Life Sciences Commons^™

Genetic Effects Mediated Through Epistatic Networks Onto Metabolic Traits, Juan Francisco Macias-Velasco

Arts & Sciences Electronic Theses and Dissertations

Predicting variation in complex traits from DNA sequence is a major public health goal, but our understanding of the genotype-to-phenotype relationship is incomplete. It will remain so unless we can adequately integrate genetic, epigenetic, and environmental information into a systems level framework. In a step towards that goal, quantitative trait mapping studies have attempted to account for environmental factors such as sex and diet, and epigenetic factors such as allelic parent-of-origin effects. Several studies used an advanced intercross of the LG/J and SM/J inbred mouse strains to unravel the genetic architecture of multiple metabolic traits. These studies found that parent-of-origin …

Developments In Proteomics, Trans-Splicing Technology And Endogenous Transcript Manipulation, Justin Alexander Melendez

Arts & Sciences Electronic Theses and Dissertations

Technological innovation drives scientific discovery, unlocks new avenues of research, and allows us to ask questions in ways that were previously unavailable. With each technological advance, our ability to perturb and explore biological systems has grown in ways previously unimagined. The theme of my thesis is the development of new technologies in biology. To this end, I have worked on three technologies that contribute to the areas of protein sequencing, RNA barcoding for trans-splicing and single-cell applications, and a new method for transcriptional knockdown.

In my first project, digital analysis of proteins by end sequencing (DAPES), we set out to …

Deconvolving Genomic Regulatory Heterogeneity With Self-Reporting Transposons, Arnav Moudgil

Arts & Sciences Electronic Theses and Dissertations

A cell’s identity is a function of the genes expressed in that cell, which are in turn regulated by transcription factors. Over the last decade, single-cell RNA sequencing (RNA-seq) has emerged as a powerful class of techniques to characterize cellular diversity in heterogeneous tissues. These methods barcode transcripts by their cell-of-origin and assign them to specific genes. The resulting high-dimensional data are further processed to reveal clusters of cells sharing transcriptional states. Annotating these clusters, based on either known or discovered marker genes, offers a glimpse into the dynamic composition of an organ or biological process. While single-cell RNA-seq excels …

Regulation Of Metabolic Stress By The Snhg3 Locus, Arthur Curtis Sletten

Arts & Sciences Electronic Theses and Dissertations

Dyslipidemia and lipotoxicity are pathologic signatures of the metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not fully understood. To identify novel genes involved in metabolic stress, our group performed an unbiased forward genetic screen for lipotoxicity resistance. My studies focused on characterizing one of the mutant cell lines isolated from this screen, in which promoter trap mutagenesis disrupted one allele of the small nucleolar RNA hosting gene 3 (Snhg3).

I demonstrate that diminished expression …

Role Of Interferon Receptors In Sting-Associated Autoinflammatory Disease In Mice, William Alexander Stinson

Arts & Sciences Electronic Theses and Dissertations

Over the past decade, the cGAS-STING pathway has emerged as a principal component of innate DNA sensing. Its activation of type I interferon (IFN) elicits host immune activation and in the absence of cGAS or STING, mice are rendered critically susceptible to infection by viruses and bacteria. Breakdowns in the molecules that regulate this pathway can promote unrestrained cGAS-STING activation and the development of various forms of autoimmune disease. For instance, gain-of-function mutations in STING cause autoinflammatory lung disease in mice and STING-associated vasculopathy with onset in infancy (SAVI) in humans. However, our understanding of the mechanisms that underlie STING-dependent …

The Role Of Neuronal Atp-Sensitive Potassium Channels In Learning And Memory, Shaul Vladimir Yahil

Arts & Sciences Electronic Theses and Dissertations

ATP-sensitive potassium (KATP) channels link cellular metabolism and membrane excitability in many tissues, including brain and pancreas. Gain-of-function (GOF) mutations to KATP channels cause neonatal diabetes, with some patients exhibiting developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome. Diabetic symptoms have been attributed to loss of membrane excitability and insulin secretion in pancreatic β-cells, though the origin of neurological deficits and the effects of neuronal KATP-GOF mutations more generally remain elusive. In this dissertation, I will present evidence that mice expressing KATP-GOF mutations pan-neuronally (nKATP-GOF) demonstrated sensorimotor and cognitive deficits, whereas hippocampus-specific hKATP-GOF mice exhibited predominantly learning and memory deficits. …

