Life Sciences Commons^™

Regulation Of Gene Expression By Rna Binding Proteins And Micrornas, Kyle Cottrell

Arts & Sciences Electronic Theses and Dissertations

Regulation of gene expression is essential to life. Post-transcriptional regulation of gene expression is a complex process with many inputs that lead to changes in localization, translation and stability of mRNAs. The translation and stability of many mRNAs is regulated by cis-elements, such as mRNA-structure or codon optimality; and by trans-acting factors such as RBPs and miRNAs. Here I report on the complex interactions between RBPs, miRNAs and characteristics of their target mRNAs in respect to effects on translation and RNA stability.

Using a reporter based approach we studied modulation of microRNA-mediated repression by various mRNA characteristics. We observed the …

Binding Affinity And Specificity Of Sh2 Domain Interactions In Receptor Tyrosine Kinase Signaling Networks, Tom Ronan

McKelvey School of Engineering Theses & Dissertations

Receptor tyrosine kinase (RTK) signaling mechanisms play a central role in intracellular signaling and control development of multicellular organisms, cell growth, cell migration, and programmed cell death. Dysregulation of these signaling mechanisms results in defects of development and diseases such as cancer. Control of this network relies on the specificity and selectivity of Src Homology 2 (SH2) domain interactions with phosphorylated target peptides. In this work, we review and identify the limitations of current quantitative understanding of SH2 domain interactions, and identify severe limitations in accuracy and availability of SH2 domain interaction data. We propose a framework to address some …

The Regulation Of Extracellular Amyloid-Β Levels By Ionotropic Glutamatergic Transmission In An Alzheimer’S Disease Mouse Model, Jane Cecelia Hettinger

Arts & Sciences Electronic Theses and Dissertations

Brain extracellular concentration of the peptide amyloid-β (Aβ) is a major contributor to Alzheimer’s disease (AD) pathogenesis. High Aβ levels in the extracellular space precipitate aggregation of the peptide into soluble and insoluble toxic species. This process begins decades before cognitive impairment and triggers the cascade of pathology that eventually leads to AD. Synaptic activity is key to the regulation of extracellular Aβ levels. Presynaptic activity drives the production of Aβ, while postsynaptic receptor activation exhibits more nuanced regulation. For example, high levels of NMDA receptor (NMDA-R) activation have been shown to decrease Aβ production through the extracellular signal-regulated kinase …

Photosensing And Thermosensing By Phytochrome B Require Both Proximal And Distal Allosteric Features Within The Dimeric Photoreceptor, E Sethe Burgie, Adam N. Bussell, Shu-Hui Lye, Tong Wang, Weiming Hu, Katrice E. Mcloughlin, Erin L. Weber, Huilin Li, Richard D. Vierstra

Biology Faculty Publications & Presentations

Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream …

Type 1 And F17-Like Pili Promote The Establishment Of The Uropathogenic E. Coli Intestinal Reservoir, Caitlin Nicole Spaulding

Arts & Sciences Electronic Theses and Dissertations

Urinary tract infections (UTI) affect over 150 million individuals worldwide every year. These infections are associated with significant morbidity and have a sizeable economic impact, with $5 billion being spent on UTI treatment in the USA annually. Uropathogenic E. coli (UPEC) are responsible for 80% of community acquired UTIs and 65% of nosocomial UTI. The current standard of care for UTI is antibiotic therapy. However, 30-50% of women experience recurrent UTI (rUTI) despite receiving antibiotic therapy. The prevalence of single and multi-drug resistant UPEC strains has led to increased reliance on carbepenems, which are primarily reserved for multi-drug resistant infections, …

Mechanisms And Regulation Of Resection In Dna Damage Response, Sharad C. Paudyal

Arts & Sciences Electronic Theses and Dissertations

Deoxyribonucleic acid (DNA) encodes genetic information essential for cell survival and function. However, it is constantly under assault from endogenous and exogenous damaging agents that not only threaten our own survival but also affect the faithful transmission of genetic information to our offspring. Double-strand breaks (DSBs) are one of the most hazardous forms of DNA damage, which if unrepaired or improperly repaired could lead to plethora of systemic human diseases including cancer. To deal with this problem, cells have evolved with a mechanism called DNA damage response (DDR) to detect, signal, and repair the breaks by inducing multiple cellular events. …

