Life Sciences Commons^™

Rna Structure And Function: Biological Relevance In Neurodegenerative And Infectious Disease Pathogenesis, Joshua Imperatore

Electronic Theses and Dissertations

The studies outlined in this dissertation encompass a broad focus, relating to the pathogenesis of neurological disorders such as Alzheimer’s disease, fragile X syndrome, and amyotrophic lateral sclerosis, as well as a novel infectious disease, COVID-19. However, all four studies detailed within this dissertation contain similar elements, namely, the coordinated role of RNA structure and function in essential molecular mechanisms. The results of these investigations allow us to speculate on various disease- related mechanisms, including dysregulated microRNA processing pathways, nucleocytoplasmic shuttling via distinct RNA-protein interactions, and fine-tuned molecular switches controlling genomic dimerization and viral hijacking of host cellular microRNAs. Taken …

Examining The Function Of Protein Acyltransferase Via The Dhhc Domain Of The Paz5 Protein In The Organism Dictyostelium Discoideum, George M. Stuart-Ranchev

Electronic Theses and Dissertations

Protein S-palmitoylation plays a crucial role in many biological systems. S-palmitoylation involves the post-translational attachment of palmitate to a cysteine residue through a reversible thioester linkage. S-Palmitoylation is used to modify both integral and membrane proteins, many of which are involved in intracellular trafficking, membrane localization, and signal transduction pathways. Intracellular palmitoylation is mediated by a family of protein acyltransferases (PATs). PAT mutations are associated with neurological diseases and cancer progression. Proteins in the PAT family are defined by the presence of a 51-amino acid cysteine-rich domain (CRD), which contains a highly conserved aspartate-histidine-histidine-cysteine (DHHC) motif. The …

Live Cell Super-Resolution Microscopy Quanitifies An Interaction Between Influenza Hemagglutinin And Phosphatidylinositol 4,5-Bisphosphate, Jaqulin N. Wallace

Electronic Theses and Dissertations

Influenza virus, colloquially known as the flu, is an acute respiratory disease that infects several millions of individuals each year in the U.S. and kills tens of thousands of those infected. Yearly viral vaccines are widely available, however, due to the virus’s high mutation rate, their efficacy varies greatly. Due to the variability in vaccine efficiency against seasonal influenza, and the potential for even more pathogenic versions of influenza to emerge at any time, there is a high demand for a universal treatment option.

Influenza virus hijacks a variety of host cell components in order to replicate. The glycoprotein hemagglutinin …

Prevention Of Chronic Inflammation By Targeting Macrophage Integrin Adb2, Cady Forgey

Electronic Theses and Dissertations

Macrophage integrin a_Db₂ promotes macrophage retention and accumulation within inflamed tissue, a key event in development of chronic inflammation. Recently, the P5 peptide was identified as a specific inhibitor for integrin a_Db₂ interaction with 2-(ω-carboxyethyl) pyrole (CEP), a ligand at inflammatory sites. This thesis aims to identify integrin a_D I-domain amino acids involved in binding P5 peptide and likewise to CEP. We propose that non-conserved, basic amino acids of the integrin a_Db₂ I-domain are responsible for binding to P5 peptide and likewise to CEP. Eight amino acids were analyzed by …

Structural Characterization And Selective Drug Targeting Of Higher-Order Dna G-Quadruplex Systems., Robert Chandos Monsen

Electronic Theses and Dissertations

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to …

Investigating The Disorder And Compaction Of Designed Minielastin Using Nuclear Magnetic Resonance., Ma Faye Charmagne Aquino Carvajal

Electronic Theses and Dissertations

Minielastins are elastin-based proteins with alternating hydrophobic and cross-link modules similar to tropoelastin. Tropoelastin is the ~70 kDa soluble monomeric precursor of elastin. The extracellular matrix protein, elastin provides elasticity to tissues and organs such as lungs, arteries and ligaments. The elastic properties of natural elastin are believed to be entropic in origin. In vivo, the elastin matrix is approximately 50% water by weight. Without water, elastin is brittle and hard. Minielastins, like tropoelastin, undergo a liquid-liquid phase transition upon an increase in temperature. Factors such as hydrophobicity, chain length and concentration affect the coacervation temperature, T_c. The …

Triclosan Disrupts Immune Cell Function By Depressing Ca2+ Influx Via Acidification Of The Cytoplasm, Suraj Sangroula

