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 …

Novel Mechanisms In The Sorting Of Proglucagon To The Secretory Granules Of The Regulated Secretory Pathway, Leonard M. Guizzetti

Electronic Thesis and Dissertation Repository

The prohormone proglucagon encodes for multiple peptide hormones, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, produced through tissue-specific processing by prohormone convertase (PC) 1/3 and PC2. In alpha cells, PC2 yields glucagon, the major counter-regulatory hormone to insulin, which together, control glucose homeostasis. In contrast, GLP-1 and GLP2 are mainly produced in intestinal L-cells by PC1/3. GLP-1 stimulates insulin secretion following a meal, and therefore has opposing function to glucagon regulating glucose homeostasis; in contrast, GLP-2 enhances gut nutrient absorption. Efficient sorting of proglucagon to secretory granules is required for nutrient-regulated secretion. The aim of this thesis is to discover …

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 …

Acquisition And Reconstruction Techniques For Fat Quantification Using Magnetic Resonance Imaging, Abraam S. Soliman

Electronic Thesis and Dissertation Repository

Quantifying the tissue fat concentration is important for several diseases in various organs including liver, heart, skeletal muscle and kidney. Uniquely, MRI can separate the signal from water and fat in-vivo, rendering it the most suitable imaging modality for non-invasive fat quantification. Chemical-shift-encoded MRI is commonly used for quantitative fat measurement due to its unique ability to generate a separate image for water and fat. The tissue fat concentration can be consequently estimated from the two images. However, several confounding factors can hinder the water/fat separation process, leading to incorrect estimation of fat concentration.

The inhomogeneities of the main …

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 …

Elucidating The Signalling Pathway Of Mer Tyrosine Kinase Receptor In Efferocytosis, Ekenedelichukwu Azu

Electronic Thesis and Dissertation Repository

Efferocytosis is the clearance of apoptotic cells and is necessary for homeostasis. Mer Tyrosine Kinase (MerTK) is a crucial efferocytic receptor whose loss is associated with chronic inflammatory diseases and autoimmunity. While previous studies have shown that MerTK mediates efferocytosis through a unique mechanism that requires integrins, MerTK signalling pathway remains unknown. Given this unusual internalization mechanism, I hypothesized that MerTK signals and engages integrins through a novel signalling pathway different from that used by other phagocytic receptors. Therefore, this study aimed to identify the signalling pathways activated by MerTK, utilizing conventional cell biology and pharmacological approaches.

I found that …

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 …

Megatevs: Single-Chain Dual Nucleases For Efficient Gene Disruption, Jason M Wolfs, Matthew Dasilva, Sarah E Meister, Xu Wang, Caroline Schild-Poulter, David R. Edgell

Biochemistry Publications

Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, …

Automating Dicentric Chromosome Detection From Cytogenetic Biodosimetry Data., Peter K Rogan, Yanxin Li, Asanka Wickramasinghe, Akila Subasinghe, Natasha Caminsky, Wahab Khan, Jagath Samarabandu, Ruth Wilkins, Farrah Flegal, Joan H Knoll

Biochemistry Publications

We present a prototype software system with sufficient capacity and speed to estimate radiation exposures in a mass casualty event by counting dicentric chromosomes (DCs) in metaphase cells from many individuals. Top-ranked metaphase cell images are segmented by classifying and defining chromosomes with an active contour gradient vector field (GVF) and by determining centromere locations along the centreline. The centreline is extracted by discrete curve evolution (DCE) skeleton branch pruning and curve interpolation. Centromere detection minimises the global width and DAPI-staining intensity profiles along the centreline. A second centromere is identified by reapplying this procedure after masking the first. Dicentrics …

Investigating The Role Of Retinoblastoma Protein And Transforming Growth Factor-Beta In Growth Inhibition, Mehdi Amiri

Electronic Thesis and Dissertation Repository

The Retinoblastoma protein (pRB) is a key regulator of cell proliferation in the G1 phase of the cell cycle. The LxCxE binding cleft is a highly conserved region of pRB. Using a knock-in mouse model, called Rb1^∆L, with disrupted pRB and LxCxE interactions, our lab has shown that epithelial cells from Rb1^∆L/∆L mice do not respond to TGF-β1 mediated growth arrest. Using shRNAs to deplete the expression of components of LxCxE motif containing complexes, data showed that SAP18 is not involved in TGF-β1 mediated growth arrest. However, depletion of SAP30 and MTA2 compromised TGF-β1 mediated growth arrest. …

Oriented Collagen And Applications Of Waveguide Evanescent Field Scattering (Wefs) Microscopy, Qamrun Nahar

