Molecular Biology Commons^™

Deconstructing Brown Adipocyte Neogenesis In Brown And White Adipose Tissue, Rayanne Burl

Wayne State University Dissertations

Global incidence of Type 2 Diabetes (T2D) has reached epidemic proportions, and increasing evidence indicates that dysfunctional adipose tissue is an important contributor to the pathogenesis of T2D. Expanding brown adipocyte (BA) populations within adipose tissues through adrenergic activation improves energy balance and insulin sensitivity. In order to exploit this remodeling of adipose tissue for therapeutic benefit, we need to understand the mechanisms by which adrenergic signaling expand populations of BAs in vivo. These studies utilized single-cell RNA-sequencing and transgenic mouse models, in combination with single-molecule fluorescence in situ hybridization (smFISH) and immunoistochemical analysis, to study BA neogenesis in vivo. …

Hormonal Regulation Of Glycine Decarboxylase And Its Metabolic Outcomes, Ruta Milind Jog

Wayne State University Dissertations

The amino acid glycine is involved in generation of multiple critical metabolites including glutathione, heme, and creatinine. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance, type II diabetes and non-alcoholic fatty liver disease. The glycine cleavage system is the predominant glycine degradation pathway in humans. The rate-limiting enzyme of glycine cleavage system is glycine decarboxylase (GLDC), and loss-of-function mutations of GLDC cause hyperglycinemia. Here, we show that GLDC gene expression is upregulated in livers of mouse models of diabetes and diet-induced obesity as well as in the fasted state in normal animals. …

Novel Insights Into The Critical Role Of Cardiolipin In Cellular Metabolism And Mitochondrial Physiology, Jiajia Ji

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondria. CL and its remodeling exert critical roles in biological processes both inside and outside of mitochondria. CL abnormalities have been associated with various mitochondrial disorders and aging. Understanding the role of CL in mitochondrial physiology and cellular metabolism could provide valuable insights into cell biology and human health. Several metabolic alterations have been reported in CL-deficient cells, including accumulated lactate, decreased PDH activity, and decreased TCA cycle function. This dissertation connected these findings by showing abnormal NAD+ metabolism in various models lacking CL. Importantly, it shows that NAD+ supplementation improves mitochondrial function …

The Role Of The Cell-Surface Protease Tmprss13 In Colorectal Cancer, Fausto Alexander Varela

Wayne State University Dissertations

Colorectal cancer (CRC) is one of the most common and deadly cancers in both men and women in the United States. Extracellular proteolysis is often dysregulated in cancer including (CRC), resulting in degradation of extracellular matrix, as well as cleavage, processing, or shedding of cell adhesion molecules, growth factors, and cytokines. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression; however, many family members have not yet been characterized in malignancy. We identified TMPRSS13 transcript to be upregulated in CRC compared to normal colon. This increase was confirmed …

The Balance Between Prostaglandin E2 Ep3 And Ep4 Receptors Determines Severity Of Cardiac Damage In Myocardial Infarction And An Angiotensin Ii-Induced Model Of Hypertension, Timothy Dean Bryson

Wayne State University Dissertations

According to the center for disease control about 610,000 people die every year in the United States from heart disease, of which, coronary heart disease is the most common form. One major risk factor for heart attack is hypertension, which affects nearly half of all Americans [472, 473]. PGE2 plays an important role in regulating cardiovascular function and mediating inflammation, both of which contribute to the development of hypertension and/or heart disease. Prostaglandin E2 can act as a vasodilator or vasoconstrictor depending on which of its receptor subtypes are activated.

In general, activation of the EP1 and EP3 receptors is …

Effect Of Endoplasmic Reticulum Stress On Vascular Smooth Muscle Cells And Its Regulation Of Sm22Α, Neeraja Priyanka Annam

Wayne State University Dissertations

Background: The vascular smooth muscle cells(VSMC) possess the ability to differentiate into a synthetic phenotype in response to stress. This phenotypic modulation may be accompanied by inflammatory or osteogenic response in chronic stress. The synthetic state is characterized by low levels of contractile markers unlike the differentiated state.