Electronic Theses and Dissertations

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca²⁺ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca²⁺ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca²⁺ dynamics in mast cells, and here we show that TCS also inhibits …

Differential Expression Of Rna In The Rat Peripheral Nervous System Following Nerve Injury And Treatment With Pain-Relieving Celecoxib-Loaded Nanomedicine, Andrea Stevens

Electronic Theses and Dissertations

The neuroinflammatory response to peripheral nerve injury is associated with chronic pain and significant changes in the expression profiles of RNAs in neurons, glia and infiltrating immune cells: a neuro-immune triad. Chronic constriction injury (CCI) of the rat sciatic nerve provides an opportunity to mimic neuropathic injury and quantitatively assess behavior and differential gene expression in individual animals. Macrophages that phagocytose intravenously injected nanoemulsion carrying the non-steroidal anti-inflammatory, NSAID, Celecoxib, naturally accumulate at the site of injury resulting in relief of CCI behavioral hyper-sensitivity. It is not known beyond the inhibition of cyclooxygenase-2 (COX-2) activity and the reduction in prostaglandin …

The Relationship Between Cell-Free Dna And Resistance Training, Henry Lang

Electronic Theses and Dissertations

The primary purposes of this dissertation were to explore relationship between cell free DNA (cf-DNA), creatine kinase (CK), C-reactive protein (CRP), vertical jump testing delayed onset muscle soreness (DOMS) in response to a high-volume resistance training protocol, and to assess the sensitivity of cf-DNA to different resistance training volume loads. The secondary purpose was to examine the relationship between cf-DNA and relative strength. Study 1 was an exploratory attempt to discover relationships between cf-DNA, CK, CRP, delayed onset muscle soreness, and performance variables. Seventeen resistance trained males were recruited, 9 were randomly assigned to receive BCAAs while 8 received a …

Testing The Potential Of Environmental Dna: Genetic Monitoring Of Estuarine And Coastal Bottlenose Dolphin (Tursiops Truncatus) Stocks In South Carolina, Kathryn Greiner-Ferris

Electronic Theses and Dissertations

Environmental DNA (eDNA) analysis is a non-invasive monitoring technique that can detect and potentially monitor elusive marine mammals. To date, the majority of eDNA studies have been performed in freshwater environments, partially due to methodological challenges posed by higher salinities and increased dilution effects of large water masses in marine environments. The objective of this study was to design and optimize species-specific oligonucleotide PCR primers to accurately detect and quantify common bottlenose dolphin (Tursiops truncatus) eDNA collected from the marine environment and to evaluate potential trends between eDNA concentration and dolphin abundance and seasonality. Primer pairs were designed to target …

The Physiology And Evolution Of Selenite Respiration In Bacteria, Michael Wells

Electronic Theses and Dissertations

The selenium oxyanions selenate (Se(VI)) and selenite (Se(IV)) can be utilized by some bacteria and archaea as terminal electron acceptors in anaerobic respiration. Se(VI) and Se(IV) respiration is mediated by a phylogenetically and ecologically diverse array of organisms, suggesting that selenium respiration is ubiquitous in natural environments. Several respiratory Se(VI) reductases have been characterized in bacteria, revealing that Se(VI) respiration has evolved independently several times in this domain. Se(IV) respiration, in contrast, has yet to be characterized. I have purified and characterized the first respiratory Se(IV) reductase from Bacillus selenitireducens MLS10. The Se(IV) reductase appeared to purify as a single …

The Retinoblastoma Protein Mediates Metabolic Reprogramming In Lung Cancer., Lindsey R. Conroy

Electronic Theses and Dissertations

Lung cancer is among the most frequently diagnosed cancers and is the leading cause of cancer-related deaths worldwide. One of the hallmark events in lung cancer pathogenesis is deregulation of the cell cycle. The retinoblastoma protein (pRb) is a tumor suppressor that is deleted, mutated, or inactivated in most lung cancer cases. Canonically, pRb functions to regulate cell cycle progression by repressing the transcriptional activity of the E2F family of transcription factors, inhibiting S phase entry. Although the cell cycle functions of pRb have been well established, recent studies have highlighted a functional role for pRb in controlling cellular metabolism. …

Quantification Of Interactions Between Influenza Hemagglutinin And Host Cell Phosphoinositides By Super-Resolution Microscopy, Matthew T. Parent

