Hcpe, A Potential Immuno-Modulatory Protein From Helicobacter Pylori That Is Dependent On The Disulfide Bond Protein Dsbhp, Jeff Lester

Electronic Thesis and Dissertation Repository

H. pylori is a human gastric pathogen that colonizes ~ 50% of the world’s population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. H. pylori produces Helicobacter cysteine rich protein HcpE, a secreted protein which may play a role in virulence. In this study we show that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles, and may play a role in the modulation of H. pylori inflammatory responses. We identified that Dsb_HP is necessary for HcpE production and secretion in H. pylori, and …

The Role Of The Ku70 Vwa Domain In The Response To Dna Double Strand Breaks, Victoria L. Fell

Electronic Thesis and Dissertation Repository

Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, and is best characterized for its central role as the initial DNA end binding factor in the “classical” non-homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. In this work we …

Regulation Of Igfbp-1 Phosphorylation In Hypoxia Via Mtor Signaling, Ian Damerill

Electronic Thesis and Dissertation Repository

In this study, we provide novel evidence for a role of fetal liver mTOR signaling in regulating IGF-I bioavailability by modulating IGFBP-1 phosphorylation due to hypoxia – a key factor in the development of reduced fetal growth in utero. We utilized HepG2 cells in vitro and demonstrated a link between mTOR inhibition and hypoxia-induced IGFBP-1 phosphorylation. Using a biological assay for IGF-I receptor autophosphorylation, we demonstrated a functional significance for hypoxia-induced IGFBP-1 phosphorylation in reducing IGF-I bioactivity in vitro. Further, we have implicated a mechanistic link to increased CK2 activity within this regulation. We demonstrate that mTOR inhibition …

Post-Translational Control Of Retinoblastoma Protein Phosphorylation, Paul M. Stafford

Electronic Thesis and Dissertation Repository

The retinoblastoma tumor suppressor protein (pRB) functions through multiple mechanisms to serve as a tumor suppressor. pRB has been well characterized to be inactivated through phosphorylation by CDKs. pRB dephosphorylation and activation is a much less characterized aspect of pRB function. In this thesis, I detail work to study the post translational control of pRB phosphorylation. Here I present work detailing efforts to generate a gene targeted mouse which disrupts PP1 binding to the C-terminus of pRB, allowing for detailed study of the mechanisms of pRB dephosphorylation. This work also details an examination of acetylation in the C-terminus of pRB, …

Regulation Of Saga By The N-Terminus Of Spt7 In Saccharomyces Cerevisiae, Dominik Dobransky

Electronic Thesis and Dissertation Repository

Spt7 is a 1,332 residue protein critical for maintaining structural integrity of the SAGA complex. I demonstrated that the extreme N-terminus of Spt7 plays an important role in SAGA function. Deletion of the first 73 (Spt7_73-1332) and 121 (Spt7_121-1332) N- terminal residues resulted in slow growth, decreased transcriptional activation at PHO5 and INO1, and a partial decrease in acetylation at lysine 18 of histone H3 at PHO5. The Spt7_121-1332 mutant did not affect Spt7’s association with Gcn5 or Tra1, or its localization within the cell. Mutation of the first four positively charged residues …

The Role Of The Pre-Sensor 1 Β Hairpin In Minichromosome Maintenance 2-7 Function, Simon K. W. Lam

Electronic Thesis and Dissertation Repository

The pre-sensor 1 (PS1) hairpin is found in helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding. To determine whether the PS1 b hairpin is required in the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the PS1 hairpin in each of the S. cerevisiae Mcm subunits to alanine. Only the PS1 hairpin of Mcm3 was essential for viability, while mutation of the PS1 hairpin in the remaining Mcm subunits resulted in minimal phenotypes, with the exception …

The Roles Of Matrix-Associated Periostin In An In Vitro Model Of Hypertrophic Scarring, Justin D. Crawford