Hypothesis: Endoplasmic reticulum (ER) stress causes phenotypic modulation in VSMCs leading to apoptosis. Many transcription factors induced by ER stress contribute to the downregulation of Sm22α. Perturbation in cytoskeletal dynamics exacerbates the ER stress response.

Methods: Ex-vivo culture was used to establish importance of Sm22 in ER stress. In vitro analysis was …

Role Of Alström Syndrome 1 (Alms1) In Nkcc2 Endocytosis, Thick Ascending Limb Function, Blood Pressure Regulation And Metabolic Function, Ankita Bachhawat Jaykumar

Wayne State University Dissertations

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na+/K+/2Cl- co-transporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to salt sensitive hypertension in rodents and humans. NKCC2 endocytosis is important for maintaining surface NKCC2 such that blocking NKCC2 endocytosis increased NKCC2 surface abundance and NKCC2-mediated NaCl reabsorption. Despite its importance, NKCC2 endocytosis has been poorly studied and a part of the reason may be attributed to the lack of availability of methods with good time resolution. Hence, we developed a method to image apical NKCC2 to monitor its endocytosis in real-time by Total Internal …

Identification Of Oxygen Optima For Mouse Trophoblast Stem Cells And Human Embryos And The Stress Responses Upon Departing Optima, Yu Yang

Wayne State University Dissertations

Low level of oxygen (O2) occurs physiologically during in vivo embryo development. As developing embryos moving from fallopian tube to uterus, oxygen level gradually decreases to ≤ 5% at the time of blastocyst implantation. Blastocysts are made of two major cell populations, trophoblast cells and inner cell mass, from which trophoblast stem cells (TSCs) and embryonic stem cells (ESCs) are derived respectively. TSCs serve as placental stem cells that later on proliferate and differentiate into placenta. Previous study has shown that 2% O2 is the optimal O2 level for mTSC in vitro growth and potency maintenance, which agrees with their …

Studies Of Sumoylation In Regulating Mif Stability And Rangap1 Nucleo-Cytoplasmic Shuttling In Controlling Its Sumo Modification, Progga Sen

Wayne State University Dissertations

SUMOylation is an essential post-translational modification that regulates a variety of critical cellular pathways ranging from nuclear transport to protein stability. Accumulating lines of evidence have shown that a perturbation of the SUMOylation pathway is associated with human diseases, especially various types of cancer. Our recent proteomic studies revealed a drastic increase in levels of SUMO2/3 modification on the proinflammatory cytokine MIF in the metastatic breast cancer cell line compared to the non-metastatic control cell line. Interestingly, the increase in levels of both MIF and global SUMO-2/3 modification in the metastatic cells are positively correlated to that of unmodified MIF …

Proteasome Inhibition As A Potential Anti-Breast Cancer Therapy: Mechanisms Of Action And Resistance-Reversing Strategies, Rahul Rajesinh Deshmukh

Wayne State University Dissertations

AMPK activation and Ubiquitin Proteasome System (UPS) inhibition have gained great attention as therapeutic strategies for the treatment of certain types of cancers. While AMPK serves as a master regulator of cellular metabolism, UPS regulates protein homeostasis. Although the crosstalk between them is suggested, the relationship between these two important pathways is not very clear. We observed that proteasome inhibition leads to AMPK activation in human breast cancer cells. We report that a variety of proteasome inhibitors activate AMPK in all of the tested cancer cell lines. Our data using Liver Kinase B1 (LKB1)-deficient cancer cells suggests that proteasome inhibitor-induced …

Hrd1 Partners In Endoplasmic Reticulum-Associated Degradation, Aaron Alexander Burr

