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IMPACT OF HIV ANTIRETROVIRAL THERAPY ON THE EUKARYOTIC GUT VIROME AND HOST HEALTH
No full textAntiretroviral therapy (ART) is prescribed to people living with HIV (PLWH) or to individuals taking it as pre-exposure prophylaxis (PrEP). In PLWH, ART can significantly reduce viral loads to undetectable levels and prevent Acquired Immune Deficiency Syndrome (AIDS). Despite ART efficacy, PLWH are still at elevated risk of multiple chronic diseases, including metabolic syndrome (MetS). The microbiome of PLWH has been shown to differ from that of HIV-negative individuals, even when receiving ART, suggesting a potential role for an ART-modulated gut microbiome in chronic disease pathogenesis. However, whether ART itself can directly modulate gut microbiome/virome, and specifically endogenous gut eukaryotic viruses, remains unknown. Here, we raise the hypothesis that ART itself has direct collateral impacts on gut eukaryotic viruses and that this modulation contributes to the increased risk of non-infectious disease. In this thesis work, I assessed the impact of antiretrovirals (ARVs) given individually (abacavir (ABC), dolutegravir (DTG), lamivudine (3TC), emtricitabine (FTC), tenofovir alafenamide (TAF), and tenofovir disoproxil fumarate (TDF)) or in combination (cART, TDF/FTC/DTG) on the metabolic health of HIV-naïve mice. Individual ARVs, as well as cART, exacerbated diet-induced weight gain (3TC, DTG, and TAF) and glucose intolerance (3TC, TAF, and FTC). Preliminary profiling revealed that DTG and FTC altered the gut microbiome while DTG and TDF altered the virome. To explore the mechanisms through which ART alters the virome, specifically eukaryotic viruses, I aimed to determine how different ARVs impact eukaryotic virus-host cell interactions in vitro. A universal concentration of ARVs (ABC, DTG, 3TC, FTC, TAF, and TDF) that does not affect Caco-2 and BHK-21 cell viability was determined. Viral plaque assays were also calibrated and preliminary experiments with ARV treatment was performed. Overall, this work suggests an association between the direct impact of ARVs on the gut microbiome/virome and MetS, and provides a framework to explore the mechanisms of eukaryotic virus modulation by ART
PRENATAL DEVELOPMENT OF THE RIBS AND INTERCOSTAL MUSCULATURE OF THE RED-EARED SLIDER TURTLE (TRACHEMYS SCRIPTA) WITH HISTOLOGICAL IMAGING
Get PDFThe turtle shell is one of the most characteristic features in the animal kingdom and has long been considered a classic example of an evolutionary novelty. The early 21st century saw a resurgence of research regarding the development of the turtle body plan via emerging experimental and molecular techniques that have since generated novel developmental models. These models have unfortunately failed to become integrated with evolutionary hypotheses of turtle origins, due in large part to the historic sparsity of informative fossils. The recent discovery of new early turtle fossils, however, has provided a framework through which developmental data can enter a reciprocally informative relationship with the fossil record. Through intensive data collection we generated alcian blue hematoxylin/orange G stained histology slides of entire embryos allowing us to capture complete tissue dynamics throughout almost the entirety of the prenatal development of Trachemys at an unprecedented level of paired spatial and cellular resolution. This detailed histological description of turtle carapace development outlines the morphogenetic timing of specific transformations of the ribs, muscles, and neurovasculature, revealing novel observations of tissue replacement and interaction at the tissue and cellular level. We find that turtles possess a highly organized mechanism of immune mediated muscular breakdown and tissue replacement throughout ontogeny, occurring primarily between Greenbaum stages 14-21. These dynamics take place in a lateral to medial direction in certain muscles groups with discrepancy in timing between the hypaxial derived intercostal muscles and the epaxial derived dorsal back muscles The role of the intercostals as a signaling center that drives rib morphology and directionality has previously been established in chick and mouse models, and therefore our findings represent a call for a deeper understanding of the morphogenetic potential of the intercostal muscles in turtle carapace development. Our findings are also consilient with previous fossil-based hypotheses of turtle evolution, most notably that the earliest known turtle Eunotosaurus africanus possessed the developmental mechanics responsible for the loss of intercostal muscles, however it did not possess the suite of complex morphogenetic factors that are active in the development of the modern turtle carapace
Examining Cleaner Cookstove Research in the Peruvian Andes
Get PDFIndoor air pollution (IAP) poses a threat to the health of rural women and children, who are disproportionately affected through exposure to biomass cookstoves. However, little is known about how we have come to know that IAP impacts their health, and the mechanisms through which we continue to acquire this knowledge.