A Study On The Hormonal Regulation And Novel Role Of Na+ Leak Channel, Non-Selective (Nalcn) In Human Myometrial Smooth Muscle Cells, Chinwendu Amazu

Arts & Sciences Electronic Theses and Dissertations

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are progesterone (P4) and estrogen (E2), which promote quiescence or contraction, respectively, by acting on the myometrial smooth muscle cells (MSMCs). While these hormones regulate uterine contractions, it is unclear how they affect electrical activity of MSMCs, which underlies uterine contractile activity. Our lab recently identified Na+ leak channel, non-selective (NALCN) as a component of the leak current in human MSMCs and showed that mice lacking NALCN in the uterus have dysfunctional labor. In …

Structural Variants Are A Major Source Of Gene Expression Differences In Humans And Often Affect Multiple Nearby Genes, Alexandra Jane Scott

Arts & Sciences Electronic Theses and Dissertations

Structural variants (SVs), including copy number variants (CNVs), balanced rearrangements, and mobile element insertions (MEIs), are an important source of diversity in the human genome but their functional effects are not well understood. SVs are technically difficult to detect and genotype1, and mapping is dependent on deep whole genome sequencing (WGS) which was, until recently, unaffordable for large cohorts. For these reasons SVs are not included in most genome-wide studies of functional variants, despite the fact that SVs are known causal agents in multiple clinical disorders2-16. However, recent advancements in high-throughput sequencing technologies that allow for widespread use of WGS, …

Dissecting The Molecular Mechanism Of Familial Cardiomyopathies, Sarah Ruth Clippinger Schulte

Arts & Sciences Electronic Theses and Dissertations

Familial cardiomyopathies, including hypertrophic (HCM), restrictive (RCM) and dilated cardiomyopathy (DCM), are the leading cause of sudden cardiac death in young people. These diseases, which are characterized by altered cardiac contractility and remodeling of the heart, can lead to heart failure. These diseases are primarily caused by point mutations in sarcomeric proteins that generate or regulate heart contraction, such as troponin T. In the heart, the troponin complex together with tropomyosin lie along the actin filament and regulate myosin’s ability to bind actin and produce force. Here I show how mutations in troponin T affect contractility at the molecular level …

Genetic Risk Factors For Neurodevelopmental Disorders: Insights From Hipsc-Cerebral Organoids, Michelle L. Wegscheid

Arts & Sciences Electronic Theses and Dissertations

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder (NDD) characterized by remarkable phenotypic variability, where affected children manifest a spectrum of central nervous system (CNS) abnormalities, including brain tumors, impairments in attention, behavior, learning disabilities, and an increased incidence of autism spectrum disorder (ASD). A significant barrier to the implementation of precision medicine strategies for children with NF1 is a lack of prognostic risk factors to guide clinical management. However, emerging population-based genotype-phenotype association studies have suggested that the germline NF1 gene mutation may represent one clinically actionable risk factor for NF1-associated neurodevelopmental abnormalities. As a critical step in …

Regional Reprogramming And The Small Intestine: Analysis And Modeling Of Adaptive Regeneration Of The Epithelium, Sarah Elizabeth Waye

Arts & Sciences Electronic Theses and Dissertations

The small intestine in homeostasis is capable of regular regeneration, but in cases of massive injury like Short Bowel Syndrome, the innate human response often fails to fully compensate for the loss of nutrient absorptive surface area that accompanies bowel resection. Murine models display an active compensatory reaction deemed “adaptation” in which the surface area of the bowel is increased to accommodate nutrient absorptive needs. This observation has highlighted several gaps in knowledge regarding bowel adaptation. Firstly, what occurs on a molecular level in murine models during adaptation? Secondly, how can the findings in mice be applied to humans in …

Deep Multi-Omics Investigations Elucidate Novel Oncogenesis Paradigms, Therapeutic Targets, And Mechanisms Of Treatment Resistance In Cancer, Daniel Cui Zhou

Arts & Sciences Electronic Theses and Dissertations

No abstract provided.

