Molecular Biology Commons^™

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 …

Imbalance Of Uracil Dna Glycosylase And Activation-Induced Cytidine Deaminase Expression In Folate Depleted Human Lymphoblastoids, Elizabeth Zanley

Wayne State University Theses

Background: The DNA base excision repair (BER) pathway is responsible for processing of genomic uracil lesions however, in some tissue types the excisional and gap-filling steps performed by UNG2 and POLβ, respectively, are impaired by folate deficiency in human and murine models in vitro. Genomic uracil damage can be acquired by inadequate conversion of uracil to thymine nucleotide precursors resulting from insufficient folate cofactors, or through activation induced cytosine deaminase (AID) activity during antibody diversification in B-cells in the context of adaptive immunity. The immunoglobulin (Ig) diversification methods in B-cells depend on the coordinated interaction between AID and UNG2, and …

Defining The Effect Of Environmental Perturbation On The Male Germline, Molly Estill

Wayne State University Dissertations

Periconceptional environment, according to the Developmental Origins of Health and Disease (DOHaD) theory, influences offspring phenotype, primarily via epigenetic mechanisms. Although the paternal component in humans is poorly understood, both maternal and paternal peri-conceptional environment are now believed to contribute to this phenomenon. Manipulation of the early embryo for treating human infertility, is suspected of contributing to offspring abnormalities through epigenetic mechanisms. To directly address the effects of common assisted reproductive technology procedures on the offspring epigenome, the DNA methylation profiles of newborns conceived naturally, or through the use of intrauterine insemination (IUI), or in vitro fertilization (IVF) using Fresh …

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 …

Novel Insights Into The Use Of Ercc1 As A Biomarker For Response To Platinum-Based Chemotherapy In Lung Cancer, Joshua Ryan Heyza

Wayne State University Dissertations

ERCC1/XPF is a DNA endonuclease with variable expression in primary tumor specimens, and has been investigated as a predictive biomarker for efficacy of platinum-based chemotherapy in non-small cell lung cancers where up to 30-60% of tumors harbor low to undetectable ERCC1 expression. The failure of an international, randomized Phase III clinical trial utilizing ERCC1 expression to predict response to platinum-based chemotherapy suggests additional mechanisms underlying the basic biology of ERCC1 in the response to platinum-DNA damage remain unknown. In this work, we aimed to characterize a panel of ERCC1 knockout cell lines generated via CRISPR-Cas9 where we identified a synthetic …

Perturbation Of Energy Metabolism At The Center Of The Mechanism Of Action Of Valproate, Michael Ghassan Salsaa

Wayne State University Dissertations

Bipolar disorder (BD) is a common and debilitating psychiatric disorder. Valproic acid (VPA) is one of the major drugs used to treat BD patients. However, it is not universally effective and, in addition, causes severe side effects. Its mechanism of action is not known, which complicates efforts to develop more effective drugs. Studies have established that VPA perturbs metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. However, the mechanism whereby VPA causes these perturbations is not understood. To address this knowledge gap, I investigated the acute and chronic effects …

Validating Functional Mechanisms For Non-Coding Genetic Variants Associated With Complex Traits, Cynthia Ann Kalita

Wayne State University Dissertations

Genome-wide association studies (GWAS) have identified a large number of genetic variants associated with disease as well as normal phenotypic variation for complex traits. However challenges remain in determining the functional relevance of human DNA sequence variants. Even after fine mapping, most variants are located in non-coding regions making it difficult to infer mechanisms linking individual genetic variants with the disease trait. In addition, we do not know under which environmental conditions the sequence variants have a functional impact, and whether they become one of many factors involved in complex phenotypes at the organismal level.

Chapter 1 describes computational methods …

Role Of Sirna Pathway In Epigenetic Modifications Of The Drosophila Melanogaster X Chromosome, Nikita Deshpande

Wayne State University Dissertations

Eukaryotic genomes are organized into large domains of coordinated regulation. The role of small RNAs in formation of these domains is largely unexplored. An extraordinary example of domain-wide regulation is X chromosome compensation in Drosophila melanogaster males. This process occurs by hypertranscription of genes on the single male X chromosome. Extensive research in this field has shown that the Male Specific Lethal (MSL) complex binds X-linked genes and modifies chromatin to increase expression. The components of this complex, and their actions on chromatin, are well studied. In contrast, the mechanism that results in exclusive recruitment to the X chromosome is …

Chchd10, A Novel Bi-Organellar Regulator Of Cellular Metabolism: Implications In Neurodegeneration, Neeraja Purandare