Electronic Theses and Dissertations

The influenza viral membrane protein hemagglutinin (HA) forms dense nanoscale clusters on host cell plasma membranes (PM), but the mechanisms that direct HA clustering are not well understood. Previous studies have observed HA associated with actin rich regions of the PM, but there are no known direct interactions between HA and actin. Phosphatidylinositol 4,5-biphosphate (PIP2) is a signaling lipid in the PM which can regulate the actin cytoskeleton, and actin comets initiated by PIP2 are known to be exploited by HA to reach the PM of infected cells. PIP2 is also used by other viruses, such as HIV and Ebola, …

Protection Against Atherosclerosis By A Non-Native Pentameric Crp That Shares Its Ligand Recognition Functions With An Evolutionarily Distant Crp, Asmita Pathak

Electronic Theses and Dissertations

C-reactive protein (CRP) is an acute phase protein of the innate immune system that has been evolutionarily conserved. Human CRP is known to exist in two different pentameric conformations; native CRP and non-native CRP that possess differential ligand recognition functions. The structure of CRP evolved from arthropods to humans, in terms of subunit composition, disulfide bonds, and glycosylation pattern. Along with change in structure, the gene expression pattern of CRP also evolved from a constitutive protein in lower invertebrates to an acute phase protein in humans. The objective of this study was to determine the function of a non-native pentameric …

The Role Of Slc7a11 In Controlling Extracellular And Intracellular Redox Environments Of Lung Fibroblasts - Potential Targets For Intervention In Aging And Idiopathic Pulmonary Fibrosis., Yuxuan Zheng

Electronic Theses and Dissertations

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition by fibroblasts. Aging and oxidative stress increase the susceptibility to IPF. Redox couples, cysteine/cystine (Cys/CySS) and glutathione/glutathione disulfide (GSH/GSSG), and their redox potentials (E_h) quantify oxidative stress. Fibroblasts from old mice maintain more oxidized extracellular E_h(Cys/CySS) than young mice. Microarray shows down-regulation of Slc7a11 potentially mediates this age-related oxidation. Slc7a11 is the key component of system X_c^-, an antiporter that imports CySS and exports glutamate. The first aim of this dissertation is to investigate the mechanistic link between Slc7a11 …

Understanding The Implications Of Anandamide, An Endocannabinoid In An Early Land Plant, Physcomitrella Patens, Md Imdadul Haq

Electronic Theses and Dissertations

Endocannabinoid signaling is well studied in mammals and known to be involved in numerous pathological and physiological processes. Fatty acid amide hydrolase (FAAH) terminates endocannabinoid signaling in mammals. In Physcomitrella patens, we identified nine orthologs of FAAH (PpFAAH1 to PpFAAH9) with the characteristic catalytic triad and amidase signature sequence. Kinetics of PpFAAH1 showed specificity towards anandamide (AEA) at 37°C and pH 8.0. Further biophysical and bioinformatic analyses revealed that, structurally, PpFAAH1 to PpFAAH4 were closely associated to the plant FAAH whereas PpFAAH6 to PpFAAH9 were more closely associated to the animal FAAH. A substrate entry gate or ‘dynamic paddle’ …

A Novel Mode Of Action Of C-Reactive Protein In Protecting Against Streptococcus Pneumoniae Infection And Synergy With Antibiotics, Donald Ngwa

Electronic Theses and Dissertations

C-reactive protein (CRP) is a part of the innate immune system, is synthesized in the liver, its blood level increases in inflammatory states, and it binds to Streptococcus pneumoniae. The conformation of CRP is altered under conditions mimicking an inflammatory milieu and this non-native CRP also binds to immobilized/aggregated/pathogenic proteins. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection. For protection, CRP must be injected into mice within two hours of administering pneumococci, thus, CRP is protective against early-stage infection but not against late-stage infection. It is unknown how CRP protects …

Examining Artifacts Of The Watershed Segmentation, Emily Jo Armitage

Electronic Theses and Dissertations

The watershed segmentation is an algorithm used to systematically track cell intercalary behaviors during germ band extension of the Drosophila embryo. Neighboring cells share a contracting vertical interface, called a T1, which continues contracting to a single point, a T2, and extending in the horizontal direction to create what is called a T3 interface (Fig. 1). Additionally, higher order vertices called rosettes occur when five or more cells meet at a common vertex. Simulated T2 events demonstrate that cell angle and not noise level in the image contributes to the incorrect detection of artifactual T1s in more acute angled cells …

