Investigating The Potential Of A Cell-Based Gene Editing Therapy For Inherited Metabolic Liver Disease, Ilayda Ates

All Dissertations

Inherited metabolic diseases (IMDs) affecting the liver are relatively rare but collectively have a prevalence of 1 in 800 live births. These diseases result from autosomal recessive single-gene mutations, leading to organ dysfunction and potentially fatal consequences if left untreated. One potential therapeutic strategy for IMDs of the liver involves using CRISPR-Cas9-induced loss of function mutations. However, translating this approach into the clinic is limited by the need for safe and effective CRISPR delivery methods. Adeno-associated viral vectors (AAVs), commonly used for CRISPR delivery, are associated with significant safety and efficacy concerns, including risks for immunogenicity, off-target mutagenesis, and genotoxicity …

A Systems Genetics Approach To Drosophila Melanogaster Models Of Rare And Common Neurodevelopmental Disorders, Rebecca Macpherson

All Dissertations

Fetal Alcohol Spectrum Disorders are a group of disorders resulting from prenatal alcohol exposure, presenting with neurodevelopmental and facial abnormalities of varying severity. SSRIDDs and CdLS are rare disorders of chromatin modification, resulting in patients with a wide range of craniofacial, digit and/or neurodevelopmental abnormalities. All of these disorders have a wide range of clinical phenotypes and disease severity, yet the role of potential genetic modifiers and gene-gene or gene-environment interactions in disease pathogenesis is largely unknown and cannot be studied in humans. Insufficient numbers of patients with a single rare disorder prevent investigation of genetic factors beyond the focal …

Investigating The Role Of Cnag_05113 In The Carnitine Biosynthesis Pathway In Cryptococcus Neoformans., Jasmine Meltzer, Rodrigo Catalan-Hurtado, Perry Kezh, Kerry Smith

Honors College Theses

Cryptococcus neoformans, the leading cause of fungal meningitis, is a fungal pathogen that causes severe infection of the central nervous system in patients with compromised immune systems, typically caused by HIV/AIDS. C. neoformans infections are present in developed countries including the United States, but most fatalities occur in sub-Saharan Africa where antiretroviral therapy, the treatment for HIV/AIDS, is less accessible. Current treatments for severe cryptococcal infections are extensive and outdated. There is a critical need for an improved understanding of the fungus and new targeted therapies. Our goal is to identify metabolic pathways important to the survival of C. …

Genetic Investigation Of Spontaneous Harlequin Coat Patterning In A Family Of Australian Shepherds, Katherine Grace Singleton

Honors College Theses

In dogs, a white base coat with black patches is termed harlequin patterning. In the Great Dane, harlequin is caused by a mutation in the ubiquitin-proteasome system, a highly complex process through which damaged proteins are degraded1. All harlequins are also heterozygous for the Merle allele of SILV, which harbors a retrotransposon that leads to production of abnormal protein2. In melanocytes with impaired protein degradation, aberrant SILV is thought to cause cell death, resulting in the characteristic white base coat of a harlequin dog1. We identified a novel, spontaneous form of harlequin patterning in a family of Australian shepherds. To …

Identification Of Sva-Mediated 3’ And 5’ Transductions In Human Genome Sequences, Emily C. Golba

Honors College Theses

Transposable elements (TEs) are sequences of DNA that can move, or transpose, within a genome. Retrotransposons are TEs that propagate via a “copy and paste” mechanism where the elements are transposed to a new genomic location via an RNA intermediate. Short interspersed element (SINE)-VNTR-Alu elements (SVAs) are non-autonomous retrotransposons that use long interspersed element 1 proteins to mobilize. SVAs are currently active in the human genome and often are characterized by the mobilization of sequences adjacent to the 3’ and 5’ ends of insertions, known as transduction events (TDs). TDs were a focus of this study due to their contribution …

The Role Of Fto, A Human Rna Demethylase In Perennial Grass Development And Abiotic Stress Responses, Andrew Fiorentino, Qian Hu, Xiaotong Chen, Zhaohui Chen

Honors College Theses

The integration of the human fat mass and obesity associated (FTO) gene into turfgrass is a novel approach at improving cell proliferation and abiotic stress resistance. The FTO protein is an RNA demethylase responsible for epigenetic regulation of the genome. In related rice, the gene is associated with increased crop yield, tiller number, and aerial biomass. It is proposed to work via demethylation of repeat RNA associated with chromatin remodeling, causing widespread transcriptional activation. In this study, the feasibility of using FTO for plant trait modification in perennial grasses is being investigated. Potentially transformed embryogenic calli of creeping bentgrass with …