Human Plcg2 Haploinsufficiency Results In A Novel Immunodeficiency, Joshua Brandon Alinger

Arts & Sciences Electronic Theses and Dissertations

NK cells are critical for the recognition and lysis of herpesvirus-infected cells. Patients with NK cell immunodeficiency may suffer from unusually severe and/or recurrent herpesvirus infections; however, the genetic cause is frequently unknown. PLCG2 encodes a signaling protein in NK cell and B cell receptor signaling, in which dominant-negative or gain-of-function mutations may cause cold urticaria, antibody deficiency, or autoinflammation. However, loss-of-function mutations and PLCG2 haploinsufficiency have never been reported in human disease. We examined 2 families with autosomal dominant NK cell immunodeficiency with dual high-dimensional techniques, mass cytometry and whole-exome sequencing, to identify the cause of disease. We identified …

The Effects Of Molecular Chaperone Modulation On Protein Folding, Prion Formation, And Prion Propagation In Saccharomyces Cerevisiae, Leeran Blythe Dublin Ryan

Arts & Sciences Electronic Theses and Dissertations

Proper and efficient protein folding is vital for cell survival. Many factors affect protein folding fidelity and prion formation, including molecular chaperone availability and activity. Research has shown that modulating chaperone availability and function can affect protein misfolding and aggregation, as well as de novo prion formation and propagation. However, the factors involved and underlying mechanisms influencing prion formation and protein folding are largely unknown. The following work aims to elucidate these areas. The Nascent Polypeptide-Associated Complex (NAC) is the first point of chaperone contact for nascent polypeptides. Previous work has shown that disruption of the NAC leads to improved …

Quantitative Characterization Of Microbial Ecologies In Dysbiosis And Infection, Eric Keen

Arts & Sciences Electronic Theses and Dissertations

In 1973, Theodosius Dobzhansky famously wrote that nothing in biology makes sense except in the light of evolution. Today, nearly 50 years later, little in microbiology – or in biology, for that matter – makes sense except in the light of genomics. Microbial genomics populates the field with innumerable testable hypotheses for evaluation in vitro and in vivo, allows us to monitor microbial populations in real time and at a massive scale, and underpins our approach to entire domains of microbiology, including microbial evolution. In this Thesis, I present three studies from my graduate research united by their common theme …

Computational Methods For Analysis Of Data For Conformational And Phase Equilibria Of Disordered Proteins, Jared Michael M Lalmansingh

Arts & Sciences Electronic Theses and Dissertations

Intrinsically disordered proteins and regions (IDPs / IDRs) are a class of proteins with diverse conformational heterogeneity that do not fold into a tertiary structure due to the lack of a native structural state. Consequently, disordered proteins are remarkably flexible and exhibit multivalent properties that enable them to adopt myriad functional roles within the cell such as: signaling transduction, transcription, enzymatic catalysis, translation, and many more. Due to their multivalency, some IDPs undergo monomeric and heterotypic interactions which can drive phase separation. Such IDPs can form membraneless organelles with specific regulatory roles within the cell which include, but are not …

Slo2.1 Channels: A New Molecular Mechanism To Regulate Uterine Excitability, Juan Jose Ferreira

Arts & Sciences Electronic Theses and Dissertations

At the end of pregnancy, the uterus transitions from a non-contractile state to a highly contractile state. Two processes primarily drive this transition. First, from the 28th week of pregnancy until labor, the resting membrane potential of uterine (myometrial) smooth muscle cells (MSMCs) gradually becomes more positive (depolarizes) (Parkington et al. 1999). Second, at the end of pregnancy, MSMCs express more oxytocin receptors and become more sensitive to oxytocin (Kimura et al. 1996). However, the detailed mechanisms by which these processes occur have not been determined. My central hypothesis was that the Na+-activated K+ channel SLO2.1 plays a key role …

On The Challenges And Rewards Of Analyzing Molecular Dynamics At The Terabyte And Millisecond Scale, Justin Roy Porter

Arts & Sciences Electronic Theses and Dissertations

Molecular Dynamics (MD) and Markov state models (MSMs) are powerful tools for estimating and concisely representing the conformational ensemble accessible to biological macromolecules, particularly proteins. Conformational ensembles are of special importance biological function, both in health and disease, because biology derives from molecules’ entire conformational distribution rather than any single structure. Consequently, MD is poised to become a powerful tool for personalized medicine and for the study of molecular sequence-function relationships generally. However, because of their hyperdimensionality and size, just generating MD datasets and Markov state models (MSMs) that represent biologically relevant molecules is a substantive technical challenge. Then, even …

Weedy Rice As A Model System For The Study Of Microevolutionary Interactions In Agricultural Contexts, Marshall Jon Wedger

Arts & Sciences Electronic Theses and Dissertations

Just under one-half of the global population relies on cultivated rice (Oryza sativa) astheir primary source of calories, making the optimization of rice agriculture immensely important. One of the primary constraints to rice agriculture is the de-domesticated (feral) form of rice known as ‘weedy rice’ that aggressively competes for space, soil nutrients, and light. Heavy infestation can reduce crop yields by as much as 80%. As a closely-related weedy descendant of cultivated rice, chemical control is difficult in rice fields, and physical weeding is labor intensive, time consuming, and largely ineffective due to early life-stage mimicry of the crop.