Using archival analysis, I explain the historical creation of rural women as the subjects of research on IAP. I use ethnographic methods - interviews and participant observation – to study a cookstove trial in the Peruvian Andes. I examine who these rural women are and how they formed relationships with field workers attempting to recruit them. I explore how field workers acquired high-quality data given the context.
I show that IAP rose to become a global priority in the early 2000s. Global Burden of Disease estimates positioned it as a top risk factor for disease, mobilizing scholarly interest and funding. Despite this interest, the ability to produce definitive causal evidence of the exposure-response relationship has remained elusive partially due to failures of adopting IAP mitigation technologies (i.e., improved cookstoves). Newer research sought to overcome this failure by using simpler clean cooking technologies, or gas burning cookstoves. In a study set in the Peruvian Andes which overcame adoption challenges, I found that rural women mistrusted the field workers during enrollment encounters, requiring the field workers to sell them on the project in ways that spoke to deficits in their economic and health realities. I show that alongside the pressure of suppressing mistrust, the field workers were under pressure to collect high quality data. When the study ended, the participants lamented the loss the relationships built with the field workers, and the free cooking fuel they received. They were learning to cope with the anxieties of reverting to a polluting but affordable way of cooking.
Through this study I show that the production of knowledge was not a value-neutral endeavor for the study participants and the field workers. The end of the study produced challenges, which require further exploration and consideration for future research
EXPLORING NOVEL CELLULAR THERPEUTICS FOR IMMUNE CHECKPOINT INHIBITOR-ASSOCIATED MYOCARDITIS: INVESTIGATING TIGIT OVEREXPRESSION IN T CELLS
Get PDFImmune checkpoint inhibitors (ICIs) represent a promising class of monoclonal antibodies designed to target immune checkpoints (ICs) and their ligands, thereby counteracting cancer-induced T-cell suppression. Their application in various cancer types has demonstrated significant potential in enhancing anti-tumor responses and improving patient outcomes. However, the widespread use of ICIs has led to the emergence of serious side effects known as immune-related adverse events (irAEs) affecting various organs such as the lung, liver, intestine, skin, and cardiovascular system. Among these, cardiovascular complications including myocarditis, atherosclerosis, pericarditis, arrhythmias, and cardiomyopathy have been reported as having high potential as leading causes of death. Notably, ICI-associated myocarditis stands out due to its high mortality rates, yet specific clinical treatments for this condition are currently lacking. This thesis aims to address this gap by developing novel cellular therapeutic approaches tailored specifically for ICI-associated myocarditis.
The first aspect of this research involves a computational analysis of inhibitory receptor expression levels in cardiovascular diseases in humans and experimental autoimmune myocarditis (EAM) mouse models. Utilizing single-cell RNA sequencing (scRNA-seq) analysis, we identified expression levels of various ICs and their ligands in hearts. From the data, we
found elevated expression levels of T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT), a novel IC, in these cardiac conditions.
