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

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Modulation of Neuronal Insulin Signaling Rescues Axonal Transport Defects in an Alzheimer’s Disease Model

Date created: 
2013-07-19
Abstract: 

Defective brain insulin signaling contributes to the cognitive deficits in Alzheimer's disease (AD). Oligomeric amyloid-β peptides (AβOs), the neurotoxin implicated in AD, induce a variety of cellular insults, including dysregulation of intracellular signaling cascades and disruption of fast axonal transport. I show that modulation of insulin signaling prevents AβO-induced defects of brain-derived neurotrophic factor (BDNF) transport in wild type (tau+/+) and tau knockout (tau-/-) primary hippocampal mouse neurons. Tideglusib, an inhibitor of glycogen synthase kinase-3β (GSK3β), an insulin signaling intermediate implicated in AD, rescues BDNF transport in tau+/+ and tau-/- neurons. Furthermore, Exendin-4, an anti-diabetes agent, activates the insulin signaling pathway through glucagon like peptide-1 receptor stimulation to also rescue BDNF transport defects similarly to Tideglusib. These results indicate a protective link between insulin signaling and tau-independent transport regulation. By establishing links between insulin signaling and AβO action, my results allow for establishing novel directions for AD therapeutics.

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

Evolution and characterization of the fatty acid-binding proteins (fabps) in Atlantic salmon (Salmo salar)

Author: 
Date created: 
2012-04-20
Abstract: 

It is suggested that gene or genome duplication is the driving force in evolution that leads to speciation. Two models, the classical model and the duplication- degeneration-complementation (DDC) model, have been proposed on the fates of gene duplicates resulting from either a gene or a genome duplication event. The classical model suggests that one of the gene duplicates might result in loss of function (non- functionalization) or gain of a new function (neo-functionalization) depending on whether the accumulated mutations over the years are deleterious or beneficial to the organism. In the DDC model, it is proposed that each of the gene duplicates might accumulate different deleterious mutations in the regulatory region of the gene, such that these genes partition the ancestral gene function (sub-functionalization). Combinations of the phylogenetic analysis of many gene families support that salmonids have undergone two additional whole genome duplications compared to the mammals, one occurred in the common ancestors of teleosts and another happened in the common ancestor of salmonids approximately 25-120 million years ago. In this thesis, the evolution of the fatty acid-binding protein (fabp) family in fish and salmonids was examined. I have characterized eighteen unique fabp genes in Atlantic salmon. These include the seven fabp sub-families described previously in fish. Phylogenetic analyses and conservation of synteny support the two whole genome duplication events in the common ancestors of teleosts and salmonids and indicate when gene losses occurred. Genetic mapping of fabp gene duplicates to homeologous chromosomes in Atlantic salmon also support that they arose by the 4R genome duplication. I also searched for the signatures of neo-functionalization and sub-functionalization by calculating dN/dS ratios, examining the nature of amino acid substitutions and expression patterns, and suggested the fates of fabp gene duplicates in Atlantic salmon. Overall, the findings of this project provide insight into the evolutionary processes at play in salmonid genomes.

Document type: 
Thesis
File(s): 
Supervisor(s): 
William Davidson
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

Integration of transport pathways in Yeast

Author: 
Date created: 
2012-03-22
Abstract: 

Cell polarity is maintained via a balance of exocytosis and endocytosis; the protein machinery that mediates these transport processes must be co-ordinated with membrane lipid signals. This lipid signalling is, in part, dependent on the establishment of membrane domains through lipid transport. Cholesterol is transported via a poorly defined route that is independent of vesicle-mediated secretory protein transport. This “non-vesicular” sterol transport is postulated to involve the conserved family of Oxysterol binding protein (OSBP) Related Proteins (ORPs), which are proposed to be sterol lipid transport proteins (LTPs). To test if ORPs primarily act as sterol LTPs or alternatively as sterol-responsive signalling proteins, the function of Saccharomyces cerevisiae OSBP Homologues (OSH1-OSH7) were analyzed. Depletion of all Osh proteins in yeast cells inhibited growth, and defects in endocytosis, polarized exocytosis, and sterol homeostasis, were observed. Consistent with a direct role in exocytosis, Oshdepletion disrupted the polarized localization of vesicle transport regulators (Rho- and Rab-GTPases, and exocyst complex subunits) and the Osh protein Osh4p was observed to travel on exocytic vesicles to sites of polarized growth. Osh4p also formed complexes in vivo with specific Rho- and Rab-GTPases, and exocyst complex subunits. Contrary to the postulated role of ORPs as LTPs, a designed mutation in Osh4p that disrupts its ability to bind and thereby transport sterols, did not inactivate the protein but caused a gain-of-function phenotype affecting exocytosis. Our experiments suggested that ORPs are not sterol LTPs and implied that sterols act as signalling ligands that repress Osh4p, and potentially other ORPs. To understand how Osh proteins might simultaneously affect both exocytosis and endocytosis, I tested whether the regulation of the exocytic and endocytic machinery are directly coupled. I found that the Rab GTPase Sec4p, which is an integral component of exocytosis, directly interacted with specific endocytic proteins at actin patches. SEC4 was required for proper endocytic site assembly and actin patch polarization, indicating that Sec4p links exocytosis and endocytosis to maintain cell polarization. Because these novel mechanisms involving sterol signalling and cell polarization are likely to be conserved, I propose these studies have broader medical implications applicable to cancer cell growth and metastasis.