Designing Epigenome Editing Tools To Understand The Functional Role Of Dna Methylation Changes In Cancer, James Mcdonald

Arts & Sciences Electronic Theses and Dissertations

DNA methylation is known to silence gene expression in the context of imprinting, X-chromosome inactivation, and retrotransposon silencing. However, the role of DNA methylation in silencing gene expression outside of these contexts is not fully understood. This is especially true in diseases such as cancer, where normal DNA methylation patterns are significantly altered. In breast cancer as well as nearly all cancer types, most of the genome loses DNA methylation while small regions of the genome gain methylation. DNA methylation generally correlates with decreased gene expression when present at a gene promoter. Therefore, these regions of hypo- and hyper-methylation may …

Effects Of Nucleosome Structure On Dna Photoproduct Formation And Deamination, Kesai Wang

Arts & Sciences Electronic Theses and Dissertations

Cyclobutane pyrimidine dimers (CPDs) are DNA photoproducts linked to skin cancer, whose mutagenicity depends in part on their frequency of formation and deamination. Nucleosomes modulate CPD formation, favoring outside facing sites, and disfavoring inward facing sites. A similar pattern of CPD formation in protein-free DNA loops suggest that DNA bending causes the modulation of photoproduct formation in nucleosomes. To systematically study the cause and effect of nucleosome structure on CPD formation and deamination, we had developed a circular permutation synthesis strategy for positioning a target sequence at different superhelix locations (SHLs) across a nucleosome in which the DNA has been …

Sequence Determinants Of The Individual And Collective Behaviour Of Intrinsically Disordered Proteins, Alexander S. Holehouse

Arts & Sciences Electronic Theses and Dissertations

Intrinsically disordered proteins and protein regions (IDPs) represent around thirty percent of the eukaryotic proteome. IDPs do not fold into a set three dimensional structure, but instead exist in an ensemble of inter-converting states. Despite being disordered, IDPs are decidedly not random; well-defined - albeit transient - local and long-range interactions give rise to an ensemble with distinct statistical biases over many length-scales. Among a variety of cellular roles, IDPs drive and modulate the formation of phase separated intracellular condensates, non-stoichiometric assemblies of protein and nucleic acid that serve many functions. In this work, we have explored how the amino …

Determining The Molecular Mechanisms Of Huntington’S Disease Through Multi-Scale Modeling, Kiersten Ruff

Arts & Sciences Electronic Theses and Dissertations

Huntington’s disease (HD) is associated with a mutational CAG repeat expansion within exon 1 of the huntingtin (Htt) gene. Post-transcriptional processing leads to the generation of N-terminal Htt protein fragments (Htt-NTFs), including those that encompass exon 1 (Httex1). Within Httex1, the CAG-repeat encoded polyglutamine (polyQ) tract is flanked N-terminally by a 17-residue amphipathic stretch (N17) and C-terminally by a 50-residue proline rich (PR) domain. Htt-NTFs, including Httex1, are among the smallest fragments that recapitulate HD pathology in mouse models. However, the direct link between Htt-NTFs with polyQ expansions and neurodegeneration that leads to HD remains unresolved. Despite being a monogenic …

Exploring Host-Virus Interactions In Caenorhabditis Nematodes, Kevin Chen

Arts & Sciences Electronic Theses and Dissertations

Caenorhabditis elegans is a powerful model organism that has elucidated many biological questions in the fields of genetics, development, and neurobiology. In addition, C. elegans has been used in the past decade to investigate host-pathogen interactions with bacteria and fungi. The recent identification of nematode viruses that naturally infect C. elegans and Caenorhabditis briggsae provides a unique opportunity to define host-virus interactions in these model hosts.