Methyltransferase, Glucose Adaptation, And Import Complex In Trypanosoma Brucei, Emily Knight

All Dissertations

Trypanosoma brucei is a kinetoplastid parasite responsible for human African trypanosomiasis (HAT) and nagana, a livestock wasting disease, which both endemic to sub-Saharan Africa. Unique to kinetoplastids are the specialized peroxisomes, named glycosomes, which compartmentalize the first several steps of glycolysis and gluconeogenesis, nucleotide sugar biosynthesis, and many other metabolic processes. Kinetoplastids are unique in that they have a single mitochondrion. In this work, I present the first study into SET domain proteins in any kinetoplastid parasites. We have characterized a predicted SET domain protein, TbSETD3, that localizes to the mitochondrion and a depletion of the protein results in growth …

Genetic Investigation Of Spontaneous Harlequin Coat Patterning In A Family Of Finnish Collies, Megan Patterson

Honors College Theses

The merle coat pattern is a pigmentary phenotype of dogs characterized by a dilute background with black patches. Merle is caused by a SINE insertion in PMEL17, a pigmentation gene expressed in melanocytes. The mutation causes aberrant splicing of transcripts and production of an abnormal protein. Harlequin is a dominant modifier of merle that further dilutes the background to white. Harlequin Great Danes have a heterozygous mutation impairing the ubiquitin-proteasome system, suggesting that the inability to degrade aberrant PMEL17 results in melanocyte death. Harlequin is not a recognized coat pattern of the Collie; however, a harlequin phenotype spontaneously appeared in …

Integrating Omim And Intact Data For The Analysis Of Gene-Phenotype Interactions In Complex Diseases: A Linux-Based Computational Tool For Network Analysis, Devin Keane

All Theses

The field of genetics is constantly evolving. New advances in bioinformatics and computational approaches are leading to exciting new developments in our ability to treat and prevent diseases. Computational genetics provides valuable insights into the complex mechanisms and layers of biological communication that shape an organism's phenotype. Understanding these mechanisms is critical to advancing human health.

The study of diseases in genetics requires a comprehensive understanding of the interactions between various biological processes, including gene expression, protein synthesis, RNA, metabolism, and cell-cell communication. To effectively address the root causes of such diseases, multi-disciplinary approaches that integrate information from different levels …

Acetate Metabolism In The Fungal Pathogen Cryptococcus Neoformans, Oly Ahmed

All Dissertations

Cryptococcus neoformans is an environmental basidiomycetous fungus with a worldwide distribution and a wide range of habitats. Inhalation of the desiccated yeasts or spores of C. neoformans often leads to opportunistic pulmonary infections in immunocompromised individuals, and in severe cases causes lethal meningitis following hematogenous dissemination. During infection, depending on the tissue and disease state, the invading fungi experience a range of nutrient microenvironments within the host body. As a result, rapid metabolic adaptations geared towards efficient utilization of carbon sources alternative to glucose become one of the prime determinants of survival and growth for the pathogen. Incidentally, cryptococcal infection …

Assesment Of Structure, Function, And Microevolutionary Dynamics Of Extrachromosomal Circular Dna In Chinese Hamster Ovary Cells, Dylan Chitwood

All Dissertations

Chinese hamster ovary (CHO) cell lines are among the most popular expression hosts used in biopharmaceutical manufacturing due to relative ease of culture, capacity to perform human-like post-translational modifications, and non-susceptibility to viruses. However, the intrinsic plasticity of the CHO genome can lead to undesired genetic rearrangements, phenotypic shifts, reduced product quality, and early culture termination that prevents continuous biomanufacturing. A characteristic of plastic and unstable genomes that is poorly understood in CHO cells is extrachromosomal circular DNA (eccDNA). EccDNAs are focal amplifications of the genome that reside in the extranuclear space. These plasmid-like entities are structurally complex and are …

Large Genomes Assembly Using Mapreduce Framework, Yuehua Zhang

All Dissertations

Knowing the genome sequence of an organism is the essential step toward understanding its genomic and genetic characteristics. Currently, whole genome shotgun (WGS) sequencing is the most widely used genome sequencing technique to determine the entire DNA sequence of an organism. Recent advances in next-generation sequencing (NGS) techniques have enabled biologists to generate large DNA sequences in a high-throughput and low-cost way. However, the assembly of NGS reads faces significant challenges due to short reads and an enormously high volume of data. Despite recent progress in genome assembly, current NGS assemblers cannot generate high-quality results or efficiently handle large genomes …

