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Papillomaviruses (PVs) are a diverse family of small (5.6kb-8.6kb) double stranded DNA viruses. PVs are non-enveloped viruses with histone wrapped genomes. To date over 700 PVs have been identified in a wide range of hosts including mammals, birds, reptiles, and fish. Over 400 HPVs have been identified and these viruses fall into 5 genera termed Alpha-papillomaviruses, Beta-papillomaviruses, Gamma-papillomaviruses, Mu-papillomaviruses, and Nu-papillomaviruses. The Alpha-papillomaviruses genus contains a subset of HPVs adapted to infect the epithelium of the genitalia, oropharynx, and anus with high efficiency. Alpha-papillomaviruses infections are ubiquitous in the global population, and HPV represents the most common sexually transmitted infection. Most Alpha-papillomaviruses cause benign infections. However, 12 Alpha-papillomaviruses (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59) are recognized as carcinogens by the International Agency for Research on Cancer. A further 13 Alpha-papillomaviruses are suspected carcinogens based on limited data. Oncogenic HPVs are responsible for 4.5% of the global cancer burden and are the etiological agents of cervical, oropharyngeal, anal, vaginal, vulvar, and penile squamous cell carcinomas. Almost all cervical and anal cancers, 15-48% of vulvar cancers, 53% of penile cancers, 78% of vaginal cancers, and 13-60% of oropharyngeal cancers are HPV driven. Interestingly, two HPV types have higher oncogenic potential than all others. Combined HPV16 and HPV18 account for over 72% of HPV driven cervical carcinomas. HPV18 is the main driver of a less common type of cervical cancer, cervical adenocarcinoma. HPV16 accounts for over 90 percent of HPV driven tumors that occur in tissues other than the cervix. Due to the high mortality of oncogenic HPVs, and the high morbidity of genital wart HPVs, preventative HPV vaccines have been developed. These vaccines are made of viral like particles containing on

Chromatin accessibility profiling is a cornerstone in epigenomic exploration, providing insights into the regulatory mechanisms that orchestrate gene expression. Understanding these mechanisms is critical as they establish the nexus between genotype and phenotype, influencing cellular identity, fate determination, and responses to environmental cues. Genome-wide methods relying on high-throughput sequencing, such as ATAC-seq (Assay for Transposase Accessible Chromatin sequencing), have emerged as powerful tools in this domain, enabling the identification of open chromatin regions where transcription factors and other regulatory proteins interact with DNA to modulate gene activity. In this dissertation, we present significant improvements in the measurement of chromatin accessibility by optimizing bulk ATAC-seq protocols for both native and fixed samples and developing a large-scale single-cell ATAC-seq method. For bulk protocol optimization, we performed a thorough analysis of 24 experimental conditions, testing all possible combinations of three reaction buffers, two enzymatic temperatures, and two enzyme sources on different nuclear preparations. This systematic evaluation, conducted on a well-characterized cell line and extended to primary mouse lung tissue, reveals intricate dependencies between protocol choices and data quality, particularly highlighting the sensitivity of chromatin accessibility data to enzymatic reaction temperatures and the physical state of nuclei. Our findings underscore the need for a multi-faceted evaluation of ATAC-seq data to mitigate protocol-driven biases and accurately represent the functional genomic elements, thereby enhancing the fidelity of chromatin profiling. Advancing beyond protocol refinement, we develop a massive-scale single-cell ATAC-seq method called txci-ATAC-seq (Ten(10)X-compatible Combinatorial Indexing ATAC Sequencing) by incorporating combinatorial indexing into a droplet-based microfluidic system. This approach s