The second contribution of this thesis involves the optimization of transfection methods to develop cellular therapeutics for ICI-associated myocarditis. We explored numerous in vitro transfection techniques, including chemical transfectants, electroporation, and viral transfection methods to induce TIGIT overexpression in mouse primary T cells. Our findings provide insights into efficient strategies for overexpressing TIGIT in T cells, laying the groundwork for the development of targeted therapies for ICI-associated myocarditis
DECIPHERING MELANOMA DYNAMICS: INTEGRATING MOUSE TUMOR MODELS & IHC PROFILING
Get PDFAcral lentiginous melanoma (ALM), an aggressive malignancy occurring on relatively stiff skin sites such as the palms, foot soles, and nail beds, presents a unique challenge to immunotherapy due to its intricate behavior and biological microenvironment. ALM is notable for its complex etiology, poorer prognosis compared to superficial spreading melanoma, and unique tumor immune microenvironment (TIME). Understanding the key players of the ALM immune landscape could better improve the development of novel therapeutic strategies to overcome immunosuppression and expand potential targets for immunotherapy. Our findings aim to establish an orthotopic mouse model for melanoma to delineate differences in the TIME of stiffer surfaces such as the footbed (foot) compared to softer surfaces such as the back. By conducting immunohistochemical (IHC) profiling and investigating mouse cancer models, we have identified variations in the TIME between the foot and back regions of mice injected with ALM, specifically regarding the infiltration of immune cells and their possible consequent effects on cancer invasiveness. Our IHC experiments, examining CD3, CD8, CD4, FOXP3, and cathepsin K, coupled with the in vivo transplantation of cell lines in the foot compared to the back, suggest a predominantly immune-regulated microenvironment with unique localization patterns in the stiffer footpad region that may contribute to a more invasive tumor phenotype. These findings offer promising insights for enhancing immunotherapy in the context of ALM, which typically exhibits poor responses to immunotherapy
THE FUTURE OF CLIMATE CHANGE IMPACTS ON THE AVIATION INDUSTRY: A PREDICTION
Get PDFClimate change effects many different aspects of the aviation industry and will continue to do so in the near future. Certain airports are more vulnerable than others, and airlines that have large operations at these airports are left vulnerable as well. Extreme weather like abnormally high max temperatures, long-lived thunderstorms and heavy rain, or large hurricanes can end up considerably costing an airline in lost revenue from delay or cancellation expenses, especially if these events occur during high volume hours. Through a detailed literature review of examples of these extreme weather events impacting large airports, this research shows how an airline reacts to these events in an effort to limit loss. Using climate scenarios outlined by the IPCC, an estimation was determined for future extreme weather and how much this will end up costing the airlines as well as how much of those costs can be attributed to climate change. The final predictions show an increased number of weather impacts, but determining the cost per occurrence is difficult since no weather event is the same, and many different parameters factor into an airline’s reaction to extreme weather
Investigating the use of single cell RNA-seq imputed genotypes toward eQTL calling
No full textThis thesis presents a pipeline for reliably genotyping and identifying expression quantitative trait loci (eQTLs) from single-cell RNA sequencing (scRNA-seq) data. There is a need for context specificity in eQTL studies, particularly regarding the variability of eQTL effects across different cell types, which can be better elucidated by scRNA-seq data. Since most single-cell datasets lack genotype information, we aim to test whether genotypes imputed from scRNA-seq reads are sufficiently accurate for downstream QTL analyses, and on par with bulk sequencing studies. Using pseudo-bulked reads across cells per individual, we tested 3 genotyping pipelines - Gencove, Monopogen and our custom pipeline that implements GATK (McKenna et al., 2010; Li et al., 2021; Dou et al., 2023). Gencove is a proprietary SaaS that performs end-to-end variant calling and imputation, our custom pipeline follows standardized variant calling procedure using GATK for variant calling and Beagle for imputation, and Monopogen is a recently developed dedicated pipeline for single cell SNV calling that claims to have better performances than traditional variant callers like GATK. Initial findings from an iPSC smart-Seq2 dataset shows promising results for 10 samples, with Pearson correlations with ground truth genotypes being above 0.9 for both Gencove and GATK but not Monopogen (with few samples having correlations above 0.88). We also explored factors contributing to improved correlations and have determined effective variant call filtration and proximity to gene regions to have the best performing genotypes. Our study lays the foundation for leveraging the vast amount of publicly available scRNA-seq data for eQTL calling pipelines
Multi-organ mapping of age-related changes to the female mouse reproductive system at cellular resolution
