Life Sciences Commons^™

Genetic And Biochemical Studies Of Human Apobec Family Of Proteins, Priyanga Wijesinghe

Wayne State University Dissertations

The AID/APOBEC family of proteins in higher vertebrates converts cytosines in DNA or RNA into uracil. These proteins have essential roles in either innate immunity or adaptive immunity. Recently, AID has also been implicated in DNA demethylation in the context of early embryogenesis in mammals. This is partly based on the reported ability of AID to deaminate 5-methyl cytosine to thymine (5mC to T). I reexamined this proposed new role of AID (5mC deamination) with two members of the APOBEC family in a novel Escherichia coli based genetic system. My results confirmed that while all three enzymes are strong cytosine …

Use Of Methionine Sulfoximine To Dissect The Role Of Glutamine Synthetase And Glutamine In Progression Of Acute Liver Failure, Amruta Anil Jambekar

Wayne State University Dissertations

Methionine sulfoximine (MSO) is a modified amino acid and a well characterized irreversible inhibitor of glutamine synthetase (GS) enzyme. Glutamine is synthesized by GS enzyme and it is the most abundant amino acid in the body. Glutamine is required by immune cells for generation, propagation and maintenance of an immune response.

To induce acute liver failure (ALF) in mice, animals were given intraperitoneal injections of E. coli lipopolysaccharides (LPS) and sugar D-galactosamine (D-GalN). When these animals were pretreated with MSO, 80% of the animals were completely rescued from liver failure. Moreover, when we characterized the immune response generated during ALF …

The Methuselah Family Of G Protein Coupled Receptors, Meghna V. Patel

Wayne State University Dissertations

G protein-coupled receptors (GPCRs) are one of the largest class of transmembrane signaling proteins that regulate essential developmental and physiological processes in a cell. GPCR success is illustrated by their abundance across both invertebrate and vertebrate genomes. Phylogenetic analyses show that GPCR families have undergone a lot of gene gain and loss during insect evolution. In Drosophila melanogaster, the fifteen Methuselah/Methuselah-like (Mth/Mthl) genes are in fact an insect specific family of GPCRs. In our study, we conducted a phylogenetic analysis using receptor sequences from five Drosophila species and two related insects, including Tribolium and Anopheles to examine the evolution of …

Phylogenetic Utility Of Mitochondrial And Nuclear Genes: A Case Study In The Diptera (True Flies), Jason Caravas

Wayne State University Dissertations

The value of mitochondrial versus nuclear gene sequence data in phylogenetic analysis has received much attention without yielding definitive conclusions. Theoretical arguments and empirical data suggest a lower phylogenetic utility than equivalent nuclear gene sequences, but there are also many examples of important progress made using mitochondrial sequences. We therefore undertook a systematic performance analysis of mitochondrial and nuclear sequence partitions taken from a representative sample of dipteran species. For phylogenetic tree reconstruction, mitochondrial genes performed generally inferior to nuclear genes. However, the mitochondrial genes resolved branches for which nuclear genes failed. Moreover, the combined use of mitochondrial and nuclear …

The Role Of Cad,Flash And Fam129b In Cancer Cell Survial And Apoptosis, Song Chen

Wayne State University Dissertations

Apoptosis is a normal process in the human body. However, apoptosis is desregulated in cancer cells. Most cancer cells gain resistance to apoptosis, leading to uncontrolled proliferation. In this dissertation, we identified three proteins, associated with apoptosis pathway. 1) CAD, a large multifunctional complex that is invariably elevated in tumor cells, 2) FLASH, a large protein with multiple growth related functions and 3) FAM129B. We demonstrate that CAD could interact with FLASH by using yeast two hybrid, co-immunopreciptation and fluorescence microscopy. In addition, functional analysis using siRNA technology further indicated that CAD could co-operate with FLASH and play roles in …

Gene Duplication And The Evolution Of The Higher Diptera, Riyue Bao

Wayne State University Dissertations

Gene duplication is an important source of evolutionary innovation. Using the publicly available genomic information, I studied lineage-specific gene duplications in Drosophila melanogaster (fruit fly), Anopheles gambiae (mosquito), Tribolium castaneum (red flour beetle), and Apis mellifera (honey bee) at three scales: eye-specific genes, developmental genes, and genome-wide. All three studies consistently show that the Drosophila genome contains an exceptionally high number of lineage-specific yet ancient gene duplicates, the majority of which must have originated during the early diversification of the higher Diptera (Brachycera) at least 100 million years ago. Genetic data suggest that gene duplication played an important role in …

Cellular Plasticity In White Adipose Tissue: In Vivo Identification Of Bipotent Adipocyte Progenitors In Adult White Adipose Tissue, Yun-Hee Lee

