Molecular Biology Commons^™

Centrosomal And Non-Centrosomal Microtubule Networks Coordinate To Drive Nuclear Dynamics In Developing Epithelium, Rashmi Budhathoki

Electronic Theses and Dissertations

The morphogenesis of developing tissues relies on extensive cellular rearrangements in shape, position, and identity. A key process in reshaping tissues is cell intercalation-driven elongation, where epithelial cells align and intercalate along a common axis. Typically, analyses focus on how peripheral cortical forces influence cell shape changes. Less attention is given to how inhomogeneities in internal structures, particularly the nucleus, impact cell shaping. Here, we examine how pulsed contractile and extension dynamics interact with the nucleus in elongating Drosophila embryos. Our data show that tightly packed nuclei in apical layers hinder tissue remodeling/oscillatory behaviors. We identify two mechanisms for resolving …

Role Of Nucleic Acids As Chaperones In Protein Folding, Zijue Huang

Electronic Theses and Dissertations

Many proteins have slow folding times in vitro that are physiologically untenable. To combat this challenge, ATP-dependent chaperonins are thought to possess the unique ability to catalyze protein folding. Performing quantitative model selection using protein folding and unfolding data, we here show that short nucleic acids containing Gquadruplex (G4) structure can also catalyze protein folding. Performing the experiments as a function of temperature demonstrates that the G4 reshapes the underlying driving forces of protein folding. To understand the structural basis of this catalytic activity, we introduce NMR method to solve the structures, at base-level resolution, of a multiconformer G4 with …

Effect Of Histidine Tag On The Enzymatic Activity Of Pseudolysin And Its Interaction With The Streptomyces Metalloprotease Inhibitor (Smpi) Protein, Adewale Adeboye Adewole

Electronic Theses and Dissertations

Pseudolysin (PLN) protein is a major virulent factor in Pseudomonas aeruginosa infection. PLN belongs to the family of zinc-dependent metalloproteases. It is naturally inhibited by a smaller protein called streptomyces metalloprotease inhibitor (SMPI), secreted by Streptomyces nigrescens. Previous and current studies demonstrate that the interaction between PLN and SMPI offers unique mechanistic insights for drug development against infections caused by P. aeruginosa. This study investigated the effect of N-terminal Histidine (His) tag on the enzymatic activity of PLN and its interaction with SMPI. His-tagged PLN and SMPI proteins were expressed in bacteria and purified by affinity chromatography …

Analysis Of An Ethanol Sensitive Bmp-Nkx2.3-Fgf Signaling Pathway In Pouch Morphogenesis., Hieu Dai Le Vo

Electronic Theses and Dissertations

Craniofacial malformations lie at the heart of Fetal Alcohol Spectrum Disorders (FASD). While there is growing evidence for a genetic component to FASD, little is known of the cellular mechanisms underlying these ethanol-sensitive loci in facial development. Bone Morphogenetic Protein (Bmp) signaling pathway dependent pouch formation is a key mechanism in facial development. We have previously shown that multiple Bmp mutants are sensitized to ethanol-induced facial defects. However, ethanol does not directly impact Bmp signaling. This suggests that downstream effectors, like nkx2.3 and Fibroblast Growth Factor (Fgf) signaling, may mediate the impact of ethanol on Bmp mutants. Here, I …

Metabolic Regulation Of Cardiac Fibroblast Phenotype And Function., Collin Kleis Wells

Electronic Theses and Dissertations

Cardiac fibroblasts are central effectors of cardiac repair after myocardial infarction (MI). In response to signaling cues, they differentiate to a range of phenotypes with robust capacities to synthesize and secrete extracellular matrix (ECM) and signaling molecules. Although activated fibroblast phenotypes are associated with pronounced changes in metabolism, it remains unclear how the metabolic network upholds the effector functions of fibroblasts in the post-infarcted heart. Here, we identified that critical enzymes in the phosphoenolpyruvate (PEP) cycle, i.e. pyruvate kinase muscle-2 (PKM2) and phosphoenolpyruvate carboxykinase-2 (PCK2), are elevated in the heart after MI and examined their role in regulating post-MI remodeling …

Investigation Into Mycorrhizae Associated With Native Venus' Flytrap Root Tissue, Anna Carnaggio

Electronic Theses and Dissertations

Dionaea muscipula, a plant commonly known as the Venus' flytrap, is found native to the southeast corner of North Carolina and the northeast corner of South Carolina. Several populations of native Venus' flytraps are known to exist in the nutrient-poor peat bog conditions in and around Lewis Ocean Bay Heritage Preserve (LOBHP) in Northeastern South Carolina. Anthropogenic pressures in Horry County, specifically surrounding LOBHP, have resulted in a loss of habitat for several native and endangered species. As a result, conservation efforts are focused on relocating native Venus' flytraps into adjacent habitats to ensure survival. This is only a temporary …

Discovery Of Photosynthetic Oxic N2-Fixation In Cyanobacteria Using Wet Lab And Machine Learning Approaches, James A. Young Iii

Electronic Theses and Dissertations

No abstract provided.