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

Regulation of Dlg-containing adhesion complexes during epithelial and synaptic plasticity in Drosophila

Author: 
Date created: 
2013-02-28
Abstract: 

Discs large (Dlg) is a multi-PDZ domain-containing protein belonging to the MAGUK superfamily of scaffolding proteins. In epithelia, Dlg serves as an apicobasal polarity determinant, where its disruption leads to tumor formation. Dlg also plays roles in maintaining synaptic structure and function, and has been implicated in neurodegeneration. Thus, understanding Dlg regulation can provide insights into human diseases including cancers and neurological disorders. In this thesis, we characterize novel Dlg regulators during epithelial and synaptic plasticity events in Drosophila development through two separate but overlapping projects. Project 1: Dorsal closure, which is a wound healing model, occurs when a hole in the embryonic epidermis is closed due to surrounding epidermal flanks that migrate towards each other over the amnioserosa occupying the hole. During migration, the leading edges of the epidermal cells abutting the hole exhibit a breakdown in apicobasal polarity as adhesions are severed with the amnioserosa. At the end of migration, however, apicobasal polarity is re-established as adhesions must form between the opposing epidermal flanks in order to seal the hole shut. Mammalian studies indicate that the Dlg-containing Scribble complex recruits p21-activated kinases (Paks), effectors for Rho GTPase signalling, to the leading edge during cell migration. We show that this interaction can act in the opposite direction as Paks can recruit the Scribble complex back to the leading edge upon dorsal closure completion. We propose that the bidirectional relationship between Paks and Dlg may allow epithelia to toggle between migratory and adhered states. Project 2: Previous studies have shown that adducin, a membrane cytoskeletal protein that regulates actin, is hyperphosphorylated in spinal cord tissue taken from patients who died with the motor neuron disease, Amyotrophic Lateral Sclerosis. To further explore the roles of adducin in the nervous system, we decided to study the Drosophila orthologue encoded by hu-li tai shao (hts). We show that Hts regulates larval neuromuscular junction morphogenesis by controlling Dlg postsynaptic targeting via indirect phosphorylation. This process is partially supressed when Hts phosphorylation in the MARCKS domain is blocked. We propose that Hts is a signalling-responsive cytoskeletal protein that contributes to synaptic growth through Dlg-mediated adhesion.

Document type: 
Thesis
File(s): 
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
See Appendix A: Movies for legend.
Supervisor(s): 
Nicholas Harden
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.

Comparative Genome Analysis of Malaria Parasite Species

Author: 
Date created: 
2013-04-11
Abstract: 

With over 200 million infections and up to one million deaths every year, malaria remains one of the most devastating infectious diseases affecting humans. Over the last few years, complete genome sequences of both human and non-human malaria parasite species have become available, adding comparative genomics to the toolbox of molecular biologists to study the genetic basis of human virulence. In this thesis, I computationally compared the published genomes of seven malaria parasite species with the aim to gain new insights into genes underlying human virulence. This comparison was performed using two complementary approaches. In the first approach, I used whole-genome synteny analysis to find genes present in human but not non-human malaria parasites. In the second approach, I first clustered virulence-associated genes into gene families and then examined these gene families for species-specific differences. Both comparisons resulted in interesting gene lists. Synteny analysis identified three key enzymes of the thiamine (vitamin B1) biosynthesis pathway to be present in human but not rodent malaria parasites, indicating that these two groups of parasites differ in their ability to synthesize vitamin B1 de novo. My gene family classification exposed within the largest and highly divergent surface antigen gene family pir a group of unusually well conserved orthologs, which should be considered as high-priority targets for experimental characterization and vaccine development. In conclusion, this thesis highlights genes and pathways that are different between human and non-human malaria parasites and therefore could play important roles in human virulence. Experimental studies can now be initiated to confirm virulence-associated functions and to explore their potential value for drug and vaccine development.

