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

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Regulation of T Cell Death in Alloimmune-Mediated Vascular Rejection

Author: 
Date created: 
2015-03-20
Abstract: 

Organ transplantation remains the most important therapeutic option for end-stage organ failure, but success of this procedure is limited by the eventual rejection of almost all grafts. T cells reject grafts by recognizing genetic differences between recipient and donor. Activation of T cells by donor antigens drives clonal expansion of graft-reactive T cells. The magnitude of the response depends on a balance between T cell activation and death. Understanding the mechanisms that regulate cell death may result in new approaches to prevent graft failure. I found that human CD8 T cells activated in the presence of IL-1, IL-6, and IL-23 were protected against activation-induced cell death (AICD) as a result of increases in c-FLIPS expression induced by IL-6. I then used an aortic interposition model of transplant arteriosclerosis (TA), a form of arterial rejection that is the main cause of heart transplant failure, to determine the effect of IL-6 on T cell death. Grafted arteries that lacked IL-6 developed less severe rejection as compared to IL-6+/+ arteries, and this correlated with an increase in T cell death. These findings indicate that IL-6 inhibits T cell death after organ transplantation. I also examined the intrinsic regulation of T cell responses by Bcl-2 family proteins. Bim is a pro-apoptotic protein known to down-regulate immune responses. Besides inducing T cell death, I found that Bim was unexpectedly required for proliferation of T cells in response to alloantigen stimulation in vitro. A partial reduction in Bim expression was sufficient to attenuate activation whereas a complete elimination of Bim was required to prevent CD4 T cell death in response to cytokine withdrawal. In vivo, there was significantly less vascular rejection in Bim+/-, but not Bim-/-, graft recipients. T cell proliferation in response to allograft arteries was significantly reduced in both Bim+/- and Bim-/- mice, but cell death was attenuated only in Bim-/- animals. These findings indicate that Bim regulates not only T cell death but also allogeneic T cell activation. In summary, my work has provided insight into the mechanisms by which extrinsic and intrinsic regulators of cell death affect immune responses in transplantation. These pathways are potential targets in the development of novel therapies to manage TA.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Jonathan Choy
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Establishment of a bipartite ciliary signaling compartment in a C. elegans thermosensory neuron

Date created: 
2014-04-17
Abstract: 

Signaling proteins are often sequestered into cellular domains, where different modulator proteins, and potentially lipid environments, ensure efficient signal transduction. How such domains form represents an important, largely unexplored question. Known as the antennae of the cell, cilia are organelles required for many signaling pathways, presenting an unique opportunity to explore how signal transduction is arranged spatially and regulated dynamically in the cells. Using the AFD thermosensory neurons of C. elegans as the model system, my research investigated the roles of ciliary proteins in regulating a signaling cascade closely associated with the cilium – the cGMP signaling pathway. I showed that different functional categories of ciliary proteins help establish two contiguous, yet distinct cGMP signaling compartments in the sensory end of AFD neurons. One compartment, a bona fide cilium, is delineated by Bardet-Biedl syndrome (BBS), Meckel syndrome (MKS) and nephronophthisis (NPHP) associated proteins at its base, and requires Inversin/NPHP-2 to anchor a cGMP-gated ion channel within the proximal ciliary region. The other, a subcompartment characterized by profuse microvilli and different lipid environment, is separated from the dendrite by a cellular junction and requires BBS-8 and DAF-25/Ankmy2 for correct localization of guanylyl cyclases needed for thermosensation. Consistent with a requirement for a membrane diffusion barrier at the subcompartment base, my data revealed the unexpected presence of ciliary transition zone proteins where no canonical transition zone ultrastructure (Y-links) is observed. My results also showed that the ciliary mutants have a reduced ability in moving toward favorable temperatures, a behavior known as thermotaxis. Finally, using a novel conditional knockout method developed during this research, I showed that the cilium acts cell-autonomously for the function of AFD neurons in thermotaxis. Based on the similarities with mammalian photoreceptors, my research suggests that differential compartmentalization of signal transduction components using different classes of ciliary proteins is important for the functions of ciliated sensory neurons.