Cellular And Developmental Insights Into The Early Evolution Of Muscle, Jeffrey J. Colgren

Electronic Theses and Dissertations

Whereas a great deal has been learned about the molecular underpinnings of morphological evolution in animals, much less is known about the origin of novel cell and tissue types. During the time in which the earliest animal lineages were diversifying, fundamental cell and tissue types, such as muscles, arose. Sponges are one of two animal lineages that lack muscles, yet they undergo coordinated full body contractions. Whereas the signaling processes have been studied, the physical mechanisms of contraction are completely uncharacterized. The main purpose of this work is to understand the primary contractile tissue of the sponge Ephydatia muelleri, …

Characterization Of The Dimerization And Salt Dependent Aggregation Of The Als-Linked Protein Tdp-43, Elizabeth Ehrhardt

Electronic Theses and Dissertations

Trans-active response (TAR) DNA-binding protein 43 (TDP-43) is essential for RNA processing but can also form toxic cytoplasmic inclusions in neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA-binding has been shown to have the potential to decrease or inhibit the aggregation of TDP-43, indicating that supplying RNA therapeutics may be a solution to treat these neurodegenerative disorders. However, the mechanism of aggregation, transitioning from TDP-43’s native dimeric state to small oligomers to toxic aggregates, is still relatively unknown. This needs to be established before determining how to target and disrupt this aggregation. Using GFP-TDP-43, …

Quantification Of Mitochondrial Zinc Homeostasis And Analysis Of Zinc And Polyamine-Mediated Axonal Trafficking, Dylan Hunt Fudge

Electronic Theses and Dissertations

The zinc ion is an important emerging signaling molecule for biological processes. In this work we engineered improved zinc sensors based on our previously developed fluorescent sensor GZnP1 to provide sensors with a higher fluorescent readout, faster kinetics, and a superior sensitivity to zinc. We utilized these zinc sensors and further developed the sensors to answer questions pertaining to biological zinc. We showed the labile zinc concentration in the mitochondrial matrix was less than 1 pM while the labile zinc concentration in the cytosol and mitochondrial IMS were comparable (~100 pM) across four different cell lines. Using these zinc sensors …

Sorting Of Cargo Proteins Within The Regulated Secretory Pathway: The Peripheral Membrane Protein Hid-1 As A Sorting And Vesicle Biogenesis Factor, Blake H. Hummer

Electronic Theses and Dissertations

Large dense core vesicles (LDCVs) form at the trans-Golgi network (TGN) and mediate the regulated release of neuropeptides and peptide hormones. Despite their central role to physiology, the mechanisms controlling biogenesis and sorting to LDCVs is not well understood. Optimizing the retention using selective hooks (RUSH) method in neuroendocrine cells, we show it is possible to visualize sorting to the constitutive and regulated secretory pathways in real-time and that the bulk of transmembrane LDCV cargoes do not sort directly onto LDCVs, but exit the TGN into non-regulated vesicles to be incorporated to LDCVs at a later step. Additionally, we characterize …

Quantification Of Dynamic Epithelial Sheet Architecture In Botryllus Schlosseri Using 2-D & 3-D Image Analysis, Roopa Madhu

Electronic Theses and Dissertations

Epithelial tubules form critical structures in various body tissues; how- ever, since they are difficult to access experimentally, their architecture and dynamics are not well understood. Here we examine the dynamic remodeling of epithelial tubes in vivo using a novel and uniquely accessible model system: the extracorporeal vasculature of Botryllus schlosseri (sea squirt). In Botryllus, massive retraction of blood vessels can be triggered without loss of barrier function, through (i) disrupting collagen crosslinking in the basement membrane using β-aminopropionitrile (BAPN); or (ii) disrupting the integrin pathway through inhibition of focal adhesion kinase (FAK). We performed stereographic projections of 3-dimensional …

Cellular And Organismal Ramifications Of De Novo Purine Synthesis Dysregulation, Randall Craig Mazzarino

Electronic Theses and Dissertations

Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, …

Rab39 And Klp98a Are Required For Furrow Formation During Early Drosophila Embryogenesis, Megan R. Millage