No full textAdvanced maternal age is associated with infertility and a greater risk of adverse health outcomes, particularly after menopause. The reduction in muscle content in this stage results in a loosening of the vaginal wall, cervix, and uterine muscles. Additionally, the accumulation of scarring and inflammation heightens the risk of various abnormalities in the reproductive organs, such as ovarian cysts, uterine fibroids, fallopian tube blockages, and a higher likelihood of developing gynecological cancers. While previous research has primarily focused on abnormalities in individual organs, there is a need for a holistic quantitative mapping of the entire reproductive system. In this study, we use a quantitative tissue mapping approach to investigate the relationship between age-related changes in the reproductive system of female mice. We utilized CODA, a deep-learning-based technique to reconstruct microanatomy of reproductive tissues at subcellular resolution from serially sectioned hematoxylin and eosin (H&E)-stained tissue, aiming to map the entire reproductive system of 8 female mice. Each mouse's reproductive tissues were serially cut into an average of 868 serial sections, which were then reconstructed using a nonlinear image registration algorithm. Deep learning semantic segmentation was applied to quantitatively label 22 distinct tissue structures, covering both normal and pathological structures of the gynecological system, achieving an overall segmentation accuracy of 93.6%. Our analysis uncovered significant post-menopausal changes in the reproductive system, notably an increase in the total volume of reproductive organs, primarily due to the growth of abnormal structures such as cysts. Specifically, our findings highlighted that ovarian cysts often develop on one side of the ovary and are associated with an increase in cysts and vacuoles in the adjacent fallopian tubes and uteruses. Moreover, our immunohistochemistry studies indicated a higher concentration of immune hotspots - including T cells, B cells, and macrophages—around non-cystic ovaries, suggesting that immune activity could play a significant role in mitigating the development or progression of cysts in the ovaries. Overall, this research reveals the complex multi-organ changes that occur within the human reproductive system as it ages, enhancing our understanding and potentially guiding future interventions to address age-related reproductive pathologies
DESIGN AND SYNTHESIS OF SELF-ASSEMBLING PEPTIDE-PACLITAXEL CONJUGATES
No full textPeptide-based hydrogels are supramolecular networks formed through the self-assembly of rationally designed peptides via noncovalent interactions in aqueous environment, and they have been extensively explored across various biomedical fields such as regenerative medicine, tissue engineering and drug delivery. This type of hydrogels is particularly appealing due to its inherent biocompatibility, biodegradability, and versatile functionalities. The conjugation of peptides with therapeutic drugs via a cleavable linker not only modulates the activity and potency of the drug in response to its microenvironment but also enhances chemical stability and enables controlled drug release. However, developing peptide-drug hydrogelators that exhibit robust self-assembly capabilities remains a significant challenge. This study explores the role of middle peptide sequences, termed as self-assembly modulating motifs (SAMMs) in determining the self-assembly and hydrogelation outcomes of designed peptide-drug conjugates. Strategic modifications to the SAMM have led to improved formation of filamentous nanostructures, as well as enhanced stability and responsiveness of the hydrogels to physiological stimuli. This study highlights the potential of peptide-drug hydrogels to improve therapeutic outcomes and minimize adverse effects in localized cancer treatments
Leveraging Existing Immunization Programs for COVID-19 Vaccine Introduction
Get PDFThis dissertation responds to the question: Do routine immunization systems contribute to pandemic preparedness and response? Chapter 1 provides a brief overview of the literature. Chapter 2 pilots a quantitative metric to categorize the 2020 immunization program global landscape. Chapter 3 provides regression analyses to understand what national characteristics were associated with higher COVID-19 vaccination program outcomes. Chapter 4 includes national vignettes and resources to support policy makers in taking action. The final chapter explains the significance, limitations, and next steps to advance this research.
For these analyses, I gathered and cleaned publicly available data for the 194 WHO Member States and used STATA to conduct descriptive analyses (Chapter 2 and 3) and regression analyses (Chapter 3). For Chapter 4, I reviewed publications, grey literature, and existing transcripts to identify the common global issues and potential actions.
Key findings included that 55% of the 194 WHO Member States had policies for all three childhood vaccines included in this analysis (DTP , MCV , and PCV ) compared to 60% for HPV (proxy for adolescent vaccination program) and 52% for seasonal influenza (proxy for adult vaccination program). Childhood vaccination programs (e.g., MCV and DTP) had higher maturity than seasonal influenza and HPV programs globally. Yet, after adjusting for World Bank income status, adult seasonal influenza vaccination programs were associated with both higher COVID-19 vaccination capacities and coverage. Trust in government was also associated with higher COVID-19 vaccination coverage.
Countries are now reviewing and acting upon COVID-19 pandemic learnings. More than 100 countries have already started updating their national pandemic plans in line with WHO guidance. Countries have the opportunity now to maintain the COVID-19 adult vaccination momentum, reduce vaccine preventable deaths, and prepare for the future