pluripotent stem cell (iPSC) technology. However, resetting the epigenetic landscape and aging clock during reprogramming results in iPSC-differentiated cells resembling fetal cell types instead of adult or aged cells. The lack of cellular aging in iPSC-based models presents a significant drawback in the investigation of age-associated diseases such as Alzheimer’s disease. My thesis aims to introduce proper cellular aging to improve iPSC-based modeling of neurodegeneration. Toward this goal, I created an inducible system to trigger senescence in iPSC-based cell models, as recent studies showed senescence playing a crucial role in aging and neurodegeneration. I utilized CRISPR-interference (CRISPRi) to suppress telomere repeat factor 2 (TERF2), a significant component of the telomere-protecting Shelterin complex. I demonstrated that suppression of TERF2 in iPSCs robustly activated DNA damage response (DDR), p53/p21 signaling, and cellular senescence in an inducible and synchronized manner. The inducible approach allows temporal control of senescence activation throughout differentiation from iPSCs to desired cell types. I applied the CRISPRi-TERF2 approach to iPSC-differentiated neural progenitor cells (NPCs) and showed that suppression of TERF2 efficiently activated DDR, p53/p21 signaling, and cellular senescence in differentiated NPCs. This inducible model of cellular senescence generated in this study will enable the investigation of cellular senescence using isogenic comparisons in the progression of age-associated neurodegeneration and improve disease modeling with a proper cellular aging context to facilitate drug discovery.
  • Home Diagnostics, Viral Dynamics, and Post-acute Sequelae of SARS-COV-2

    Herbert, Carly (2024-02-09)
    Introduction: The emergence of the novel coronavirus, SARS-CoV-2, has necessitated prompt evaluations of diagnostic technologies, viral dynamics, and long-term complications of COVID-19 to inform clinical and public health strategies. Methods: Using data from the RADx Clinical Studies Core collected from October 2021-February 2022, we evaluated the longitudinal performance of reverse transcriptase polymerase chain reaction (RT-PCR) and antigen-detecting rapid diagnostic tests (Ag-RDT) by day past symptom onset and close-contact exposure and compared performance by sex, age, vaccination status, and variant (Aim 1). We further examined the association between SARS-CoV-2 viral load, BMI, and sex (Aim 2). Lastly, we conducted a follow-up survey in August 2023 regarding Long COVID. We then modeled the relationship between viral clearance of SARS-CoV-2 and Long COVID (Aim 3). Results: RT-PCR and Ag-RDT showed the highest percent positivity two days past symptom onset (RT-PCR: 91.2%; Ag-RDT: 71.1%) and six days past exposure (RT-PCR: 91.8%; Ag-RDT: 86.2%). Performance did not differ by vaccination status, variant, age category, or sex. In males, increasing BMI was associated with higher viral load in a dose-response fashion, and males had significantly lower viral load than females for BMI>29. Lastly, the risk of long COVID with 3-4 symptoms and 5+ symptoms increased by 2.90 times (95% CI: 1.09-7.74) and 4.54 times (95% CI: 1.84-11.2) per viral load slope-unit increase, respectively. Conclusion: Understanding SARS-CoV-2 viral dynamics is critical to identify effective diagnostic strategies for COVID-19, explain differences in COVID-19 outcomes across sex and BMI, and understand mechanistic contributors of Long COVID.
  • Protein and Guide RNA Engineering of a Compact Cas9 for Enhanced Precision Genome Editing

    Bamidele, Nathan (2024-02-06)
    CRISPR-Cas technologies enable robust manipulation of genetic material, and have been instrumental in advancing a wide range of fields across the life sciences. Specifically, with the ability to correct or alter faulty genes, genome editing tools promise to transform the field of genetic medicine. Current CRISPR-based editors [nucleases, base editors (BEs), and prime editors (PE)] can be programed to induce efficient mutagenesis/repair, conversion, and polymerization, respectively. Presently, nucleases - the most clinically advanced genome editors - suffer from inadequate control of genome editing outcomes. Over time, the field has focused on precision editors such as BEs and PEs that do not rely on double-strand breaks and greatly improve the safety and control of genome editing outcomes. Despite these advances, challenges such as targeting scope, accuracy and in vivo delivery represent major hurdles for the therapeutic application of next-generation editing systems such as BE and PE. In this thesis, I focus on alleviating some of the key obstacles associated with effective genome editing by improving the unique properties of a compact Cas9 orthologue (Nme2Cas9 from Neisseria meningitidis). The bulk of my thesis consists of protein engineering efforts to improve the activity and targeting scope of Nme2Cas9-derived editing systems. My later work focuses on the development of chemically stabilized guide RNAs, providing a path to facilitate in vivo delivery in a variety of formats. Overall, the advances presented in this thesis contribute to the versatility of CRISPR-based genome editing systems for a variety of therapeutic and research applications.
  • An exploration of influenza A virus entry factors using CRISPR-based gene editing