Weedy …

Regulatory Effects Of The E. Coli Recbcd Nuclease Domain On Dna Unwinding Kinetics, Nicole Fazio

Arts & Sciences Electronic Theses and Dissertations

I have examined the effects of deleting the nuclease domain of the E. coli helicase RecBCD on the rates of ATP-independent DNA melting, single stranded (ss) DNA translocation, and double stranded (ds) DNA unwinding by RecBCD. The canonical role of the nuclease domain is DNA degradation, but the removal of this domain showed unexpected effects on other RecBCD activities including DNA binding, melting, and unwinding. This thesis presents a mechanistic study of DNA unwinding by RecBCD and a RecBCD variant with the nuclease domain deleted (RecBΔnucCD). I examined the rates of ssDNA translocation and dsDNA unwinding by RecBCD and RecBΔnucCD …

Surveillance And Dynamics Of Bacteria And Resistance Genes In Humans And Hospitals, Alaric Wences D'Souza

Arts & Sciences Electronic Theses and Dissertations

Bacteria live in, on, and around us. These bacteria affect human health through beneficial commensal interactions that promote normal development and through harmful infections or perturbations leading to disease. Understanding the prevalence, spread, and resilience of these bacteria in humans and human environments is critical to clinical medicine and epidemiology. I investigated environmental and human resident bacteria to understand dynamics in bacterial communities and how these dynamics may impact human health.Hospitals are one critical location of human-bacterial interactions. Bacteria can contaminate hospital surfaces from patients or environmental sources and subsequently transmit from surfaces to vulnerable patients. I analyzed bacteria from …

Cancer Epigenome Reprogramming, Jennifer Ann Karlow

Arts & Sciences Electronic Theses and Dissertations

The identification of recurrent genetic mutations in cancer and their functionalcharacterization has provided a strong foundation for our understanding of tumorigenesis. The more recent observation of recurrent and specific epigenetic changes also present in cancer has widened this view, now establishing cancer as a disease of both genetic and epigenetic misregulation. Enhancers, genomic regions primarily responsible for tissue-specific gene expression, have been shown to be frequent targets of both genetic and epigenetic abnormalities. The observation that DNA methylation within regulatory regions has traditionally correlated with reduced gene expression, coupled with the known role of enhancers in regulating tissuespecific gene expression, …

Structural Analysis And Vaccine Efficacy Of Hla Mutants, Kelly Tomaszewski

Arts & Sciences Electronic Theses and Dissertations

Staphylococcus aureus is a commensal of the human skin and also a major human pathogen. Currently, there has been no successful vaccine despite many approaches over the last two decades. S. aureus α-hemolysin (Hla), a potent cytotoxin, plays an important role in the pathogenesis of S. aureus diseases, through the activation of its receptor, ADAM10. We utilized three distinct Hla mutants with differing structural and ADAM10 binding properties to examine for vaccine efficacy. Our studies have demonstrated immunization with each vaccine candidate antigens provided significant protection against S. aureus skin infection yet elicited distinguishable immune responses. We have also generated …

C. Elegans Response To Cadmium Toxicity, Brian James Earley

Arts & Sciences Electronic Theses and Dissertations

Cadmium is an environmental pollutant and significant health hazard that is similar to the physiological metal zinc. Residing in the same group of the periodic table, cadmium and zinc share chemical characteristics that are important for their industrial uses in electroplating, batteries, pigments, and metal alloys. The similarities of ionic cadmium and zinc have significant repercussions on biological systems. While it has long been clear that cadmium is toxic to biological systems, the mechanisms of cadmium toxicity remain poorly understood. In contrast, mechanisms of zinc homeostasis have been elucidated in growing detail. In C. elegans high zinc homeostasis is regulated …