The lung is a complex organ that requires proper development, maintenance, and renewal of specialized cell types to support every stage of life. A better understanding of the biological and molecular processes that regulate normal lung cell function is essential for identifying etiologies of respiratory disease that may prove as useful therapeutic targets. In order to study this intricate multicellular system we need tools, such as single-cell RNA-sequencing (scRNA-seq), that can capture cell specific expression. Here, I expand the utility of scRNA-seq to airway samples which may prove as a useful research and clinical tool. Additionally, I apply scRNA-seq to evaluate a commonly used in vitro model system of airway regeneration. Rapid technology advancements have greatly expanded the accessibility and resolution of genomic tools. The research presented in this dissertation extends the single-cell gene expression profiling toolkit to clinical samples and an experimental model.Chapter 2 outlines methods that can be used to generate high-quality scRNA-seq data from induced sputum samples. Induced sputum is a non-invasive sampling method, making for a desirable technique in large-scale and/or longitudinal studies. Despite challenges presented by working with these samples, such as mucus contamination and dead cells, we established a processing method with superior quality metrics. Importantly, we found cells isolated from sputum with this protocol could be cryopreserved, enabling flexible study design. With this processing method, we identified major cell types like alveolar macrophages (AMs) and rarer immune cells, highlighting the utility of these methodologies for understanding lung homeostasis, development, and disease. Chapter 3 presents the first, to our knowledge, single-cell study of mouse tracheal epithelial cells in an air-liquid interface culture. Our data emphasizes the importance of evaluating established model systems with high-resolution tools like scRNA

Club Cell Secretory Protein (CC16) is a homodimeric pneumoprotein secreted by club cells and other non-ciliated epithelial cells in the distal airways. Although the complete biological functions and mechanisms of CC16 have not been elucidated, several studies have shown that CC16 has anti-inflammatory and antioxidant properties in the lungs. Studies have shown that low circulating levels of CC16 are correlated to accelerated declines in lung function, increased inflammation, and increased disease severity in asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) patients. CC16 has also been shown to directly decrease pathogen burden, pulmonary inflammation, and airway hyperresponsiveness during a variety of bacterial and viral lung infections; thereby, further supporting its role in regulating inflammation and immune responses. The airway epithelium is the first line of defense against inhaled pathogens and environmental toxicants; therefore, making it an important mediator of inflammation and immune responses. Airway epithelial cells are important regulators of normal lung homeostasis, which includes lung fluid balance, metabolism, mucociliary clearance of trapped particulates, activation of immune cells during infection or injury, and regulation of smooth muscle function. The four major epithelial cells observed within the lungs are basal, club, ciliated, and goblet cells, all of which perform different functions within the lung. Dysregulation of epithelial cells contributes to the respiratory conditions manifested in pulmonary diseases including asthma, COPD, or CF. In these studies, we provide data showing that CC16 is important for regulating protective properties within the pulmonary epithelium during naïve conditions and during infection with Mycoplasma pneumoniae (Mp). During early life Mp infection, we show that CC16 protects the lungs by decreasing airway hyperresponsiveness, remodeling, inflammation, and Mp burden within the lung i

While most individuals with asthma experience similar symptoms, the time of onset, severity, and underlying mechanisms of disease vary greatly. This has led to the development of unique asthma endotypes and the characterization of asthma into varying subtypes such as allergic asthma, exercise induced asthma, late-onset neutrophilic asthma, and others as discussed in Chapter 1. These endotypes often require precise therapies that target the distinct cellular and molecular mechanisms driving the disease. Menopause associated asthma is a subtype of asthma that has been described in clinical settings but has yet to be characterized as a unique asthma endotype. Individuals experiencing menopause-associated asthma tend to have severe exacerbations, often requiring hospitalization, and respond poorly to standard treatment. Current literature describing menopause associated asthma is sparse, and researchers have been unable to develop an appropriate animal model to study the disease. The studies reported here describe the development of a novel mouse model of menopause associated asthma, as well as insights into the cellular and molecular mechanisms at play. Chapter 2 details the development and assessment of this new model in which we combined the Vinylcyclohexene Diepoxide (VCD) model of menopause with the house dust mite (HDM) model of asthma, to develop a novel model of menopause associated asthma in mice that closely mimics findings that have been detailed in the human condition. In this model, the mice demonstrated significant decreases in airway function in menopausal asthmatic mice, as well as increased neutrophil recruitment in the BALF, infiltration of inflammatory cells in the alveolar spaces, and excessive collagen production surrounding the airways, indicative of airway remodeling. In addition, as compared to asthmatic mice only, menopausal asthmatic mice had increased levels of non-Th2 cytokines, specifically Th-17, and elevated Th-9 T-cells in their lungs, su

