Molecular Biology and Biochemistry - Theses, Dissertations, and other Required Graduate Degree Essays

Receive updates for this collection

Gfat1 and Gfat2 encode functionally equivalent enzymes in Drosophila melanogaster: a molecular, genetic, and evolutionary analysis

Author: 
Date created: 
2018-10-26
Abstract: 

The hexosamine biosynthesis pathway (HBP) diverts 2-5% of glucose from glycolysis, ultimately producing uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important substrate in protein glycosylation. This pathway is of particular importance in Drosophila melanogaster because chitin, the primary component of insect cuticle, is composed of N-acetylglucosamine polymers. I report that the rate-limiting enzymes of the HBP, GFAT1 and GFAT2 (glutamine:fructose-6-phosphate aminotransferase), are functionally equivalent by genetic rescue using cDNA transgenes of both genes. I also report that neither transgenically upregulating the enzymes of the HBP, nor those of the hexosamine signalling pathway (HexSP), extends the lifespan of D. melanogaster. Evolutionary analyses using fluorescence in situ hybridization in six species of Drosophila support a model that would place the Gfat1 relocalization event from euchromatin to heterochromatin after the melanogaster group diverged from the rest of Drosophila.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Barry Honda
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Investigating the role of HIV-1 Nef during reactivation of latent viral reservoirs

Author: 
Date created: 
2018-09-21
Abstract: 

A major barrier to development of a cure for HIV-1 is the ability of the virus to establish latent infection that reactivates to cause disease if antiretroviral therapy is discontinued. The HIV-1 protein Nef displays multiple in vitro and in vivo functions, including the ability to modulate T cell signaling events that may alter the activation status of infected cells. Nef is critical for viral pathogenesis, but its role during latency remains unclear. To investigate this, I generated a novel panel of latent T cell clones (C-Lat) harbouring a single integrated copy of HIV-1 encoding functional or defective nef genes. By assessing the location and genomic features of the proviral DNA integration sites in these clones, I observed that the presence of functional Nef variants was associated with a broader repertoire of inducible latent T cell lineages. By characterizing the reactivation phenotypes of these clones following stimulation with latency reversing agents (LRAs), I observed higher early and late viral protein expression in C-Lat clones encoding functional nef compared to those encoding defective nef. I confirmed these observations by disrupting the functional nef gene in C-Lat clones using CRISPR/Cas9 strategies. Variable viral reactivation phenotypes were observed in Nef knock-out (NefKO) clones following stimulation with LRAs, but the efficiency of early and late viral protein expression was consistently lower in NefKO clones compared to their corresponding parental isolates. My research highlights the ability of Nef to modulate HIV-1 reactivation from latency. Results indicate that Nef may play an important role in determining the breadth and diversity of inducible viral reservoirs following infection. If so, a better mechanistic understanding of Nef’s impact may uncover new strategies to enhance viral reactivation from latency that are clinically beneficial.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Mark Brockman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Regulation of inducible nitric oxide synthase expression in human cells

Author: 
Date created: 
2018-06-28
Abstract: 

Nitric oxide (NO) is a bioactive gas that has multiple roles in innate and adaptive immune responses and dysregulated expression of its production by inducible nitric oxide synthase (iNOS) is implicated in the pathogenesis of various inflammatory diseases and cancer. The mechanisms by which the expression of iNOS is regulated in human cells is incompletely understood. I show that NO positively regulates iNOS expression through a positive feedback mechanism that involves S-nitrosylation and activation of the small Ras GTPase, which in turn activates downstream PI3K/Akt and mTOR pathways. This feedback mechanism acts in a post-translational manner to increase iNOS protein levels by reducing its ubiquitination and proteasomal degradation. In addition to examining the feedback regulation of iNOS protein expression, I also studied how gene expression of this enzyme is controlled. I focused on the hypoxia inducible factor (HIF) because of the central role of this transcription factor in controlling many aspects of cell biology. The human iNOS gene promoter has three predicted HIF binding sites and mutation of one of these sites at -4.9 kb reduced the induction of iNOS gene promoter activity by cytokines. Further, HIF-1a gene editing by CRISPR/Cas9 eliminated HIF activity and reduced the induction of iNOS mRNA and protein expression by cytokines. Altogether, I discovered two mechanisms whereby iNOS expression is positively regulated at the protein and gene expression levels in an inflammatory setting. The role of NO and HIF in augmenting iNOS expression may provide insight into new therapeutic strategies for inflammatory diseases and cancer.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Jonathan C. Choy
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Investigating the neuroprotective role of OGA inhibition by Thiamet-G against Alzheimer\'s disease