This dissertation first explored the transcriptional response of C. elegans and C. briggsae to virus infection by RNA-seq. I identified a total of 320 differentially expressed genes (DEGs) in C. elegans following Orsay virus …

Nuclear Export Factor 3 Regulates The Localization Of Small Nucleolar Rnas, Melissa Wanling Li

Arts & Sciences Electronic Theses and Dissertations

Metabolic diseases, such as obesity and diabetes, are associated with excess levels of lipids, which can lead to organelle dysfunction, cell death and eventually organ dysfunction. This process, termed lipotoxicity, is still not completely understood. In a genetic screen used to identify genes critical for lipotoxicity, the Schaffer lab has identified small nucleolar RNAs (snoRNAs) within the ribosomal protein L13a (Rpl13a) locus that mediate the cellular response to lipotoxic and general metabolic stress. These snoRNAs are non-canonical in that they accumulate in the cytosol after metabolic stresses like lipotoxicity and oxidative stress, suggesting that cells have specific mechanisms for regulating …

Dna Replication Challenges: Telomeres And R Loops, Shankar Parajuli

Arts & Sciences Electronic Theses and Dissertations

Faithful DNA replication and repair are essential for maintaining genome stability and preventing various diseases including cancer. Both processes are executed by numerous redundant mechanisms to ensure that these processes are uninterrupted even when a primary mechanism fails. Despite this, they are not immune to challenges and failures leading to DNA damage and genome instability. These problems are more evident at the difficult-to-replicate regions of the genome such as the telomeres that cap and protect linear chromosome ends. Additionally, topological structures such as RNA:DNA hybrids, commonly referred to as R loops, can also present severe challenges to the DNA replication …

Specificity Determination By Paralogous Winged Helix-Turn-Helix Transcription Factors, Adam Joyce

Arts & Sciences Electronic Theses and Dissertations

Transcription factors (TFs) localize to regulatory regions throughout the genome, where they exert physical or enzymatic control over the transcriptional machinery and regulate expression of target genes. Despite the substantial diversity of TFs found across all kingdoms of life, most belong to a relatively small number of structural families characterized by homologous DNA-binding domains (DBDs). In homologous DBDs, highly-conserved DNA-contacting residues define a characteristic ‘recognition potential’, or the limited sequence space containing high-affinity binding sites. Specificity-determining residues (SDRs) alter DNA binding preferences to further delineate this sequence space between homologous TFs, enabling functional divergence through the recognition of distinct genomic …

Robust Algorithms For Detecting Hidden Structure In Biological Data, Roman Sloutsky

Arts & Sciences Electronic Theses and Dissertations

Biological data, such as molecular abundance measurements and protein

sequences, harbor complex hidden structure that reflects its underlying

biological mechanisms. For example, high-throughput abundance measurements

provide a snapshot the global state of a living cell, while homologous

protein sequences encode the residue-level logic of the proteins' function

and provide a snapshot of the evolutionary trajectory of the protein family.

In this work I describe algorithmic approaches and analysis software I

developed for uncovering hidden structure in both kinds of data.

Clustering is an unsurpervised machine learning technique commonly used

to map the structure of data collected in high-throughput experiments,

such …

Antiviral Nucleoside Inhibitors Of Leishmania Rna Virus 1: Discovery And Mechanism, John Isaac Robinson

Arts & Sciences Electronic Theses and Dissertations

Some Leishmania parasites in the Viannia sub-genus are persistently infected with Leishmania RNA virus 1 (LRV1), a single-segmented double-stranded RNA virus belonging to the family Totiviridae. Infected parasites cause greater pathology and reach higher populations in mouse models of Leishmania infection. In human disease, LRV1+ parasites are correlated with increased frequency of treatment failure and relapse. Efficient methods to detect LRV1 and eliminate it from parasites are required to better understand the role of LRV1 in Leishmania infection. We optimized multiple techniques to measure LRV1 levels in parasites, most notably using flow cytometry to measure the amount of viral capsid …

Mass Spectrometry-Based Structural Proteomics: Methodology And Application Of Fast Photochemical Oxidation Of Proteins (Fpop), Ben Niu

Arts & Sciences Electronic Theses and Dissertations

The dissertation will be solely focused on using mass spectrometry to characterize protein high order structures (HOS), it emphasizes the use of hydroxyl radical footprinting (FPOP) coupled to bottom-up MS approach. A detailed background information about FPOP, and the corresponding method developments as well as applications will be covered.