    Kyawe, Pyae Phyo (2023-12-18)
    Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that is capable of causing epidemics and pandemics. During the 2021-2022 influenza season, approximately 9 million people in the U.S. were infected with influenza, resulting in an estimated 5,000 deaths. The error-prone nature of the IAV polymerase results in antigenic drift and antigenic shift which contribute to low vaccine efficacy and escape from antivirals. Furthermore, the host factors required for the complete IAV infectious cycle have not been fully identified. The aim of this dissertation is to examine the host factors that may contribute to IAV infectivity in human lung cells. My goal is to understand how changes in the expression levels of host factors can impact influenza infection by CRISPR-mediated knockout or overexpression of target genes. Utilizing CRISPR screens, several candidates, whose up- or down-regulation resulted in reduced IAV infection in the human A549 cell line were identified. I confirmed that the knockout of CMAS or overexpression of B4GALNT2 inhibited IAV infection. In addition, I tested whether overexpression of two candidates from the CRISPR activation screen – DEFB127 and ADAR1 – would inhibit IAV and non-IAV viruses. Surprisingly, overexpression of the two candidates had minimal impact on IAV in A549 cells, but overexpression of ADAR1 had a pro-viral effect on other viruses. Taken together, these data provide insight into host factors modulating IAV infection and how CRISPR-mediated gene modulation can be utilized to further understand the IAV life cycle and for development of therapeutic agents for flu.
  • Experimental and Computational Methods for Identifying Death-Regulatory Genes from Chemo-Genetic Profiles

    Honeywell, Megan E (2023-12-18)
    A common approach to understanding how drugs induce their therapeutic effect is identifying the genetic determinants of drug sensitivity. This can be achieved following systematic loss- or gain-of-function genetic perturbations with CRISPR/Cas9. Because these “chemo-genetic profiles” are generally performed in a pooled format, inference of gene function is subject to several confounding influences, including variation in growth rates between clones or variation in the degree of coordination between growth and death. To overcome these issues, we developed an analysis method called MEDUSA (Method for Evaluating Death Using a Simulation-assisted Approach). MEDUSA uses time-resolved measurements and model driven constraints to reveal the combination of growth and death rates that generated the drug-treated clonal abundance. We find that MEDUSA is uniquely effective at identifying death regulatory genes, and we apply MEDUSA to determine how DNA damage-induced lethality varies in the presence and absence of p53. We find that loss of p53 switches the mechanism of DNA damage-induced death from apoptosis to a non-apoptotic form of death called MPT-driven necrosis. We find that activation of MPT by DNA damage requires high respiration, and that cell death can be exacerbated by modulating NAD+ in p53-deficient cells. These findings demonstrate the accuracy and utility of MEDUSA, both for determining the genetic dependencies of lethality and for revealing opportunities to promote the lethality of chemotherapies in a cancer specific manner.
  • Advancing RNA-Targeting Therapeutics by Oligonucleotide Engineering: RNA Activation, Off-Target Effect, and Co-Targeting Nuclear and Cytoplasmic RNA

