Biochemistry, Biophysics, and Structural 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 …

Characterization Of Ranbpm Subcellular Localization And Function In Hdac6 Regulation, Louisa M. Salemi

Electronic Thesis and Dissertation Repository

RanBPM has been shown to interact with numerous proteins implicating it in a variety of cellular processes including apoptosis, transcription regulation, cell migration, adhesion and morphology and has been shown to have tumour suppressive functions. RanPBM has been hypothesized to be a scaffolding protein and is part of a large protein complex termed the CTLH complex. Although the homologous complex in yeast has been demonstrated to have E3 ubiquitin ligase activity, whether the evolutionarily conserved human complex retains this activity remains to be determined. In this work we aim to characterize the regions of RanBPM that regulate its subcellular localization …

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 …

Using Mutual Information To Reprogram Dna Specificity Of Laglidadg Endonucleases, Marcon Laforet

Electronic Thesis and Dissertation Repository

A systematic approach to reprogram protein-DNA interactions has yet to be discovered. This study investigates the ability of co-variation analyses to identify potential protein-DNA contacts that regulate specificity. Here, 27 LAGLIDADG Homing Endonucleases (LHEs) and their 22-basepair DNA targets were collated into a Multiple Sequence Alignment (MSA) that was subjected to pairwise co-variation calculations. Using the LHE I-OnuI as a reference, an amino acid-DNA pair, lysine (K) 231 and adenine +3, generated the highest score. To test if the K231/A3 score was biologically relevant we tested protein mutants for altered nuclease specificity at +3 DNA point mutants. Randomizing the 231 …

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 …

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 …

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …

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. …

The Mechanism Of Activation Of Potassium Dependent And Potassium Independent Asparaginases From Common Bean (Phaseolus Vulgaris), Ebenezer Oluwafemi Ajewole

Electronic Thesis and Dissertation Repository

L-asparaginases play an important role in nitrogen mobilization in plants. This study investigated the biochemical and biophysical properties of potassium dependent (PvAspG1) and potassium independent (PvAspG-T2) L-asparaginases from P. vulgaris. Previous studies revealed that PvAspG1 requires potassium for catalytic activation and crystal structure analysis suggested that Ser-118 in the activation loop plays a critical role in alkali metal coordination. This amino acid residue is replaced by an isoleucine in PvAspG-T2. Reciprocal mutants of the enzymes were produced and the effect of the amino acid substitution on the kinetic parameters, secondary structure conformation, allosteric effector binding and pH profile were studied. …

Structural Analysis Of Tpr Ligand Complexes Of Stip1 Implicated In Alzheimer's Disease, Andrzej Maciejewski

Electronic Thesis and Dissertation Repository

Amyloid-beta oligomers (AbOs) induce neurological dysfunction in part through the cellular prion protein (PrP^C) resulting in deregulation of Ca²⁺ homeostasis in Alzheimer’s disease (AD). Stress inducible phosphoprotein 1 (STIP1), a cochaperone of Hsp70 and Hsp90, protects neurons from AbO-induced cell death. As well, STIP1 interacts with the Ca²⁺ sensor S100A1, which is an important biomarker upregulated in AD and regulates STIP1 and other cochaperone association with Hsp70 and Hsp90. While the molecular details of STIP1-Hsp complexes are well studied, little information is available concerning alternate STIP1 binding partners. Here, we investigated the structural details of STIP1 …

Domains Of Stip1 Responsible For Regulating Prpc-Dependent Amyloid-Β Oligomer Toxicity., Andrzej Maciejewski, Valeriy G Ostapchenko, Flavio H Beraldo, Vania F Prado, Marco A M Prado, Wing-Yiu Choy

Biochemistry Publications

Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AβO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) …

Assessment Of Post-Treatment Imaging Changes Following Radiotherapy Using Magnetic Susceptibility Techniques, Jean-Guy Belliveau

