• 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.
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  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.
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  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.
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  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.
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  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.
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  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.
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  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.
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  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.
• TAL1 and LMO2 Promote Leukemia-Initiating Cell Quiescence and Chemotolerance in T-ALL

  O'Connor, Kevin W (2023-08-21)

  Relapse remains a major barrier to the successful treatment of children with T-cell acute lymphoblastic leukemia (T-ALL) and may represent a failure to eliminate leukemia-initiating cells (L-ICs) that possess distinguishing biological features from the bulk leukemic population. TAL1 and LMO2 are often coordinately misexpressed in T-ALL patients and their ectopic expression cooperates to transform thymic progenitors in mice. In this model, double negative-3 (DN3) stage thymic progenitors harbor L-ICs, yet only a subset of DN3 leukemic cells have L-IC activity. We interrogated L-IC heterogeneity in our Tal1/Lmo2 mouse T-ALL model using a combination of single cell RNA-sequencing (scRNA-seq) and H2B-GFP nucleosome labeling. We identified a cell cycle restricted DN3 subpopulation with high Notch1 activity and enrichment of Tal1/Lmo2 targets and T-cell quiescence genes. This dormant DN3 population significantly increased during leukemogenesis, exhibited chemotolerance and was enriched for genes associated with patient minimal residual disease (MRD). In vivo studies using the Tet-inducible H2B-GFP model revealed that Tal1 and Lmo2 cooperate to promote quiescence in DN3 cells. Examination of TAL1/LMO patient samples revealed that the L-IC enriched CD7+CD1a- thymic progenitors (hL-IC) were also chemotolerant and were also variably associated with quiescence. Collectively, our results document the emergence of dormant and chemotolerant L-ICs during Tal1/Lmo2-induced leukemogenesis in mice and relapsed T-ALL patients.
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  The Interactions Between Fluoropyrimidines and the Gut Microbiome that Lead to Drug Resistance in Bacteria and Can Alter the Drug Efficacy in the Host

  Rosener, Brittany (2023-08-21)

  Bacterial metabolism of host-targeting drugs can impact the success of host treatment. It has been found that host-targeting drugs may not only interact with the host cells but can inhibit growth of bacteria. With repeated exposure, this inadvertent impact on microbes can apply selective pressure leading to genetic adaptation of the host microbiome. This adaptation can, in turn, alter the bacterial metabolism of the drug and lead to a change in drug availability and toxicity in the host. One such set of drugs we explored are the fluoropyrimidines 5-fluorouracil (5-FU) and 5-fluoro-3’-deoxyuridine (FUDR) used to treat tumors. Using E. coli loss-of-function screens, we identified 5-FU resistant strains that decreased drug toxicity on the C. elegans host. Furthermore, the mechanisms of resistance developed after repeated exposure to 5-FU and FUDR converged to a select set of resistance mechanisms involving the nucleotide synthesis and salvage pathway. We also found bacteria evolved in nutrient-poor media reduced the host drug toxicity whereas bacteria evolved in nutrient-rich media did not alter the drug toxicity on the host. Next, we identified similar mechanisms of resistance in Comamonas aquatica. 5-FU evolved C. aquatica but not FUDR evolved C. aquatica decreased drug toxicity in a C. elegans host. Lastly, we explored the selective pressure of 5-FU treatment on the gut microbiome Using a murine model and the E. coli knock-out library, we identified that selection by the gut environment matches previously studied mechanisms in which some E. coli strains preferentially colonize the gut. Then, we found 5-FU treatment enriches for mutants known to provide 5-FU resistance in vitro. Overall, we found that bacteria can become resistant to fluoropyrimidines leading to changes in drug efficacy for the host. Additionally, 5-FU treatment in mice can also select for genotypes in the gut that provide resistance to 5-FU exposure.
• The Post-Transcriptional Regulation Mechanism and Functional Importance of a Key Maternal mRNA, GLP-1

  Coskun, Peren (2023-08-17)