Wayne State University Dissertations

CHCHD10 (Coiled-coil Helix Coiled-coil Helix Domain containing protein 10) and MNRR1 (Mitochondrial Nuclear Retrograde Regulator 1, also known as CHCHD2), have been shown by us to be stress regulators of mitochondrial function that act both in the mitochondria and in the nucleus. Both are members of the twin CX9C family, but CHCHD10 in particular, has been found in mutant form to be linked to a myriad of neurodegenerative conditions. In mitochondria, both activate cytochrome c oxidase (COX) whereas in the nucleus, both act as transcription regulators of a subset of genes that contain a 13-bp sequence termed as the oxygen …

Phosphorylation Of Cytochrome C Threonine 28 Regulates Electron Transport Chain Activity In Kidney: Implications For Amp Kinase, Gargi Mahapatra, Ashwathy Varughese, Qinqin Ji, Icksoo Lee, Jenney Liu, Asmita Vaishnav, Christopher Sinkler, Alexandr A. Kapralov, Carlos T. Moraes, Thomas H. Sanderson, Timothy L. Stemmler, Lawrence I. Grossman, Valerian E. Kagan, Joseph S. Brunzelle, Arthur R. Salomon, Brian Fp Edwards, Maik HüTtemann

Biochemistry and Molecular Biology Faculty Publications

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc iso- lated from kidneys is phosphorylated on Thr28, leading to a par- tial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing supe- rior behavior regarding protein stability and its ability to degrade …

Patholigical And Prognostic Role Of Mdig In Pancreatic Cancer, Srinivas Ashok Kumar

Wayne State University Theses

Pancreatic cancer is a highly aggressive malignant disease having very limited therapeutic options that ultimately results in its poor prognosis. It is still elusive on the etiology and tumorigenic mechanisms of pancreatic cancer. In the present report, we provide evidence showing involvement of the mineral dust-induced gene (mdig) in the pathogenesis and prognosis of the pancreatic cancer. Using immunohistochemistry approach on human pancreatic cancer tissue microarray, we found differential expression of mdig in pancreatic adenocarcinoma and normal pancreas. Based on the staining intensities of mdig in these tissue samples, we found that 12% of the cancer tissues were strongly positive …

The Effect Of Acetylation Of Cytochrome C On Its Functions In Prostate Cancer, Viktoriia Bazylianska

Wayne State University Theses

Prostate cancer is the second leading cause of cancer death among men in America. The progression of cancer goes along with the Warburg effect, a metabolic switch from depending primarily on mitochondrial respiration to glycolysis. In addition, cancer cells manage to evade apoptosis. Cell signaling, via posttranslational modifications (PTMs), is one of the most important means of regulation, and most commonly dysregulated in cancer. In prostate cancer, androgen signaling plays a crucial role in driving cell proliferation.

Mammalian Cytochrome c (Cytc) is a multifunctional protein involved in cellular life and death decision. It is an essential component of the electron …

Human Cytomegalovirus Cytoplasmic Virion Assembly Complex: Structure In Vivo And Role Of Pul103 In Its Biogenesis, Ma Christina Raye Lim

Wayne State University Theses

Human Cytomegalovirus (HCMV) is a linear, double stranded DNA virus that causes severe disease in the immunocompromised, and is one of the common cause of congenital disease. Antivirals that are currently available for treatment target the DNA replication of the virus cycle, and are highly toxic. Finding new drug targets, such as proteins responsible for virion assembly and egress, would help to alleviate the disease burden.

HCMV remodels the host cell to form a structure called the cytoplasmic virion assembly complex (cVAC), a site of virion maturation and egress. The first objective of this work is to study the structure …

Fam129b Phosphorylation And Its Effect On Membrane Localization In Confluent Hela Cells, Lakshmi Thompil Somasekharan

Wayne State University Theses

Phosphorylation and de-phosphorylation of many proteins is a key regulator of cellular life. It maintains cellular activity through an array of signaling pathways like cell division, proliferation and growth. However, Overexpression or mutations by the constitutive activation of phosphorylation machinery disrupt the balance in the cell, driving the inappropriate activation or deactivation of the cellular processes it controls. FAM129B is a protein whose activity is partly maintained by phosphorylation and dephosphorylation at the six serine residues at the C-terminal. FAM129B is expressed highly in many forms of cancer and its main function is to suppress apoptosis and enhances cancer cell …

Elucidating Structure, Function, And Small Molecular Interactions Of Human Immunodeficiency Virus And Chikungunya Virus, Kristin Nicole Slater

Wayne State University Theses

Abstract HIV-1:

Human immunodeficiency virus-1 (HIV-1) is a widespread, incurable retrovirus known to cause

immunodeficiency and a shortened life span. Despite successful treatment methods, HIV-1

frequently mutates, resulting in antiviral resistance. Many therapies target the HIV-1 protease

(PR), which is responsible for cleaving the viral polyprotein essential for its life cycle. HIV-1 PR

often evades treatment by way of mutations and less commonly through residue insertions. We

have identified a clinical isolate with a five residue insertion between residues 28 and 29.