Interactions Of The Sars-Cov-2 Viral Genome 3’-Untranslated Region With Viral And Host Rnas, Caleb Frye, Mihaela Rita Mihailescu

Electronic Theses and Dissertations

This dissertation focuses on the characterization of RNA-RNA interactions within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and with host microRNAs. As the causative agent of coronavirus disease 2019 (COVID-19), SARS-CoV-2 has evolved rapidly since its appearance. This has warranted prompt characterization of the virus particularly of its single stranded RNA (ssRNA) genome. By using a combination of bioinformatics, biophysics, and/or biological assays, we analyzed the SARS-CoV-2 viral genomic RNA and uncovered interactions of genomic RNA with host RNAs, highlighting an underutilized method of targeting RNA viruses. We showed here that the conserved elements in the viral genomic …

Dysfunction Of Polyamine Metabolism In Down Syndrome Related To Cellular Senescence And Β-Amyloid Production, Andres D. Sola

Electronic Theses and Dissertations

Polyamines (PA) are ubiquitous in cells and highly involved in several important cellular functions and thus the dysregulation of their biosynthetic pathway is potentially associated with the progression of age-related diseases like Alzheimer’s’ disease (AD). Though there is research describing how PA content changes in organisms as they age or in those experiencing rapid aging from increased rates of senescence, as in Down syndrome (DS), it is not clear how this connection occurs and whether modulating this pathway may reveal downstream effects on related disease progression. This study examines how the modulation of the PA biosynthetic pathway by inhibition of …

Unraveling The Role Of Hid-1: Insights Into Sgii Regulation, Breanna L. Sellers

Electronic Theses and Dissertations

The regulated secretory pathway is important for the secretion of hormones and neuropeptides from large dense core vesicles (LDCVs). Dysregulation of the biogenesis of LDCVs can result in metabolic and neurological diseases. Recent studies have suggested that the peripheral membrane protein HID-1 plays a role in the sorting and storage of granins which contribute to LDCV biogenesis. To further understand how HID-1 influences the regulation of granins, we investigated potential HID-1 interactors. We determined that while HID-1 is likely functioning in the same pathway as USP25, a deubiquitinating enzyme (DUB), the cause of reduced cellular SgII levels is likely not …

Sequence Specificity Of Non-Structural Protein 15 From Sars-Cov-2, John Vincent Mcguire

Electronic Theses and Dissertations

The SARS-CoV-2 virus was recently able to cause a global pandemic, largely due to the asymptomatic period that occurs during initial infection. During this period, patients are infectious but do not exhibit symptoms, resulting in the unintentional spread of the virus. This occurs because the virus effectively avoids detection by the immune system during the early stages of infection, and as symptoms are the result of immune system activation, they never manifest. Non-structural Protein 15 (Nsp15) is one of the tools the virus uses to remain undetected, via its degradation of Pathogen-Associated Molecular Patterns (PAMPs) that would otherwise be recognized …

Furthering Ascidian Taxonomy Using Molecular Biology, Nicholas Gulnick

Electronic Theses and Dissertations

Ascidians are our closest invertebrate relatives and comprise nearly 3,000 species separated into three orders: Aplousobranchia (most speciose), Stolidobranchia, and Phlebobranchia (least speciose). Ascidians can be classified as either solitary or colonial organisms. Species delimitation using morphological characters alone has had varied results. Well known, widely distributed, morphological species have turned out to be catch all species comprised of several cryptic species. Molecular markers can help mitigate some of the issues presented by strictly using morphological observations, including resolving the status of cryptic species, and accessing the expert knowledge required to identify a species. By incorporating molecular markers and pairing …

Unraveling The Complex Dynamics Of Cardiac Fibrosis: Ccn1 As A Novel Regulator Of Scar Collagen Architecture And Structural Integrity In Myocardial Infarction., Annalara Garland Fischer

