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

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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.

Examination of lipid phase behaviour in drug delivery systems using X-ray scattering and nuclear magnetic resonance

Date created: 
2017-12-14
Abstract: 

The use of lipid nanoparticles (LNPs) for drug delivery offers exciting new avenues for gene therapy. The LNP’s cationic lipid (XTC2) and anionic endosomal lipids such as lysobisphosphatidic acid (LBPA) are hypothesized to form non-bilayer phases which promote cargo release into the cytoplasm. However, this release is inefficient in current formulations. Computer simulations based on accurate lipid models could be used to predict LNP formulations having improved release efficacy. The inverted hexagonal (Hii) phase of dioleoylphosphatidylethanolamine was studied with small angle X-ray scattering (SAXS) and deuterium nuclear magnetic resonance (NMR) to provide lattice repeat spacing and acyl chain order parameters for simulations. Physical characteristics of LBPA and XTC2 forming non-bilayer phases were measured as functions of temperature and pH using SAXS and phosphorus NMR. In particular, under acidic conditions, an equimolar mixture of LBPA/XTC2 exhibited Hii and cubic phases that could enhance the release of cargo from the LNP.

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

Integrating regulatory mechanisms of Wnt signaling in development and tissue homeostasis

Author: 
Date created: 
2017-10-24
Abstract: 

Evolutionarily conserved signal transduction pathways mediate the ability of cells to respond to their environment and coordinate with each other for proper development and homeostasis of an organism. The Wnt/Wingless (Wg) pathway is required for proliferation, differentiation, stem-cell renewal and homeostasis, and when disrupted leads to disease. Wnt signaling does not control all these processes alone, its activity is extensively regulated by interaction with other signaling pathways and cellular mechanisms. This is mediated predominantly through phospho-regulation of the key pathway components by kinases and phosphatases. Our lab conducted an in vivo RNAi screen designed to identify novel kinase and phosphatase regulators of the Wnt pathway. In my PhD thesis research I further characterized three potential regulators: Downstream of Raf1 (Dsor1), Protein phosphatase 4 (PP4), and myosin phosphatase. Knockdown of Dsor1 reduced Wnt target gene expression and decreased stabilized β-catenin, the key effector protein of the Wnt pathway. Dsor1 and β-catenin had a close physical interaction, and catalytically inactive Dsor1 caused a reduction in active β-catenin, suggesting that Dsor1 counteracts destruction of β-catenin. Additionally, Ras-Dsor1 activity was independent of EGFR, and likely activated by the insulin-like receptor to promote Wnt. This work demonstrates novel crosstalk between Insulin and Wnt signaling via Dsor1. The reduction of PP4 inhibited Wg pathway activity, by reducing Notch-driven wg transcription. PP4 was found to promote Notch signaling within the nucleus of the receiving cell. Furthermore, PP4 regulates proliferation independently of its Notch interaction. This study identified a new role for PP4 in Notch signaling, and subsequently transcriptional regulation of wg. Reduced myosin phosphatase inhibited Wnt signaling by causing increased non-muscle myosin II (NMII) activation and cellular contraction. NMII activation stabilizes cortical F-actin resulting in accumulation of E-cadherin to the adherens junctions (AJ). E-cadherin titrates available β-catenin to the AJs in order to maintain cell-cell adhesion under contraction. The decreased cytoplasmic β-catenin results in insufficient nuclear translocation for full Wnt target gene transcription. This work elucidates that the dynamic activation of actomyosin contractility refines patterning of Wnt target gene expression. These studies identified three novel regulatory mechanisms for controlling Wnt signaling in development and homeostasis.

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

Metagenomic analysis of river microbial communities

Author: 
Date created: 
2017-09-22
Abstract: 

As concern over the availability of freshwater increases, so does the interest in river microorganisms due to their importance in drinking water safety and signalling environmental contamination. However, foundational understanding of their variability in rivers is lacking, especially for viruses. Here, I present work to improve the understanding of planktonic microbial communities in rivers over time in the context of varying environmental conditions and contrasting land use. DNA-sequencing based metagenomic and phylogenetic marker gene (16S, 18S, g23) approaches were used to profile microbial communities, coupled with measures of environmental and chemical conditions. I analysed microbial community profiles from monthly samples collected over one year from three watersheds with agricultural, urban, or minimal land use. Viral, bacterial, and microeukaryotic planktonic communities were synchronous overall, but had contrasting geographic patterns and the strength of their synchrony, as well as their relationships with environmental conditions, were heterogenous across sampling sites. These differences illustrated that bacteria are important yet insufficient representatives of microbial community dynamics despite their prevalence in microbiome research. However, this emphasis on bacteria has produced richer reference databases, which enabled a gene-specific analysis. Using a reference-based approach, I found that communities with lower water quality due to agricultural activity had higher abundances of nutrient metabolism and bacteriophage gene families. Based on these water quality associated findings and on complementary analyses, I identified potential biomarkers to demonstrate that bacterial river metagenome data could feasibly support the development of new assays for water quality monitoring. To complement these studies of anthropogenic contamination, I studied bacteria in river biofilms across a natural gradient of metal concentrations at a potential mining site. Clear relationships among metal concentrations, pH, and microbiomes were evident and this study provided fundamental knowledge of microbial communities at a potential mine site before disruption from development. Throughout these studies, the scarcity of reference information for microbial communities in lotic freshwater provided an opportunity to identify weaknesses in popular microbiome analysis methods and present approaches better suited to poorly characterised environments. Overall, my work aims to improve the understanding of planktonic river microbial community variability, both for the advancement of basic science and to support future development of more effective water quality monitoring approaches.