    Wang, Feng (2023-12-15)
    Traditional protein-targeting therapeutics by small molecules can target ~3000 proteins, representing <2% of the human genome. In comparison, ~80% of human genome is transcribed into RNAs, which are mostly targetable by oligonucleotides. Thus, RNA-targeting therapeutics enable a much bigger spectrum of drug targets. With nearly 20 FDA-approved oligonucleotide drugs and numerous ongoing clinical trials, RNA-targeting therapeutics by oligonucleotides have significantly addressed unmet medical needs. However, challenges persist in gene expression enhancement and comprehensive dual-RNA modulation. Here, I set out to address these two challenges. Firstly, I identified two ASOs designed to target intron 1 of FXN pre-mRNA, leading to a ~2-fold increase in FXN mRNA levels in multiple cell models. Despite rigorous controls such as two RNA quantification assays and normalization by multiple housekeeping genes, the FXN activation by these two ASOs wasn’t driven by direct binding to the FXN pre-mRNA or a mutual off-target transcript. Surprisingly, it’s found dependent on guanosine-rich motifs in the full PS backbone, suggesting non-base-paired off-target effects. Secondly, we developed siRNASO, an oligonucleotide scaffold integrating the functionalities of siRNA and ASO into a single molecule. siRNASOs demonstrated potent single RNA silencing and comprehensive dual-RNA modulation, including RNA silencing, splicing modulation, and RNA editing in vitro and in vivo. Notably, siRNASO exhibits excellent tolerability in the mouse CNS, suggesting its potential as a therapeutic platform for CNS disorders. Overall, the research in this thesis will serve as a valuable foundation for future research and therapeutic applications, significantly contributing to the advancement of RNA-targeting therapeutics.
  • The Role of Binding Sequence, Position, and Promoter Strength on the Regulatory Modes of E. coli Transcription

    Guharajan, Sunil (2023-12-11)
    The activity of Transcription Factors (TFs) is essential to gene regulation, facilitating the transfer of information encoded in cis-regulatory elements into temporal and spatial control of gene expression. Despite progress in mapping the genomic binding sites of TFs, the regulatory role of TFs once bound is often convoluted. Here, we utilize a synthetic biology approach that allows for precise manipulation of TF concentration in vivo and interrogation of the role of promoter features on the regulatory function of E.coli TFs. Using thermodynamic models of gene regulation, we decouple TF occupancy from its maximal regulatory output and probe its regulation on two steps of the transcription process: stabilization of RNA polymerase (RNAP) binding and modulation of transcription initiation. Profiling the CpxR activator, we discover universal stabilization across regulated positions, with differences in strong and weak activation set primarily by regulation of the initiation rate. Formulating a high-throughput approach, we probe the regulation of 93 E.coli TFs to find that the relative contributions of binding sequence and position on the mode of action are decoupled, with position playing a dominant role for select factors. Building on this information, we assessed the interplay between binding position, TF identity, and basal promoter strength - uncovering a conserved mode of stabilizing regulation across TFs with diverse regulatory outcomes. Taken together, our work delineates the effect of promoter architecture on the quantitative regulatory activity of TFs, with implications for design of synthetic gene circuits and understanding natural promoters
  • Microfluidic High-Throughput Methods for the Induction and Characterization of Repeatable, Titratable Traumatic Neural Injury in the Nematode Caenorhabditis elegans

    White, Hamilton (2023-12-06)
    Traumatic brain injury (TBI) causes polymodal trauma leading to persistent changes in brain function, behavior, cellular structure, and is a known risk factor for neurodegenerative disease. Current injury models correlate the presence and duration of injury conditions with animal behavior, but they do not reveal underlying effects on brain function at the cellular and subcellular scale in a continuous, longitudinal manner. To identify underlying mechanisms relating acute brain injury with functional outcomes, we sought to develop a reliable, scalable TBI model in C. elegans to directly observe injury progression at behavioral, neurofunctional and structural levels, both immediately and over hours to days. Previously, ultrasonic shock waves and vortex-induced blunt force trauma caused paralysis in thrashing animals, with broad population variability. We investigated ultrasonic cavitation as a repeatable and titratable TBI induction method using a bath sonicator modified for precise, sub-second timing control. Video recordings during sonication revealed animals near a rigid surface were injured in a dose-dependent manner, whereas those in bulk liquid were relatively unharmed. Using an expanded and flexible optogenetic and chemical stimulation platform, repeated assessment of neural function of up to 24 hours allowed examination of structural degeneration and neurofunctional recovery. To show the platform’s usefulness, we identified sexually dimorphic outcomes in injury response, a channel inhibitor that modulates neural activity recovery post-injury via genetic and pharmacological means, and assess an ortholog of FHM1 for sensitizing animals to injury. Overall, sonication-induced TBI provides repeatable assays for real-time, in vivo recording of neuronal structure, function, and behavior, before and after single or repeated injury, enabling further study on injury mechanisms, progression, and potential therapies to minimize damage and enhance recovery.
  • Addressing Bottlenecks of Prime Editing Through Improved pegRNA Designs and Rationally Engineered Prime Editor Variants

