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

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Minor pilins play a major role in pilus dynamics and functions

Author: 
Date created: 
2015-08-17
Abstract: 

Type IV pili (T4P) in Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) represent the simplest of all pilus systems whereby all the proteins needed for pilus assembly are encoded within a single operon. These systems are unusual for their lack of a retraction ATPase and each operon encodes only one minor pilin instead of several. How can pili retract without a retraction ATPase? The only minor pilin in the operon is key to understanding retraction in these systems. V. cholerae T4P, the toxin co-regulated pilus (TCP), produces the minor pilin TcpB which shares N-terminal homology with the major pilin but possess a larger C-terminal domain. TCP can assemble in a ∆tcpB mutant but at much lower levels than the wild type strain. We show that the minor pilin is required for efficient pilus assembly and pilus-related functions. We quantified the stoichiometry between the major and minor pilins and established this ratio is critical to maintaining optimal pilus functions. We show by immunodetection and immunogold electron microscopy that the minor pilins are incorporated into surface-displayed pili at low levels. Moreover, we determined minor pilin incorporation at the base of an assembling filament is necessary to induce pilus retraction and this mechanism is mediated by a conserved glutamate at position 5. This residue is conserved in all major pilins and some minor pilins, and is hypothesized to be critical for stabilizing pilin:pilin interactions by charge complementarity during assembly when new pilins are incorporated at the base of the filament. We characterized the ETEC minor pilins and achieved similar findings. We propose a new model to explain pilus extension and retraction by which the minor pilins have dual functions in priming pilus assembly as the first subunit in assembly and inducing retraction by incorporating into assembling filaments to stall assembly and cause spontaneous depolymerization of the pilin subunits. Our results have implications in understanding pilus dynamics in the more complex T4P systems and in the related bacterial virulence factor, the type II secretion system.

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

RNA: A JACK OF ALL TRADES. Studying the regulatory role of 6S RNA in E. coli and the impact of exosomal RNA in parasite pathogenesis

Date created: 
2015-07-24
Abstract: 

During the past two decades we have seen an explosion in our understanding of RNA dependent gene regulation. We now know that RNA is involved in every major event in the life of cells, from the Okazaki fragments involved in DNA replication to programmed cell death. The work described here explores two situations in which RNA plays an important role; how 6S RNA helps ensure bacterial survival and the role of RNA in helping the intracellular parasite, Leishmania, escape the immune system by taking refuge inside mammalian macrophages.6S RNA is a non-coding RNA that regulates bacterial transcription by sequestering the RNA polymerase holoenzyme (Eσ70) in low nutrient conditions. In high nutrient environments, Eσ70 is released by the synthesis of a short product RNA (pRNA) using the 6S RNA as a template. A range of 6S RNA release-defective mutants were selected and characterized from a highly diverse in vitro pool. There is complex crosstalk between regions of the 6S RNA large open bubble that interact with Eσ70 in a cooperative manner so as to ensure efficient pRNA-dependent release. When a group of 6S RNA mutants was over-expressed in E. coli, they significantly delayed growth and decreased cell survival indicating that 6S RNA release rate plays a key role in regulating normal transcriptional dynamics and ultimately cell division. Interestingly, cells resumed normal growth rates approximately 6 hours after mutant 6S RNA overexpression. This growth pattern might be correlated with the accumulation of a protein factor that binds strongly to the 6S and mutant 6S RNA, and data suggest that 6S RNA also might bind to an RNase.RNA may contribute directly to parasite pathogenesis in trypanosomatids. Leishmania spp. uses exosomes to weaken mammalian host cells. Exosomes are known to be involved in intercellular communication. We examined the use of exosomes and their RNA from two species of Leishmania and how that RNA reprograms host cells. Exosome RNA cargo is delivered to host cell cytoplasm during in vitro studies. Sequencing of exosomal RNA indicated that the majority of cargo sequences were derived from non-coding RNA, while Northern blotting confirmed the specific and selective enrichment of tRNA-derived small RNAs in exosomes. We also identified a number of novel transcripts, which appeared to be specifically enriched in exosomes compared to total cell RNA. To our knowledge this is the first report that exosomes are used by a pathogen to invade new host cells. These findings also open up a new avenue of research on non-canonical, small RNA pathways in trypanosomatid parasites, which may elucidate pathogenesis factors and identify novel therapeutic targets.

Document type: 
Thesis
Supervisor(s): 
Peter J Unrau
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Genomic selection for improvement of Atlantic salmon aquaculture

Date created: 
2015-04-17
Abstract: 

Since the beginning of aquaculture, breeders have practiced selection of the most suitable specimens for the improvement of their population and at the same time their production. Atlantic salmon (Salmo salar) aquaculture is not an exception and selection efforts have taken place since the beginning of their culture during the early 1970s in Norway. As expected, most of the selection during the early years was done through phenotypic observations of fish and their response to farming conditions. Initial stages of selection in Atlantic salmon targeted traits like growth, timing of sexual maturation and some disease resistance and the results were very positive. Through approximately 40 years of farming (~12 generations), the time to produce a standard market-size 4 kg fish has halved and the rate of food conversion has been amazingly improved. Today, genomic tools promise to be able to develop more efficient selection systems to improve traditional phenotype-based selection systems. Selection could be improved with the identification of genomic regions controlling specific traits of interest, and the currently available 6.5K SNP array that was developed for Atlantic salmon provides an opportunity to do so. In my study I analyzed a Canadian farmed population (Cermaq), which originates from the Norwegian Mowi strain. I aimed to identify genomic regions controlling traits of interest such as growth and sexual development, but I also analyzed the genomic status of the population to learn how selection has affected its genomic diversity. By performing QTL and GWAS analyses I was able to identify multiple genomic regions potentially controlling growth and sexual maturation, one of these regions associated to Maskin, a gene involved in gonad development that shows a high expression in Atlantic salmon ovaries. Additionally, analysis to identify regions under selection allowed me to identify many genomic regions that seem to be selected in the Cermaq population. The polygenic nature of the traits makes it difficult to postulate particular regions to be controlling specific traits; the most likely scenario is that many regions are controlling a particular trait, each of these regions having some effect on the phenotypic outcome.

Document type: 
Thesis
Supervisor(s): 
William Davidson
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

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.