Data-Driven Biomarker Panel Discovery In Ovarian Cancer Using Heterogenous Data Fusion On Exosomal And Non-Exosomal Microrna Expression Data, Paritra Mandal

All Dissertations

Ovarian cancer (OC) is an aggressive gynecological cancer and is currently the 5th leading cause of deaths due to cancer in women. High mortality rates are attributable to the vague pathogenesis and asymptomatic nature of the early stages. The development of a liquid biopsy for routine OC screening could help identify the disease at an earlier stage, making treatments more likely to be effective thereby increasing survival rates. Exosomes, small (~100nm) extracellular vesicles present in body fluids, have been shown to contain cancer-progression, onset, and related factors, making them good candidates for use in liquid biopsies. However, to date, only …

Heat Stress Response And Excystation In Entamoeba Histolytica, Irem Bastuzel

All Dissertations

Entamoeba histolytica is a water- and food-borne intestinal protozoan parasite that causes amoebiasis and liver abscess and is responsible for symptomatic disease in approximately 100 million people each year leading to ~ 100,000 deaths. The most common disease transmission follows the oral-fecal route, but it can also be transmitted by mechanical vectors such as animals carrying the amoeba from contaminated sources to water systems. In rare cases, disease transmission has been recorded in some patients in which men-to-men sexual practices were preferred.

The life cycle of E. histolytica starts through ingestion of infectious cysts, which are non-dividing, quadri-nucleated structures surrounded …

Modeling Electrostatics In Molecular Biology And Its Relevance With Molecular Mechanisms Of Diseases, Mahesh Koirala

All Dissertations

Electrostatics plays an essential role in molecular biology. Modeling electrostatics in molecular biology is complicated due to the water phase, mobile ions, and irregularly shaped inhomogeneous biological macromolecules. This dissertation presents the popular DelPhi package that solves PBE and delivers the electrostatic potential distribution of biomolecules. We used the newly developed DelPhiForce steered Molecular Dynamics (DFMD) approach to model the binding of barstar to barnase and demonstrated that the first-principles method could also model the binding. This dissertation also reflects the use of existing computational approaches to model the effects of Single Amino Acid Variations (SAVs) to reveal molecular mechanisms …

Investigating The Biochemical Properties Of A Novel Mutation, A194v, In Human Rad51, Briana Vollbeer

All Theses

DNA double-strand breaks (DSB) are one of the most serious DNA lesions because improper repair of a DSB can lead to loss of heterozygosity, aneuploidy, and cancer. One of the primary pathways to repair DSBs is homologous recombination (HR). HR resects the DNA around the DSB and then uses homologous DNA as a template to restore the broken sequence. RAD51 has a vital function in this pathway by forming a nucleoprotein filament on a resected end of the DSB. The nucleoprotein filament searches for homology within the homologous DNA. Once homology is located, strand invasion followed by strand exchange occurs. …

Deciphering Medicago Truncatula Nodulation Using Time-Series Transcriptomic Data At Multiple Levels Of Resolution: Organ, Tissue, And Single-Cell, Yueyao Gao

All Dissertations

Use of chemical nitrogen fertilizers has environmental repercussions such as global warming, soil contamination, and aquatic eutrophication. Legumes form a symbiotic association with nitrogen-fixing bacteria (rhizobia sp.) to obtain atmospheric nitrogen through the formation of a specialized root structure called a nodule. Understanding the transcriptional reprogramming during nodulation is a powerful approach to decipher the genetic control of nodulation, with the goal of engineering nitrogen-fixing symbiosis into non-leguminous crops. This dissertation focuses on the analytics of bulk, tissue-specific, and single-cell RNA-seq technologies and how I utilized them to discover a collection of genes to aid in deciphering nodulation mechanisms in …

Clinical And Molecular Characterization Of Key Features Of Phelan-Mcdermid Syndrome And Potential Approaches To Clinical Trials, Bridgette Moffitt

All Dissertations

Phelan-McDermid Syndrome (PMS) is a rare neurodevelopmental disorder that involves chromosomal abnormalities on 22q13.3 and pathogenic variants with the SHANK3 gene. SHANK3 encodes for a critical scaffolding protein that is important in post-synaptic density, function, and maintenance of excitatory synapses. Haploinsufficiency of SHANK3 is considered the major cause of the neurological phenotype of PMS. These studies focused on further characterizing the neurologic phenotype of sleep disturbances in individuals with PMS.

