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

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The immunological and vascular effects of IL-6 signalling in transplant arteriosclerosis

Author: 
Date created: 
2019-07-08
Abstract: 

Transplant arteriosclerosis (TA) is a pathological vascular condition that is a main cause of chronic rejection and eventual failure of cardiac transplants. TA is caused by the activation of T cells towards allogeneic antigens expressed by vascular cells, which results in T cell-mediated injury and dysfunction of allograft arteries. T cells also support the activation and secretion of donor specific antibodies (DSAs) by B cells that contribute to the pathogenesis of TA. Interleukin-6 (IL-6) is secreted within hours of surgical transplantation and plays an important role in activation of allogeneic immune responses and regulation of vascular processes that influences TA. There are two main mechanisms by which IL-6 can signal to cells, classic and trans, that are distinguished by whether it binds to membrane bound or soluble forms of its receptor (IL-6R). In classic signalling IL-6 binds to IL-6R on the surface of cells. In trans-signaling IL-6 binds to soluble IL-6R. In both scenarios, the IL-6/IL-6R complex associates with the signalling subunit gp130 on cell membranes to transduce intracellular signalling events. Interestingly, IL-6 classic and trans-signalling have distinct biological outcomes. I examined the mechanism by which IL-6 classic and trans signalling contributes to immune activation in TA and characterized the biological outcomes of IL-6 signalling in endothelial cells. IL-6 classic and trans signalling were redundant for the activation of peripheral T cells that cause TA. However, eliminating IL-6R expression in T cells significantly reduced the development of DSAs in the serum of graft recipients, suggesting that IL-6 classic signalling in T cells may be required for antibody-mediated pathology in TA. In addition to activating immune cells, IL-6 acts on endothelial cells to induce inflammation and protect the vasculature from injury. IL-6 trans signalling in ECs significantly induced STAT-3, ERK1/2 and Akt activation. This signalling mechanism was needed for ICAM upregulation and the secretion of inflammatory cytokines by IL-6, indicating that IL-6 trans-signaling drives inflammatory activation of ECs. IL-6 classic signalling induced ERK1/2 and Akt activation but not STAT3 and was sufficient to stimulate the secretion of IL-8 and to protect ECs from cell death caused by serum deprivation but did not induce other inflammatory processes in ECs. My results suggest that IL-6 trans and classic signalling differentially affect inflammatory and survival responses in endothelial cells, which may have implications for understanding the vascular effects of IL-6R blockade in patients. Overall, my findings provide new insight into the immune and vascular effects of IL-6 in transplantation.

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

Mechanism of eubacterial 6S RNA release and Development of RNA Mango toolkit to study Ribonucleoprotein complexes

Date created: 
2017-04-13
Abstract: 

Ribonucleic acids (RNA) perform diverse biochemical functions in cells ranging from being an intermediate in the flow of genetic information, catalyst performing crucial reactions and as regulators of several processes. In bacteria, 6S RNA is a global transcription regulator that binds and inhibits house keeping RNA polymerase holoenzyme (core polymerase + σ70) under low nutrient conditions and rescues transcription in high nutrient conditions by the synthesis of a short product RNA (pRNA) using itself as a template. I show that a kinetic intermediate containing 6S RNA:core enzyme complex, that is enhanced by the formation of a phylogenetically conserved ‘release’ hairpin arises during 6S RNA release. Using nucleotide feeding experiments to slow down the release and a 6S RNA mutant which precludes the hairpin formation, I found the release process involves intrinsic ‘scrunching’ type mechanism that is modulated by the ability to form a release hairpin during the process of 6S RNA release. Given the importance of RNA in regulating various cellular processes, a fluorescence tool to track RNA in real time is limiting as RNA lacks intrinsic fluorescence. The Unrau lab has in vitro selected RNA Mango aptamer that binds thiazole orange with nanomolar affinity while increasing its fluorescence up to 1100 fold. To elucidate how this small aptamer exhibits such properties, which make it particularly well suited for studying low-copy cellular RNAs, we, in collaboration with D′Amaré’s lab, determined its co-crystal structure, discovering a three-tiered G-quadruplex connected to a duplex through a GAAA tetraloop-like junction. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb) ligand I have created a Mango toolkit that simultaneously enables the purification and characterization of endogenous cellular RNPs in vitro.