Through molecular dynamics simulations we analyzed the protease protein structure and

determined that the residue insertion created a …

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 …

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 …

Clinicopathology And Molecular Determinants Underlying Benign Breast And Breast Cancer Lesions, Andreana Holowatyj Holowatyj

Wayne State University Dissertations

Despite converging incidence rates for breast cancers by race, disparities in mortality persist where black women suffer from poorer prognosis compared to white counterparts. To understand the clinical, demographic, and molecular characteristics underlying these disparities, we examined differences among patients with breast cancer to understand the role of human epidermal growth factor receptor 2 (HER2) status, age, and race/ethnicity among women diagnosed with hormone receptor-positive breast cancer, and disparities in surgical therapy among female patients with early stage young-onset breast cancer. Benign breast disease, another known risk factor for breast cancer, includes a histological spectrum of lesions, could contribute to …

Analysis Of The Secondary Neurodegenerative Consequences Of Primary Oligodendrocyte Stress Through The Use Of The Novel Obiden Mouse Model, Daniel Zdzislaw Radecki

Wayne State University Dissertations

The work of this project was to develop, test and characterize a potential novel mouse model of the neurodegenerative disease Multiple Sclerosis (MS). Historically, MS has been identified as a primary autoimmune disease of the central nervous system (CNS). However, treatments based on this view have met with limited success, and in most cases, fail to prevent progression of MS from mild to moderate and severe forms. Original observations regarding axonal and neuronal pathology in the white and gray matter of the CNS were rediscovered in the 1990s. These observations indicated that even in the absence of the immune system, …

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 …

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 …

Navigating Human Cytomegalovirus (Hcmv) Envelopment And Egress, William Longeway Close

Wayne State University Dissertations

Human cytomegalovirus (HCMV) is a ubiquitous viral pathogen. In individuals with fully functioning and mature immune systems, HCMV is associated with mild symptoms prior to establishing latency. In individuals with naïve or compromised immune systems, HCMV is capable of causing severe organ damage. HCMV is the leading infectious cause of congenital birth defects and a major non-genetic cause of hearing loss. Unfortunately, antiviral treatment options lack diversity due to limited knowledge of virion replication. If HCMV replication were better understood, new antiviral treatments could be developed.

In this work, we describe the development and implementation of new tools to study …

Expression, Purification And Characterization Of Lysine Methyltransferase Smyd5, Wen Xue

Wayne State University Theses

Methylation of histones and non-histone proteins play vital roles in numerous cellular processes including gene expression regulation and DNA damage response. The identifications of methyltransferase SMYD protein family are not well characterized. SMYD5 is a unique and critical member of SMYD family involved in immune response, stem cell renew, hematopoiesis regulation and cancer metastasis. Understanding its function and structure is monumental to human disease. With the achievement of SMYD5 expression and purification, the association between SMYD5 and its substrate histone H4 has been investigated. While possessing a poly-E tail instead of the TPR domain included in other members, SMYD5 is …

New Open Conformation Of Smyd3 Implicates Conformational Selection And Allostery, Nicholas Spellmon, Xiaonan Sun, Wen Xue, Joshua Holcomb, Srinivas Chakravarthy, Weifeng Shang, Brian Fp Edwards, Nualpun Sirinupong, Chunying Li, Zhe Yang

Biochemistry and Molecular Biology Faculty Publications

SMYD3 plays a key role in cancer cell viability, adhesion, migration and invasion. SMYD3 promotes formation of inducible regulatory T cells and is involved in reducing autoimmunity. However, the nearly “closed” substrate-binding site and poor in vitro H3K4 methyltransferase activity have obscured further understanding of this oncogenically related protein. Here we reveal that SMYD3 can adopt an “open” conformation using molecular dynamics simulation and small-angle X-ray scattering. This ligand-binding-capable open state is related to the crystal structure-like closed state by a striking clamshell-like inter-lobe dynamics. The two states are characterized by many distinct structural and dynamical differences and the conformational …