    Ponnienselvan, Karthikeyan (2023-11-15)
    Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. With the versatile ability to introduce point mutations, deletions and insertions, prime editors have the ability to correct around 89% of known genetic variants associated with human diseases. However, there are several bottlenecks currently restricting prime editing activity that need to be addressed to further their use as therapeutics. In the first half of this thesis, we address the auto-inhibitory interaction between the PBS and the spacer sequence that affects pegRNA binding efficiency and target recognition. We show that destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency. These design parameters were initially fueled by our goal to improve prime editor ribonucleoprotein activity where the auto-inhibitory interaction of the pegRNA is more prominent, but we show that they can be applied to multiple prime editing formats to increase editing rates. In the case of end-protected pegRNAs, we discover that a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, we show that a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. In the first study, we noticed that the prime editor protein had the tendency to aggregate during purification procedures and that the editing rates were still modest in primary cells. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. Prior optimization of the system has been performed in rapidly dividing cell lines like HEK293Ts where dNTP concentration is not a limiting factor. Primary cells that are quiescent or slowly proliferating have tightly regulated intracellular dNTP levels that could limit the reverse transcription process. Therefore, in the second half of this thesis, we address two more bottlenecks of prime editing - solubility of the prime editor protein and the intracellular dNTP concentration. To address that, in the reverse transcriptase domain, we introduced the L435K mutation that improves the solubility of the protein. Additionally, we introduced a V223M mutation that changes the active site of the reverse transcriptase to resemble a lentiviral enzyme that is more efficient in non-dividing cells. We show that this rationally engineered prime editor variant with increased solubility and lower Km to dNTPs, increases editing rates across diverse cell types and in vivo. Finally, we show that targeted SAMHD1 degradation by co-delivery of VPX to increase dNTP concentration in the cell further increases prime editing rates. We believe that addressing these bottlenecks, with the recommendations we describe in this thesis, will contribute to the advancement of prime editor ribonucleoproteins and mRNA for in vivo and ex vivo therapeutics.
  • Investigating effects of environmentally acquired epigenetic factors on the mammalian embryo transcriptome

    Krykbaeva, Marina (2023-10-20)
    The major aim of this work is to shed light on epigenetic effects on embryonic development. To this end, we implemented two experimental paradigms. First, we investigated the effect of maternal diet on the embryonic transcriptome. We used in vitro fertilization to isolate gamete-carried factors and single-embryo RNA-Seq to produce a high-resolution data set in 4-cell, morula, and blastocyst embryos, as well as oocytes. We found that although differential expression was observed in most stages of development, these changes were fairly small in size. Likewise, offspring created using an embryo transfer procedure did not exhibit phenotypic differences as a result of maternal diet. However, alterations in gene expression of mitochondrial respiration and lipid and cholesterol metabolism genes were detected in offspring tissue with a clear sex bias. Second, we compared transcriptomes of embryos produced using three methods of fertilization – natural mating (NM), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) as well as parthenogenesis. The largest differences were detected in IVF embryos, largely in the categories of translation and ribosome biogenesis. ICSI embryos exhibited a small deviation in differentiation-associated gene expression. Parthenogenesis, an embryo-like system with no paternal contributions, resulted in vast expression changes encompassing ~20% of expressed genes and was further used as a model system to confirm a role for sperm-carried RNAs in regulating embryo gene expression. Lastly, this single-embryo data set was used to characterize stochasticity in gene expression and confirm the presence of both “quiet” and “noisy” genes. Overall, we provide two large-scale data sets comprised of hundreds of embryos, which serves as a systematic approach to investigating the effect of epigenetic factors on the embryonic transcriptome.
  • Identifying vulnerabilities in sugar nucleotide metabolism of cancer cells