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

Integration of transport pathways in Saccharomyces cerevisiae

Author: 
Date created: 
2017-02-28
Abstract: 

Plasma membrane (PM) homeostasis is essential for viability and depends on maintaining a constant balance between the amount of membrane material arriving at the cell cortex and the amount recycled. This membrane transport is mediated by two distinct mechanisms: (i) vesicular transport, utilizing membrane-enclosed vesicles for bulk transport of membrane proteins and lipids; and (ii) non-vesicular lipid transport. While the molecular mechanisms of these processes are well defined, they are generally considered independent events but how they are integrated is poorly understood. To gain insight into how these transport pathways are coordinated at the PM, three avenues of research were conducted using a combination of genetic, biochemical and live-cell microscopy assays. First, I showed that the Oxysterol-binding protein-related protein (ORP) Osh4p, implicated in non-vesicular sterol transfer, was found to associate with exocytic vesicles and formed complexes with regulators of polarized exocytosis, including the small GTPases Sec4p, Cdc42p, and Rho1p. Second, I tested the function of the evolutionarily conserved endoplasmic reticulum (ER)-associated protein Arv1p, also suggested to be involved in non-vesicular sterol transfer. Ultimately Arv1p was found to be dispensable for sterol exchange between the ER and PM but instead it was shown to play an important role in maintaining ER ultrastructure; Arv1p might be involved in regulating insertion of tail-anchored proteins into membranes. Finally, the essential yeast Rab GTPase Sec4p, principally known to be a key regulator of exocytosis, was shown to mechanistically couple polarized exocytosis with cortical actin polymerization, which activates Las17p (the yeast Wiskott–Aldrich syndrome [WASp])-dependent endocytosis. Las17p activation results in actin filament nucleation, which pulls the PM inward for endocytic vesicle biogenesis. Sec4p thereby represents the first direct regulatory link that couples exocytosis and endocytosis, which we termed \"yeast compensatory endocytosis.\" By identifying novel mechanisms that coordinate intracellular transport pathways, these studies provided important new insights into how PM homeostasis is regulated and maintained.

Document type: 
Thesis
File(s): 
S1 Video. Photobleaching enables single particle tracking of GFP-Sec4p at the cell cortex.
S2 Video. Las17p-RFP and GFP-Sec4p particles co-localize at cortical actin patches.
S3 Video. Sla1p-RFP and GFP-Sec4p co-localize at cortical actin patches.
S4 Video. GFP-Sec4p particles co-localize with Abp1p-RFP at cortical actin patches.
S5 Video. SEC4 and SEC2 function are required for normal actin patch polarization and dynamics.
Senior supervisor: 
Christopher Beh
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Mechanisms and regulation of plasma membrane-endoplasmic reticulum Membrane Contact Sites in Saccharomyces cerevisiae

Author: 
Date created: 
2018-12-13
Abstract: 

Membrane tether proteins staple the endoplasmic reticulum (ER) to other cellular membranes at Membrane Contact Sites (MCSs), as possible conduits to coordinately regulate lipid metabolism and membrane exchange. To determine the role of ER-PM MCSs in membrane regulation, I generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tether proteins (the yeast E-Syts Tcb1p-3p; VAP homologs Scs2p and Scs22p; and the TMEM16 homolog Ist2p), as well as the presumptive tether Ice2p. Although Δ-s-tether cells lack direct ER-PM MCSs they were viable, albeit with severe defects in lipid homeostasis and membrane organization. To determine the role of MCSs in non-vesicular sterol transport, the bi-directional exchange of sterols between the ER-PM in Δ-s-tether cells was directly assayed in vivo. No significant defects in non-vesicular sterol transport were detected though lipidomic analysis revealed major dysfunction in phospholipid and sphingolipid synthesis. Based on these findings I hypothesized that Δ-s-tether growth defects might be rescued either metabolically, by increasing phosphatidylcholine synthesis through choline addition, or by overexpressing the phospholipid methyltransferase OPI3. These modifications did restore growth, but not by re-establishing ER-PM MCSs, suggesting that these membrane attachments are not physically required for lipid transport but rather as regulators of lipid metabolism. Phosphatidylinositol-4-phosphate (PI4P) accumulated in the PM of Δ-s-tether cells, consistent with the observed synthetic lethality between Δ-s-tether mutations and mutations in either the SAC1 PI4P phosphatase gene or OSH4, which encodes a PI4P binding protein. Even though transport assays indicated that MCSs do not mediate ER-PM sterol exchange, we tested if sterols played a role in contact site generation. Cells depleted of sterols by repressing ERG9, which encode the sterol specific biosynthetic enzyme squalene synthase, exhibited nearly complete coverage of the inner cytoplasmic PM surface with cortical ER. These results support a model for ER-PM MCSs in which they collectively act as a regulatory nexus for coordinating multiple lipid metabolic pathways to control cell membrane composition.