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

Functional analysis of the E3 ubiquitin ligase HECTD1, and its relationship with the TRABID deubiquitinase

Date created: 
2014-04-07
Abstract: 

HECTD1 is a conserved ubiquitin ligase essential for cellular migration and development of the vertebrate neural tube. Here I show that HECTD1 accumulates along cell-cell contacts and mitotic spindle, and localizes to the centrosome in a dynein-independent manner. Likewise, I reveal that TRABID, a deubiquitinase and suspected HECTD1 interaction partner, enriches along spindle microtubules during cytokinesis, and at the centrosome. Previously documented knock-down phenotypes of HECTD1 and TRABID suggest that the proteins may participate in cytoskeletal dynamics. Interestingly, HECTD1 and TRABID were also shown to interact with the Adenomatous polyposis coli (APC) protein, modulating its K63 polyubiquitination. Redistribution of APC occurs in HECTD1 and TRABID knock-down cells, leading me to hypothesize that the ligase-deubiquitinase system controls cytoskeletal organization through APC trafficking. Given the importance of HECTD1 in mammalian development, I generated a hectd1 knock-out cell line using TALENs that will permit further detailed analysis of the microtubule-associated roles of HECTD1.

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

Hemagglutinin-esterase (HE) from Infectious Salmon Anemia virus (ISAv): Characteristics and attempted expression

Date created: 
2015-04-02
Abstract: 

Infectious salmon anemia virus (ISAv) is a pathogen that mainly affects Atlantic salmon (Salmo salar), which are commonly grown in the aquaculture industry. The resulting disease, infectious salmon anemia, has caused large financial losses for this industry. ISAv is a member of the Orthomyxoviridae family, as are the influenza type A, B and C viruses, and it belongs to the genus Isavirus. ISAv has a single dual-functional surface protein that is involved in the interaction with the host cells, namely: a hemagglutinin-esterase (HE). The HE protein binds preferentially to 4-O,5-N-diacetylneuraminic acid residues that are present on the target cells, which in this case are salmon erythrocytes. As is the case for influenza, the binding of HE protein to 4-O-acetylsialosides (receptor function) triggers infection in salmon, whereas the esterase active site, which possesses the 'receptor destroying' activity hydrolyzes the 4-O-acetyl groups, aiding the release of the viral progeny to infect neighbouring cells. This thesis reports the cloning and the attempted expression of the haemagglutinin-esterase from two different ISAv strains, that is a Canadian and a Norwegian strain, using baculovirus expression systems.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Andrew Bennet
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Optimizing Drug Delivery: Characterization of DLin-KC2-DMA/Distearoylphosphatidylserine by 31P and 2H NMR Spectroscopy

Author: 
Date created: 
2013-12-10
Abstract: 

Lipid nanoparticles (LNPs) are used to deliver siRNA to hepatocytes via endocytosis and subsequent endosomal release. With 99% of the LNPs taken up by the cell via endocytosis, only 1% of the siRNA is actually released into the cell cytosol. To improve the effectiveness of LNPs association with and disruption of endosomal membranes, the biophysical properties of a model system composed of 1:1 molar ratio of anionic lipid 1,2-distearoyl(d70)-sn-glycero-3-[phospho-L-serine] (DSPS-d70) and the cationic lipid DLin-KC2-DMA are characterized by 2H and 31P NMR spectroscopy. The bilayer to inverted hexagonal (HII) phase transition of the model system was shown to be influenced by temperature, pH and salt concentration. The order parameter profiles are obtained, revealing the extent of acyl chain movement of DSPS-d70 in bilayer and HII phases. The results help provide insights into computational simulation and eventually optimized LNPs design.