Author: 
Date created: 
2018-02-23
Abstract: 

The glycosylation of nucleocytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) is important for regulation of protein function and cellular signaling. Addition of GlcNAc monosaccharide unit to target proteins requires the enzyme O-GlcNAc transferase (OGT), and removal of O-GlcNAc depends on the enzyme O-GlcNAcase (OGA). Previous works have shown that OGA inhibitors and enhancers of autophagy both reduced cognitive impairment as well as Aβ and tau aggregation in Alzheimer\'s disease (AD) mouse models. Here, it was shown that OGA inhibition enhanced autophagy in neuro-2a cells and AD mouse brain through an mTOR independent pathway. These data suggest that OGA inhibition provides neuroprotection by promoting autophagy dependent clearance of protein oligomers. To investigate this relationship, we established inducible cellular models of tauopathy to show that OGA inhibition decreased levels of pathological tau species. These results suggest OGA inhibition is a possible therapeutic strategy against AD that may involve the enhancement of autophagy.

Document type: 
Thesis
File(s): 
Senior supervisor: 
David Vocadlo
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Directed evolution of a bacterial sialidase and characterization of mechanism based inactivation of glycosidases

Date created: 
2017-12-07
Abstract: 

Sialic acids are often found at the terminal positions on the glycan chains that adorn all vertebrate cells and glycoproteins. This prominent position confers an essential role to sialic acid residues in biology, evolution and disease propagation. The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn, which is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid. Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycoside hydrolases (GHs). These enzymes have been categorized into more than 130 different families. Glycoside hydrolase family 33 (GH33) contains exo-sialidases (E.C. 3.2.1.18, neuraminidases), from both eukaryotes and prokaryotes, which catalyze the hydrolysis of sialic acid from glycoconjugates. Interestingly, subtle differences exist in both the structure of the particular sialic acid and its position of attachment to glycoconjugate chains between humans and other mammals. These differences are indicators of the unique aspects of human evolution, and are relevant to understanding an array of human conditions. The present thesis reports on routes that we explored to further unravel the importance of sialic acids. We developed tools to probe for various sialic acid structures such as Kdn. To this end, we constructed a random mutant library of the neuraminidase from the soil bacterium Micromonospora viridifaciens (MvNA) and identified a number of recurring mutations in the sialidase gene which lead to a more efficient hydrolysis of synthetic natural substrate analogues such as 8FMU α-Kdn-(2→6)- β-D-Galp. We also using the available structure of wild type MvNA bound to the natural inhibitor, DANA, to identify amino acids potentially involved in recognition and binding to acetylated sialic acids and generated genetic libraries which we used along with positive and negative evolutionary screens to identify several clones capable of hydrolyzing Kdn glycosides more efficiently than Neu5Ac substrates. Kinetic studies on these clones allowed for determination of enzyme efficiencies and specificities. We also report our study of covalent inhibition of α-glucosidase from Saccharomyces cerevisiae (GH13). The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of carbenium ions in the active site that are trapped rapidly by an enzymatic residue.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Andrew Bennet
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Comprehensive Genomic Characterization of Mantle Cell Lymphoma

Author: 
Date created: 
2017-06-29
Abstract: 