    Doshi, Mihir B (2023-10-20)
    Cancer cells exhibit elevated metabolic demands, imposing a need for metabolic reprogramming. The aim of the thesis is to identify a targetable metabolic vulnerability using an approach that leverages the altered pathways in cancer cells to induce the accumulation of inherently toxic metabolites to eliminate cancer cells selectively. Through a systematic analysis of transcriptomics and cancer dependency data, we identified UXS1, a Golgi enzyme responsible for converting UDP-glucuronic acid (UDPGA) to UDP-xylose that is conditionally essential in cells expressing high levels of its upstream enzyme UGDH. Here, we demonstrate that UGDH high cancer cells are dependent on UXS1 to prevent excess buildup of UDPGA, generated by UGDH. Excess UDPGA causes disruption of the structure and function of the Golgi, leading to aberrant protein glycosylation and improper protein trafficking of critical glycoproteins within cancer cells. We find that UGDH expression is elevated in various cancers, including lung adenocarcinoma and breast carcinoma. Furthermore, elevating UGDH expression is beneficial to cancer cells, because UDPGA functions as a substrate in the detoxification of chemotherapeutic agents. Therefore, chemo-resistant cells upregulate UGDH expression, enhancing their susceptibility to UXS1 ablation. Consequently, this study reveals the therapeutic potential of targeting UXS1 in cancer treatment, offering a novel approach to exploit the metabolism of sugar nucleotides in cancer cells.
  • mRNA Sequence Features Determine the Efficiency of Translation Termination and Association of the Nonsense-Mediated mRNA Decay Machinery with Elongating Ribosomes

    Mangkalaphiban, Kotchaphorn (2023-10-20)
    Translation of mRNA into protein is terminated when the ribosome encounters one of the three stop codons (UAA, UAG, and UGA) at the end of an open reading frame (ORF). Infrequently, stop codons are decoded by a near- cognate tRNA, allowing “readthrough” of the stop codon and synthesis of an extended polypeptide. When termination occurs prematurely, the mRNA is degraded by the nonsense-mediated mRNA decay (NMD) pathway. Premature and normal termination appear to differ in their efficiency, but the exact “rules” of how NMD distinguishes them mechanistically remain to be elucidated. Using ribosome profiling and bioinformatics analyses, this study aims to understand, at a transcriptome-wide level, the cis-acting elements that influence termination efficiency and how premature termination is recognized by Upf1, a key NMD factor. Analyses of yeast and human mRNA sequences in both normal and readthrough- inducing conditions revealed largely conserved roles of identities of the stop codon, the following nucleotide, P-site codon, and 3’-UTR length in readthrough efficiency regulation. The analyses of yeast mRNAs associated with Upf1-bound ribosomes demonstrated that Upf1 binds ribosomes in two distinct complexes across all mRNA ORFs, suggesting that Upf1 associates with the ribosome during translation elongation before premature termination takes place. Together, these results provide insights into the regulation of termination and the early steps of NMD at the transcriptome-wide level.
  • Investigating the Role of Chronic Exercise and Autophagy in a Poly(GR) Mouse Model of Frontotemporal Dementia

    Learnard, Heather (2023-09-30)
    Understanding how exercise can attenuate social and cellular deficits seen in frontotemporal dementia (FTD) could provide a unique insight and potential for new therapeutic approaches to help patients suffering from FTD and other devastating FTD associated diseases such as Amyotrophic Lateral Sclerosis (ALS). This project helped study the effect of chronic exercise in a poly(GR)-specific mouse model of FTD, highlighting the specific response exercise has on autophagy and poly(GR) toxicity in this experimental system. The goal of this work was to identify the amount/type of exercise that would be most beneficial to reduce poly(GR) load or other neurotoxicity indicators; as well as, identifying the mechanism underlying autophagy disfunction with GR80 overexpression, and if exercise could also alleviate this disruption. Additionally, to compliment the cortical neuron FTD specific mouse model being used to investigate exercise, we also wanted to make a motor neuron mouse model to better understand poly(GR)’s contribution specifically to ALS. In doing this we used Homeobox Protein 9 (HB9) as a promoter due to its lack of expression in sensory and interneurons, in hopes to study motor neurons only. With a understanding of how poly(GR) impacts both FTD and ALS respectively we can better understand the disease pathology progression and hopefully uncover novel ways to intervene, treat, or prevent these disease more effectively.
  • High Throughput Tools for Tickborne Disease Surveillance and Investigation of Tick, Pathogen, and Commensal Microbiome Association at Single-Tick Resolution