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

Effects of Interleukin-17 on Endothelial Nitric Oxide Synthase and Transplant Arteriosclerosis

Author: 
Date created: 
2014-09-30
Abstract: 

Transplant arteriosclerosis (TA), a vascular condition characterized by intimal thickening and vasomotor dysfunction of allograft arteries, is a leading cause of solid organ transplant failure. Properties of the endothelium such as the activity of eNOS control the structural and functional changes that occur in arteries with TA. We have examined the effect of IL-17 on eNOS expression in endothelial cells. Up-regulation of eNOS by IL-17 occurred through a post-translational mechanism because there was no effect of IL-17 on eNOS mRNA levels and inhibition of mRNA translation with cycloheximide did not prevent eNOS induction; but IL-17 treatment of ECs prolonged eNOS protein half-life. To begin examining the role of IL-17 signaling in graft cells in the development of TA, aortic segments from WT and IL-17RA-KO mice were interposed into allogeneic recipients. There were no significant changes in terms of TA development in IL-17RA-KO transplants compared to WT transplants. In summary, these results begin to define the cellular mechanisms by which IL-17 induces eNOS and the relevance of this cytokine to the pathogenesis of TA.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Jonathan Choy
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

Investigating the localization and function of the C. elegans HAM-1 protein

Author: 
Date created: 
2014-12-02
Abstract: 

In C. elegans, HAM-1 is an asymmetrically localized protein that regulates several asymmetric neuroblast divisions. Although the protein contains a putative DNA binding domain, nuclear localization of HAM-1 has never been observed through immunostaining of embryos with anti-HAM-1 antibodies. However, a GFP::HAM-1 fusion protein has been detected in the nucleus of transgenic animals under direct fluorescence microscopy. Through a biochemical subcellular fractionation of embryonic extracts, I determined that endogenous HAM-1 is primarily a nuclear protein. I have also examined HAM-1 localization in different ham-1 mutant embryos. This analysis has revealed several key residues in the N-terminus and a region in the C-terminus that are required for proper cortical localization of HAM-1. Finally, I have shown that Tyrosine residue 369 is crucial for HAM-1 function. Thus, my work has lent insight into HAM-1 sequences that contribute to function and localization of the protein.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Nancy Hawkins
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

The organization, regulation and functions of genes in the pericentric heterochromatin of the Drosophila melanogaster third chromosome

Date created: 
2013-11-22
Abstract: 

Alterations to chromatin through the post-translational modification of histone proteins or the binding of non-histone proteins is important for the tight control and specification of nuclear processes (e.g. transcription) occurring at most chromosomal regions. In this thesis, I report on the roles of enzymes responsible for adding methyl groups to histone H3 at lysine 4 (H3K4)—a modification pattern which is found at most active genes. I focus on a functional characterization of dSet1, which I show is responsible for most di- and trimethylation of H3K4. I also present evidence that dSet1 interacts with members of an evolutionarily conserved complex and that complex members are collectively required for H3K4 methylation. This work establishes a model system for better understanding functions of methyl-H3K4 in metazoans. Given that the dSet1 gene was found in pericentric heterochromatin, I became interested in the regulation, functions and organization of other genes in this specialized genomic compartment. Pericentric heterochromatin encompasses approximately 1/3 of the Drosophila melanogaster DNA sequences, yet contains just over 1% of Drosophila genes. Since pericentric heterochromatin has a low gene density, remains condensed throughout the cell cycle, and ‘silences’ most genes ectopically placed within it, it has in the past been mistakenly regarded as transcriptionally inert. However, there are still approximately 200 transcribed genes present in pericentric regions, and many of these genes are essential and/or expressed ubiquitously throughout development. Here I report a broad requirement for structural components of heterochromatin (Su(var) proteins) in the expression of genes in Drosophila pericentric heterochromatin, expanding on previous work reporting that Su(var) proteins and a heterochromatic environment are required for the expression of genes in this region. Due to difficulties arising in sequencing, mapping and genetically manipulating heterochromatic regions, much work remains in order to fully characterize the heterochromatic genome. I have assisted in these efforts by identifying and characterizing essential functions in 3rd chromosome pericentric heterochromatin by sequencing genetic mutants and through gene-targeting using RNA interference (RNAi). Together my data will contribute to collaborative efforts to obtain a basic understanding of the regulation, organization and functions of genes in pericentric heterochromatin.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Barry Honda
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Identification, Evolution, and Expression of the Trace Amine-Associated Receptor (TAAR) Gene Family in Atlantic Salmon (Salmo salar)