Electronic Theses and Dissertations

The formation of a plasma membrane furrow is an essential process during development. Furrow formation is necessary for successful cell division and cytokinesis in addition to the ability to create multicellular tissues. Here, I will explore the role of the Golgi-associated Rab protein Rab39 in furrow formation during early Drosophila embryogenesis. Rab39 is one of eight Rab proteins that has been shown to localize to discrete puncta by live imaging in early Drosophila embryos, but its function and pathway have not been well characterized. In this thesis will I show that Rab39 forms dynamic, tubular structures that colocalize with trans-Golgi …

Profiling Transcription And Retrotransposition Of Mouse L1 Subfamilies, Lingqi Kong

Electronic Theses and Dissertations

Transposable elements, also called jumping genes, comprise almost 45% of the human genome. In contrast, only 1% of the human genome is protein-coding sequences. The function and advantages of maintaining such massive copies of transposable elements in the human genome are still unclear. Long interspersed element 1 (L1), the most substantial group and the only active autonomous transposable element in the human genome, has revealed its unique roles in many diseases. The insertional mutagenesis induced by L1 retrotransposition events could threaten human genomic stability and generate unexpected mutations. L1 overexpression has been documented in both somatic and germline cells and, …

Analysis Of Coevolving Residues Of Xcdxcdx-Phd, A Distinct Type Of Phd-Finger, Shraddha Basu

Electronic Theses and Dissertations

Plant homeodomain (PHD), a Zinc-finger scaffold, is a conserved protein module in eukaryotes that typically recognizes unstructured histone tails, and thus, PHDs play a crucial role in chromatin signaling. The sequences of Zinc-fingers, in general, diversify during the course of evolution often giving rise to subtypes (e.g., RBR-RING (Dove 2017) or zf-CxxC subtypes (Long 2013) who typically contain specific sequence signatures. We recently discovered that PHD fingers also contain a distinct subtype, namely the xCDxCDx-PHD. xCDxCDx-PHD has a distinct composition of specific amino acids that coevolved (coevolving residues) in the course of evolution, and xCDxCDx-PHD also shows a unique mechanism …

Genetic Study Of Alkane Production And Oxic Nitrogen Fixation In Anabaena Sp. Pcc 7120, Jaimie L. Gibbons

Electronic Theses and Dissertations

Anabaena sp. PCC 7120 is a filamentous, nitrogen-fixing cyanobacterium that uses spatial separation to perform photosynthesis and nitrogen fixation simultaneously. Under fixed nitrogen limiting conditions, Anabaena 7120 forms specialized cells, called heterocysts, to fix nitrogen. In this research, I sought to add to the knowledge surrounding the process of heterocystous nitrogen fixation in Anabaena 7120. Cyanobacteria universally produce alkanes, which have been suggested to play a role in helping the organism adapt to abiotic stress, such as diazotrophic conditions. In my first study, I sought to identify the genes required for production of the hydrocarbon heptadecane (C17H36). Through using a …

Molecular Facet Of Host-Pathogen Interactions In Fusarium Head Blight In Wheat ( Triticum Aestivum L.), Bimal Paudel

Electronic Theses and Dissertations

Fusarium head blight (FHB) is a severe disease of wheat (Triticum aestivum L.). It not only reduces the quantity of the harvested grains but also decreases the grain quality due to mycotoxins contamination, especially Deoxynivalenol (DON). Qfhb1 (or simply called Fhb1) is the most important quantitative trait locus (QTL) for FHB resistance. Our lab has previously identified wheat gene WFhb1-1 (aka. Wfhb1_c1) as a candidate for FHB resistance gene. Here we report that WFhb1-1 has been cloned. The gene (GenBank # KU304333.1) consists of a single exon, encoding a putative membrane protein of 127 amino acids. WFhb1-1 protein produced in …

Scavenger Hunt: The Search For Sulfur Mustard-Neutralizing Compound In 2-Chloroethyl Ethyl Sulfide (Cees) Treated Human Keratinocyte (Hacat) Epithelial Cells, Tanner Diemer

Electronic Theses and Dissertations

Sulfur mustard is a highly toxic and dangerous vesicant that has been utilized as a chemical warfare agent (CWA) since World War I. Despite its extensive history, an effective antidote to sulfur mustard exposure still does not exist. With detectable levels of unreacted sulfur mustard lasting days after initial contact, a window of opportunity exists to administer a “scavenger” to affected individual immediately and in the days following exposure in hopes of reducing harm by neutralizing unreacted sulfur mustard. For this strategy to be effectively implemented, it is essential to identify a candidate compound with excellent affinity towards sulfur mustard …