    Chauhan, Gaurav (2023-09-25)
    The prevalence of tickborne diseases worldwide is increasing virtually unchecked due to lack of effective control strategies. The transmission dynamics of tickborne pathogens are influenced by the tick microbiome, tick co-infection with other pathogens. Understanding this complex system could lead to new strategies for pathogen control, but will require large-scale, high-resolution data. Here we present a strategy that combines citizen science with new molecular strategies to provide the single-tick resolution data urgently needed to inform management of tickborne pathogens. Our citizen science-based initiative, Project Acari, harnessed the power of volunteers across the US to collect more than 3,000 ticks. To assay collected ticks, we developed a high-throughput screening method using Molecular Inversion Probes (MIPs) that identify tick species, associated pathogens, and the species on which the tick most recently fed. Applying MIPs to 853 individual ticks successfully identified the species of 715 ticks, of which 85 were infected with pathogens of 12 different species. We also detected host DNA in 60 ticks. We also generated the first comprehensive data on both prokaryotic and eukaryotic microbiome of individual ticks using full-length 16S and 18S sequencing. Our findings corroborate reports of the influence of tick species, sex, and geography on the tick prokaryotic microbiome. We also identify novel associations between the carriage of B. burgdorferi and specific microbial taxa. Our work underscores the power of citizen science, paired with high-throughput processing, to elucidate the ecology of tickborne disease and to guide pathogen-control initiatives.
  • Bacteria-Tumor-Drug Interactions: Investigating Bacterial Tumor Colonization and Bacterial Evolved Resistance to Anti-Cancer Therapy

    Sayin, Serkan (2023-09-25)
    The human microbiome has been extensively studied, yet remains elusive due to its complexity. Recent findings showed that many solid tumors may harbor a microbiome. Bacterial presence in tumors may cause cancer progression, modify the chemical structures of anti-cancer treatments or alter the immune responses. Basic principles of how bacteria initiate a population and expand in tumors, and how they adapt to anti-cancer therapies is an underexplored area. For instance, gamma-proteobacteria found in pancreatic ductal adenocarcinomas cause chemoresistance by converting gemcitabine to its inactive form by the cytidine deaminase enzyme. Here, I first focused on this drug-bacteria interaction to understand bacterial evolution to gemcitabine and how it could affect existing bacteria-drug interactions. Using a genome-wide genetic screen, I showed that many loss-of-function mutations can cause gemcitabine resistance. I found that one-third of the resistance mutations increase or decrease bacterial drug breakdown, which can decrease or increase the gemcitabine load in the local environment. I also found that the adaptation of E. coli to gemcitabine resulted in the inactivation of the nucleoside permease NupC, which increased the drug burden on co-cultured cancer spheroids. Secondly, I focused on exploring the bacterial colonization of tumors in vivo. Using an isogenic barcoded E. coli library, I showed the presence of a narrow bottleneck during tumor colonization and skewed bacterial dissemination in the tumor environment. Overall, this study sheds light on quantitative bacterial colonization principles in tumors and intra-species bacterial adaptation to anti-cancer drugs with implications to the cancer cells.
  • Investigating the Role of MicroRNAs in Regulating Hematopoietic Stem Cell Aging

    Kadungure, Tatenda (2023-09-14)
    Aging is associated with a functional decline of tissue-specific stem cells. MicroRNAs (miRNAs) have been implicated in regulating hematopoietic stem cell (HSC) function, but their roles in HSC aging have not been extensively studied. The goals of my thesis were to understand how aging impacts miRNA expression in HSCs and how altered miRNA expression impacts HSC function during aging. First, I used RNA sequencing to compare miRNAs expressed in young versus old HSCs, and identified 112 upregulated and 72 downregulated miRNAs with HSC aging. Binding motifs of ATF1 were found to be enriched in the promoter regions of the upregulated miRNAs, consistent with prior findings of ATF binding motifs enrichment in the open accessible chromatin regions associated with HSC aging. Furthermore, predicted protein-coding targets of upregulated miRNAs significantly overlapped with downregulated genes in old HSCs identified in previously published studies, suggesting that these miRNAs underlie some of the age-related gene expression changes in HSCs. Second, I carried out an in vivo CRISPR/Cas9-based loss of function screen targeting 172 HSC-expressed miRNAs to identify miRNAs that negatively regulate HSC function. Among seven identified candidates, the expression of miR-542 was increased in old HSCs. I further demonstrated that deletion of miR-542 stimulated expansion of old HSCs in vitro and enhanced bone marrow reconstitution capacity of old HSCs in vivo. Taken together, my dissertation highlights a potential causal role of miR-542 in HSC aging and reports other candidate miRNAs that might play a role in HSC aging.
  • Host Responses Toward Influenza Associated Pulmonary Aspergillosis