Wayne State University Dissertations

Protein Quality Control (PQC) comprises cellular pathways that regulate the turnover of short-lived, misfolded proteins. A main component of PQC is Endoplasmic Reticulum (ER)-Associated Degradation (ERAD), which controls the degradation of proteins synthesized in the ER. Aberrations in ERAD have been linked to malignancies such as sarcomas, breast, and pancreatic carcinomas, as well as neurodegenerative disease. The machinery in this system is complex and while significant progress has been made to understand ERAD, it is not clear how the different components come together, or how they are regulated. HRD1 is a resident ubiquitin ligase that has been proposed as a …

Soy Isoflavones Mediate Radioprotection Of Normal Lung Tissue By Modulating The Radiation-Induced Inflammatory Response, Lisa Marie Abernathy

Wayne State University Dissertations

Radiation-induced lung injury (RILI) is caused by an early inflammatory process triggered by damage to lung parenchyma, epithelial cells, vascular endothelial cells and stroma. Initially, oxidative injuries after radiation induce altered expression of pro-inflammatory cytokines. Infiltrating inflammatory cells are stimulated and activated, producing additional mediators, resulting in a cytokine cascade. The expansion and perpetual activation of inflammatory cells, as well as lung parenchyma, lead to clinical pneumonitis. Activated cells produce molecular mediators and growth factors that affect the proliferation and gene expression of lung fibroblasts. This process leads to increased collagen synthesis and deposition, eventually leading to the development of …

A Novel Function For 12-Lipoxygenase In C-Met And Integrin Β4 Axis Crosstalk, Elizabeth Tovar

Wayne State University Dissertations

Cancer cell metastasis is the single most threatening occurrence of tumor progression and predicts patient prognosis as well as survival. Invasion can be regulated by the Met receptor tyrosine kinase (c-Met), integrin beta4, and the lipid enzyme, 12-Lipoxygenase (12-LOX). Therefore we sought to determine if beta4, c-MET and 12-LOX comprise a signaling axis. c-Met is implicated in cancer cell dissemination through regulation of invasion in EMT where cell-cell junctions are disturbed to allow motility. Furthermore, beta4 promotes cellular adhesion to the extracellular matrix through hemidesmosomes. However, the homeostatic signaling functions of beta4's cytoplasmic tail can be hijacked by growth factor …

Regulation Of Nkcc2 Trafficking By Vesicle Fusion Proteins Vamp2 And Vamp3 In The Thick Ascending Limb, Paulo Sebastian Caceres Puzzella

Wayne State University Dissertations

The thick ascending limb (TAL) in the kidney regulates extracellular fluid volume and blood pressure. The Na/K/2Cl cotransporter NKCC2 plays a central role in NaCl absorption by the TAL and blood pressure. NKCC2 trafficking to the apical membrane is a major mechanism to control NKCC2 activity. However, little is known about the proteins that mediate NKCC2 trafficking. Inhibition of the vesicle fusion proteins VAMP2 and VAMP3 blunts the increase in surface NKCC2 expression and NaCl absorption in response to stimulation by cAMP. In other cells, VAMPs mediate fusion of exocytic vesicles with the plasma membrane. Whether VAMP2 and VAMP3 mediate …

Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate, Rania M. Deranieh

Wayne State University Dissertations

Inositol is a six-carbon cyclitol that is ubiquitous in biological systems. It is a precursor for the synthesis of numerous biologically important compounds, including inositol phosphates and phosphoinositides that are essential for cell function and viability. Inositol compounds play a role in membrane formation, gene regulation, signaling, regulation of ion channels, and membrane trafficking. Furthermore, inositol regulates hundreds of genes, including those involved in the biosynthesis of inositol and phospholipids. While transcriptional regulation of inositol biosynthesis has been extensively studied and well characterized, regulation of inositol biosynthesis at the enzymatic level has not been addressed. The current study shows that …

The Transcriptional Regulation Of Flagellin-Induced Innate Protection Of The Cornea: Role Of Irf1 And Atf3, Gi Sang Yoon

Wayne State University Dissertations

Pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against infections by reprogramming gene expression. This study explored the flagellin-induced modifications of transcription factor expression and function, specifically of IRF1 and ATF3 in corneal epithelial cells to elucidate the transcriptional mechanisms underlying the protective function of flagellin on the cornea.

