Life Sciences Commons^™

Functional Characterization Of Threonine 49 Phosphorylation Of Cytochrome C, Antoine Khobeir

Wayne State University Theses

Cytochrome c (Cytc) is a pivotal multifunctional mitochondrial protein that serves as a single electron carrier between complexes III and IV of the electron transport chain. It has important roles in both cellular respiration and apoptosis. The novel Thr49 (T49) phosphorylation of Cytc likely affects mitochondrial respiration, membrane potential, ROS production, ATP production, and apoptosis. Based on the functional characterization of previously mapped phosphorylation sites (Tyr97, Tyr48, Thr28, Ser47, Thr58) of the lab, we hypothesize that T49 phosphorylation will lead to controlled respiration, optimal intermediate mitochondrial membrane potential, lower ROS production, and inhibition of apoptosis compared to unphosphorylated Cytc. Here …

Novel Role Of Endoplasmic Reticulum-Associated Degradation In The Regulation Of Ceruloplasmin, Stephen William Hippleheuser

Wayne State University Theses

Ceruloplasmin (Cp) is a secreted ferroxidase produced by the hepatocytes that assists in the transport of iron throughout the human body. In human aceruloplasminemia patients, due to Cp deficiency excess intracellular iron buildup leads to ailments like liver cirrhosis, neurodegeneration, and blindness. We recently found that the biogenesis of Cp in the endoplasmic reticulum (ER) is regulated by a principal ER quality-control process, ER-associated degradation (ERAD). ERAD clears misfolded ER proteins for cytosolic proteasomal degradation, with the Sel-1 suppressor of lin-12-like (Sel1L)-HMG-CoA reductase degradation 1 (Hrd1) protein complex representing the most conserved branch in mammals. Interestingly, we found that Sel1L-Hrd1 …

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 …

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 …

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 …

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 …

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 …

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 …

Contribution Of Cx3cr1/Fractalkine (Cx3cl1) Axis To The Progression Of Non-Small Cell Lung Cancer (Nsclc), Rhonda Prince

Wayne State University Theses

A thesis is presented on the characterization of the contribution of the chemokine axis, CX_3CR1/Fractalkine, to the progression of Non-Small Cell Lung Cancer (NSCLC). Cancer is the second leading cause of death in the United States, and Lung cancer is one of the four leading causes of the disease. Carcinoma cells, unlike their normal counterparts, gain the ability to express chemokine receptors. Understanding if/how this gain of expression contributes to the progression of cancer could provide a lucrative therapeutic target. Goals of the study include investing if the NSCLC cell line A549, an adenocarcinoma cell line, express the chemokine receptor …

Synergetic Effect Of Permethrin And Dmh On Anti-Oxidation And Damage Response Genes, Karim Mohamed

Wayne State University Theses

The objective of this study was to evaluate the Synergetic Effect of Permethrin and 1,2-Dimethylhydrazine (DMH) on Anti-oxidation and Damage Response genes. The animal models used for this study were 8 week old C57/blk6 female mice. The mice were fed a Folate Adequate (FA) diet. At 8 weeks, the mice were intraperitoneally injected with pesticides. Three 8-week old female mice were injected intraperitoneally with a mix of cis and trans permethrin (75%:25% respectively) at 30% median lethal dose (96 mg/kg body weight). Permethrin was dissolved in corn oil and injected 24 hours before sacrifice. In three other mice, DMH was …

Rates And Modes Of Sequence Evolution In Various Lineages Within Chenopodiaceae, James Andrew Naeger

Wayne State University Theses

Sexual dimorphism in domesticated spinach, Spinacia oleracea, is thought to be determined by differential expression of floral organ identity genes. We examined the floral organ morphologies of seven species in the Chenopodiaceae, including two wild species of spinach, in order to gain insight into the evolution of floral reproductive strategy in this clade. The species within the Anserineae demonstrate extensive evolution in floral morphology and reproductive strategy, and Spinacia is unique for having been domesticated rather recently and for being dioecious. We found C. album to be hermaphroditic, C. foliosum and M. nuttalliana to be gynomonoecious, while C. bonus-henricus …