The first chapter will be a comprehensive review regarding the FPOP. Following this, chapter 2, 3, and 4 will be focused on the method developments. Chapter 2 describes an isotope dilution GC-MS method to quantitate OH radicals in FPOP; chapter 3 describes the incorporation of Leu-enkephalin as reporter peptide for a …

The Impact Of The Mitochondrial Metabolism Of Induced Pluripotent Stem Cells Upon Differentiation, Stefanie T. Shahan

McKelvey School of Engineering Theses & Dissertations

Induced pluripotent stem cells (iPSCs) can be differentiated into any cell type found in the body. The derivation of a stem cell derived β cell (SC-β) capable of responding to glucose by secreting insulin was hugely significant for diabetes research and opened up the possibility of cell replacement therapy to combat this widespread disease (Pagliuca et al. 2014). The optimization of differentiation procedures such as this could improve yield, function, cost, and efficiency of a stem cell-derived product. Current approaches to improve differentiation are primarily focused on signal transduction pathways, while the metabolic state of the cells has received little …

Mechanisms Of G Protein Regulation By Rgs Proteins And Small Molecule Inhibitors, Stanley Michinobu Kanai

Arts & Sciences Electronic Theses and Dissertations

G protein coupled receptors transduce diverse extracellular signals like hormones, neurotransmitters, and photons to specific cellular responses through heterotrimeric G proteins. G proteins activate numerous effectors and signal transduction pathways, and therefore the regulation of G proteins is crucial for faithful propagation of specific cellular and physiological responses. A better understanding of the mechanisms that regulate G proteins should provide new insight into signaling pathways that govern healthy and disease states, and also provide opportunities for discovery of novel therapeutic targets.Regulator of G protein signaling (RGS) proteins are crucial regulators of G proteins, for they control amplitude and duration of …

Structural, Biophysical, And Functional Studies Of Trem2 In Neurodegenerative Disease, Daniel L. Kober

Arts & Sciences Electronic Theses and Dissertations

Alzheimer's disease (AD) and other neurodegenerative diseases present a large and growing challenge to global health. The immune system, particularly the innate immune system, is increasingly recognized as having a major role in these pathologies. The innate immune system is responsible to contain disease and promote healing. However, immune misregulation exacerbates disease. The innate immunomodulatory receptor Triggering receptor expressed on myeloid cells-2 (TREM2) is expressed on myeloid cells such as dendritic cells, macrophages, and in the brain, on microglia. TREM2 is a single-pass transmembrane receptor with an extracellular Ig domain that mediates ligand binding. This protein regulates inflammation in vitro …

The Rpl13a Snorna U33 Forms Novel Snornps During Lipotoxicity, Miquia Sherree Henderson

Arts & Sciences Electronic Theses and Dissertations

Lipid overload contributes to the pathogenesis of diabetic complications, causing tissue damage and cell death in a number of organ systems. This process is termed lipotoxicity. Animal and cell culture studies have demonstrated that oxidative stress and endoplasmic reticulum stress are major pathways engaged in the lipotoxic response. However, the molecular mechanisms of lipotoxicity are not well understood. A genetic screen revealed that small nucleolar RNAs (snoRNAs) encoded in the introns of the Rpl13a locus are critical for cell death in response to lipotoxicity. Initial studies have suggested that the Rpl13a snoRNAs function in this pathway through non-canonical modes of …

Disordered Proteins: Connecting Sequences To Emergent Properties, Tyler Scott Harmon

Arts & Sciences Electronic Theses and Dissertations

Many IDPs participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding …

Mass Spectrometry-Based Structural Analysis Of Photosynthetic Protein Assemblies, Yue Lu

Arts & Sciences Electronic Theses and Dissertations

This dissertation focuses on using mass spectrometry-based techniques to study photosynthetic protein assemblies. Photosynthesis is a process that converts light energy into chemical energy, the basis of most life on Earth. The two most crucial protein machineries involved in this process are reaction center and light harvesting complexes. They are usually giant protein complexes with different numbers of co-factors. In a more expanded sense, photosynthesis is not just about the utilization of solar energy, the regulation of light energy is also essential as excess light energy is detrimental to photosynthesis organisms. Again, protein assemblies play an indispensable role in this …