Mantle cell lymphoma (MCL) is a rare, aggressive and incurable type of cancer with a high incidence of systemic dissemination and a median survival time of 3-4 years. Understanding the genetics and biology of MCL are necessary for the development of better biomarkers and novel targets of therapies. A limited number of sequencing studies were performed on MCL recently, which improved the knowledge on causal mutations and molecular mechanisms of MCL. However, the small cohort sizes of these studies and use of outdated computational tools and databases limited the findings of these studies. The objective of our study was to identify the genetic alterations and molecular pathways that are associated with the development, progression, and dissemination of MCL by using leading-edge bioinformatics tools, up-to-date molecular biology databases, and a large sample size. Presented here is a meta-analysis of whole exome sequencing data from tumor biopsies of 67 MCL patients, which resulted in the identification of several novel gene targets of recurrent mutations in MCL such as SP140, S1PR1, PTPRD, HNRNPH1, LRP1B, FAT1, MAP3K14, and DST. Our analysis revealed four mutation hotspots in the first exons of MAP3K14 with predicted activating impact on the protein in MCL, and five other types of B-cell lymphoma and leukemia. We further proposed that the recurrent mutations of MAP3K14 are potentially associated with a higher likelihood of relapse, because of their emergence in relapse biopsies of four patients. This finding suggests that the recurrent mutations of MAP3K14 may have application for prediction of disease outcome, response to therapy, and transformation of the indolent variant of MCL to the more aggressive subtype.Finally, we detected a significant accumulation of mutations in signaling pathways with roles in mechanisms of tumor metastasis, such as Rho GTPase mediated signaling, focal adhesion, G-protein coupled receptor signaling, cAMP-PKA signaling, ERK-MAPK signaling, ROBO-Slit signaling, and JAK-STAT signaling. These findings offer new insights into the understanding of driver mutations and molecular mechanisms underlying the aggressive clinical course of MCL and may have implications for the development of therapies.

Document type: 
Thesis
Senior supervisor: 
Mark Paetzel
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Methods for chemical mapping of O-GlcNAc in the Drosophila genome

Author: 
Date created: 
2017-07-10
Abstract: 

O-linked N-acetylglucosamine (O-GlcNAc) is an important protein modification installed onto hundreds of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). Here, I discuss the development of an antibody-free metabolic feeding approach, which enables unbiased mapping of O-GlcNAcylated proteins in a genome-wide manner. This mapping method is detailed in Drosophila and compared to other O-GlcNAc mapping methods related to chromatin immunoprecipitation followed by sequencing (ChIP-seq), in order to demonstrate its overall efficacy. Using a combination of experimental and bioinformatics methods, I define new genes regulated by OGT. I also report on the development of robust software used to process and analyse time course ChIP-seq data, and prove its versatility and proficiency using both simulated and published data sets. This software is then applied to the analysis of a time course O-GlcNAc chemical mapping experiment in Drosophila larvae, generating the first ever time course ChIP-seq experiment performed on both a protein modification and in a living organism. Using this approach I am able to distinguish between loci that are more sensitive to O-GlcNAc cycling and those that are affected more by protein turnover. These studies provide an improved understanding of the regulation of gene expression by O-GlcNAc, while providing the wider community with new computational tools for time resolved analysis of genome-wide binding by proteins.

Document type: 
Thesis
File(s): 
Senior supervisor: 
David Vocadlo
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Regulation and conservation of caspase-activated autophagy

Date created: 
2017-06-27
Abstract: 

Autophagy is an evolutionarily conserved cellular process that recycles proteins and organelles to maintain cellular homeostasis or provide an alternative source of energy in times of stress. While autophagy promotes cell survival, it can also be regulated by proteins associated traditionally with apoptosis. In an effort to better understand the complex intersections of these disparate cell fates, previous studies in Drosophila identified an apoptotic effector caspase, Dcp-1, as a positive regulator of starvation-induced autophagy. Further, the Drosophila heat-shock protein, Hsp83, was identified as a Dcp-1 interacting protein and a putative negative regulator of autophagy. The aims of my thesis were to investigate the relationship between Dcp-1 and Hsp83 in the context of autophagy, and to determine if caspase-regulated autophagy was functionally conserved in humans. In vivo analyses of Hsp83 loss-of-function mutants in fed conditions showed increases in both autophagic flux and cell death. Hsp83 mutants also had elevated levels of pro-Dcp-1, which was attributed to reduced proteasomal activity. Analyses of an Hsp83/Dcp-1 double mutant revealed that the caspase was not required for cell death in this context but was essential for the ensuing compensatory autophagy, female fertility, and organism viability. These studies not only demonstrated unappreciated roles for Hsp83 in proteasomal activity and new forms of Dcp-1 regulation, but also identified an effector caspase as a key regulatory factor for sustaining adaptation to cell stress in vivo by inducing compensatory autophagy. To address whether effector caspases also regulate starvation-induced autophagy in human cells, caspase-3 (CASP3), a human homolog of Dcp-1, was examined in several human cell lines. These studies showed that CASP3 was required for the upregulation of starvation-induced autophagy in most cell lines examined, but was not required for maintaining basal levels of autophagy. In human cells, another heat-shock family member, HSP60, was identified as a CASP3-interacting protein. HSP60 was shown to negatively regulate autophagy by controlling the subcellular localization of CASP3 in response to nutritional status. Epistasis analyses suggest that the increase in autophagy observed from loss of HSP60 was dependent on the accumulation of cleaved CASP3 in the cytosol. This work highlights a novel function for CASP3 in starvation-induced autophagy in human cells and illustrates how its response is regulated by HSP60-controlled subcellular localization. Altogether, my studies provide novel insights into stress adaptive relationships between heat-shock proteins and caspases in Drosophila and human cells.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Sharon Gorski
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Sequence analysis of ctDNA in NHL to monitor tumour progression and evolution