In Vitro Characterization Of Serine 47 Phosphorylated Cytochrome C, Ashwathy Mary Varughese

Wayne State University Theses

Cytochrome c is a 12.4kDa ubiquitously expressed protein known to be involved in many physiological processes of the cell such as respiration and apoptosis. The goal of our lab is to increase our knowledge of the regulation of cytochrome c in these opposite activities, and our working model posits that cytochrome c is decisively regulated by phosphorylation. When phosphorylated, cytochrome c leads to an "optimal" functioning in the electron transport chain by lowering electron flux, preventing harmful high mitochondrial membrane potentials and thus ROS production under healthy conditions. However, under cellular stress cytochrome c might be dephosphorylated favoring high mitochondrial …

The Nh2-Hypervariable Region Modulates The Binding Affinity Of Troponin T For Tropomyosin, Chinthaka Kaushalya Amarasinghe

Wayne State University Theses

The troponin complex plays a central role in the allosteric function of sarcomeric thin filaments by enacting conformational changes during the Ca2+-regulated contraction and relaxation of striated muscle. The troponin subunit T (TnT) has two binding sites for tropomyosin (Tm) and is responsible for anchoring the troponin complex to the thin filament. Although the C-terminal and middle regions of the TnT polypeptide chain are highly conserved among the three muscle type isoforms, the hypervariable N-terminal region has evolutionarily diverged significantly among isoforms. Previous studies have shown that the N-terminal variable region fine-tunes Ca2+ regulation of muscle contractility via modulation of …

Effect Of Folate Deficiency And Aging On Mtor Signaling Network In The Liver Of Dna Polymerase B Haploinsufficient Mice, Amanda Lynn Arrabi

Wayne State University Theses

EFFECT OF FOLATE DEFICIENCY AND AGING ON mTOR SIGNALING NETWORK IN THE LIVER OF DNA POLYMERASE B HAPLOINSUFFICIENT MICE

by

AMANDA ARRABI

August 2013

Advisor: Dr. Ahmad R. Heydari

Major: Nutrition and Food Science

Degree: Masters of Science

The interaction between the dietary nutrients and the human genome causes some alterations in the molecular pathways that could significantly impact the development of cancer. The main approach to such concerns is to closely observe how the diet modifies the gene expressions that control several cellular processes in the human body. Folate deficiency (FD) has established an important role in modulating the …

Investigating The Metal Binding Affinity Of Bacterial Zinc Transporters: Zupt And Truncated Znta (Znta-Pina), Fei Nie

Wayne State University Theses

Zinc is essential trace elements for all living organisms and serves as cofactor and structural cofactors for more than 300 enzymes. ZupT is one of the members of ZIP super family of transporter proteins, which were identified as iron and zinc transporter in Eukaryotes at first. However, recent evidence showed that some of members can also transport other metal ions, such as manganese or cadmium, and ZIP proteins also exist in bacteria. ZupT is responsible for zinc uptake in E. coli; however, its full metal selectivity is not yet characterized. In addition, the mechanism of transport is also unknown. Therefore, …

Characterizing Cyclin J By Identifying Conserved Protein-Protein Interactions, Phillip Jacob Selman

Wayne State University Theses

Cyclins are proteins that bind to Cyclin-dependent kinases, or Cdks, through a conserved domain called the Cyclin Box. Many Cyclins regulate the cell cycle. A few Cyclins impact cellular processes outside of the cell cycle. Also, a few Cyclins have poorly understood functions.

Cyclin J is a member of the Cyclin superfamily of proteins. Cyclin J is conserved among all metazoans, but is presently not well understood. All the research done on Cyclin J has been done in Drosophila.