  Translational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. C. elegans Notch homolog glp-1 maternal gene is necessary for germ cell proliferation, and embryonic fate determination. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3’ UTR. When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3’ UTR reporter transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of pos-1 increases the average polyA tail length of endogenous glp-1 transcripts in embryos. In this dissertation, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection. This result rules out alternative polyA site selection as a mechanism of regulation. We also show that wild-type glp-1 transgenic reporter embryos have a shorter average polyA tail length compared to transgenic reporters with mutated GLD-1 or POS-1 binding motifs. We have studied the effect of cytoplasmic polymerases, deadenylases and translation initiation factors on our transgenic reporters. Our RNAi experiments show that two cytoplasmic polyA polymerases, GLD-2 and GLD-4, have strikingly different effects on the expression of reporter transgenes harboring GLD-1 or POS-1 binding motif mutations. By contrast, none of the deadenylases affect the transgenic reporter expression. We also observed strong derepression of all reporters upon reduction of the translation initiation factor ife-3. The results reveal that POS-1 and GLD-1 exert their repressive effects in different ways through cytoplasmic polyA polymerase activity, while IFE-3 mediated translation repression is independent of both POS-1 and GLD-1. Lastly, we have examined the biological significance of glp-1 3’UTR to the worm reproduction by using CRISPR/Cas9 mutagenesis to generate glp-1 3’UTR mutations in the endogenous locus. Characterization of a 71 base pair mutation that deletes the GLD-1 and POS-1 binding sites in the glp-1 3’UTR reveals a 2-fold reduction in the number of embryos produced and a 4-fold reduction in the hatch rate. Imaging results show that the mutant embryos appear to have patterning defects. Together, our results show that the glp-1 3’UTR contributes to reproductive health but is not essential to fertility.
• Suppression of Sphingolipid Catabolism by a Nuclear Hormone Receptor Promotes Pathogen Resistance in C. elegans

  Nasrallah, Mohamad (2023-07-20)

  Sphingolipids are key structural components of cell membranes and function as signaling molecules that are required for diverse biological functions in all metazoan animals. Here we characterize a novel immunometabolic pathway that regulates sphingolipid catabolism to promote resistance to bacterial infection. From an RNAi screen for transcriptional regulators of pathogen resistance in the nematode C. elegans, we identified the nuclear hormone receptor nhr-66, a ligand-gated transcription factor homologous to human HNF4. Tandem chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) experiments revealed that NHR-66 is a transcriptional repressor, which directly targets sphingolipid catabolism and stress response genes. Transcriptional de-repression of two sphingolipid catabolic enzymes in nhr-66 loss-of-function mutants drives the breakdown of sphingolipids, which enhances host susceptibility to infection with the bacterial pathogen Pseudomonas aeruginosa. Genetic epistasis analysis revealed that nhr-66 functions with the PPARɑ homolog nhr-49 for host defense against P. aeruginosa and in the regulation of sphingolipid catabolism genes. These data define transcriptional control of sphingolipid catabolism in the regulation of cellular sphingolipid and ceramide levels, revealing an immunometabolic axis that is required for host survival during pathogen infection.
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  A Systems-Level Study of Transcriptional Regulation of C. elegans Metabolism

  Nanda, Shivani (2023-07-14)

  Metabolism is controlled to ensure organismal development and homeostasis. Several mechanisms regulate metabolism, including allosteric control and transcriptional regulation of metabolic enzymes and transporters. Until now, metabolism regulation has mostly been described for individual genes and pathways, and the extent of transcriptional regulation of the entire metabolic network largely remains largely unknown. In the first part of this thesis, I used variation in mRNA levels as a proxy for transcriptional regulation of metabolism and found that three-quarters of all metabolic genes are transcriptionally regulated in the nematode Caenorhabditis elegans. I also found that many annotated metabolic pathways are coexpressed. With the aid of gene expression data and the iCEL1314 metabolic network model, I defined coregulated sub-pathways in an unbiased manner. By utilizing a large gene expression compendium, I determined the conditions under which sub-pathways exhibit strong coexpression. Additionally, we developed 'WormClust', a web application that facilitates a gene-by-gene query of genes to view their association with metabolic (sub)- pathways. In the second part of this thesis, I addressed the "missing" annotations in C. elegans metabolism. Through the analysis of various datasets spanning space, time, and conditions, I associated orphan metabolic genes, transporters, transcription factors, and RNA-binding proteins with the existing metabolic network. Overall, this study illuminates the ubiquity of transcriptional regulation of metabolism and provides a blueprint for similar studies in other organisms, including humans.
• Minimally Invasive Analysis of Allergic and Irritant Contact Dermatitis Identifies Distinguishing Biomarkers and Intercellular Signaling Pathways

  Frisoli, Michael L. (2023-07-07)

  Allergic contact dermatitis is one of the most common inflammatory skin diseases, yet identification of environmental triggers can be challenging, and treatment options are limited for patients with frequent exposures. The chemical patch test has been used for over a century to diagnose patient-specific allergen sensitivities; however, limited test sensitivity and subjective interpretability motivates the use of modern scientific tools to improve the patch test diagnostic process. Additionally complicating diagnosis, chemical patch application can cause skin inflammation through both allergic sensitivity as well as nonspecific irritation. To discover allergy-specific biomarkers, I induced both irritant and allergic contact dermatitis in human volunteers, sampled skin lesions by non-scarring methods, and quantified transcriptomic and proteomic changes. Using single cell RNA sequencing together with a newly adapted method for ligand-receptor cell signaling estimation, I identified allergy-specific, irritant-specific, and allergy/irritant-shared cell responses of estimated disease significance. Quantification of inflammatory proteins within skin interstitial fluid also identified several biomarkers that were capable of distinguishing allergic from irritant contact dermatitis with 100% specificity and over 80% sensitivity.
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  The Therapeutic Potential of Targeting Hepatic MCT1 in Non-Alcoholic Steatohepatitis

