Molecular Biology Commons^™

The Association Between Oxidative Stress, Cellular Differentiation And Galectins In Human Promyelocytic Leukemia Cells (Hl-60), James R. Vinnai

Electronic Thesis and Dissertation Repository

Galectins are a group of β-galactoside-binding proteins involved in different cellular processes including stress responses and differentiation. The role and expression of galectins under oxidative stress and during neutrophilic differentiation was examined in HL-60 cells. Galectin gene (LGALS), and galectin protein expression were determined using RT-qPCR and immunoblotting, respectively. Neutrophilic differentiation was measured via a spectrofluorometric assay. DNA methylation and JNK signaling were investigated as galectin regulatory mechanisms. Menadione-induced oxidative stress, DMSO-induced differentiation, DNA hypomethylation and JNK signaling all promoted similar galectin expression profiles. Antioxidant N-acetylcysteine attenuated the menadione-induced galectin expression but only partially attenuated DMSO-induced galectin expression. …

Comprehensive Molecular Characterization Of Human Nodal, Scott D. Findlay

Electronic Thesis and Dissertation Repository

Nodal and related ligands are highly conserved members of the TGF-beta superfamily with well-established and essential roles in the early embryonic development of vertebrates, and in cell fate decisions in human embryonic stem (hES) cells. Aberrant NODAL signaling also generally promotes pro-tumourigenic phenotypes and the progression of a wide array of human cancers. Despite being pursued as a potential therapeutic target, many aspects of NODAL’s molecular biology remain poorly understood. This thesis provides a comprehensive characterization of gene expression from the human NODAL locus at multiple levels. First, an intronic NODAL SNP known as rs2231947 was found to be functional …

Riboregulation Of Bacterial Transposons, Michael J. Ellis

Electronic Thesis and Dissertation Repository

Bacterial transposons typically exist in a mutually beneficial relationship with the host cell. Limited transposition can benefit the host while also ensuring the survival of the element. An important component of this relationship is that transposition must be tightly regulated. In this thesis I explore ways that the host and transposon each control transposition in E. coli and provide evidence that a transposon can also control host gene expression in S. enterica Typhimurium. Post-transcriptional regulation with small non-coding RNAs (sRNA) has emerged as a key way that bacteria respond to stress and regulate many cellular processes. The RNA-binding protein Hfq …

Freeze Tolerance Of Cyphoderris Monstrosa (Orthoptera: Prophalangopsidae), Jantina Toxopeus, Jacqueline Lebenzon, Alexander H. Mckinnon, Brent J. Sinclair

Biology Publications

The great grig, Cyphoderris monstrosa Uhler (Orthoptera: Prophalangopsidae), is a large (20-30 mm, >1 g), nocturnal ensiferan that in habits montane coniferous forests in northwestern North America. C. monstrosa overwinters as a late-instar nymph, but its cold tolerance strategy has not previously been reported. We collected nymphs from near Kamloops, British Columbia, in late spring to determine their cold tolerance strategy. C. monstrosa nymphs were active at low temperatures until they froze at -4.6 ± 0.3 °C. The nymphs survived internal ice formation (i.e. are freeze tolerant), had a lethal temperature between -9 and -12 °C, and could survive for …

Molecular Characterization Of Viruses Infecting Greenhouse Vegetables In Ontario, Bin Chen

Electronic Thesis and Dissertation Repository

Greenhouse vegetable production is a very important and successful agricultural sector in Ontario. However, newly emerging viral or virus-like diseases have become a major limiting factor in greenhouse vegetable production. To determine the identity of the viral pathogens in the three major greenhouse vegetables in Ontario, we conducted in-depth sequencing of small RNAs isolated from virally infected plants used next-generation sequencing technology. Subsequent bioinformatics analyses revealed six viral pathogens including Bell pepper endornavirus (BPEV), Parietaria mottle virus (PMoV), Cucumber green mottle mosaic virus (CGMMV), Tobacco streak virus (TSV), Pepper mild mottle virus (PMMoV), and Pepino mosaic virus (PepMV). The full-length …

Development And Performance Evaluation Of An Antibody-Based Technology For Detection Of E. Coli O157 In Meat Samples And Its Potential Evolution Using Antibody Engineering, Yadira Tejeda Saldaña

Electronic Thesis and Dissertation Repository

Escherichia coli O157 is a persistent pathogen linked to food and waterborne infectious outbreaks with severe health consequences such as hemorrhagic colitis and hemolyticuremic syndrome (HUS). Because it is considered one of the major pathogens that contributes to the global burden of foodborne disease, its early detection within the food chain is an important milestone towards reducing foodborne diseases and economic losses due to contaminated food. Herein, the development and validation of a lateral flow immunoassay (LFIA) point-of-care (POC) device is described. Application of the LFIA test kit was focused on detection of E. coli O157 in raw meat products …

Characterization Of The Catalytic Ck2 Subunits With Substitutions At Residues Involved In Inhibitor Binding, Paul Desormeaux

Electronic Thesis and Dissertation Repository

CK2 is a constitutively active, ubiquitously expressed and pleiotropic serine/threonine protein kinase that is implicated in many cellular functions including tumorigenesis. CK2 has two catalytic subunits, CK2a and CK2a’, that carry out its function in the cell. Previous studies have indicated that inhibitor-refractory mutants have been effective in recovering residual CK2 activity, in the presence of inhibitors, when compared to wild type CK2. Based on these observations, inhibitor-refractory mutants were created for both CK2a and CK2a’ and tested with various concentrations with two CK2-specific inhibitors, CX-4945 and inhibitor VIII. The CK2a triple mutant (V66A/I174A/H160D) was tested in inducible U2OS Flp-In …