Its mRNA is present in the early embryo, then disappears, only to reappear in adult females. When probing protein extracts with …

Optimization Of Labeling Techniques; Determination Of Best Parameter For Olfactory Mucosal Progenitor Cell Delivery And Study Of Effects Of Methylene Blue And Polyethylene Glycol In An Animal Model Of Spinal Cord Injury, Kiran Kumar Rokkappanavar

Wayne State University Theses

In the United States, the incidence of TSCI is about 40 per million persons per year, with approximately 250,000 living survivors of TSCI in the United States in July 2005. A number of human clinical trials (85) are ongoing using stem cells to evaluate the methods to reduce the injury following TBI. Among the stem cells, olfactory mucosal progenitor cells have several advantages with respect to ease of obtaining, fate of cells, etc. Our study was intended to optimize the labeling technique and to determine the best parameter to deliver the stem cells to the site of injury. We found …

Role Of Phosphoylation Of The Mycobacterium Tuberculosis Proteasome In Oxidative Stress Response And Regulation Of Homoserine Kinase (Thrb) In Corynebacterium Glutamicum, Heather Baun

Wayne State University Theses

Part 1:

One-third of the world's population is infected with the latent form of Mycobacterium tuberculosis. M. tuberculosis must be able to control this latent even in response to nitrosative and oxidative stress conferred by the host. It is not well known how M. tuberculosis controls this state.

One possibility for this control is through its eleven eukaryotic-like serine/threonine protein kinases. Our lab has focused research on two eukaryotic-like ser/thr protein kinases, PknA and PknB. It has been found that PknA expression may affect the stability or the assembly of the proteasome complex, which is essential for M. tuberculosis to …

Investigation Of Bacterial Rna-Directed Dna Methylation Via Dcm And Hfq, Dandan Li

Wayne State University Theses

Bacterial small RNAs and the RNA chaperone Hfq play crucial roles in post-transcriptional gene regulation, often as parts of stress-response pathways, but little is known about their roles in regulation of gene transcription. A recent report showed that changes in methylation patterns caused by DNA cytosine methyltransferase (Dcm) were linked to gene regulation occurring during the transition to stationary phase. Here, we show that Dcm involves in the stress responses under nutrient starvation and cold stress. Dcm and Hfq together mediate gene expression under cold stress. Hfq promotes Dcm-catalyzed cytosine methylation at specific sites near the rpoS promoter, which is …

Cysteine Desulfurase And Isd11: A Drosophila Model, Rahul Ravindran Nair

Wayne State University Theses

ABSTRACT

Cysteine desulfurase and Isd11: A Drosophila model

Iron-sulfur clusters are cofactors with evolutionary origins that date back to the pre-biotic world. Ever since life originated, these cofactors have intermingled with proteins to play vital roles in sustaining life. My research focuses on one such protein, the cysteine desulfurase (Nfs) that has the PLP cofactor incorporated in its active site and avails of the catalytic property of PLP to provide sulphur for Iron-sulfur cluster biogenesis and assembly in a cell. Interestingly, in a eukaryotic cell, despite the versatility of PLP, cysteine desulfurase's role as a "sulphur-extractor" is incomplete without another …

Studies Of Regulated Exocytosis From Neuroendocrine Cells, Madhurima Das

Wayne State University Theses

In this thesis we study cargo release and fusion pore dilation during calcium triggered exocytosis and the co-localization of calcium sensing proteins essential for exocytosis, in neuroendocrine cells.