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

The minor pilin TcpB is located at the tip of the toxin co-regulated pilus of Vibrio cholerae and is the receptor for the filamentous phage CTXφ

Date created: 
2017-07-19
Abstract: 

Type IV pili are polymers of the major pilin subunit found on the surfaces of many Gram negative bacteria. They act like grappling hooks, undergoing cycles of polymerization, adhesion, and retraction, to mediate a diverse array of functions, including twitching motility, DNA uptake and adhesion. T4P possess several minor pilins, which are homologous to the major pilin but are produced in much lower quantities. Minor pilins prime Type IV pili assembly and have been proposed to localize to the tip of the pilus, but this has not been shown definitively. The Vibrio cholerae toxin co-regulated pilus (TCP) is a T4P that mediates microcolony formation, which is critical for the development of the gastrointestinal disease cholera. TCP is the primary receptor for the filamentous cholera toxin phage CTXφ, which binds to the pilus via its tip-associated protein, pIII. TCP possess a single minor pilin, TcpB, which initiates pilus assembly as well as retraction. We hypothesized that TcpB is located at the tip of the pilus and forms the binding site for CTXφ pIII. Here I use direct and competition ELISA to show that recombinantly expressed soluble TcpB and pIII interact. I show that CTXφ phage infection of V. cholerae is reduced 90 % in the presence of soluble TcpB or anti-TcpB antibody. Furthermore, gold-labeled anti-TcpB antibody binds to the tip of purified TCP, providing the first direct localization of a minor pilin to the tip of a T4P. Finally, I show that phage uptake is reduced 98 % in a retraction-deficient V. cholerae strain, demonstrating the role of pilus retraction in this process. My results define a two-step mechanism for CTXφ infection of V. cholerae, which involves (i) binding of CTX via its tip-associated pIII protein to its receptor, TcpB, at the tip of the pilus, and (ii) retraction of the pilus, which pulls CTXφ into the bacterial periplasm as if it were an extension of the pilus.

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

Characterization of signaling pathways enabling coordinated morphogenesis of tissues during Drosophila dorsal closure

Author: 
Date created: 
2017-06-30
Abstract: 

Drosophila dorsal closure (DC) is the best-characterized model system for studying wound healing. During DC, a hole in the dorsal epidermis, covered by an epithelium called the amnioserosa (AS), is sealed by migration of the epidermal flanks. Seamless closure is achieved through coordinated morphogenesis of the AS and epidermis, which is facilitated by communication between the two tissues via bidirectional signaling networks. To better understand this crosstalk, three diffusible signals present during DC were analyzed, and their signaling roles were identified: 1.) Folded gastrulation (Fog), which may act as an upstream activator of a JNK pathway in the epidermis; 2.) the TGF-β ligand, Decapentaplegic (Dpp), which regulates production of the steroid hormone, 20- hydroxyecdysone (ecdysone) in the AS; 3.) ecdysone, which interacts with the transcription factor AP-1 to regulate gene transcription in the AS. Signaling via these molecules ultimately regulates myosin contractility necessary for morphogenesis of both tissues during DC.

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

Pan-Cancer Identification and Prioritization of Cancer-Associated Alternatively Spliced and Differentially Expressed Genes: A Biomarker Discovery Application

Author: 
Date created: 
2016-11-21
Abstract: 

Tumour cells arise through aberrant expression of genes and the proteins they encode. This may result from a direct change to DNA sequence or perturbations in the machinery responsible for production or activity of proteins, such as gene splicing. With the advent of massively parallel RNA-sequencing (RNA-seq), large-scale exploration of changes at the stage of transcription and posttranscriptional splicing has the potential to unravel the landscape of gene expression changes across human cancers. Aberrantly expressed genes in cancer can serve as molecular biomarkers for discrimination of tumour and normal cells if localized to the cell surface and therefore can be used as targets for targeted antibody-based cancer therapy. In the current study, I devised an analysis pipeline to identify and rank such events from human cancer RNA-seq datasets. Using my pipeline, I conducted a pan-cancer analysis in the RNA-sequencing data of more than 7,000 patients from 24 different cancer types generated by the cancer genome atlas (TCGA). I identified abnormally expressed and alternatively spliced genes, which seemed to be cancer-associated in comparison to a large compendium of transcriptomes from non-diseased tissues gathered from Genotype-Tissue Expression (GTEx) and TCGA. My analysis revealed 1,503 putative tumor-associated abnormally expressed genes and 1,142 novel cancer-associated splice variants occurring in 694 genes. In order to rank identified candidate genes, I performed an extensive literature search and studied known therapeutic antibody targets to collect the characteristics of an ideal antibody target in cancer. I developed an R package, Prize, based on the Analytic Hierarchy Process (AHP) algorithm. AHP is a multiple-criteria decision making solution that allows a user to prioritize a list of elements based of a set of user-define criteria and numerical score that express the importance of each criterion to achieving the goal. I built an AHP model to depict cancer biomarker target properties for ranking and prioritizing the genes. Using this model, Prize was able to successfully recognize and rank known tumour biomarker targets among the top 25 ranked list along with other novel candidates.

Document type: 
Thesis
Senior supervisor: 
Dr. Steven J.M. Jones
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.