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

Fluorogenic RNA aptamers: Exploring applications for nucleic acid detection

Author: 
Date created: 
2019-08-16
Abstract: 

Worldwide concern for disease detection has led to demand for sensitive, specific, and simple pathogen detection methods. In recent years, methods for detecting nucleic acids, including molecular beacon reporters and the isothermal amplification of nucleic acids have become popular. Highly fluorescent RNA Mango aptamers have been developed that can operate in salt concentrations required for in vitro systems. Presented here are two novel applications of RNA Mango aptamers for the detection of nucleic acids. First, by engineering RNA Mango, a molecular beacon like switch was developed that maintains minimal fluorescence in the absence of RNA target, and that dramatically increases in fluorescence upon target addition. Second, by incorporating Mango aptamers into an existing isothermal amplification method (NASBA), attoMolar concentrations of RNA can be detected with high sensitivity and specificity. These Mango tools improve upon current RNA detection technologies and provide simple fluorescent methods for detecting RNA.

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

Exploring and exploiting the minor pilins of Type IV pili

Author: 
Date created: 
2019-07-17
Abstract: 

Gram-negative bacteria are naturally resistant to large antibiotics in large part because of their impermeable outer membrane. Type IV pili (T4P) are surface-displayed filamentous polymers of major pilin proteins that have the ability to pull substrates through the outer membrane via retraction. T4P have one or several low-abundance minor pilins that localize to the tip of the pilus, mediating functions such as DNA binding and uptake. We hypothesize that minor pilins can be exploited as targets for drug delivery, facilitating the uptake of drugs across the bacterial outer membrane. Here I expressed and purified putative tip-associated minor pilins from important human pathogens and collected x-ray diffraction data on one of these, PilX from Neisseria gonorrhoeae. Additionally, we conclusively identify the Vibrio cholerae minor pilin, TcpB, at the tip of its pilus and use it to screen a phage display library in search of potential carrier molecules.

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

Development of single-molecule assays for proteolytic susceptibility: Force-induced collagen destabilization

Author: 
Date created: 
2019-04-26
Abstract: 

Collagen’s unique triple helical structure allows for mechanical support and stability at all hierarchical levels including single molecules, fibrils, fibers and tissues. Surprisingly, the structural stability of the triple helix under force is not well known. I explored its response to applied force with single molecule proteolytic susceptibility assays. To perform the experiments I designed, developed and constructed a low-cost and accessible single-molecule force spectroscopy instrument called the Mini-Radio Centrifuge Force Microscope or MR.CFM. Centrifuge force microscopy allows for a wide dynamic force range, massively parallel single-molecule force measurements, and external field-free application of force. Collagen’s triple helix is resistant to proteolytic cleavage due to steric hindrance. By comparing trypsin’s cleavage rate of collagen with and without force in the MR.CFM, I monitored the triple helix’s force stability. I showed an increase of cleavage rate with force, suggesting a force-enhanced destabilization of the triple helix. One of the most challenging components of single-molecule force spectroscopy is surface chemistry; unsatisfied with available approaches I developed a surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer, F127-NHS. I created an easy-to-use assay called the microsphere adhesion by gravity, inversion, then counting, or “MAGIC” assay, to quantitatively measure the interactions between a surface and microsphere. Guided by data from the MAGIC assay I reduced nonspecific interactions with a multistep labelling protocol for the microspheres. I achieved 94% of DNA microspheres singly tethered to the F127-NHS surface. In addition, I showed the surface’s utility in fluorescence and force spectroscopy assays. Taken together my work has enabled the advancement of accessible force spectroscopy and furthered the understanding of collagen’s response to force.