  Min, Kyounghee (2023-06-29)

  Emerging evidence supports substrate overload to the liver as an initial driver of non-alcoholic steatohepatitis (NASH) progression. Metabolic disease patients have increased plasma lactate levels. Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases. Indeed, monocarboxylate transporter 1 (MCT1) haploinsufficiency in mice causes resistance to diet-induced obesity, steatosis, and hepatic inflammation. However, little is known about the therapeutic potential of hepatic MCT1 and its contribution to NASH fibrogenesis. In this study, we developed novel adeno-associated virus (AAV) lecithin-retinol acyltransferase (Lrat)-Cre vectors that selectively target hepatic stellate cells, as well as two types of hepatic MCT1-targeting fully chemically modified siRNA compounds with distinct cell delivery capabilities: Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs. We demonstrated that stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. Similarly, MCT1 silencing by Chol-siRNA decreased liver collagen 1 level in mice, and decreased LX2 stellate cell collagen expression. Interestingly, however, hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis, without alleviating triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 does not appear to be an attractive therapeutic target for NASH. Furthermore, combining our previously developed chemically modified siRNA compound (DGAT2-1473) with Chol-MCT1-siRNA was more effective than Chol-MCT1-siRNA alone in preventing NASH fibrosis, indicating that a dual targeting approach may be most effective for alleviating NASH.
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  Biochemical Investigations into the S. cerevisiae Chromatin Remodeling Enzyme SWR1C

  Baier, Alexander S (2023-06-28)

  Chromatin refers to the higher-order organization of DNA in the nucleus of the cell, and this architecture fundamentally underpins essential cellular processes such as response to environmental change, differentiation of cell type, and more as well as enables the incredible information density of nuclear DNA contents. At its simplest, chromatin consists of approximately 147 base pairs of DNA wrapped around an octamer of histones, termed the nucleosome. Cells further discern between nucleosomes via means such as highly regulated spacing along genes, inter-nucleosome positioning, post-translational histone modifications, or histone variations, all of which are regulated by a class of nuclear enzymes called chromatin remodeling enzymes. My thesis focuses on the S. cerevisiae chromatin remodeling enzyme SWR1C, which exchanges canonical histone H2A for variant H2A.Z. This histone variant is enriched proximal to important sites such as actively transcribed genes, centromeres, and double strand DNA breaks. Prior in vitro characterization of SWR1C has been limited by the inability to produce complex nucleosomal substrates. My research reports the first use of asymmetrically assembled nucleosomes to probe the activity of SWR1C. Herein I deliver concrete confirmation of SWR1C preference for linker distal H2A eviction and novel insights regarding the dual essentiality of the nucleosomal acidic patch for SWR1C activity, as well as stimulation of activity by the contralateral H2A.Z dimer and linker DNA. This work brings forth new insights into the behavior, structure, and function of SWR1C and reveals its unique features as compared to other closely related chromatin remodeling enzymes.
• Adherence to Clinical Practice Guidelines for Screening and Management of Pediatric High Blood Pressure: A Mixed Methods Study

  Goulding, Melissa (2023-06-28)

  Background Prevention and early detection of pediatric hypertension are crucial to promote cardiovascular health across the life course. As such, the American Academy of Pediatrics recommends regular blood pressure (BP) screening and follow-up. However, the extent to which their 2017 guidelines have been implemented into practice is unknown. Methods This dissertation included three projects conducted in a large health system in Central, Massachusetts. (1) Electronic health record data from children aged 3-17 years were used to determine the prevalence of guideline adherent screening and differences across child- and clinic-level factors related to social vulnerability; (2) the prevalence of guideline adherent follow-up and differences across these child- and clinic-level factors; and (3) semi-structured interviews were conducted with pediatric and family medicine providers. Main Results Aims 1 and 2 included 19,695 and 4,563 children respectively. The prevalence of adherence was 89% for screening and 18% for follow-up. Children with obesity and public insurance had lower odds of receiving guideline adherent screening. Children who were patients at clinics with larger Medicaid populations and larger patient panels had lower odds of receiving both guideline adherent screening and follow-up. Qualitative interviews revealed a lack of trust in BP readings, lack of resources, and logistical challenges were barriers to guideline adherence. Conclusions This mixed methods dissertation identified disparities in pediatric BP screening and sub-optimal rates of guideline concordant BP follow-up. Furthermore, through its description of differences in adherence and contextualization of provider perceived barriers, it identified important targets for interventions to improve pediatric hypertension prevention and care.

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