Initially we used Superarray to screen for transcription factors and identified Interferon Regulatory Factor (IRF) 1 and Activating Transcription Factor (ATF) 3 as the most drastically affected genes by flagellin pretreatment in P. aeruginosa challenged human corneal epithelial cells (CEC). However, flagellin pretreatment had opposite effects …

Acidic Pericellular Ph: Effects On Proteolysis And Gene Expression As Determined In 3d Models Of Breast Carcinoma, Jennifer M. Rothberg

Wayne State University Dissertations

Among the non-cellular microenvironmental factors that contribute to malignancy of solid tumors is an acidic peritumoral pH. The first objective was to determine if an acidic extracellular pH observed in vivo (i.e., pHe 6.8) affects the activity of proteases, such as cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional cultures. At pHe 6.8 there were increases in pericellular active cysteine cathepsins and in degradation of DQ-collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of …

Identifying Sm22 As A Key Player In Arterial Diseases, Jianbin Shen

Wayne State University Dissertations

Background : Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and osteochondrogenesis are present. However, the role of this downregulation in arterial pathogenesis is unknown. Hypothesis : Downregulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout (Sm22^-/-) mice and their wild type littermates were subjected to carotid artery denudation, an artery injury model. Analyses were conducted on carotid arteries 2 weeks after injury. Primary VSMCs were isolated from mouse aortas and investigated individually at passage 2 to 4. Sm22 knockdown was …

Identification Of Cellular Functions Of Cardiolipin As Physiological Modifiers Of Barth Syndrome, Amit Shridhar Joshi

Wayne State University Dissertations

Cardiolipin (CL) is an anionic phospholipid synthesized in the mitochondrial inner membrane. Perturbation of CL metabolism leads to Barth syndrome (BTHS), a life threatening genetic disorder. I utilized genetic, biochemical and cell biological approaches in yeast to elucidate the cellular functions of CL. Understanding the functions of CL is expected to shed light on the pathology and possible treatments for BTHS.

BTHS is caused by mutations in TAZ1, which encodes a CL remodeling enzyme called tafazzin. BTHS patients exhibit a wide range of clinical presentations, indicating that physiological modifiers influence the BTHS phenotype. A targeted synthetic lethality screen was performed …

Hdm2 Small-Molecule Inhibitors For Therapeutic Intervention In B-Cell Lymphoma, Angela Sosin

Wayne State University Dissertations

Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, compromising p53 activity. Therefore, lymphoma is a suitable model for studying therapeutic value of disrupting HDM2-p53 association by small-molecule inhibitors (SMIs). HDM2 SMIs have been developed and are currently under various stages of preclinical and clinical investigation. This study examined various molecular mechanisms associated and biological effects of two different classes of HDM2 SMIs: the spiro-oxindoles (MI-219) and cis-imidazoline (Nutlin-3) in lymphoma cell lines and patient-derived B-lymphoma cells. Surprisingly, results revealed significant quantitative and qualitative differences between these two agents. At the molecular level, effect of Nutlin-3 was generally more …

Cardiac Calsequestrin Phosphorylation And Trafficking In The Mammalian Cardiomyocyte, Timothy Mcfarland

Wayne State University Dissertations

Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significant quantities of Ca2+ and altering ryanodine receptor activity at the junctional sarcoplasmic reticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site of biosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multiple antibodies specific to classic rough ER markers, and with the creation of CSQ-DsRed tetramer fusion protein, we were able to establish a juxtanuclear localization of rough ER in cardiomyocytes. Using fluorescence confocal microscopy, the translocon complex …

Human Trophoblast Survival And Invasion In The Developing Placenta: Autocrine Regulation By Hbegf, Philip Jessmon