Development Of A Novel Class Of Chemicals For Labeling Abasic Sites In Cellular Dna And Killing Cancer Cells, Shanqiao Wei

Wayne State University Dissertations

Abasic (AP) sites are the most common type of lesions in DNA. Numerous endogenous and exogenous agents and cellular processes can induce the formation of AP sites in DNA. If left unrepaired, the deleterious AP sites cause mutagenesis and cytotoxicity. Methoxyamine is known to react with AP sites and block base excision repair. Another alkoxyamine, aldehyde-reactive probe (ARP) tags AP sites with a biotin and has been widely used to quantify these sites. In this study, I have combined both these abilities into one alkoxyamine, AA3, which reacts toward AP sites with better reactivity than ARP at physiological pH. Additionally, …

Identification Of Lipolysis-Derived Lipid Mediators And The Activation Of A Pro-Inflammatory Cyclooxygenase Pathway, Via Cyclooxygenase-2, In Adipose Tissue, Allison Christine Gartung

Wayne State University Dissertations

Adipose lipolysis triggers pro-inflammatory responses that play critical roles in insulin resistance and associated metabolic syndrome. However, pro-inflammatory mediators generated by adipose lipolysis, particularly in the context of lipid mediators, are poorly defined. In this study, the activation of the beta-3 adrenergic receptor (ADRB3)/hormone sensitive lipase (HSL) pathway, a well-employed model system, was utilized to characterize the pro-inflammatory lipid mediators generated by adipose lipolysis. Cultured adipocytes were treated with an ADRB3 agonist and the media was analyzed for eicosanoids using the LC-MS/MS lipidomic method. Among the characterized eicosanoids, I found that approximately 43 metabolites generated by cyclooxygenase (COX), lipoxygenase, and …

A Critical Role Of Cxcr2 Pdz Motif-Mediated Interactions In Endothelial Progenitor Cell Homing And Angiogenesis, Yuning Hou

Wayne State University Dissertations

Bone marrow-derived endothelial progenitor cells (EPCs) participate in postnatal vascularization in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl terminus. The PDZ motif has been reported to regulate cellular signaling and functions. Here we investigated the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We have found that introducing exogenous CXCR2 C-terminus significantly attenuated in vitro EPC migration and angiogenic activities in response to CXCR2 ligands, as well as in vivo EPC …

The Effects Of Courtship And Pairing Behavior On The Nonapeptide And Noradrenergic Systems Of Adult Male And Female Zebra Finches, Erin Lowrey Ondercin

Wayne State University Dissertations

Social relationships are complex and likely involve the multiple neural circuits, including those involved in learning, memory, motivation, and attention. Two neurotransmitter pathways highly involved in these neural circuits are norepinephrine (NE) and the nonapeptides, vasopressin (AVP) and oxytocin (OT). There is extensive research implicating a role for the nonapeptides in trust, sociality, parental care, and romantic relationships. There is little direct evidence for the role of nonapeptides in monogamous relationships in any species other than the prairie vole (Goodson 2013). However, there is evidence that nonapeptides are important in pair bonding for both male and female zebra finches (Lowrey …

Investigating The Role Of Dna Polymerase Beta In The Aging Phenotype Of Down Syndrome, Aqila Ahmed Ahmed

Wayne State University Dissertations

Down syndrome (DS) is a chromosomal condition characterized by accelerated aging that has yet to be directly linked to a DNA repair defect. Reduced PolB and unrepaired damage from oxidative stress observed in DS, point toward defective base excision repair (BER). In this study, we report that low PolB transcript correlates with increased markers of senescence. The gene dosage effect of Trisomy 21 is likely the source for PolB downregulation. We show that the HSA21-localized miR-155 overexpression correlates with a decrease in Creb1 and PolB, thus establishing a putative regulatory pathway. Data from the DS mouse model, Ts65Dn, reveal low …

An Investigation Into The Molecular Basis Underlying Enhancement Of Transcription By The Intron In Budding Yeast, Neha Agarwal Agarwal

Wayne State University Dissertations

It is now quite evident that the introns, which are removed from the primary transcript by the process of splicing, are involved in a variety of important functions in eukaryotic cells. One of the evolutionarily conserved functions of introns is their role in regulating transcription of genes that harbors them. This effect of a splicing-competent intron on transcription is known as ‘Intron-Mediated Enhancement of transcription’ (IME). It has been observed that the intron-containing genes are often transcribed more efficiently than non-intronic genes. However, the molecular mechanism underlying IME in budding yeast and higher eukaryotes is not entirely clear, and that …