Mapping Analyte-Signal Relations In Lc-Ms Based Untargeted Metabolomics, Nathaniel Guy Mahieu

Arts & Sciences Electronic Theses and Dissertations

The goal of untargeted metabolomics is to profile metabolism by measuring as many metabolites as possible. A major advantage of the untargeted approach is the detection of unexpected or unknown metabolites. These metabolites have chemical structures, metabolic pathways, or cellular functions that have not been previously described. Hence, they represent exciting opportunities to advance our understanding of biology. This beneficial approach, however, also adds considerable complexity to the analysis of metabolomics data - an individual signal cannot be readily identified as a unique metabolite. As such, a major challenge faced by the untargeted metabolomic workflow is extracting the analyte content …

Biophysical Studies Of The Intracellular Domains Of The Egfr Family Of Receptor Tyrosine Kinases, Kwabena A N Sarpong

Arts & Sciences Electronic Theses and Dissertations

The epidermal growth factor receptor and its three orthologues, HER2, HER3 and HER4 have been the subject of intensive basic, clinical and translational research due to their involvement in cancers. These proteins are part of elaborate networks that interact with a myriad of other molecules to effect diverse signaling pathways and affect cellular processes such as migration, apoptosis, cell differentiation and so on. In addition to the formation of preformed dimers, it is well established that ligand engagement leads to receptor dimerization in all family members, except HER2. In the past decade, it has been shown that their intracellular domains …

Tissue Damage Quantification In Alzheimer's Disease Brain Via Magnetic Resonance Gradient Echo Plural Contrast Imaging (Gepci), Yue Zhao

Arts & Sciences Electronic Theses and Dissertations

Alzheimer’s disease (AD) affected approximately 48 million people worldwide in 2015. Its devastating consequences have stimulated an intense search for AD prevention and treatment. Clinically, AD is characterized by memory deficits and progressive cognitive impairment, leading to dementia. Over the past two to three decades, researchers have found that amyloidbeta (Aβ) plaques and neurofibrillary tau tangles occur during a long pre-symptomatic period (preclinical stage) before the onset of clinical symptoms. As a result, identification of the preclinical stage is essential for the initiation of prevention trials in asymptomatic individuals. Currently, Positron Emission Tomography (PET) imaging with injected 11C or 18F …

The Role Of Rna Interference In The Control Of Leishmania Rna Virus 1 Infection, Erin Acino Brettmann

Arts & Sciences Electronic Theses and Dissertations

The presence of Leishmania RNA virus 1 (LRV1) in parasites of the Leishmania (Viannia) subgenus increases the virulence of the parasite in mouse models of leishmaniasis and is correlated with treatment failure, relapse, and the development of mucocutaneous disease in humans. LRV1 is not shed or infectious; rather, the infection is persistent, and as yet it is unknown how the parasite controls virus levels. Many eukaryotic organisms use RNA interference (RNAi) to limit virus replication, and Leishmania (Viannia) parasites have an active RNAi pathway. To determine whether Leishmania are capable of using RNAi to control LRV1, we sequenced sRNAs from …

The Localization And Function Of Novel Tetrahymena Thermophila Cytoskeletal Genes Bbc29 And Bbc39, Emily K. Moore, Nicole C. Zanolli

Undergraduate Research Symposium Posters

Ciliary function is known to play an important role in many human conditions, including chronic sinus and pulmonary diseases and problems with infertility. Cilia are cytoskeletal structures that protrude from the cell body to facilitate movement. Ciliary structure is conserved throughout eukaryotes, from unicellular to multicellular organisms, including humans. A clear understanding the proteins that compose cilia and how they interact with one another will increase our knowledge about important cytoskeletal elements. Because cilia are difficult to study in multicellular organisms, the unicellular eukaryote Tetrahymena thermophila serves as a useful model for the study of cytoskeletal genes, due to their …