Electronic Thesis and Dissertation Repository

In this thesis, Waveguide Evanescent Field Scattering (WEFS) microscopy is developed as a non-invasive, label-free live cell imaging technique. This new high-contrast imaging can be employed to study the first hundred nanometers from the surface as it utilizes the evanescent field of a waveguide as the illumination source. Previously, waveguide evanescent field fluorescence (WEFF) microscopy was developed as a fluorescence imaging technique comparable to the total internal reflection fluorescent (TIRF) microscopy. Both the WEFF and WEFS technique utilizes the same fundamental concepts except in WEFS microscopy imaging is accomplished without the application of any fluorescent labeling. In this work, bacterial …

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 …

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 …

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 …

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 …

Sirna Targeting Of Thymidylate Synthase, Thymidine Kinase 1 And Thymidine Kinase 2 As An Anticancer Therapy: A Combinatorial Rnai Approach, Christine Di Cresce

Electronic Thesis and Dissertation Repository

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular …

Validation Of Predicted Mrna Splicing Mutations Using High-Throughput Transcriptome Data, Coby Viner, Stephanie Dorman, Ben Shirley, Peter Rogan

Biochemistry Publications

Interpretation of variants present in complete genomes or exomes reveals numerous sequence changes, only a fraction of which are likely to be pathogenic. Mutations have been traditionally inferred from allele frequencies and inheritance patterns in such data. Variants predicted to alter mRNA splicing can be validated by manual inspection of transcriptome sequencing data, however this approach is intractable for large datasets. These abnormal mRNA splicing patterns are characterized by reads demonstrating either exon skipping, cryptic splice site use, and high levels of intron inclusion, or combinations of these properties. We present, Veridical, an in silico method for the automatic validation …

Splicing Mutation Analysis Reveals Previously Unrecognized Pathways In Lymph Node-Invasive Breast Cancer., Stephanie N Dorman, Coby Viner, Peter K Rogan

Biochemistry Publications

Somatic mutations reported in large-scale breast cancer (BC) sequencing studies primarily consist of protein coding mutations. mRNA splicing mutation analyses have been limited in scope, despite their prevalence in Mendelian genetic disorders. We predicted splicing mutations in 442 BC tumour and matched normal exomes from The Cancer Genome Atlas Consortium (TCGA). These splicing defects were validated by abnormal expression changes in these tumours. Of the 5,206 putative mutations identified, exon skipping, leaky or cryptic splicing was confirmed for 988 variants. Pathway enrichment analysis of the mutated genes revealed mutations in 9 NCAM1-related pathways, which were significantly increased in samples with …

Interpretation Of Mrna Splicing Mutations In Genetic Disease: Review Of The Literature And Guidelines For Information-Theoretical Analysis., Natasha Caminsky, Eliseos J Mucaki, Peter K Rogan

Biochemistry Publications

The interpretation of genomic variants has become one of the paramount challenges in the post-genome sequencing era. In this review we summarize nearly 20 years of research on the applications of information theory (IT) to interpret coding and non-coding mutations that alter mRNA splicing in rare and common diseases. We compile and summarize the spectrum of published variants analyzed by IT, to provide a broad perspective of the distribution of deleterious natural and cryptic splice site variants detected, as well as those affecting splicing regulatory sequences. Results for natural splice site mutations can be interrogated dynamically with Splicing Mutation Calculator, …

Localized, Non-Random Differences In Chromatin Accessibility Between Homologous Metaphase Chromosomes., Wahab A Khan, Peter K Rogan, Joan Hm Knoll

Biochemistry Publications

BACKGROUND: Condensation differences along the lengths of homologous, mitotic metaphase chromosomes are well known. This study reports molecular cytogenetic data showing quantifiable localized differences in condensation between homologs that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy DNA probes used in fluorescence in situ hybridization.

RESULTS: Fourteen probes (from chromosomes 1, 5, 9, 11, 15, 17, 22) targeting genic and intergenic regions were developed and hybridized to cells from 10 individuals with cytogenetically-distinguishable homologs. Differences in hybridization between homologs were non-random for 8 …

Validation Of Predicted Mrna Splicing Mutations Using High-Throughput Transcriptome Data., Coby Viner, Stephanie N Dorman, Ben C Shirley, Peter K Rogan

Biochemistry Publications

Interpretation of variants present in complete genomes or exomes reveals numerous sequence changes, only a fraction of which are likely to be pathogenic. Mutations have been traditionally inferred from allele frequencies and inheritance patterns in such data. Variants predicted to alter mRNA splicing can be validated by manual inspection of transcriptome sequencing data, however this approach is intractable for large datasets. These abnormal mRNA splicing patterns are characterized by reads demonstrating either exon skipping, cryptic splice site use, and high levels of intron inclusion, or combinations of these properties. We present, Veridical, an in silico method for the automatic validation …