Pancreatic beta cells secrete several hormones, the most studied one being insulin. C-peptide is a protein which is co-stored with and secreted from the same vesicles as insulin. It is found in the soluble phase unlike insulin, which is found in the dense core. The pancreatic beta cells also secrete the Chromogranin B (CgB) which is mostly found in the dense cores of secretory vesicles. In chapter 1, we found that CgB, …

Modulation Of Anti-Tumor Immune Response By Tgf-Β-Inducible Early Gene 1 (Tieg1), Andi Cani

Wayne State University Theses

Cancer immunotherapy has had limited clinical efficacy partly because regulatory T cells (Treg) suppress the immune response to tumor-associated antigens. Inducible regulatory T cells (iTreg), which are converted from naïve CD4 T cells by TGF-β, an abundant cytokine in the tumor microenvironment, may contribute to this immune suppression. Induction of Foxp3 by TGF-β is mediated by the transcription factor TIEG1 and abrogation of this protein prevents Foxp3 expression. We are testing the hypothesis that blockade of TIEG1 to prevent iTreg conversion will enhance immune response in DNA vaccination to the tumor associated antigen Her-2. Wild type and TIEG1 knockout mice …

Investigation Of A 16s Rna Central Domain Pseudoknot, Jenna Marie Jasinski-Bolak

Wayne State University Theses

X-ray crystallography of the prokaryotic 30S ribosomal subunit revealed a myriad of complex RNA-RNA, RNA-protein, and protein-protein interactions. Among these are several phylogenetically conserved RNA pseudoknots. Pseudoknots are structurally and functionally diverse RNA secondary structures. They are generally formed by two short complimentary sequences separated by many bases of single stranded regions or loops. These relatively simple folds are often yield complex structures that are key components of functionally important conformational changes in RNA structure. One such pseudoknot is located in the central domain of the 16S rRNA.

The central domain pseudoknot is formed by Watson-Crick base pairing between G570-C866 …

Disrupting Cxcr2 Macromolecular Complex Pdz-Domain Interactions During Inflammatory Chemotaxis, Marcello Castelvetere

Wayne State University Theses

Neutrophils are the body's first responders to inflammation, being the most abundant white blood cell type in circulation and they quickly initiate an immune response through chemokine signaling. Inflammatory chemokines signal via their receptor CXCR2, which initiates an inflammatory response, recruiting leukocytes to sites of inflammation. Chemokine signaling is important for proper host protection, yet uncontrolled activity is responsible for a variety of pathological conditions: including rheumatoid arthritis, ischemia-reperfusion injury, arteriosclerosis, multiple sclerosis, psoriasis, inflammatory bowel disease, and allergic reactions.

In this report I show a CXCR2 macromolecular signaling complex exists in neutrophils, containing NHERF1 and PLCβ2. I also demonstrate …

The Role Of Zip Superfamily Of Metal Transporters In Chronic Diseases, Purification & Characterization Of A Bacterial Zip Transporter: Zupt., Iryna King

Wayne State University Theses

In mammals zinc is the second most abundant essential trace metal. Since Zn² is a small, hydrophilic, and a highly charged ion, it cannot be transported across the plasma or intracellular organelle membrane by passive diffusion. Different types of cells require a different constant concentration of zinc at all times. Presence of excess free Zn ions can be toxic to the cell. All cells must have tightly regulated homeostatic mechanisms in order to preserve healthy levels and proper compartmentalization of zinc. This is accomplished through the actions of specialized proteins that facilitate zinc uptake, efflux and compartmentalization. If the …

Identification Of Deletion Mutants Of Inositol Kinases And Phosphatases Hypersensitive To Valproate, W-Mudiyanselage Manoj Senaka Bandara

Wayne State University Theses

ABSTRACT

IDENTIFICATION OF DELETION MUTANTS OF INOSITOL KINASES AND PHOSPHATASES HYPERSENSITIVE TO VALPROATE

by

WELLEWATTA MUDIYANSELAGE MANOJ SENAKA BANDARA

MAY 2010

Advisor: Dr. Miriam Greenberg

Major: Biological Sciences

Degree: Master of Science

Bipolar disorder (BD) is a chronic psychiatric illness affecting at least 1% of the world population. BD is ranked as the sixth greatest cause of death or disability globally. The cause of BD is unknown. Although the anticonvulsant valproate (VPA) is widely used as a mood stabilizer to treat BD, VPA is not completely effective and causes numerous side effects. Hence, it is important to develop more effective …