Sleep disturbances are a common feature of PMS, however there is little research available on the subject matter. We reviewed the literature on sleep disturbances and used the Phelan-McDermid Syndrome …

Genetics And Genomics Education Among Physician Assistants, Wesley Patterson

All Dissertations

This dissertation comprises five chapters to describe genetics and genomics education among physician assistant/associate (PA) students and practicing PAs. Chapter I introduces the gap in supply and demand of genetic services, the need for non-genetics healthcare providers to fill the gap, and the PA profession as a solution.

Chapter II is a rapid literature review that summarizes the available literature regarding genetics and genomics education for PAs. A paucity of literature exists to describe the current state of PA genetics-genomics education. The few studies retrieved describe content being taught in PA programs, the number of genetics-genomics contact hours PA students …

Characterization Of Mef2c-Related Disorders: Genotype, Phenotype, And Gene Pathway Dysregulation, Jessica Ann Cooley Coleman

All Dissertations

MEF2C­-related disorders are characterized by intellectual disability, developmental delay, lack of speech, seizures, stereotypic movements, hypotonia, and brain abnormalities and are caused by pathogenic alterations involving the MEF2C gene. Despite published cases, MEF2C­-related disorders are difficult to recognize clinically. These studies sought to further characterize MEF2C­-related disorders by investigating the genotypes, phenotypes, and gene functions (or dysfunctions) associated with the disorder.

Tremors have been reported in some patients with MEF2C­-related disorders, but the concept of tremors has been complicated by vague definitions and numerous categorization methods. We performed a concept analysis following the Walker and Avant …

Effects Of Continuous In Situ Low-Dose Ionizing Radiation On Microorganisms, Molly E. Wintenberg

All Dissertations

Precise detection and monitoring of nuclear fuel cycle, enrichment, and weapon development activities are critical for supporting warfighter preparation in chemical, biological, radiological, nuclear, and explosives (CBRNE) operations, clandestine activities, and nuclear compliance. A biological sensing system could serve as an alternative to traditional detection methods by using organic material naturally present in the environment to discreetly detect residual trace nuclear material. Microorganisms provide an optimal platform for an alternative sensing system; however, their response to low levels of ionizing radiation is poorly characterized. Combining the power of next-generation sequencing and transcriptomic analysis, this dissertation takes an approach to obtain …

Identification Of Genetic Factors Underlying Megaesophagus And Startle Disease In Dogs, Sarah M. Bell

All Dissertations

Dogs provide an excellent model for human hereditary disease research; thus, the development of canine genomic tools has been prioritized in recent years. Today, SNP arrays, multiple genome assemblies, and multi-breed reference panels containing whole genome resequencing (WGS) data from hundreds of canids are available to facilitate genome-wide genotyping in the dog. Herein, a variety of genome-wide techniques are employed to identify the genetic factors underlying congenital idiopathic megaesophagus (CIM) in two breeds, German shepherd dogs (GSDs) and Great Danes, and startle disease in Spanish greyhounds.

CIM is a complex canine esophageal motility disorder characterized by ineffective peristalsis and esophageal …

Unraveling The Genetic Architecture Of Somatic Embryogenesis In Upland Cotton, Adam M. Canal

All Theses

Somatic embryogenesis is the de novo development of asexual embryos because of the plasticity of the plant cell. In tissue culture, the biochemical and genetic mechanisms of dedifferentiated callus tissues can be reprogrammed to transdifferentiate into developed, polarized embryos, which can ultimately regenerate into whole plants. Although this rarely occurs in nature, scientists have exploited this process for decades to regenerate whole plants following gene transformation or for micropropagation. While some species are amenable to in vitro regeneration, upland cotton is particularly recalcitrant, with regenerative potential being confined to only several genotypes. The lack of elite, regenerable genotypes greatly restricts …

Characterization Of A Potential Glucose Transporter In Trypanosoma Brucei, Matthew Morgan

All Theses

Trypanosoma brucei, the African trypanosome, is an organism heavily dependent on glucose for ATP production during the infectious stage of its life cycle. Here, we have explored the role of an uncharacterized protein designated “novel glucose transporter” (NGT) as a potential glucose transporter. Sequence analyses suggests that NGT shares similarities (either at the primary sequence level or structurally) with Trypanosome Hexose Transporters 1 (TbTHT1), and human GLUT3, both of which are membrane sugar transporters. NGT was localized by fluorescence microscopy to subcellular structures consistent with lysosomes. Silencing NGT expression with RNA interference in parasites resulted in a growth defect …

Identification Of Genetic Variants Underlying Maxillary Canine-Tooth Mesioversion And Small Body Size In Shetland Sheepdogs, Sydney R. Abrams