Electronic Thesis and Dissertation Repository

Abnormal scarring is a type of benign fibrosis of the skin that can restrict mobility, dexterity and quality of life. There are few, if any, truly effective treatment options for these conditions. Hypertrophic scarring is a common form of abnormal scarring characterized by increased fibroblast proliferation and differentiation of apoptosis-resistant and hyper-contractile myofibroblasts that promote excessive deposition and contracture of the extracellular matrix (ECM). Periostin, a secreted ECM protein, is transiently expressed during normal cutaneous wound repair, but is abnormally abundant and persistent in abnormal scars and other benign fibroses that display enhanced fibroblast proliferation and myofibroblast differentiation. The objectives …

Mechanistics Of Prothymosin Alpha And Nrf2 In The Keap1-Nrf2 Mediated Oxidative Stress Response, Halema Khan

Electronic Thesis and Dissertation Repository

In an effort to dissect the mechanism of interaction of IDPs, in this thesis we focus on Prothymosin a (ProTa) and nuclear factor erythroid 2-related factor 2 (Nrf2), intrinsically disordered proteins, in the Nrf2 mediated oxidative stress response. Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of Nrf2, a key transcription factor of cytoprotective genes. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses Nrf2 activity. During oxidative stress, Nrf2 is released from Keap1 and is shuttled to the nucleus, where it initiates pro cell survival gene transcription. ProTa also interacts with the …

Characterizing The Domain- And Phosphorylation-Requirements Of The Interaction Between Peptidyl Prolyl Isomerase Pin1 And Mitotic Phosphatase Cdc25c, Dana Onica

Electronic Thesis and Dissertation Repository

The enzyme Pin1 is a peptidyl-prolyl cis-trans isomerase consisting structurally of two domains, an N-terminal WW protein interaction domain and a C-terminal PPIase catalytic domain. Both domains bind a phosphorylated serine/threonine-proline motif, however, a precise mechanism regarding how binding to interactors is coordinated by both domains has not yet been determined. Although multiple models exist to explain this process, it appears that the interactions may be substrate-specific. With regards to a well–studied Pin1 interactor, CDC25C, we hypothesize that binding occurs via the simultaneous model. This model suggests that two binding sites, each having low affinity, may bind in concert producing …

The Role Of The Arginine Methyltransferase Carm1 In Global Transcriptional Regulation., Niamh Coughlan

Electronic Thesis and Dissertation Repository

Arginine methylation is a prevalent post-translational modification that is found on many nuclear and cytoplasmic proteins, and has been implicated in the regulation of gene expression. CARM1 is a member of the protein arginine methyltransferase (PRMT) family of proteins, and is a key protein responsible for arginine methylation of a subset of proteins involved in transcription. In this thesis I examine some of the mechanisms through which CARM1 contributes to global transcriptional regulation.

Using a ChIP-DSL approach, we show that the p/CIP/CARM1 complex is recruited to 204 proximal promoters following 17β-estradiol (E2) treatment in MCF-7 cells. Many of the target …

Targeted Proteomics Of Human Pluripotent Stem Cells, Kevin Gregory Kania

Electronic Thesis and Dissertation Repository

Human pluripotent stem cells (hPSCs) exhibit two unique characteristics: pluripotency and self-renewal. These properties are maintained by a series of complex signaling pathways, however, quantitative data for the respective proteins is lacking. Selected reaction monitoring (SRM) is a targeted, quantitative technique in mass spectrometry that is highly sensitive in peptide detection. In this thesis, an SRM protocol was developed in order to detect and quantify a defined set of proteins responsible for maintaining stem cell pluripotency. Two hESC differentiation protocols were validated for use as model systems within which to measure differential protein expression by SRM. SRM assays were generated …

Maternal Control Of Genomic Imprinting: Effects Of Infertility And Ovarian Stimulation In A Mouse Model, Michelle M. Denomme

Electronic Thesis and Dissertation Repository

Gametogenesis and early embryogenesis are important stages in which genome-wide epigenetic transitions required for early mammalian development are orchestrated. This is exemplified by the occurrence of genomic imprinting, where epigenetic mechanisms lead to the monoallelic expression of a subset of genes. Parental-specific DNA methylation in the gametes results in the distinct nonequivalence of the parental genomes in the early embryo. Changes from normal gamete and embryo development by impaired fertility or assisted reproductive technologies (ARTs) may disrupt the processes of imprint acquisition and imprint maintenance. My hypothesis is that aberrant imprinted methylation arises from impaired maternal fertility or ovarian stimulation …