Method Development For Enhancing Sensitivity Of Dynamic Nuclear Polarization Nuclear Magnetic Resonance Spectroscopy For Structural Studies Of Pkc-Drug Interactions, Patrick Terrence Judge

Arts & Sciences Electronic Theses and Dissertations

To perform the most relevant structural studies on biological systems, experiments need to be carried out when the target proteins are in their endogenous cellular environment. Nuclear magnetic resonance (NMR) is well-suited to probe the structure and dynamics of a wide variety of systems, including biologically relevant proteins. However, NMR suffers from an inherent lack of sensitivity. Dynamic nuclear polarization (DNP) NMR is a powerful technique that is used to enhance NMR sensitivity by transferring the greater polarization of exogenously doped electron spins to nuclear spins of interest though the use of a high-power microwave source. Solid effect radicals offer …

Precision Diffusion Imaging To Analyze Individual-Specific Plasticity, Nicole Andrea Seider

Arts & Sciences Electronic Theses and Dissertations

Diffusion weighted imaging (DWI) is used to non-invasively infer and characterize the structure and integrity of the brain’s white matter fibers. Individual-specific precision diffusion imaging can identify additional organizational detail important for understanding basic brain connectivity and for advancing clinical applications of DWI in neuromodulation and neurosurgical planning. The reliability of individual specific DWI and data requirements for various analytic methodologies must first be systematically assessed. The reliability and accuracy of precision diffusion imaging was evaluated as a function of data quantity and analysis method, using both simulations and highly sampled individual-specific data (Chapter 2). Parameter estimation methods that allowed …

Association Of Structural Variation (Sv) With Cardiometabolic Traits In Finns, Lei Chen

Arts & Sciences Electronic Theses and Dissertations

Cardiovascular diseases (CVDs) are known to be associated with a variety of quantitative risk factors such as cholesterol, metabolites, and insulin. Understanding the genetic basis of these quantitative traits can shed light on the etiology, prevention, diagnosis, and treatment of disease. However most prior trait-mapping studies have focused on single nucleotide variants (SNVs) and Indels, with the contribution of structural variation (SV) remaining unknown. In this thesis, we present the results of a study examining genetic association between SVs and cardiometabolic traits in the Finnish population. In the first chapter, we used sensitive methods to identify and genotype 129,166 high-confidence …

Chemical Damage To Mrna And Its Impact On Ribosome Quality-Control And Stress-Response Pathways In Eukaryotic Cells, Liewei Yan

Arts & Sciences Electronic Theses and Dissertations

Ribosome often faces defective adducts that disrupt its movement along the mRNA template. These adducts are primarily caused by chemical damage to mRNA and are highly detrimental to the decoding process on the ribosome. Hence, unless dealt with, chemical damage to RNA has been hypothesized to lead to the production of toxic protein products. Even more detrimental is the ability of damaged mRNA to drastically affect ribosome homeostasis through stalling. This in turn would lead to greatly diminished translation capacity of cells. Therefore, the inability of cells to recognize and resolve translational-stalling events is detrimental to proteostasis and could even …

Vestibulospinal Circuit In The Larval Zebrafish, Zhikai Liu

Arts & Sciences Electronic Theses and Dissertations

The vestibular system sense gravity and self-motion to help animals maintain body balance. Although vestibular signals inform the brain of the directions and speed of our body movements, it still remains unclear how these sensory information are processed and organized in the central nervous system. My thesis aims to illustrate neural computation underlying central vestibular tuning and the topographic organization of the vestibular circuits. First I established a novel approach to perform whole-cell recording of synaptic inputs in vivo during multi-axis movements in the central vestibular neurons. This technical advance allowed me to simultaneously measure presynaptic and postsynaptic tuning, along …

Regulation Of Transcription Factor Binding Specificity: From Binding Motifs To Local Dna Context, Jiayue Liu

Arts & Sciences Electronic Theses and Dissertations

Regulation of transcription factor (TF) binding specificity lies at the heart of transcriptional control which governs how cells divide, differentiate, and respond to their environments. TFs are known to bind to DNA in a sequence specific manner, and such short sequence is known as transcription factor binding site (TFBS). However, the in vivo TF bound regions do not always contain a TFBS, and additionally, there are often excessive non-functional TFBSs with binding potential in the regulatory regions that are unbound for a given TF. This dissertation focuses on understanding the principles of TF binding specificity and is divided into two …