Wayne State University Dissertations

HBEGF is a multifunctional protein in early pregnancy that induces cytotrophoblast (CTB) cell differentiation to an invasive phenotype, protects against apoptosis, and is involved in an autocrine signaling mechanism that leads to its own protein synthesis. CTBs exist in a low O₂ environment during the first 10 weeks of implantation, during which they invade the decidualized uterine stroma. Inhibitors of intracellular signaling pathways demonstrated that at 20% O₂ HBEGF induces an increase in cell migration through the ERK, MAPK14, JNK, or PIK3 pathways downstream of signaling through its ERBB receptors. Also downstream of these four pathways, HBEGF induces …

Palmitoylation And The Yeast Casein Kinase Yck2, Irene Papanayotou

Wayne State University Dissertations

Palmitoylation is a post-translational lipid modification that allows proteins to interact with membranes. In the yeast Saccharomyces cerevisiae, the casein kinase Yck2 is palmitoylated twice at its two C-terminal palmitoyl-accepting cysteine residues, by the palmitoyl-transferring enzyme Akr1. Once palmitoylated, Yck2 traffics through the well characterized secretory pathway to the plasma membrane where it participates in many cellular functions, including bud morphogenesis, cytokinesis, nutrient sensing, and receptor internalization. While the hydrophilic Yck2 is presumably synthesized on cytosolic ribosomes, it gains access to the membrane system by interaction with the six transmembrane-spanning Golgi-localized Akr1. Since palmitoylation occurs at membranes and the palmitoyl-transferases …

Amphiregulin (Areg) And Epidermal Growth Factor (Egf): Disparate In Egfr Signaling And Trafficking, Andrea Jacqueline Baillo

Wayne State University Dissertations

We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the …

The Role Of The Sparc Acidic Domain And Egf-Like Module In Glioma Migration, Invasion, And Signaling, Heather M. Mcclung

Wayne State University Dissertations

THE ROLE OF THE SPARC ACIDIC DOMAIN AND EGF-LIKE MODULE IN GLIOMA MIGRATION, INVASION, AND SIGNALING

HEATHER M. MCCLUNG

Advisor: Sandra A. Rempel, Ph.D.

Major: Pharmacology

Degree: Doctor of Philosophy

We have previously shown that Secreted Protein Acidic and Rich in Cysteine (SPARC) is upregulated in all astrocytoma grades and increases tumor cell migration and invasion. It is thought that different domains within the protein may regulate SPARC functions, suggesting domain-specific targeting to inhibit invasion. To enhance our understanding of SPARC-mediated invasion, we first confirm, at the protein level, our previous cDNA array results, that SPARC increases expression of the …

Frazzled And Abelson Interact To Regulate The Actin Cytoskeleton In Drosophila, Bridget Elsa Varughese

Wayne State University Dissertations

Guidance receptors such as Frazzled affect cell shape and motility by directly or indirectly modulating the cytoskeleton. Fra is particularly needed for the formation of the posterior commissures in a developing Drosophila embryo. The cytoplasmic tyrosine kinase, Abelson Kinase (Abl) enhances the loss of commissures observed in fra mutant. Abl physically interacts with Frazzled to help guide commissural axons across the midline. Furthermore, the loss of commissural axons is only seen when the actin dynamics are perturbed. Abl is also known to regulate actin-dependent processes underlying formation of filopodia, microspikes and membrane ruffles. So, we established a Drosophila S2 cell …

Regulatory And Functional Aspects Of Foxo3a Transcription Factor And Their Implications In Prostate Cancer, Melissa Elise Dobson

Wayne State University Dissertations

The P13K/Akt pathway is a critical mediator of growth factor signaling involving many cellular functions. The deregulation of this pathway has been shown to be involved in the development of various cancers. One of the main targets of this pathway is FoxO3a, a transcription factor whose target genes are involved in important cellular processes such as apoptosis, cell cycle control, and glucose metabolism. FoxO3a is regulated by various post translational modifications including acetylation, ubiquitination and phosphorylation. The transcription factor is directly phosphorylated by Akt on 3 residues: Threonine 32, Serine 253 and Serine 315. Phosphorylation by Akt generates binding sites …