Electronic Theses and Dissertations

Expansion of the extracellular matrix (ECM), known as fibrosis, accompanies nearly all forms of cardiac disease; however, its most significant and complex manifestations arise following a myocardial infarction (MI). This process, principally led by fibroblasts, constitutes a necessary and adaptive response acutely to fill the void from cardiomyocyte necrosis following ischemic injury due to the heart’s lack of regenerative capacity. This acute response never fully resolves and contributes to ongoing collagen accumulation in non-infarcted regions, leading to diminished ventricular compliance, ongoing myocyte loss, and attrition in function—culminating in heart failure. Further, recent research demonstrates that concomitant with progressive increases in …

The Role Of Edc3 In Drosophila Melanogaster Neuronal Development, Keelan Zius

Electronic Theses and Dissertations

Enhancer of mRNA-decay 3 (Edc3) is a component of multiple decapping complexes that regulates the transcriptome by targeting a subset of transcripts for decapping for 5’-to-3’ messenger RNA (mRNA) decay. A missense mutation in the conserved Lsm domain of Edc3 has been reported in two siblings presenting with mild, non-syndromic intellectual disability (ID), however, the role of Edc3 is neuronal development has never been evaluated. Here, we use the Drosophila melanogaster model system to investigate the effects of Edc3 disruption on neuronal development. We found Edc3 reduction leads to significant hyperplasia at the larval neuromuscular junction (NMJ), providing the first …

Morphological Remodeling Of The Endoplasmic Reticulum During Mitosis And Its Influence On The Integrity Of The Spindle Apparatus, Katie R. Rollins

Electronic Theses and Dissertations

The Endoplasmic Reticulum (ER) is the largest organelle in the cell by surface area and forms contacts with other organelles throughout the cell. In recent work, the ER has been implicated in numerous processes within the cell beyond its canonical roles of protein synthesis and calcium storage. The ER also undergoes a remarkable transition in morphology during cell division, with mitotic divisions requiring a massive remodeling of various membranous organelles such as the nuclear envelope and Golgi. However, whether changes in ER behaviors modulate mitotic events is less clear. The rapid cleavage divisions in the early Drosophila embryo are a …

The Effect Of Novel Microtubule Modulators On Experimental Lewy Body Disease, Anuj S. Jamenis

Electronic Theses and Dissertations

In Lewy body disorders, α-synuclein aggregates are believed to destabilize the microtubular framework. However, microtubules are a challenging therapeutic target, as excessive rigidity or flexibility afforded by microtubule modulators may be neurotoxic. Thus, the objective of the present study was to evaluate dual-acting microtubule- stabilizing/destabilizing agents in cellular and animal models of early-stage Lewy body disease.

In Aim 1 we tested the dual-acting tricylic pyrimido[4,5-b]indole analogs AG161-47 and AG161-41 in an in vitro preformed fibril induced model of Lewy body disease. We found that both compounds reduced preformed fibril-seeded α-synucleinopathy without toxicity in primary hippocampal cultures. AG161-47 subtly impacted microtubule …

Omani Camels From A Cultural And Genomics Perspective, Al Muatasim Al Zadjali

Electronic Theses and Dissertations

The Dromedarian camel, Camelus dromedarius, is native to the Arabian Peninsula, including the Sultanate of Oman. These camels are used for food, milk, as well as show and racing competitions. Despite their economic and cultural importance research on camels in Oman is limited. The goal of this study was to examine their genomic variation, relationship with camels in other parts of the Arabian Peninsula, and to determine if selective breeding has led to the establishment of distinct breeds in Oman. Information was compiled from multiple sources to produce a comprehensive review on the breeding, management, economic and cultural use, …

Impact Of Glycerophosphocholine Metabolism On Phenotypes In Saccharomyces Cerevisiae And Schizosaccharomyces Pombe, Victoria Hrach

Electronic Theses and Dissertations

Glycerophosphocholine (GPC) is an abundant metabolite in eukaryotes. In baker’s yeast, Saccharomyces cerevisiae, GPC can be produced via deacylation of the major membrane glycerophospholipid phosphatidylcholine (PC). My lab has identified a novel pathway to re-synthesize PC from GPC by acylating it at the sn-1 and sn-2 positions; this pathway has been termed the PC Deacylation-Reacylation Pathway (PC-DRP). We previously characterized the acyltransferase Gpc1 and its ability to synthesize one-tailed lyso-PC from GPC, as well as that noting that GPC1 is upregulated in inositol-free conditions and results in more saturated PC. Here, I add to this basic characterization by establishing …

Advancing Biological Applications Through Microfluidic-Based Tool Development, K.M. Taufiqur Rahman

Electronic Theses and Dissertations

This research undertook an interdisciplinary approach, integrating bioengineering, microbiology, molecular biology, and systems biology to investigate bacterial dynamics behavior. Specifically, it delved into the development of microfluidic devices for biological applications such as bacterial cell counts, real-time observation of plant roots (here, specialized lectin-coated microbeads are used that mimic root characteristics), and soil microbe interactions. Furthermore, Next-Generation Sequencing and systems biology methodologies were employed to explore the intricate, multifaceted survival mechanisms of Escherichia coli persister population. Studying and quantifying persisters or testing for the existence of VBNC (viable but nonculturable) is challenging. These experiments require precise counts. It has been …

Flavonol Glucosylation: A Structural Investigation Of The Flavonol Specific 3-O Glucosyltransferase Cp3gt, Aaron S. Birchfield

Electronic Theses and Dissertations

Flavonoid glycosyltransferases (GTs), enzymes integral to plant ecological responses and human pharmacology, necessitate rigorous structural elucidation to decipher their mechanistic function and substrate specificity, particularly given their role in the biotransformation of diverse pharmacological agents and natural products. This investigation delved into a comprehensive exploration of the flavonol 3-O GT from Citrus paradisi (Cp3GT), scrutinizing the impact of a c-terminal c-myc/6x histidine tag on its enzymatic activity and substrate specificity, and successfully achieving its purification to apparent homogeneity. This established a strong foundation for potential future crystallographic and other structure/function analyses. Through the strategic implementation of site-directed mutagenesis, a thrombin …

The Interplay Between Lung Adenocarcinoma, Nutrient Availability, And The Microbiome., Alexis A. Vega

Electronic Theses and Dissertations

Lung cancer is one of the most common forms of cancer diagnosed, and has the highest lethality rate across all other forms of cancer in the U.S. While current therapeutic options include chemo-, immuno-, and radio-therapy, the benefits of caloric or nutrient restriction on cancer cells has also been investigated. Due to the many biological functions associated with methionine, many have proposed a methionine restricted diet would lead to favorable outcomes when combating cancer. Although our cells are incapable of synthesizing methionine, the bacteria found in our microbiome can. Furthermore, recent discoveries suggest an independent microbiome found within tumors that …

Spatial Structure Formation By The Post-Transcriptional Gene Regulator Rsme In Pseudomonas Fluorescens Pf0-1, Anton Evans

Electronic Theses and Dissertations

Microorganisms are often found in microbial communities we call biofilms. Organisms living in these crowded environments have significant evolutionary pressure to retain access to the resources necessary to sustain life. My research uses the bacterium Pseudomonas fluorescens Pf0-1 to study how organisms evolve strategies to solve this crowding problem as aging colonies repeatedly generate mutant patches. These mutants expand the reach of the colony resulting in decreased local density as they push themselves up to the resource rich surface. These spatial structures result from social interactions between the mutant and the parental cells mediated through extracellular secretions, resulting in the …

Multiphasic Molecular Mechanisms Regulate Expression Of Interleukin 1Β, As Well As Other Immediate-Early Genes, In Monocytes And Macrophages, David Macar

Electronic Theses and Dissertations

Interleukin 1β (IL-1β), an important cytokine in both health and disease, functions as a major proinflammatory mediator of pathological challenges, including involvement in the cytokine storm associated with Macrophage Activation Syndrome, Hemophagocytic Lymphohistiocytosis and the severe secondary effects of SARS-CoV-2 infection. Therapeutic approaches to limiting the effects of IL-1β protein have focused either on specific protein neutralization before release from activated macrophages (by general metabolic inhibitors and inflammasome inhibition) or after release by ligand and receptor obstruction (e.g., Canakinumab, Anakinra and Rilonacept). Although these approaches are efficacious, they inhibit steps after gene expression. Since a single gene expresses …

Polar Body Membrane Breakdown Occurs Independent Of Macroautophagy In C. Elegans, Shruti Kolli

Electronic Theses and Dissertations

Failure to digest cell corpses can lead to lupus-like autoimmune disorders in mammals. To better understand if LC3-associated phagocytosis (LAP) drives cell corpse breakdown in phagolysosomes, we studied the clearance of the C. elegans polar body during embryonic development. ATG16L1 has separable roles in regulating LC3 recruitment during macroautophagy and LAP. We demonstrated that the ATG16L1 C. elegans orthologs, ATG-16.1 and ATG-16.2, function redundantly to promote polar body membrane breakdown. We also discovered that truncating the LAP-specific WD40 domain of ATG-16.2 unexpectedly disrupts autophagy. Furthermore, we confirmed that polar body membrane breakdown occurs independent of macroautophagy. We also showed that …

Epitranscriptomic Regulation In Breast Cancer And Pcb-Induced Liver Disease., Belinda Petri

Electronic Theses and Dissertations

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, …

Met Alterations In Glioblastoma: Characterization Of Patient-Derived Xenografts And Therapeutic Strategies, Anna Musket

Electronic Theses and Dissertations

Glioblastoma is the most commonly diagnosed central nervous system primary malignancy and it is considered a terminal diagnosis with few treatment options available. Glioblastoma tumors frequently develop treatment resistance due in part to their highly heterogenic nature. The heterogeneity of glioblastoma is partially attributed to the presence of glioma stem-like cells (GSC), which are highly invasive and resistant to chemotherapy and irradiation treatments. Signaling of the receptor tyrosine kinase MET is a known regulator of GSC. Glioblastoma patients have an increasingly poor prognosis that corresponds with increasing MET expression. Both GSC and MET are known to contribute to treatment resistance …

Neuronal Or Intestinal Knockdown Of C. Elegans Nadk-1 Decreases Oxygen Consumption And Reactive Oxygen Species, Jake Regan

Electronic Theses and Dissertations

Reactive oxygen species (ROS) such as H2O2 can damage cellular components and are formed as a byproduct of mitochondrial oxidative metabolism. Studies using the nematode C. elegans have found that increasing ROS during development or early adulthood can extend lifespan, while increasing ROS during later adulthood normally decreases lifespan. NADPH provides the reducing power for several cellular antioxidants and is synthesized in a two-step reaction from NAD+ with the first step being catalyzed by NAD kinase (NADK). In this study, the effects of knocking down C. elegans cytoplasmic NADK, nadk-1 globally or in a neuron or intestine-specific manner starting from …

Cyclic-Di-Gmp Regulates Salmonella Typhimurium Infection Of Epithelial Cells And Macrophages, Abdulafiz Musa

Electronic Theses and Dissertations

Regulation of the bacterial second messenger cyclic-di-GMP in Salmonella Typhimurium allows it to delicately alter phenotypes to optimize invasion and survive intracellularly in epithelial cells and macrophages to become virulent and cause infection. Cyclic-di-GMP concentration is regulated by the presence of external stimuli, sensory diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), and cyclic-di-GMP binding effectors. Previous studies established that maintenance of low cyclic-di-GMP concentrations is required for survival in macrophages, and that deletion of 3 active PDEs reduces this survival. Here I showed that these 3 PDEs also influenced the infection of epithelial cells. Further studies re-established the decreased survival in …

Small Gtpase Regulated Intracellular Protein Trafficking In Endothelium, Caitlin Francis

Electronic Theses and Dissertations

Intracellular protein trafficking is the movement of membrane-bound organelles to and from requisite locations within the cell. Small GTPases are a critical component to the spatiotemporal accuracy of intracellular trafficking pathways as they determine the specificity and direction of organelle transport. There exists over 150 small GTPases categorized into 5 sub-families and are employed across all cell types. Despite their universal expression and relevance to cellular function, small GTPases remain incompletely understood across tissue types. In various instances, the trafficking pathway of a particular Rab in one cell type may belong to a completely disparate pathway in another cell type. …

Uncovering Zn2+ And Ca2+ Homeostasis And Crosstalk In Primary Hippocampal Neurons, Chen Zhang

Electronic Theses and Dissertations

Zn²⁺ has been suggested to act as an intracellular signaling molecule due to its regulatory effects on numerous protein targets including enzymes, transcription factors, ion channels, neurotrophic factors, and postsynaptic scaffolding proteins. However, intracellular Zn²⁺ concentration is tightly maintained at steady levels under natural physiological conditions. Dynamic changes in intracellular Zn²⁺ concentration have only been detected in cells exposed to pathologic stimuli or upon receptor ligand binding. In the first project of my dissertation, for the first time, we revealed that developing neurons fire spontaneous and synchronous Zn²⁺ spikes at a specific time window (from 14 …