Date created: 
2014-09-12
Abstract: 

It is widely hypothesized that Atlantic salmon are imprinted at a young age with olfactory cues, which they use as a guide in order to return to their natal streams to spawn. However, the molecular mechanism(s) behind this biological phenomenon remain unknown. Therefore, in order to better understand imprinting and homing in Atlantic salmon, it is important to characterize the repertoire of olfactory receptors in thisspecies. A search of the first assembly of the Atlantic salmon genome revealed 27 putatively functional trace amine-associated receptor (TAAR) genes and 25 putative TAAR pseudo-genes. Genetic mapping, phylogenetic analysis, binding-site prediction, and quantitative PCR were performed using the Atlantic salmon TAAR genes. The identification of this gene family in Atlantic salmon will facilitate additional studiesinvolving olfaction and homing such as determining the range of allelic variation in olfactory receptors genes of different salmon populations.

Document type: 
Thesis
File(s): 
Supervisor(s): 
William Davidson
Department: 
Science: Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Enabling systems-level analyses of the host response to infectious diseases in bovine and other mammalian species

Date created: 
2014-09-29
Abstract: 

The innate immune response is a critical branch of immunity, providing a first line of defense against pathogens and shaping subsequent adaptive immune responses. The complexity of this system necessitates the application of systems-level approaches. InnateDB is an integrated web-accessible database and systems biology platform being developed to facilitate the systems level analysis of innate immunity pathways and networks. One of the aims of this thesis was to enhance InnateDB with bovine data, thereby providing a resource for investigation of this agriculturally important model organism. Using an orthology based approach, over 70% of InnateDB’s human protein-protein interactions (PPIs), and a similar fraction of human pathways were reconstructed in cow and integrated into InnateDB. Pathway analysis, the statistical association of observations at the molecular level with processes at a more systems level, plays a crucial role in the interpretation of high-throughput experimental datasets. A widely neglected challenge in pathway analysis relates to the handling of multifunctional genes. I therefore developed SIGORA, a novel pathway analysis method that identifies genes and gene-pairs that are unique signatures of a pathway and examines their over-representation in a given list of genes of interest (e.g. the list of differentially expressed genes in an infectious condition). With several biological datasets, SIGORA outperformed traditional methods, delivering biologically more plausible and relevant results. This was also reflected in significantly lower false positive rates for simulated datasets. An additional challenge in high-throughput dataset interpretation concerns the lack of functional annotation for many genes. The guilt by association (GBA) principle was applied in a conservative manner to a large tissue expression dataset (105 Tissues, 13000 genes) to infer gene functions from co-expression data. Overall, 180 previously un-annotated bovine genes were assigned a putative function by this approach. In 20% of the cases, the inferred function was additionally supported by literature in other species. microRNAs are emerging as important innate immune response regulators and as biomarkers of disease. Determining microRNA functions requires the identification of their targets, yet computational prediction of such targets is challenging. As part of a group investigating microRNA roles in bovine mastitis, I used a combination of prediction tools to compile a list of likely targets. Here, the overall emerging picture (including pathway enrichment) is consistent with our current understanding of this condition. Collectively this work provides new tools and insights that may more broadly be used to improve systems-based analysis of bovine and other mammalian responses.

Document type: 
Thesis
Supervisor(s): 
Fiona Brinkman
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.