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

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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.

Applying metagenomics analysis towards a better understanding of freshwater microbial communities

Date created: 
2017-06-15
Abstract: 

Microbial communities may now be studied in more detail using culture-independent methods such as metagenomics (directly analyzing genomes from an environmental sample). One of the many potential applications of metagenomics is in the assessment of water quality. Current methods for detection of fecal pollution in water rely on culture-based microbial testing which is slow and can lack sensitivity and specificity. For the WatershedDiscovery.ca project, it was hypothesized that a molecular-based test, developed based on metagenomics analysis, may be more rapid, and accurate for characterizing fecal pollution. Many bioinformatics methods for metagenomics sequence classification have been developed, but when initiating this research, no comprehensive evaluation of method accuracy had yet been published. Thus, using both in silico and in vitro simulated communities, a comprehensive evaluation of metagenomics taxonomic sequence classification methods was performed. Utilizing knowledge gained from this comparative evaluation, a study was undertaken of microbial community dynamics in monthly water samples from sites in urban, protected, and agricultural watersheds collected over a one-year period. Freshwater samples collected from sites affected by agricultural activity showed distinct microbial profiles versus samples collected from unaffected sites, and a notable presence of Legionella was discovered in all watershed sampling sites (the largest study of Legionella in watersheds to date). Furthermore, biomarkers were developed that could distinguish agriculturally affected samples from pristine samples collected in our watershed study. Finally, there is a lack of methods for the prediction of subcellular localization (SCL) from metagenomics sequences—of interest for the identification of cell surface/secreted proteins for development of ELISA-based diagnostics and other applications. Thus, PSORTb, a precise bacterial and archaeal SCL program, was modified to enable the classification of metagenomics sequences, and applied to the analysis of the watershed samples. A database of protein SCL associated with PSORTb was expanded to make it suitable for a wider diversity of microbes, particularly those with atypical cell envelopes. Collectively this work expands our understanding of metagenomics software accuracy, and available analysis tools, and provides insight into freshwater microbial community dynamics, with potential application in water quality test development.

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

Dissecting the sensory roles of motility-associated ciliary genes in Caenorhabditis elegans

Date created: 
2016-08-30
Abstract: 

Cilia are microtubule-based organelles that emanate from the surface of most mammalian cell types. Motile cilia have well known roles in producing flow, while non-motile cilia play important sensory/signalling roles. Both forms are based on a similar axonemal structure, but ciliary motility requires additional components that conform to a regular arrangement along microtubules thought to be dictated by the protofilament ribbon (pf-ribbon). While pf-ribbon proteins have been implicated in ciliary motility, sensory/signalling functions in non-motile cilia have been less apparent. Although the ciliated organism Caenorhabditis elegans lacks motile cilia, orthologues of several ciliary pf-ribbon-associated proteins are present, including PACRG (Parkin co-regulated gene) and EFHC1 (EF-hand containing 1). In addition to their localisation to motile cilia, the pf-ribbon proteins show expression in neuronal cells of the brain where they may play important sensory roles. In particular, EFHC1 is mutated in the most common form of inherited epilepsy in humans and has been shown to be important for proper neuronal communication. This work investigates these motility-associated genes in C. elegans to dissect their sensory/signalling roles. We find that both PCRG-1 and EFHC-1 localise to a small subset of non-motile cilia in C. elegans, suggesting that they have been adapted to mediate specific sensory/signalling functions. We show that PCRG-1 influences a learning behaviour known as gustatory plasticity, where it is functionally coupled to heterotrimeric G-protein signalling. We also demonstrate that PCRG-1 promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signalling, and that EFHC-1 also promotes longevity, suggesting shared signalling functions for these proteins. In addition, EFHC-1 modulates dopamine signalling where it is required for ciliary mechanosensation and regulating synaptic release of dopamine in cooperation with a voltage-gated calcium channel. Our findings establish previously unrecognised sensory/signalling functions for both PACRG and EFHC1 that may be important for neuronal communication in the human brain, where both proteins are known to be present. Furthermore, our work provides important clues for understanding and ultimately providing novel avenues for intervention of disorders such as epilepsy.

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

Localization and assembly of the Vibrio cholerae type IV pilus secretin channel

Date created: 
2017-04-12
Abstract: 

Type 4 pili (T4P) are filamentous structures found on the surfaces of many Gram-negative bacteria, including Vibrio cholerae. The V. cholerae T4P are the toxin-coregulated pili (TCP), which mediate bacterial aggregation and exoprotein secretion, critical functions in colonization of the human intestine to cause the diarrheal disease cholera. TCP assemble at the inner membrane (IM), grow through a multiprotein conduit in the periplasm and through a secretin channel in the outer membrane (OM). The multimeric secretin channel is formed by secretin subunits, which are translocated across the IM by the Sec apparatus, and in most T4P systems, are transported to the OM with the help of a lipoprotein co-chaperone. In the V. cholerae TCP the secretin subunit itself, TcpC, is a lipoprotein, and its putative co-chaperone, TcpQ, is non-lipidated. Here we use mutagenesis, cellular fractionation and functional assays to investigate secretin channel assembly in V. cholerae. TcpC must be co-expressed with TcpQ to complement a ΔtcpC mutant in an assay of pilus functions, but the reciprocal is not true. TcpQ is necessary for pilus assembly but not for localization of TcpC to the outer membrane, demonstrating that TcpQ is not a co-chaperone for TcpC. The periplasmic domain of TcpC can be expressed on its own, localizes to the outer membrane, and localizes TcpQ at the outer membrane as well, provided TcpC is lipidated. When the periplasmic domain of TcpC is unlipidated it gets degraded and TcpQ accumulates in the periplasm, suggesting that the periplasmic domain of TcpC interacts with TcpQ and localizes it to the outer membrane via its lipid moiety. Our results lead to a model whereby TcpC Cys1 is lipidated at the IM by the Lgt machinery and transported to the OM in complex with TcpQ. TcpC inserts into the OM at two points: via its C-terminal portion, which forms a β-barrel channel with TcpC C-terminal domains of other secretin subunits, and via lipid moiety in its N-terminal domain, which interacts with TcpQ to link the OM channel to periplasmic pilus conduit.

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