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

Examining the function of primary HIV-1 Nef isolates in the setting of viral control

Author: 
Date created: 
2014-04-24
Abstract: 

HIV is a global pandemic. While combination anti-retroviral therapy can block viral replication, HIV establishes latent reservoirs that persist for life. A rare group of HIV-infected individuals called “elite controllers” spontaneously suppress plasma viremia to clinically undetectable levels. Understanding how elite controllers contain HIV could assist in developing more effective interventions, such as vaccines or eradication strategies. The HIV Nef protein is critical for viral pathogenesis and progression to AIDS. Nef modulates cellular proteins to increase viral replication and to evade host immunity. Functional impairment of several HIV proteins has been seen in elite controllers, but prior to my thesis a systematic analysis of Nef had not been done. To explore this, I examined Nef clones from elite controllers and progressors. I observed that Nef clones from controllers displayed significantly lower activity for multiple functions. Furthermore, I identified natural polymorphisms in Nef that contributed to attenuated function and confirmed these by testing Nef mutants. In summary, I have shown that natural sequence variation in Nef results in substantial differences in protein function. Nef clones isolated from elite controllers displayed the poorest activity for multiple functions, indicating that attenuation of Nef may contribute to reduced pathogenesis in these cases. Therapeutic targeting of the Nef domains identified in my studies could contribute to HIV eradication strategies.

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

Cytogenetic evolution of genus Salmo

Author: 
Date created: 
2019-02-21
Abstract: 

The ancestral genome duplication that occurred around 80-100 MYA in the common ancestor of extant salmonids induced a stressed auto-tetraploid genome. This was followed by expansion of transposable elements which is believed to facilitate large-scale chromosomal rearrangements. The species radiation did not occur until around 40-50 million years later when earth’s climate was going into a cooling stage. As the result of the large-scale genome reorganization, present-day salmonids share large syntenic collinear blocks in a species-specific order. In the present study, our first aim was to visualize these rearrangements by taking advantage of genus Salmo (including two sole members, namely, Atlantic salmon (S. salar) and brown trout (S. trutta)) where species not only exhibit distinct karyotypes, but also intra-species chromosomal polymorphisms. Moreover, another aim of present study was to provide cytogenetic support for confirming the identity of sex determining gene, sdY, in Atlantic salmon by revealing its physical locations in both European and North American derived Tasmanian Atlantic salmon. In the genus of Salmo, large scale rearrangements including fusions, translocations, fissions and possible chromosome arm loss were evident in the present study, giving rise to the extant karyotypes of S. salar and S. trutta. The fact that no polymorphic karyotype of the S. trutta was observed in our study and other publications suggests a relative stable genomic background. In contrast, polymorphic karyotypes due to Robertsonian rearrangements of three pairs of chromosomes were found in Canadian subpopulations of S. salar. The fixation of the homozygous translocation in most Canadian populations indicates possible fixation of advantageous mutations and suggests a mild to near-neutral underdominance of this rearrangement in its ancestral heterozygous state. On the other hand, the observation of potentially deleterious Robertsonian tandem fusions in all Canadian populations indicate the initial rearrangement likely took place in a small effective population with subsequent spreading into other river populations through colonization. In our effort to visualize the physical locations of sdY, a single-copy gene, the gene was indeed found residing on the sub-telocentric region of Ssa02q in European Atlantic salmon, consistent with results from previous SEX mapping studies. In comparison, multiple locations including Ssa02q, Ssa03p and Ssa06p were shown to be locations of sdY in a North American derived Tasmanian population, supporting a previous study suggesting a jumping gene theory.

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

Interactions among Dam, SeqA and mismatch repair proteins in Escherichia coli

Author: 
Date created: 
2019-03-29
Abstract: 

The accuracy of DNA replication is very important, and organisms have several proofreading and repair systems to prevent mutations from occurring. Lesions can be introduced by errors during replication, chemical mutagens, UV or ionizing radiation. In Escherichia coli, mismatches are detected by MutS and MutL which together activate MutH to initiate repair. Repair is dependent on GATC hemi-methylation signals on the DNA which is added by DNA adenosine methylase (Dam). SeqA acts as a regulator of DNA replication, sequestering the origin and preventing reinitiation. We hypothesize that 1) Dam and SeqA are coordinated by MutL, and (2) persistent mismatches caused by lack of polymerase proofreading will increase mismatch repair activity. Results show that Dam binds to both SeqA and MutL, and no significant increase in mismatch repair activity was detected when the error prone polymerase was induced. These data suggest the importance of temporal coordination of methylation and/or interaction of Dam and MutL in preparation for mismatch repair. Our data is consistent with previous literature that shows mismatch repair primarily works against transitions and is inefficient at preventing transversions.

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