Multi-Gene Resistance To Neonicotinoids In The Colorado Potato Beetle, Leptinotarsa Decemlineata, Emine Kaplanoglu

Electronic Thesis and Dissertation Repository

The Colorado potato beetle, Leptinotarsa decemlineata, is a significant pest of potato, and its impact on agriculture is measured on a global scale. The beetle is mainly controlled by neonicotinoid insecticides, however, resistance development is a growing concern. Resistance to neonicotinoids is thought to involve elevated activity of detoxifying enzymes and xenobiotic transporters that break-down and excrete insecticide molecules. Here, using mRNA sequencing, I identified multiple detoxifying enzyme and xenobiotic transporter genes transcriptionally up-regulated in a neonicotinoid resistant strain of beetles. I then used RNA interference to knock down the transcript levels of the ten most promising genes in …

Iron Acquisition Strategies Employed By Staphylococcus Lugdunensis, Jeremy R. Brozyna

Electronic Thesis and Dissertation Repository

Iron is crucial for many cellular processes including DNA synthesis and respiration. The majority of iron in mammals is in heme within hemoproteins, inside cells, or transported through circulation by the glycoprotein transferrin, which constitutes the greatest iron source in serum. Limiting iron availability is an important facet of nutritional immunity to help prevent infection.

Staphylococcus lugdunensis is a human skin commensal and opportunistic pathogen capable of causing a variety of infections, including particularly aggressive endocarditis. It is an emerging pathogen with elevated virulence compared to other species of coagulase-negative staphylococci. The versatility of S. lugdunensis to infect multiple niches …

Insights Into Chibby's Structural Elements And Their Interplay In Wnt Signaling Protein-Protein Interactions, Ryan C Killoran

Electronic Thesis and Dissertation Repository

The Wnt/b-catenin signaling pathway is critical to embryonic development and adult tissue homeostasis. Mutations to Wnt signaling components can cause dysregulation of the pathway, leading to various human diseases such as cancer. The partially disordered protein Chibby (Cby) is a conserved nuclear protein that acts as an antagonist in the Wnt/b-catenin signaling pathway. Cby’s antagonism is accomplished via two mechanisms. First, by competing with the Tcf/Lef family of transcription factors, Cby abrogates the b-catenin-mediated transcription of Wnt signaling genes. Moreover, upon phosphorylation on serine 20 by the kinase Akt, Cby forms a complex with the protein 14-3-3 to facilitate the …

Cal And Magi Pdz Protein Regulation Of Crfr1 And 5-Ht2ar Trafficking And Signaling, Maha Mahmoud Hammad

Electronic Thesis and Dissertation Repository

PDZ (PSD95/Disc Large/Zona Occludens) domain-containing proteins are scaffolding proteins that play important roles in regulating the activity of G protein-coupled receptors. Corticotropin Releasing Factor Receptor 1 (CRFR1) and Serotonin 2A Receptor (5-HT_2AR) are two GPCRs that are commonly associated with mental disorders. Both receptors also contain a class I PDZ-binding motif at the carboxyl terminal tail. In the first chapter, we investigate the effects of CAL (CFTR-associated ligand) on regulating the trafficking and signaling of CRFR1. We demonstrate a role for CAL in inhibiting CRFR1 endocytosis, cell surface expression, and CRF-mediated ERK1/2 signaling via the CRFR1 PDZ-binding motif. …

Regulation Of Phosphatase And Tensin Homolog Expression And Activity By Transforming Growth-Factor Beta In The Trabecular Meshwork Cells: Implications For Primary Open Angle Glaucoma, Nikoleta Tellios

Electronic Thesis and Dissertation Repository

Glaucoma is a multifactorial condition caused, in part, by fibrosis of the sieve-like trabecular meshwork (TM) tissue, which impedes drainage of aqueous humor (AH), leading to increased intraocular pressure and associated optic nerve damage and blindness. Fibrosis of the TM is mainly caused by the increased levels of active transforming growth factor-β 2 (TGFβ2) in the AH of glaucoma patients.

Previous reports have shown that TGFβ decreases the expression of Phosphatase and Tensin Homolog (PTEN) gene and that PTEN is a major regulator of ECM deposition. In this study we investigate the regulation of PTEN protein expression and …

Characterization Of Numb As A Regulator Of Anaplastic Lymphoma Kinase, Ran Wei

Electronic Thesis and Dissertation Repository

Cellular events rely on protein-protein interactions that are often mediated by modular domains which recognize particular sequence motifs in binding partners. The NUMB protein is the first described cell fate determinant and multifaceted adaptor that is involved in a wide variety of cellular events. NUMB mainly mediates protein interactions via its modular PTB domain. Here we present a systematic investigation of the NUMB-PTB interactome by employing an integrative strategy combining both protein and peptide arrays. We profiled NUMB-PTB binding specificity and interacting proteins genome-wide. The receptor tyrosine kinases (RTKs) are found highly enriched in the interactome, raising the possibility that …

Structural And Functional Studies Of The Heptose Modifying Enzymes That Play A Role In Campylobacter Jejuni Virulence., Heba Soliman Barnawi

Electronic Thesis and Dissertation Repository

Campylobacter jejuni is a major cause of gastroenteritis in humans. The capsule of some species contains unique modified heptoses. Heptose modification was elucidated for C. jejuni NCTC11168 and 18-176, and novel epimerases and reductases essential for the heptose modification were identified. We hypothesized that heptose modifying enzymes in C. jejuni have specific catalytic residues that allow for substrate and product specificity. Substrate synthesis, structural modeling, point mutations, and enzymatic analysis have been applied to map the active sites. Putative catalytic residues showed substrate and/or product specificity. The epimerases structures were solved by crystallography done by our collaborator. We also hypothesized …