All Theses

The domesticated dog (Canis lupus familiaris) exhibits a unique population structure, with high levels of genetic homogeneity within breeds due to selective breeding. These closed breeding populations can result in breed-specific inherited disorders. Maxillary canine-tooth mesioversion (MCM) is a genetically complex dental anomaly observed nearly exclusively in the small Shetland Sheepdog breed. Here, we utilized a genome-wide approach to discover a locus of major effect on chromosome 9. Using whole-genome resequencing data from a bilaterally affected dog, we identified variants in two genes: FTSJ3, encoding an RNA methyltransferase, and GH1, encoding growth hormone. Independent genome-wide association …

Identification Of Biomarker Systems Of Autism Spectrum Disorder And Uterine Cancer, Allison Hickman

All Dissertations

Complex diseases and disorders pose a challenge to scientists due to their variable and often inconsistent genetic and environmental underpinnings across affected individuals. Because of this variability, large condition-specific datasets and corresponding analytical tools and approaches are being curated as resources to investigate potential genetic trends in complex diseases and disorders. In this Dissertation, I used DNA- and RNA-based resources to discover polygenic biosignatures associated with Autism Spectrum Disorder (ASD) or uterine cancer. To explore the intersection of small-effect common DNA variants and regulation in ASD, I discovered and analyzed trends in allelic associations at eQTLs within ASD-affected individuals. Association …

Genetic And Mirna Transcriptomic Analysis Of Autoregulation Of Nodulation Signaling In Medicago Truncatula, Diptee Chauligain

All Dissertations

Legumes can source nitrogen from the air through biological nitrogen fixation inside root nodules formed in a symbiosis with rhizobia. A complex root-to-shoot-to-root signaling pathway called Autoregulation of Nodulation (AON) controls the number of nodules formed depending on the plant’s nitrogen requirements. In Medicago truncatula, the MtSUNN receptor complex in the shoot binds to the root-generated AON signals, peptides called MtCLEs, resulting in downregulation of miR2111 expression in the shoot, with the effect of decreased transport of miR2111 to the roots. Decreased miR2111 levels in the roots then cause an increase in transcript levels of the miR2111 targets MtTML1 …

Comparative Transcriptome Profiling Provides Insights Into Plant Salt Tolerance In Seashore Paspalum (Paspalum Vaginatum), Peipei Wu, Steven Cogill, Yijian Qiu, Zhigang Li, Man Zhou, Qian Hu, Zhihui Chang, Hong Luo, Rooksana E. Noorai, Xiaoxia Xia, Christopher Saski, Paul Raymer

Publications

Background

Seashore paspalum (Paspalum vaginatum), a halophytic warm-seasoned perennial grass, is tolerant of many environmental stresses, especially salt stress. To investigate molecular mechanisms underlying salinity tolerance in seashore paspalum, physiological characteristics and global transcription profiles of highly (Supreme) and moderately (Parish) salinity-tolerant cultivars under normal and salt stressed conditions were analyzed.

Results

Physiological characterization comparing highly (Supreme) and moderately (Parish) salinity-tolerant cultivars revealed that Supreme’s higher salinity tolerance is associated with higher Na+ and Ca2+ accumulation under normal conditions and further increase of Na+ under salt-treated conditions (400 mM NaCl), possibly by vacuolar sequestration. Moreover, K+ retention under …

Discovery Of Genomic Variations By Whole-Genome Resequencing Of The North American Araucana Chicken, Rooksana E. Noorai, Vijay Shankar, Nowlan H. Freese, Christopher M. Gregorski, Susan C. Chapman

Publications

Gallus gallus (chicken) is phenotypically diverse, with over 60 recognized breeds, among the myriad species within the Aves lineage. Domestic chickens have been under artificial selection by humans for thousands of years for agricultural purposes. The North American Araucana (NAA) breed arose as a cross between the Chilean “Collonocas” that laid blue eggs and was rumpless and the “Quetros” that had unusual tufts but with tail. NAAs were introduced from South America in the 1940s and have been kept as show birds by enthusiasts since then due to several distinctive traits: laying eggs with blue eggshells, characteristic ear-tufts, a pea …

Non-Enzymatic Roles Of Human Rad51 At Stalled Replication Forks, Jennifer M. Mason, Yuen-Ling Chan, Ralph W. Weichselbaum, Douglas K. Bishop

Publications

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not D-loop activity, to reveal mechanistic aspects of RAD51’s roles in the response to replication stress. Here, we find that cells lacking RAD51’s enzymatic activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly, we find that RAD51’s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication …