Cell and Developmental Biology Commons^™

Deciphering The Role Of Human Arylamine N-Acetyltransferase 1 (Nat1) In Breast Cancer Cell Metabolism Using A Systems Biology Approach., Samantha Marie Carlisle

Electronic Theses and Dissertations

Background: Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. NAT1 can additionally hydrolyze acetyl-coenzyme A (acetyl-CoA) in the absence of an arylamine substrate. NAT1 expression varies inter-individually and is elevated in several cancers including estrogen receptor positive (ER+) breast cancers. Additionally, multiple studies have shown the knockdown of NAT1, by both small molecule inhibition and siRNA methods, in breast cancer cells leads to decreased invasive ability and proliferation and decreased anchorage-independent colony formation. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. Additionally, consequences …

Unfolded Protein Response Pathways In Skeletal Muscle Homeostasis., Kyle R. Bohnert

Electronic Theses and Dissertations

Skeletal muscle mass, contractile properties, and metabolic function are regulated through the coordinated activation of multiple intracellular signaling pathways and genetic reprogramming. The endoplasmic reticulum (ER) plays a pivotal role in protein folding and calcium homeostasis in many cell types, including skeletal muscle. Disruption of calcium levels or accumulation of misfolded proteins in the ER lumen leads to stress, which results in the activation of a signaling network called the unfolded protein response (UPR). Further, recent studies have suggested that in certain conditions, UPR pathways can be activated independent of ER stress. However, the role of ER stress and the …

Origins Of Animal Cell Adhesion, Jennyfer Mora Mitchell

Electronic Theses and Dissertations

A fundamental requirement for multicellularity is cell adhesion. This includes mechanisms by which cells adhere to each other and to their secreted extracellular matrix (ECM). Two well characterized adhesion complexes in animals include: 1) adherens junctions (AJs), which are involved in cell-cell adhesion and composed of cadherin receptors, p120-, α- and β-catenin, and 2) focal adhesions (FAs), which are involved in cell-ECM adhesion and include proteins such as integrins, vinculin, paxillin, talin and focal adhesion kinase (FAK). The molecular components of AJs are widely conserved in animals, and largely absent in non-animals. In contrast, the molecular components of FAs have …

Superresolved Three-Dimensional Analysis Of The Spatial Arrangement Of The Human Immunodeficiency Virus Type-1 (Hiv-1) Envelope Glycoprotein At Sites Of Viral Assembly, Carmen Anne Buttler

Electronic Theses and Dissertations

Human Immunodeficiency Virus type 1 (HIV-1) replicates by forcing infected host cells to produce new virus particles, which assemble form protein components on the inner leaflet of the host cell's plasma membrane. This involves incorporation of the essential viral envelope glycoprotein (Env) into a structural lattice of viral Gag proteins. The mechanism of Env recruitment and incorporation is not well understood. To better define this process, we seek to describe the timing of Env-Gag encounters during particle assembly by measuring angular positions of Env proteins about the surfaces of budding particles. Using three-dimensional superresolution microscopy, we show that Env distributions …

Evaluating The Interaction Between The Human Melanocortin-2 Receptor And The Accessory Protein, Mrap1: Chimeric Receptor And Alanine Substitution Studies On Transmembrane Domain 4, Extracellular Loop 2, And Transmembrane Domain 5, Perry Victoria Davis

Electronic Theses and Dissertations

The melanocortin-2 receptor (MC2R) is the most complex due to its trafficking and ligand selectivity requirements for proper activation. The MC2R requires the melanocortin receptor accessory protein-1 (MRAP1) for proper trafficking and activation of the receptor by the melanocortin hormone, ACTH. MRAP1 is a single transmembrane-spanning domain protein that creates a homodimer with another MRAP1 protein. Furthermore, MRAP2 creates a heterodimer with the MC2R. Previous studies have shown that the MRAP1 protein contains an activation motif required for activation of MC2R and this activation motif located on the extracellular space side of the plasma membrane of the cell. The objective …

The Identification And Segmentation Of Astrocytoma Prior To Critical Mass, By Means Of A Volumetric/Subregion Regression Analysis Of Normal And Neoplastic Brain Tissue, Lyn Higgins

Electronic Theses and Dissertations

As the underlying cause of Glioblastoma Multiforme (GBM) is presently unclear, this research implements a new approach to identifying and segmenting plausible instances of GBM prior to critical mass. Grade-IV Astrocytoma, or GBM, is an aggressive and malignant cancer arising from star-shaped glial cells, or astrocytes, where the astrocytes, functionally, assist in the support and protection of neurons within the central nervous system and spinal cord. Subsequently, our motivation for researching the ability to recognize GBM is that the underlying cause of the mutation is presently unclear, leading to the operative that GBM is only detectable through a combination of …

Mechanisms By Which Mechanotransduction Promotes Proliferation In Keratinocytes, Chhavi Chaudhary

Electronic Theses and Dissertations

Chronic wounds are wounds that do not heal within 30 days and often they can last over a year. Interference in any of the wound healing stages may hinder the process. Some of the local and systemic factors such as infection, old age, diabetes, AIDS, and the regular application of corticoids may also have negative effects on the healing process. Cell proliferation is an important phase in epidermal wound healing in which surviving epithelial cells replicate independently into daughter cells through mitosis and maintain a balance between cell growth and cell loss during the cell cycle. Physical and chemical stimuli …

Identification Of Macropinocytosis Regulating Proteins And Signaling From Macropinosomes, Louise Monga

Electronic Theses and Dissertations

Understanding macrophage cell biology is important due to macrophages key roles in human health and diseases including proper immune function, wound healing, atherosclerosis, and cancer. Despite their importance, relatively little is understood about macrophage activation, growth factor signaling, and cytoskeletal regulation. This thesis presents data from investigations into mechanisms of macrophage growth factor signaling and actin polymerization for ruffling and macropinocytosis. Alt-R CRISPR-Cas9 method and dextran uptake assay were used to knock out individual genes (SHP-1, Lyn, Syk, BTK, Vav1) and determine their role in macropinocytosis and CSF-1R signaling. Dextran uptake was disrupted in SHP-1 and Lyn targeted knockout cells, …

Deciphering The Role Of Adrenergic Hormones In Embryonic Cardiac Calcium Signaling And Metabolism, Jessica Peoples

Electronic Theses and Dissertations

The adrenergic hormones norepinephrine (NE) and epinephrine (EPI) are critical regulators of mammalian cardiovascular physiology. NE and EPI mediate stress responses to enhance cardiovascular function, however dysregulation of adrenergic signaling leads to heart failure, congenital heart malformations, and sudden cardiac death. Adrenergic hormone-expressing cells were found in the early embryonic heart, and NE has been determined essential for embryonic cardiac development. Despite extensive work in adults, the regulatory roles and adrenergic targets of these hormones during embryonic cardiac development have not yet been fully determined. Prior transcriptomic studies from our lab showed that expression of signal transduction and metabolic genes …

The Dlk1-Meg3 Locus In Malignant Cells Of Proposed Primordial Germ Cell Origins., Zachariah Payne Sellers

Electronic Theses and Dissertations

Primordial germ cells (PGCs) are hypothesized to deposit hematopoietic stem cells (HSCs) along their migration route through the embryo during the early stages of embryogenesis. PGCs also undergo global chromatin remodeling, including the erasure and reestablishment of genomic imprints, during this migration. While PGCs do not spontaneously form teratomas, their malignant development into germ cell tumors (GCTs) in vivo is often accompanied by the retention of hypomethylation at the IGF2-H19 imprinting control differentially methylated region (DMR). Previous studies in bimaternal embryos determined that proper genomic imprinting at two paternally imprinted loci was necessary for their growth and development: Igf2-H19 and …

The Regulation Of Rotavirus–Infected Ht29.F8 And Ma104 Cells Treated With Arachidin 1 Or Arachidin 3, Caleb M. Witcher

Electronic Theses and Dissertations

Rotavirus (RV) infections cause severe life threatening diarrhea in young children and immunocompromised individuals. Several effective vaccines have been developed for young children but are not protective against all strains of RV, and there are no anti-RV therapeutics. Our laboratory has discovered a decrease in the number of infectious simian RV particles (SA114f) in human intestinal cell line, HT29.f8 cells with the addition of either of two stilbenoids, arachidin-1 (A1) or arachidin-3 (A3). This suggests effects on the host cell and RV replication. We examined the cellular effects of human RV strain (Wa) on a human intestinal cell line (HT29.f8) …

Identifying The Signaling Mechanisms Of Egfr-Mediated Apoptosis., Nicole Marion Jackson

Electronic Theses and Dissertations

The Epidermal Growth Factor Receptor (EGFR) is a 170-kilodalton transmembrane protein that belongs to the ErbB family of receptor tyrosine kinases. Upon ligand-mediated activation, the EGFR is responsible for cell growth, proliferation, and tissue homeostasis; however, the EGFR is overexpressed in many human malignancies, including MDA-MB-468 cells, a metastatic breast epithelial cell line. Studies within this cell line, and other cell lines characterized with high EGFR levels, have shown that EGF stimulation results in the induction of apoptosis. However, the mechanisms and signaling effectors implicated in this process have yet to be elucidated. The overarching research goal of this dissertation …

Dense Core Vesicle Heterogeneity In Anterior Pituitary Cells, Kelly Sinak

Electronic Theses and Dissertations

Peptides, which are packaged in dense core vesicles, are an integral part of the function of the endocrine and neurological systems. The dense core vesicles function as an efficient form of peptide storage prior to regulated exocytosis. Two different dense core specific transmembrane proteins traffic different when comparted to retained prolactin cores, offering evidence of heterogeneity of vesicles within a single cell. By comparing synaptotagmin 1 and 7 distribution in male rat and lactating female lactotrophs, a distinct pattern emerges. Cells that retain prolactin cores after exocytosis correspond with those that contain synaptotagmin 1. This finding is a reversal for …

Macropinosome Maturation Is A Clathrin Dependent Process In Bone Marrow Macrophages, Susmita Poudel

Electronic Theses and Dissertations

Macrophages nonspecifically take up extracellular fluids, solutes and macromolecules by macropinocytosis. Understanding the mechanisms of macropinosome maturation will inform the study of lipid uptake, viral entry, antigen processing and presentation, as well as regulation of cell growth. Colony stimulating factor-1 receptor (CSF-1R) is internalized by small vesicle endocytosis, trafficked to nascent macropinosomes and degraded. These CSF-1R positive macropinosomes mature through a sequence similar to endosomes, progressing from EEA1 and Rab5 to Rab7 positive vesicles before fusing with lysosomes. Here we report the assembly of clathrin on internalized macropinosomes shown both by live-cell microscopy of cells expressing clathrin light chain-yellow fluorescent …

Lipid-Laden Macrophages Downregulate Akt Phosphorylation And Metabolize Lipid Droplets Via Autophagy, Rifat Sultana

Electronic Theses and Dissertations

Macrophages contribute to plaque formation in atherosclerosis. Macrophages take up modified low-density lipoproteins and store excess cholesterol and triglycerides in lipid droplet organelles. Evidence of lipid-laden macrophages or “foam cells” is apparent on histology sections of diseased arteries, and this lipid-laden appearance can be recreated in cell culture upon exposure of cultured macrophages to acetylated LDL (Ac-LDL). Under nutrient stress, neutral lipids in lipid droplets are hydrolyzed by lipolysis, autophagy, or both. However, these processes are not well understood in macrophages. We created lipidladen macrophages by 24-h exposure to Ac-LDL and analyzed dynamics of lipid droplet metabolism following removal of …

Chaperonin Containing Tcp1 (Cct) As A Target For Cancer Therapy, Ana Carr

Electronic Theses and Dissertations

Treatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the CCT2 subunit is highly expressed in breast cancer and could be therapeutically targeted. To determine whether CCT is a marker of disease progression in other cancers, we analyzed CCT2 gene …

Novel Cytokine Signaling And Molecular Therapeutic Strategy In Pancreatic Cancer, Sarah Gitto

Electronic Theses and Dissertations

Pancreatic ductal adenocarcinoma (PDAC) is highly chemo-resistant and has a five year survival rate of < 8%. Risk factors of pancreatic cancer, such as chronic pancreatitis, help to elicit a pro-tumor immune response, and highly fibrotic environment that promotes tumorigenesis. To study how chronic pancreatitis promotes cancer initiation, traditional KRasG12D mice and double mutant Akt1Myr/KrasG12D mice were used to model microenvironment changes. Akt1Myr/KrasG12D mice were more susceptible to chronic tissue damage, accelerated tumor development and metastatic disease. These mice exhibited histological changes consistent with immune cell privilege, where M2 macrophages and non-cytotoxic eosinophils were co-localized with fibrotic regions. IL-5 expression was up regulated in pancreatic cells undergoing acinar to ductal metaplasia and then diminished in advanced lesions. Tumor cells treated with IL-5 exhibit increased migration and activation through STAT5 signaling. Collectively, the results suggest that eosinophils, which are responsive to IL-5, are key mediators in the pancreatic environment subjected to chronic inflammation and injury. Current therapeutics fall short in increasing patient survival. There remains an urgent need for innovative treatments and thus we tested difluoromethylornithine (DFMO) in combination with a novel polyamine transport inhibitor, Trimer44NMe, against Gemcitabine-resistant PDAC cells. Prior clinical failures when targeting polyamine biosynthesis with DFMO monotherapy may be due to tumor escape via an undefined polyamine transport system. In pancreatic tumor cells DFMO alone and with Trimer44NMe significantly reduced PDAC cell viability by inducing apoptosis or cell cycle arrest. In vivo orthotopic PDAC growth with DFMO treatment resulted in decreased c-Myc expression, a readout of polyamine pathway dysfunction. Moreover, dual inhibition significantly prolonged survival of tumor-bearing mice, and increased M1 macrophage infiltration and reduced FoxP3 expression. Collectively, these studies demonstrate that targeting polyamine pathways in PDAC is a promising immunomodulating therapy that increases survival.

Identification Of Cdks As Novel Targets Of Aspirin And Its Metabolites: A Potential Role In Cancer Prevention, Rakesh Dachineni

Electronic Theses and Dissertations

Background:
The pursuit of drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. Till date, although multiple CDK inhibitors have been identified and few are undergoing clinical trials, only two synthetic drugs have been approved by Food and Drug Administration (FDA) for use in the treatment of cancer. These two drugs are mainly used for the treatment of metastatic breast cancer in combination with other drugs; however they have toxicity associated with their use and extends patients life not more than 24 months. Therefore, there is an urgent need for …

Pharmacokinetics, Tissue Distribution, Synergistic Activity, And Antitumor Activity Of Two Isomeric Flavones, Crystal L. Whitted

Electronic Theses and Dissertations

Flavonoids are polyphenolic secondary metabolites found in plants that have bioactive properties including antiviral, antioxidant, and anticancer. Two isomeric flavone were extracted from Gnaphalium elegans and Achyrocline bogotensis, plants used by the people from the Andean region of South America as remedies for cancer. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5, 7–dihydroxy- 3, 6, 8 trimethoxy flavone/ flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3, 5–dihydroxy-6, 7, 8–trimethoxy flavone/ flavone B) have shown antineoplastic activity against colon cancer cell lines dependent upon their differentiation status. Pharmacokinetic studies reported herein were used to determine dosing for antitumor assays, as well as determine target tissue concentration. These included the …

Retinoic Acid Signaling Regulates Krt5 Independently Of Stem Cell Markers In Submandibular Salivary Gland Epithelium., Timur Maratovich Abashev

Electronic Theses and Dissertations

Vitamin A metabolism, which produces the signaling molecule Retinoic Acid (RA), has been demonstrated to be important for growth and branching morphogenesis of mammalian embryonic salivary gland epithelium. However, it is not known whether RA functions directly within epithelial cells or in associated tissues that influence morphogenesis of salivary epithelium. Moreover, downstream targets of RA transcriptional regulation have not been identified. Here we show that canonical RA signaling occurs in multiple tissues of embryonic mouse salivary glands, including epithelium, associated parasympathetic ganglion neurons, and non‑neuronal mesenchyme. By culturing epithelium explants in isolation from other tissues we demonstrate that RA influences …

Isolation Of Egfr-Containing Early Endosomes., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR undergoes clathrin-mediated endocytosis. This spatial regulation of the receptor is an important regulator of receptor expression as it mediates its degradation. Endocytosis also has implications on EGFR downstream signaling, though the details are not fully understood. The goal of this thesis is to develop a method to isolate early endosomes in order to study downstream effectors associated with activated EGFR in this compartment. HeLa cells were used to test various …

Endothelial Hspa12b Is A Novel Protein For The Preservation Of Cardiovascular Function In Polymicrobial Sepsis Via Exosome Mir-126, Xia Zhang

Electronic Theses and Dissertations

Sepsis is the most frequent cause of mortality in most intensive care units. Cardiovascular dysfunction is a major complication associated with sepsis, with high mortality rates up to 70%. Currently, there is no effective treatment approach for sepsis.

The integrity of the endothelium is fundamental for the homeostasis of the cardiovascular system. Sepsis induces endothelial cell injury which is the key factor for multiple organ failure. The increased expression of adhesion molecules and chemokines in endothelial cell promotes leukocytes infiltration into the tissue. The loss of tight junction proteins and increased permeability of the endothelial cells will provoke tissue hypoxia …

Microrna-186 And Metastatic Prostate Cancer., Dominique Zilpha Jones

Electronic Theses and Dissertations

MicroRNA (miR) dysregulation alters cancer-associated gene expression, which contributes to cancer pathogenesis. For example, miR-186 over expression lead to enhanced proliferation and migration in pancreatic cancer cell models. However, the role of miR-186 in prostate cancer (PCa) remains controversial. Previously, miR-186-5p was up-regulated in PCa patient serum (stage III/IV) compared to controls. Furthermore, miR-186-5p was up-regulated in metastatic PCa (PC-3, MDA PCa 2b, LNCaP) relative to normal prostate epithelial cells (RWPE1). We hypothesized miR-186 inhibition will reduce aggressive PCa using metastatic cell models. To test this, we evaluated whether miR-186-5p inhibition would reduce aggressive PCa behavior and overexpression induce malignant …

Investigation Of Novel Functions For Dna Damage Response And Repair Proteins In Escherichia Coli And Humans, Benjamin A. Hilton

Electronic Theses and Dissertations

Endogenous and exogenous agents that can damage DNA are a constant threat to genome stability in all living cells. In response, cells have evolved an array of mechanisms to repair DNA damage or to eliminate the cells damaged beyond repair. One of these mechanisms is nucleotide excision repair (NER) which is the major repair pathway responsible for removing a wide variety of bulky DNA lesions. Deficiency, or mutation, in one or several of the NER repair proteins is responsible for many diseases, including cancer. Prokaryotic NER involves only three proteins to recognize and incise a damaged site, while eukaryotic NER …

Identification Of Host Factors Required For Yersinia Pestis Macrophage Intracellular Survival And Their Impact On Vacuole Maturation, Acidification And Trafficking., Michael Graylin Connor

Electronic Theses and Dissertations

Y. pestis is a facultative intracellular pathogen and the causative agent of plague. This bacterium, while most noted or the Black Death during the European 14^th century, is not a historic pathogen but a re-emerging pandemic with both domestic and global impact. Y. pestis is capable of colonizing the macrophage, and actively subverts phagolysosome maturation to establish a replicative niche known as the Yersinia containing vacuole (YCV). The exploited host factors required to support the YCV are unknown. Here we identified a comprehensive list of host factors required for Y. pestis survival through a genome-wide RNAi high-throughput screen. We …

Modulation Of Cell Death Signaling And Cell Proliferation By The Interaction Of Homoserine Lactones And Paraoxonase 2., Aaron Mackallan Neely

Electronic Theses and Dissertations

Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule that functions to facilitate bacteria-bacteria communication. C12 has also been reported to affect many aspects of human host cell physiology, including evoking cell death in various types of cells. However, the signaling pathway(s) leading to C12-triggerred cell death remains unclear. To clarify cell death signaling induced by C12, we examined mouse embryonic fibroblasts (MEFs) deficient in one or more caspases. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in cells, probably through the direct induction of mitochondrial membrane permeabilization. Previous studies indicate that …

The Apoptotic And Inhibitory Effects Of Phylloquinone In The U937 Cell Line, Tesha E. Blair

Electronic Theses and Dissertations

Phylloquinone is a natural analog of vitamin K that has been shown to both inhibit cancer cell growth and induce apoptosis in several cancer cell lines. This study examined these effects in a non-Hodgkin lymphoma cell line, known as U937. Cell growth inhibition and apoptosis were assessed through the quantification of cell density and area, following treatment with several concentrations of phylloquinone. In addition, apoptosis was detected and quantified using immunofluorescent markers of apoptosis (i.e. annexin V, APO-BrdU). Treatment with phylloquinone resulted in reduced overall cell density, increased overall cell area, and an increased frequency of apoptosis in U937 cells. …

Akap150 Dynamics In Anterior Pituitary Cells, Kristen E. Dew

Electronic Theses and Dissertations

Cellular communication occurs as a result of changes in signaling pathways. A well-studied signaling pathway is through G protein coupled receptors (GPCRs). In gonadotropes, GPCR stimulation by GnRH leads to the activation of protein kinase A (PKA). Activated PKA can phosphorylate ion channels, potentially causing an influx of calcium, depolarization and secretion of hormones. A scaffolding protein known as AKAP150 anchors PKA near L-type calcium channels. In addition, AKAP150 anchors phosphatases, which provides temporal control during signaling events. It was recently shown that AKAP150 is mobile in neuronal dendrites, providing regulation to where the signaling cascade occurs in the cell. …

The Ecology And Evolution Of Rare, Soil Specialist Astragalus Plants In The Arid Western U.S., Joseph M. Statwick

Electronic Theses and Dissertations

Organisms that specialize in uncommon habitats are, by their very nature, inherently uncommon. Specialization has its advantages, namely reduced competition and predation, but it also incurs costs. Specialists often have small population sizes, narrow ranges, and fragmented habitat, all of which engender negative consequences on an evolutionary timescale. Herein, I examine benefits and costs of specialization in selenium-hyperaccumulating plants in the genus Astragalus (Fabaceae). These plants are disproportionately likely to be rare and of conservation concern. Thus, I optimized germination pretreatments for Astragalus species such that seed loss can be minimized during ex situ cultivation, and found that physical scarification …

Enhancing The Industrial Potential Of Filamentous Cyanobacteria, Tylor J. Johnson

Electronic Theses and Dissertations

The objectives of this project were to improve the industrial potential of filamentous N2-fixing cyanobacteria by increasing its biofuel tolerance, and to evaluate the economic feasibility and environmental impacts of a theoretical, cyanobacteria-based biofuel production facility. To develop a method to quantify filamentous cyanobacteria in dilute culture media, a dual-stained fluorescence assay was evaluated. While the viable cell stain (SYTO® 9) was accurate, the non-viable cell stain (propidium iodide) also bound to viable cells. Additional non-viable cell stains were evaluated, but none were accurate at quantifying viability. Thus we concluded that the viable cell stain SYTO® 9 is a reliable …