Electronic Thesis and Dissertation Repository

Radiation therapy (RT) is a common treatment for brain neoplasms and is used alone or in combination with other therapies. The use of RT has been found to be successful in controlling tumors and extending the overall survival of patients; however, there are many unanswered questions regarding radiotherapy effects in the normal brain surrounding or infiltrated by tumor. Changes to the vascular and parenchyma have been documented, and more recently inflammatory mechanisms have been postulated to play a role in radiation injury. Traditional imaging techniques used within the clinic (CT and MRI) are often lacking in their ability to differentiate …

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 …

The Mitochondrial Calcium Uniporter Is Autoregulated By Cation Binding To The Β-Grasp-Like Matrix Domain, Samuel K. Lee

Electronic Thesis and Dissertation Repository

Mitochondrial calcium (Ca²⁺) uptake plays fundamental roles in various signaling processes. Entry of Ca²⁺ into the mitochondrial matrix is tightly controlled by the mitochondrial Ca²⁺ uniporter (MCU) in a higher order complex regulated by inter- and intra-molecular protein-protein interactions. However, the precise mechanisms controlling MCU function and regulation remain largely unknown. I identified a well-folded N-terminal MCU region from residues 72-189, and its crystal structure revealed a β-grasp-like fold with a cluster of acidic residues that facilitates interactions with dibasic cations. Binding of Ca²⁺ or Mg²⁺ destabilize this fold and shift the protein towards …

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 …

Structure And Function Relationships Between Atpase Family, Aaa Domain Containing Protein 5, Proliferating Cell Nuclear Antigen, And Usp1-Associated Factor 1, Tam T. Bui

Electronic Thesis and Dissertation Repository

The genome is constantly damaged by intracellular and extracellular factors. At sites of DNA damage, replication forks are stalled, leading monoubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination of PCNA promote the switch from regular high-fidelity polymerases to Y-family polymerases for bypassing damaged DNA. Prolonged replication by these polymerases may lead to increased mutagenesis, so tight regulation of this process is required. ATAD5 recruits a deubiquitinase complex consisting of ubiquitin-specific protease 1 (USP1) and USP1-associated factor 1 (UAF1) to control PCNA monoubiquitination. The mechanism by which ATAD5 and PCNA interact has been previously unexplored. We show through biochemical and structural …

Parkin Misfolding, Dysfunction, And Degradation In Parkinson's Disease, Alexander S. Mccarton

Electronic Thesis and Dissertation Repository

Mutations in the gene encoding parkin (PARK2) result in familial early onset forms of Parkinson’s disease (PD) based on the loss of parkin’s E3 ubiquitin ligase function. Protein misfolding is a common molecular feature of most neurodegenerative diseases, including PD. To test whether parkin misfolding also plays a role in the more common spontaneous PD, we established and functionally characterized a parkin yeast model. We found that oxidative and protein folding stress, parkin point mutations and truncations, and parkin’s interaction with the PD-associated kinase PINK1 profoundly alter parkin’s subcellular localization and toxicity. Notably, these conditions also induce parkin fragmentation, degradation, …

Interaction Of Seca With Unfolded Polypeptides, Aliakbar Khalili Yazdi

Electronic Thesis and Dissertation Repository

The evolutionarily well-conserved SecA is essential for bacterial post-translational translocation. SecA uses the energy of ATP to drive preproteins through the membrane pore. The functional oligomeric state of SecA and the molecular basis for recognition of unfolded polypeptides by SecA are major unresolved questions that must be addressed to understand preprotein targeting and the molecular mechanics of SecA-mediated translocation. This thesis will address three aspects of these questions. First, the role of unstructured termini in the oligomerization and function of various SecA constructs was elucidated. By re-examining the tetramerization of a truncated SecA construct (SecA-N68), it was shown that the …