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

Identification of genes involved in heat stress in arctic charr

Author: 
Date created: 
2011-12-16
Abstract: 

I set out to identify candidate genes that can be used to develop genetic markers associated with Upper Temperature Tolerance (UTT) for use in a genomics-assisted broodstock program for Arctic charr (Salvelinus alpinus) and for examining wild populations at risk due to climate change. This was accomplished using genomic resources available for Atlantic salmon (Salmo salar), which allowed me to identify and examine genomic regions and specific genes of interest. In addition, I conducted expression profiling of Arctic charr exposed to acute and chronic thermal stress. Using comparative genomics, I identified several Atlantic salmon fingerprint scaffolds containing markers associated with UTT in Arctic charr and rainbow trout (Oncorhynchus mykiss). One of these was fully sequenced using 454 GS FLX next-generation sequencing and annotated, which identified nine genes in the putative Quantitative Trait Locus (QTL) region of the Atlantic salmon genome. This analysis also provided evidence that the 454 sequencing technology was suitable for partial assembly and gene annotation, but not for de novo whole genome sequencing of a complex salmonid genome. Next, I conducted expression profiling of phenotypically tolerant and intolerant Arctic charr. The differentially expressed genes were compared with those identified within the UTT QTL sequenced previously, which suggested COUP-TFII as a particularly interesting candidate gene. Heat shock proteins (Hsps) and hemoglobins were also significantly associated with acute thermal stress. Concurrently, I performed expression profiling of Arctic charr exposed to moderate, chronic temperature stress that mimicked a more realistic situation. Again, Hsps were identified in the thermal stress response, as well as ribosomal proteins, which were up-regulated throughout the exposure and the recovery period. Finally, I identified and fully annotated all of the hemoglobin genes in Atlantic salmon. This identified substantially more hemoglobin genes in this species than in any other fish analyzed to date, and included several non-Bohr beta hemoglobins, which may be used in emergency response situations. Combined, the findings of my research have substantial implications for developing a temperature tolerant Arctic charr broodstock and for examining wild populations of salmonids for responses to temperature stress brought by climate change.

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

Computational ortholog prediction: evaluating use cases and improving high-throughput performance

Date created: 
2013-03-08
Abstract: 

Orthologs are genes that diverged from an ancestral gene when the species diverged. High-throughput computational methods for ortholog prediction are a key component of many computational biology analyses. A fundamental premise in these analyses is that orthologs (when predicted correctly) are functionally equivalent and can be used to transfer gene annotations across species. Currently, many existing ortholog prediction methods generate a sizeable number of incorrect ortholog predictions, especially in cases of complex gene evolution. My thesis examines the functional equivalence hypothesis further and presents one solution that increases the precision of ortholog prediction. To examine the use of orthologs in computational analysis, I conducted and evaluated three projects that employ ortholog prediction in distinct ways. In these projects, orthologs were used to (1) identify conserved, unique genes in metazoan species, (2) validate predicted gene regulatory modules in Pseudomonas aeruginosa, and (3) construct a transcriptional regulatory network in Aspergillus fumigatus. I identified factors affecting ortholog prediction in these specific use cases, demonstrating how successive gene duplications, incomplete genomes and rapid evolution of gene regulation can impact the results for such analyses. To improve ortholog prediction, I evaluated and augmented an existing method called Ortholuge. Ortholuge is a computational method that increases the precision of ortholog prediction in a high-throughput setting. I evaluated the performance of Ortholuge, showing that its approach of classifying orthologs based on their relative phylogenetic divergence does identify orthologs that are more functionally equivalent. I compared Ortholuge to contemporary methods QuartetS and OMA, and showed that Ortholuge consistently identifies functionally-equivalent orthologs across a range of taxonomic distances. I also further developed Ortholuge’s functionality by reducing run-time, increasing accuracy and improving usability through a number of modifications. Lastly, to make Ortholuge results available to the research community, I developed a database of Ortholuge ortholog predictions for bacteria and archaea species. This online database provides high-level visualization of orthologs and the ability to easily run complex queries to retrieve genes that are shared or unique between specified taxa. Overall, this work contributes an enhanced method for precise high-throughput ortholog identification and increases our understanding of the functional equivalences between orthologs.

