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 …

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, …

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. …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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, …

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 …

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.

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 …

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 …

Testing Janzen’S Physiological Barrier Hypothesis Through Experimental Evolution And Biogeographic Studies On Sister Species Pairs, Vincent John Fasanello

Arts & Sciences Electronic Theses and Dissertations

Background: Janzen’s physiological barrier hypothesis suggests that variation in the effectiveness of physiological barriers to dispersal underlies global patterns of speciation and biodiversity. He noted that because a species’ physiology often evolves to match the range of conditions it has experienced in evolutionary history, it is likely that lineages that occupy habitats with wider ranges of temperature variation will tend to be better at dispersing across thermal gradients and will therefore be less likely to speciate as a result of mountain barriers. Despite decades of research into different aspects of this fundamental hypothesis, its assumptions and predictions remain largely untested. …

Linking Neuronal Protein Phosphatase 2a To The Dlk Stress Kinase Signaling Cascade, Margaret Hayne

Arts & Sciences Electronic Theses and Dissertations

Neurons are constantly responding to internal and external cues as they adapt through signaling cascades and transcriptional programs. I have identified a role for the dual leucine zipper kinase (DLK) and the transcriptional program it controls in neuronal inhibition of the protein phosphatase PP2A. PP2A is an essential phosphatase, expressed in all cell types and required for survival. There is a reduction in PP2A activity in Alzheimer’s disease patient brains which is linked to increased phosphorylation of the microtubule associated protein Tau and ultimately cell death. Here, I have demonstrated that PP2A acts to restrain the DLK stress response and …

The Role Of Excited States In Determining Β-Lactamase Function And Bacterial Fitness, Catherine Rae Knoverek

Arts & Sciences Electronic Theses and Dissertations

Proteins are macromolecular machines that play a role in nearly every biological process. They are dynamic molecules which adopt many different conformations as they fold into their 3D structures, interact with their binding partners, and perform their functions. The most probable (lowest energy) protein conformation is referred to as the ground state, and this is often assumed to be the state determined by experimental methods such as x-ray crystallography. However, proteins also adopt higher energy excited states which can have significant probabilities. As these excited states are notoriously difficult to find and study, it is unclear if excited states contribute …

Investigation Of Ifnγ-Induced Control Of Intracellular Pathogens, Michael Mcallaster

Arts & Sciences Electronic Theses and Dissertations

Genes required for the lysosomal degradation pathway of autophagy play key roles in topologically distinct cellular processes with significant physiologic importance. One of the first-described of these ATG gene-dependent processes is the requirement for a subset of ATG genes in interferon-γ (IFNγ)-induced inhibition of norovirus and Toxoplasma gondii replication. In this dissertation we identified novel components that are required for or that negatively regulate this immune mechanism. Enzymes involved in the conjugation of UFM1 to target proteins including UFC1 and UBA5, negatively regulated IFNγ-induced inhibition of norovirus replication via effects of Ern1. We identified and confirmed that IFNγ-induced inhibition of …

Testing Candidate Cerebellar Presymptomatic Biomarkers For Autism Spectrum Disorder, Zoe Wilson Hawks

Arts & Sciences Electronic Theses and Dissertations

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder diagnosed on the basis of social impairment, restricted interests, and repetitive behaviors. Contemporary theories posit that cerebellar-mediated error signaling impairments contribute to the causation of ASD. However, the relationship between infant cerebellar functional connectivity (fcMRI) and later ASD behaviors and outcomes has not been investigated. Such work is critical to establish early (presymptomatic) cerebellar correlates of ASD. Methods: Data from the Infant Brain Imaging Study (n=94, 68 male) were used to evaluate cerebellar fcMRI as a presymptomatic biomarker for ASD. Specifically, brain-behavior associations were analyzed for 6-month cerebellar connections in relation …

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 …

Functional Optical Imaging Of The Developing Mouse Cortex In Health And Disease, Rachel Marlene Rahn

Arts & Sciences Electronic Theses and Dissertations

Neurodevelopmental disorders (NDDs) are a heterogeneous family of disorders characterized by the presence of abnormal developmental trajectories. Functional connectivity (FC) neuroimaging provides a minimally-invasive method by which to investigate the progression of these disorders and identify potential biomarkers of disease-related dysfunction for use in therapeutics’ development. My thesis work therefore took several approaches to the question of how the functional connectome reflects developmental change as well as system-wide perturbations from environmental or genetic factors. I explored the use of optical fluorescence and intrinsic signal imaging to characterize FC and stimulation-derived responses in a NDD model of perinatal exposure to selective …