The Role Of Sirtuin 6 In Maintaining Vascular Integrity, Sharon Z. Leung

Electronic Thesis and Dissertation Repository

Oxidative stress is an underlying cause for vascular pathologies including inflammation, hypertension, and atherosclerosis. Sirtuins (SIRTs) are a family of NAD+ dependent deacetylases with pronounced roles in cellular metabolism and aging. SIRT6 is expressed in vascular smooth muscle cells (SMCs) and may offer protection from oxidative stress-induced damage. To study the role of SIRT6 in SMCs, we created a novel strain of SMC-specific SIRT6-deficient (SIRT6KO) mice with Cre-lox technology. Because no defects were observed in the aortas of SIRT6KO mice, they were then infused with angiotensin II (Ang II) to induce oxidative stress. Compared with vehicle controls, SIRT6KO mice developed …

A Unified Analytic Framework For Prioritization Of Non-Coding Variants Of Uncertain Significance In Heritable Breast And Ovarian Cancer, Eliseos J. Mucaki, Natasha G. Caminsky, Ami M. Perri, Ruipeng Lu, Alain Laederach, Matthew Halvorsen, Joan H. M. Knoll, Peter K. Rogan

Biochemistry Publications

Background

Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT) and prioritizing patients with large intragenic deletions.

Methods

We captured and enriched for coding and non-coding variants …

Cost-Effectiveness Of Using A Gene Expression Profiling Test To Aid In Identifying The Primary Tumour In Patients With Cancer Of Unknown Primary., M B Hannouf, E Winquist, S M Mahmud, M Brackstone, S Sarma, G Rodrigues, P Rogan, J S Hoch, G S Zaric

Biochemistry Publications

We aimed to investigate the cost-effectiveness of a 2000-gene-expression profiling (GEP) test to help identify the primary tumor site when clinicopathological diagnostic evaluation was inconclusive in patients with cancer of unknown primary (CUP). We built a decision-analytic-model to project the lifetime clinical and economic consequences of different clinical management strategies for CUP. The model was parameterized using follow-up data from the Manitoba Cancer Registry, cost data from Manitoba Health administrative databases and secondary sources. The 2000-GEP-based strategy compared to current clinical practice resulted in an incremental cost-effectiveness ratio (ICER) of $44,151 per quality-adjusted life years (QALY) gained. The total annual-budget …

Automated Discrimination Of Dicentric And Monocentric Chromosomes By Machine Learning-Based Image Processing., Yanxin Li, Joan H Knoll, Ruth C Wilkins, Farrah N Flegal, Peter K Rogan

Biochemistry Publications

Dose from radiation exposure can be estimated from dicentric chromosome (DC) frequencies in metaphase cells of peripheral blood lymphocytes. We automated DC detection by extracting features in Giemsa-stained metaphase chromosome images and classifying objects by machine learning (ML). DC detection involves (i) intensity thresholded segmentation of metaphase objects, (ii) chromosome separation by watershed transformation and elimination of inseparable chromosome clusters, fragments and staining debris using a morphological decision tree filter, (iii) determination of chromosome width and centreline, (iv) derivation of centromere candidates, and (v) distinction of DCs from monocentric chromosomes (MC) by ML. Centromere candidates are inferred from 14 image …

Prioritizing Variants In Complete Hereditary Breast And Ovarian Cancer (Hboc) Genes In Patients Lacking Known Brca Mutations., Natasha G Caminsky, Eliseos J Mucaki, Ami M Perri, Ruipeng Lu, Joan H M Knoll, Peter K Rogan

Biochemistry Publications

BRCA1 and BRCA2 testing for Hereditary breast and ovarian cancer (HBOC) does not identify all pathogenic variants. Sequencing of 20 complete genes in HBOC patients with uninformative test results (N = 287), including non-coding and flanking sequences of ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD51B, STK11, TP53, and XRCC2, identified 38,372 unique variants. We apply information theory (IT) to predict and prioritize non-coding variants of uncertain significance (VUS) in regulatory, coding, and intronic regions based on changes in binding sites in these genes. Besides mRNA splicing, IT provides a common …

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 …