Wayne State University Dissertations

Nutritional and pharmacological stimuli can dramatically alter the cellular composition and phenotype of white adipose tissue (WAT). Nonetheless, the identity of progenitors that contribute to this cellular plasticity in vivo remains poorly understood. Utilizing genetic lineage tracing techniques in combination with in situ imunohistochemical analysis, we demonstrate that brown adipocytes (BA) that are induced by beta3-adrenergic receptor activation (ADRB3) in WAT arise from the proliferation and differentiation of cells that express platelet-derived growth factor receptor alpha (PDGFRA), CD34 and Sca1 (PDGFRA+ cells). PDGFRA+ cells have a unique morphology in which extended processes contact multiple cells in the tissue microenvironment. Surprisingly, …

Exploring Conformational Variability Of An Rna Domain In The Ribosome: From Structure And Function To Potential Antibiotic Targeting, Yogo Sakakibara

Wayne State University Dissertations

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome.

Previous model studies using small hairpin H69 showed the conformational differences …

In Vivo Display: A Selection And Its Derivatives For Antimicrobial Peptide Lead Identification, Wesley David Colangelo

Wayne State University Dissertations

The rise of antibiotic resistance necessitates new approaches for the isolation of new antimicrobials with novel inhibitory mechanisms, bypassing the development of rapid resistance by modification of pre-existing resistance mechanisms. In response, we have developed a series of systems for the rapid isolation and identification of peptides that inhibit the growth of Escherichia coli and other bacteria, termed in vivo display (IVD).

IVD harnesses the cellular processes of E. coli for the expression of a library of random peptides at the terminus of a display protein. A library of 12-amino acid random peptide sequences was added to either the C- …

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 …

Examining Autoimmune Sequelae During Cancer Immunotherapy In A Combined Autoimmune Thyroiditis And Tumor Mouse Model, Suresh Kari

Wayne State University Dissertations

Some clinical trials of immunotherapeutic agents against cancers have resulted in the development of autoimmune sequelae, including autoimmune thyroiditis. It has been suggested that the immunotherapy resulted in an alteration in the immunoregulatory mechanism(s). Using experimental autoimmune thyroiditis (EAT), a mouse model of Hashimoto's thyroiditis (HT), the importance of regulatory T cells (Tregs) has been well established; inhibition of Tregs leads to enhanced thyroiditis with repeated doses of mouse thyroglobulin (mTg) without adjuvant. To simulate cancer patients, we developed a combined EAT and tumor model in our EAT-susceptible mice (CBA/J). In first establishing the tumor model, we found Treg depletion …

Membrane-Bound Immunomodulators As Adjuvants In A Cell Culture-Based Avian Influenza Vaccine, David Daniel Fischer

Wayne State University Dissertations

Inactivated viral vaccines often generate suboptimal immune responses. Adjuvants are incorporated into vaccines to increase their immunogenicity, however currently available adjuvants have shortcomings which have limited their use in human and veterinary medicine. This necessitates the development of new adjuvants and delivery systems. Cytokines have been extensively tested as adjuvants in vaccines but challenges such as diffusion from antigen, short half-lives and production costs have been encountered. To address this, we developed a technology that efficiently produces inactivated, whole-virus influenza vaccine bearing membrane-bound cytokines. Tethering the cytokine to the antigen of interest keeps the immunomodulator in close contact with the …

Vip And Host Immunity, Xiaoyu Jiang

Wayne State University Dissertations

The purpose of the current dissertation was to examine how VIP regulates host immunity and corneal healing, specifically, its control of growth factors and TLR expression in the P. aeruginosa infected cornea. Firstly, VIP treatment increased growth factor expression (EGF, GHF, FGF and VEGF) in infected cornea. Notably, treatment with a mixture of EGF, FGF and HGF prevented corneal perforation, reduced pro-inflammatory cytokines and bacterial plate count, while increasing anti-inflammatory cytokines and antimicrobials such as murine beta-defensin2 and 3.

We also investigated the expression of TLR-signaling pathways in P. aeruginosa infected corneas with or without VIP treatment. PCR array and …

A Role For Reactive Oxygen Species In Photodynamic Therapy, Michael Price

Wayne State University Dissertations

Photodynamic therapy (PDT) is based on the ability of certain photosensitizing agents to selectively localize in neoplastic cells and their vasculature. Subsequent irradiation at a wavelength corresponding to a photosensitizer absorbance band excites the photosensitizer molecules, leading to energy transfer reactions and fluorescence. It was initially concluded that the phototoxic effect occurred when energy from the excited state of the photosensitizer was transferred to dissolved oxygen to form singlet oxygen. This product has a very brief half-life and will cause cellular damage only in the immediate vicinity of its formation. But an excited-state photosensitizer can also interact with oxygen to …

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 …