Antibody responses serve a fundamental role in the clearance of active infections and the establishment of immunity post-infection or vaccination. Following either infection or vaccination, the presence or absence of neutralizing antibody titers is considered the most reliable immunological correlate of protection. As such, the production of durable post-vaccination antibody responses has long been an essential criterion in modern vaccine design. Nonetheless, much variability exists among commonly administered vaccines in their ability to generate stable, protective antibody titers.In the Winter of 2019-2020, the novel coronavirus SARS-CoV-2 began circulating in human populations and soon initiated a global pandemic. Development of reliable serological assays capable of detecting post-infection anti-SARS-CoV-2 antibody responses became a chief focus of public health and basic research efforts. Determining the dominant viral antigens targeted by the immune system, how quickly neutralizing titers were established, and the longevity of anti-SARS-CoV-2 responses, were all pressing topics. Moreover, these questions remained pertinent in the context of vaccination as the pandemic progressed and novel anti-SARS-CoV-2 vaccines became widely distributed. In many ways, the pandemic thrust these basic immunological processes into the everyday minds of both researchers and the general public. Antibody responses are mounted by terminally differentiated lymphocyte called plasma cells, which arise from activated B cell precursors. Plasma cells emerge in two waves of the B cell response. An early burst is generated within the first week post-exposure; these cells aim to clear an active infection and persist for a matter of days. A second wave of plasma cells are formed over a period of weeks as the B cell response matures. These cells secrete superior antibodies to their early plasma cell counterparts and ideally provide long-term protection. Two features are most critical for dura

Coronary artery disease is complex, arising from many genetic and environmental factors. Thedisease aetiology involves pathophysiology of multiple cell-types, which can be assayed using transcriptomic technologies. In this work, I present an investigation of vascular cell-type transcriptomes in both in vivo and in vitro models of atherosclerosis. Using a meta-analysis of singlecell RNA-seq datasets gathered from murine models of atherosclerosis, I determine that smooth muscle cell phenotypic plasticity shows a greater degree of transcriptional variability in vivo than in a previously developed cholesterol treatment assay in vitro. Furthermore, differentially expressed transcripts between smooth muscle lineage positive and smooth muscle lineage negative macrophages are identified. Human aortic endothelial cell co-expression clusters are identified in untreated and pro-inflammatory conditions and tested for association with common genetic variation, either as individual variants or as a polygenic risk score for coronary artery disease. I find little evidence that transcriptional modules are driven by common genetic variation in endothelial cells. In conclusion, transcriptomic assays and systems genetics allow for the investigation of complex disease processes, and further research is required to uncover the mechanisms governing smooth muscle phenotypic plasticity and endothelial transcriptional responses to common genetic variation.

Human complex disease is driven by a multitude of factors including individual’s genetics and environmental stimuli. To deconvolute the effect of genetics on cell biology from the rest of the factors, my work focuses on the transcriptional and epigenetic consequences of inheriting disease associated loci. Essentially, by focusing on the immediate effects of genetic variation, we can apply mechanisms to variants that were previously functionally obscure. To identify functional mechanisms of genetic variation, we collected high-throughput sequencing data including gene expression, transcription factor binding, chromatin accessibility, and histone modifications as well as genotypes of 6.7 million SNPs in 53 human aortic endothelial cell samples from donors of varying genotypes, sexes, and ancestries. We used quantitative trait locus (QTL) mapping to single out over 700,000 SNPs that were associated with gene expression in our population. Of the ~700,000 expression QTLs discovered, 8,747 were associated with an epigenetic trait as well, such as chromatin accessibility, transcription factor binding, or histone modification, providing a mechanism for the association with target gene transcription. Additionally, the expression QTLs are enriched in high significance coronary artery disease associated loci meaning that we are identifying the possible pathways that drive increased risk for disease. Through QTL analyses, we discovered loci with multiple layers of evidence suggesting function within the human genome. To verify these candidates, we performed a massively parallel reporter assay called Self Transcribing Active Regulatory Region sequencing (STARR-seq) on 34,444 variants. Through STARR-seq, we were able to functionally validate 5,711 SNPs. Additionally, we found that chromatin accessibility and transcription factor binding were highly enriched within the validated set suggesting that these measures are vital when identifying functional variation from molecular data.