Author: 
Date created: 
2018-04-09
Abstract: 

NHL (non-Hodgkin lymphoma) is the fifth and sixth most prevalent cancer in Canada diagnosed annually among men and women respectively. With current conventional treatment, the five year survival rate is 67%. However, continued observations post-treatment are needed due to the risk of patient relapse. Liquid biopsies provide an effective, non-invasive means for such observations. Here, we evaluated the efficacy and utility of circulating tumour DNA (ctDNA) in relapsed patients with NHL. We detected ctDNA in at least one plasma sample from 90.9% of patients tested. We showed a significant increase in ctDNA was associated with a lack of treatment response. We demonstrate the utility of ctDNA to facilitate genetic characterization and direct observation of tumour heterogeneity and evolution. These results support the utility of ctDNA as a biomarker for tumour progression and as a substrate to study the genetic dynamics of NHL tumours over the course of treatment.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Ryan Morin
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Investigation of B cell and T follicular helper cell responses following priming with immunogens designed to trigger VRC01-class neutralizing antibodies to HIV-1

Date created: 
2017-11-30
Abstract: 

Antibodies are one of the host’s main defences against invading pathogens. There has yet to be a vaccine that can elicit antibodies capable of neutralizing a wide array of circulating Human Immunodeficiency Virus type-1 (HIV-1) strains. The general inability of germline (gl) precursors of these antibodies, termed broadly neutralizing antibodies (bnAbs), to bind recombinant forms of the Env spike used in prospective vaccine formulations has been identified as one of the likely obstacles to achieving a bnAb response by vaccination. The design of antigens that can engage gl precursors of bnAbs, dubbed “gl targeting”, is a strategy currently being explored to elicit bnAbs. The VRC01-class of bnAbs, which target the highly conserved CD4 binding site on the HIV Env spike, are attractive templates for vaccine design owing to their tremendous neutralization potency and breadth and common mode of antigen recognition. Here, we investigated a panel of antigens, derived from the 45_01dG5 strain of HIV-1, for their ability to engage VRC01-class gl precursors. Additionally, we assessed their capacity to stimulate T follicular helper (Tfh) cell and B cell responses in C57BL/6 mice after a single immunization, using assays developed with two model immunogens. Specifically, we assessed the influence, on Tfh and B cell responses, of appending a single copy of the PanDR helper epitope (PADRE) to select immunogens. We found that several constructs bind mature and gl-reverted versions of the VRC01 bnAb, as well as one of two VRC01-class precursor antibodies tested here. The immunizations revealed that immunogens with a glycan-masked V3 elicit a very weak Tfh response, which may have led to correspondingly weak B cell responses. Appendage of the single PADRE motif was insufficient to reverse the otherwise weak Tfh cell responses observed with the V3-masked immunogens used here, supporting the need for multiple copies of the motif to adequately provide Tfh cell mediated B cell help. In sum, this work provides insight into the early immune response to priming by HIV-1 candidate immunogens as part of a first phase of explorations toward eliciting VRC01-class bnAbs.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Ralph Pantophlet
Jonathan Choy
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.