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

The evolution of mitochondrial genome structure and function in insects

Date created: 
2005
Abstract: 

The mitochondrial genomes (mt-genomes) of animals are very compact in structure, encoding thirteen protein genes involved in the production of ATP, and the key components of the translation system to express these proteins. The mitochondrial expression system, which functions separately from that of the nucleus, shows characteristics of both prokaryotic and eukaryotic expression systems, and has diverged greatly from that currently observed in the closest living relatives of mitochondria, the a-proteobacteria. Current understanding of transcript maturation is that large multi-gene transcripts are processed by the removal of intervening tRNA genes, leaving behind RNA templates to be matured into the functional mRNAs and rRNAs. One of the most striking features of insect mt-genomes has been the apparent replacement of a start codon with a stop codon for the essential mitochondrial gene cytochrome c oxidase subunit 1 (coxl). When first observed in Drosophila, Clary and Wolstenholme proposed a highly unusual four-base "ATAA" start codon. With the expanded sampling of mitochondrial sequence across the various insect orders, the data does not support the use of this aberrant initiation for coxl. At the initiation of this study, the diversity of insect groups represented by complete mt-genome sequence was very poor. To address this deficit, I undertook sequencing projects to increase the number of insect orders represented in the mitochondrial sequence databases. I report the complete mt-genome sequences for two insects, the spittlebug Philaenus spumarius, and the giant stonefly Pteronarcys princeps. The sequences are annotated and compared to other insect mt-genomes in the sequence databases. I report the cDNA sequences of Drosophila melanogaster mitochondrial mRNAs, rRNA subunits, and a population of pre-mRNA molecules that are intermediates of the RNA processing system. Models to explain mitochondrial transcript maturation in light of these new observations are proposed. Comparative analyses were undertaken to apply the information gained from the mitochondrial transcripts of D. melanogaster to the mitochondrial structure and annotations of mt-genomes from the other insects. These analyses suggest a 5' specific modification to the tRNA punctuation model for insect mitochondria. This modification may represent a further evolutionary simplification of the mitochondrial expression system.

Document type: 
Thesis
File(s): 
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (Ph.D.)

Investigation of the dimer interface of CTP:phosphocholine cytidylyltransferase and its modulation by lipids

Author: 
Date created: 
2005
Abstract: 

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme in phosphatidylcholine synthesis. CCT is an amphipathic homodimer, whose activity is regulated by membrane binding - a process resulting in conformational restructuring of the dimer interface. I used site-directed mutagenesis of domain N lysines in CCT, followed by lysine specific chemical cross-linking in the presence and absence of activating lipids, to locate sites of dimer interactions and to pinpoint where conformational rearrangement occurs. Analysis revealed that none of the lysines in the N-terminus are required for cross-linking. Thus lysinemediated cross-links involve other CCT domains. I found that CCT 236, despite lacking a membrane-binding domain, appears to undergo conformational changes that disrupt the dimer interface in the presence of activating lipids. The mechanism causing lipid-induced rearrangements of both CCT 367 and CCT 236 dimer interfaces remains unknown, but appear to be different. Moreover, the two CCTs are activated by different lipids suggesting different activation mechanisms.

Document type: 
Thesis
File(s): 
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)

Developing broodstock of Arctic Charr (Salvelinus alpinus L.)

Author: 
Date created: 
2004
Abstract: 

Most of the economically important traits in animal breeding programs are quantitative in nature. Detecting major genes and/or blocks of genes influencing these traits has been made possible by the availability of hypervariable DNA markers. In this study, phenotypic variations related to growth and body girth in the two domesticated strains of Arctic char (Salvelinus alpinus L.) at Icy Waters Ltd. (Whitehorse, Yukon, Canada) were examined and then quantitative trait loci for growth were identified using a genome wide scan approach. Twelve crosses involving the pure strains (Tree River and Yukon GoldTM), the reciprocal hybrids, and the reciprocal backcrosses were set up with ten families per cross. After 18 months of rearing in the hatchery environment under identical culture conditions, it was observed that backcrosses with a 75% Tree River genome contribution ((YGfxTRm)fxTRm) grew fastest and possessed greatest variance. A total of 198 highly polymorphic microsatellite markers, from various salmonid species, covering 41 linkage groups on the current Arctic charr linkage map were tested for a genome scan. Sixty two highly polymorphic markers were chosen to perform a genome wide scan on a hll-sib backcross family, namely 6-1 0, to detect genetic factors responsible for the variation of growth in Arctic charr. These markers cover 28 of the 46 linkage groups in the currently available, low-resolution genetic map of Arctic charr. Results from a transmission disequilibrium test (TDT) indicate a significant association (0.001

Document type: 
Thesis
File(s): 
Department: 
Department of Molecular Biology and Biochemistry - Simon Fraser University
Thesis type: 
Thesis (M.Sc.)