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

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A conserved interdomain linker of CCT mediates allosteric communication between regulatory and catalytic domains

Author: 
Date created: 
2018-12-03
Abstract: 

Folding landscapes of linkers between ligand-binding and functional domains evolved to facilitate transmission of inter-domain signals. I investigated the structure/function of a conserved linker between the catalytic and membrane-binding (M) domains of CCT, which regulates phosphatidylcholine synthesis and activates upon membrane binding. The activity of CCT is very sensitive to mutations in the linker. Recent molecular dynamics simulations revealed that upon removal of auto-inhibitory contacts between the M domain and the active site, the αE helix adjacent to the linker bends toward the active site, bringing the catalytic domain close to the membrane surface. Tryptophan fluorescence quenching revealed that the linker lies superficially on the membrane surface. FRET between engineered tryptophans and vesicles containing Dansyl-Phosphatidylethanolamine support a bent αE helix conformation that is dependent on the native linker sequence. The data suggests that the linker may communicate membrane binding signals to enhance CCT activity by directly stabilizing a bent αE.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Rosemary Cornell
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
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): 
Supervisor(s): 
Ryan Morin
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.