    Lee, Chrono K. (2023-08-31)
    Aspergillus fumigatus is a saprophytic fungus that is responsible for causing a wide range of diseases primarily affecting immunocompromised hosts. However, cases of influenza associated pulmonary aspergillosis have been reported and the cause for the lethality remains ambiguous. The aim of this dissertation is to examine the underlining immunology and pathology of influenza associated pulmonary aspergillosis. Utilizing a model of post-influenza aspergillosis, we observed 100% mortality when mice were superinfected with A. fumigatus conidia during early stages of influenza, whereas all mice survived when challenged at later stages. In addition, mice dually infected with influenza and A. fumigatus had elevated levels of proinflammatory cytokines and chemokines compared with control mice. Different than our expectations, histopathology examination did not reveal escalation of inflammation nor increased germination of conidia in the lungs of superinfected mice. Although neutrophil recruitment was dampened when influenza-infected mice were challenged with A. fumigatus, the fungal clearance ability of neutrophils remained intact as reactive oxygen species production was not affected by influenza. Furthermore, the loss of interferon α downstream signals, but not interferon ɣ, increased the lethality of secondary aspergillosis in influenza-infected mice. Taken together, our data suggest that the high mortality rate seen in mice during the early stages of influenza associated pulmonary aspergillosis is multifactorial with dysregulated inflammation being a greater contributor than uncontrollable microbial growth. This discovery opens a new paradigm for investigation and treatments that can be formulated for influenza associated pulmonary aspergillosis.
  • Advancing CRISPR-Cas Gene Editing Technologies: Engineering of Guide RNA, Donor Template, Editing Effector, and In Vivo Delivery

    Chen, Zexiang (2023-08-29)
    Gene editing technologies have revolutionized various fields, from agriculture to medicine, by providing powerful tools to modify genetic materials. Early efforts, such as gene targeting, ZFN and TALEN, have laid the foundation for this field. In the past decade, CRISPR-Cas, derived from prokaryotic adaptive immune systems, has been re-engineered as gene editing tools, including nuclease editors, base editors, and prime editors. The simplicity, effectiveness, and versatility of these CRISPR-Cas gene editing tools have rapidly propelled their widespread use in both academia and industry. Despite the tremendous potential, many challenges arise during the development of CRISPR-Cas gene editing, and this thesis focuses on tackling some of the key ones. On one hand, I have dedicated my efforts to engineering gene editing components. This includes the synthesis of long guide RNA using click chemistry, enhancing the efficiency of homology-directed repair (HDR)-based editing using chemically modified donor templates, and improving modular prime editing platform by engineering effectors. On the other hand, I have also focused on the in vivo delivery of gene editors. Specifically, I have explored the first use of lipid nanoparticles for delivering chemically modified pegRNA and prime editor effector mRNA to achieve in vivo prime editing. Additionally, I have developed a fluorescence-based mouse reporter system to assess the in vivo performance of gene editors. Overall, the work presented in this thesis will greatly contribute to the advancement of CRISPR-Cas gene editing technologies, fostering progress in future research and therapeutic applications.
  • A Bacterial Pathogen Induces Reversible Developmental Slowing by High Reactive Oxygen Species and Mitochondrial Damage in Caenorhabditis Elegans

    Mirza, Zeynep (2023-08-28)
    Host-pathogen interactions are complex by nature, and the host developmental stage increases this complexity. Development is an energetically demanding period when biomass production and cell differentiation events occur. We investigated how a developing organism copes with the additional energy-expensive burden of pathogen stress during this crucial period. We explored this question by utilizing Caenorhabditis elegans larvae as the host and the bacterium Pseudomonas aeruginosa as the pathogen. By screening 36 P. aeruginosa isolates, we found that the CF18 strain causes a severe but reversible developmental delay. CF18 slows larval development via induction of reactive oxygen species (ROS) and mitochondrial dysfunction. In response, the larvae upregulate mitophagy and antimicrobial and detoxification genes; however, mitochondrial unfolded protein response (UPRmt) is repressed. Consistent with these observations, antioxidant or iron supplementation or the removal of larvae from CF18 rescues developmental delay, mitochondrial damage, and high ROS. We examined the virulence factors of CF18 required for developmental delay via transposon mutagenesis, RNA-sequencing, and candidate gene deletion approaches. Our results showed that virulence factors regulated by quorum sensing and the GacA/S system were responsible for developmental slowing. We also demonstrated that well-studied mitochondrial toxins of P. aeruginosa, phenazines and hydrogen cyanide, are not required for CF18-induced developmental slowing. This study highlights the importance of ROS levels and mitochondrial health as determinants of developmental rate and how pathogens can attack these important features.

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