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

Receive updates for this collection

Regulation of the kinesin-3 motor, KIF1A, in a cellular model of Alzheimer’s disease

Author: 
Date created: 
2016-01-07
Abstract: 

Neurons are morphologically unique cells that rely on axonal transport for their function and viability. Amyloid-β oligomers (AβOs), a neurotoxin in Alzheimer’s disease (AD), disrupt axonal transport via dysregulation of signaling cascades. I assessed the role for glycogen synthase kinase 3β (GSK3β), a kinase implicated in AD, in the direct regulation of the kinesin KIF1A. Inhibition of GSK3β prevented transport defects in AβO-treated primary neurons, and co-immunoprecipitation studies confirmed an interaction between KIF1A and GSK3β. Mass spectrometry on KIF1A isolated from AD transgenic mouse brain (Tg2576) showed that within a regulatory domain, Ser 402 is phosphorylated and conforms to a GSK3β recognition site. The transport of a phospho-resistant (S402A) form of KIF1A was unaffected in AβO-treated neurons whereas KIF1A (S402E) transport is severely reduced. These data suggest that AβOs impair transport via GSK3β acting directly on KIF1A. Ultimately, this work may identify novel mechanisms of KIF1A regulation in AD.

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

Dysregulation of neuronal calcium signaling impairs axonal transport independent of tau in a model of Alzheimer's disease

Date created: 
2014-10-30
Abstract: 

Neurons rely on microtubule-based, fast axonal transport of proteins and organelles for development, communication and survival. FAT impairment precedes overt cellular toxicity in multiple neurodegenerative diseases, including Alzheimer’s disease (AD). Intracellular Ca2+ dysregulation is also widely implicated in early AD pathogenesis; however, its role in transport impairment is unknown. Our lab was first to demonstrate that soluble amyloid-β oligomers (AβOs), proximal neurotoxins in AD, impair vesicular transport of axonal brain-derived neurotrophic factor (BDNF). Contrary to a central paradigm, I show that BDNF transport is blocked independent of the microtubule-associated protein, tau, microtubule destabilization, and acute cell death. Significantly, BDNF transport is impaired by non-excitotoxic activation of calcineurin (CaN), a Ca2+-dependent phosphatase. Based on these findings, I investigated Ca2+-dependent mechanisms that underlie the spatiotemporal progression of AβO-induced transport defects and dysregulate KIF1A, the primary kinesin motor required for BDNF transport. Because CaN and its effectors, protein phosphatase-1 (PP1) and glycogen synthase kinase 3β (GSK3β), are present in both dendrites and axons, I investigated if postsynaptic AβO binding impairs dendritic transport prior to FAT disruption. AβOs induce dendritic and axonal BDNF transport defects simultaneously; however, maximal dendritic transport defects are observed prior to maximal impairment of FAT. I correlated the spatiotemporal progression of transport defects with Ca2+ elevation and CaN activation in dendrites and subsequently in axons. Postsynaptic CaN activation converges on axonal Ca2+ dysregulation to impair FAT. Specifically, AβOs colocalize with axonal VGCCs, and blocking VGCCs prevents FAT defects. Finally, BDNF transport defects are prevented by dantrolene, a compound that reduces Ca2+-induced- Ca2+ release through ryanodine receptors in axonal and dendritic ER membranes. Together, these mechanisms activate CaN-PP1-GSK3β signaling and lead to inhibitory phosphorylation of KIF1A at a highly conserved consensus site within its dimerization domain. Collectively, this thesis establishes novel roles for Ca2+ dysregulation in BDNF transport disruption and tau-independent toxicity during early AD pathogenesis.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Michael Silverman
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

The ‘who, where and when’ of neurogenesis during Drosophila melanogaster eye development

Date created: 
2014-10-30
Abstract: 

The development of structurally diverse and functionally complex multicellular organs is dependent on coupling the generation of cellular heterogeneity with structural organization. My thesis exploits the retina of the genetically tractable Drosophila melanogaster to address how organs develop from naïve tissues. During retinal development a wave of apical constriction, known as the morphogenetic furrow (MF), advances across the eye-disc epithelium as the leading front of photoreceptor (PR) differentiation. My thesis addresses three aspects of organogenesis from the perspective of neurogenesis: (1) ‘Who’ will develop as a PR? (2) ‘Where’ will neurogenesis initiate? (3) ‘When’ will neurogenesis occur? ‘Who?’ Neurogenesis begins with proneural factor expression, which assigns neural competence to naïve cells; this is coupled to MF progression in the retina. The Notch pathway refines neural competence to single evenly spaced PRs, in a process termed lateral inhibition. I identified nemo (nmo) as a target of proneural factors and show that Nmo promotes Notch-mediated lateral inhibition, thus contributing to the selection of single PRs and other neural cell types. ‘Where?’ A prerequisite to the initial onset of PR neurogenesis is regional-fate competence. Antennal/head fate selectors compete with and mutually antagonize eye-fate selectors to subdivide the tissue. Overlaid onto this are signalling pathways, which cooperate to promote MF initiation and neurogenesis. I find that compromising one of these pathways, the Ras/MEK/MAPK cascade, affects where neurogenesis initiates by regulating both regional-fate specification and signal transduction. This is achieved via factors that control extracellular ligand diffusion. ‘When?’ Although signalling pathways regulate gene expression to promote MF progression and neurogenesis, how this translates into epithelial morphogenesis is not known. I find that the MF’s pace is sensitive to integrin adhesion receptor levels, and that integrins are genetically downstream of the signalling events driving MF progression. Integrins within the MF stabilize microtubules to promote apical constriction and MF progression, thus genetically linking signalling events with cytoskeletal remodeling events necessary for morphogenesis and neurogenesis. My thesis supports the notion that cellular diversity follows from the interplay between signal transduction and cell competence, and that tissue structure is a consequence of signal transduction-regulated cell adhesion and cytoskeletal remodeling.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Esther Verheyen
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Comparative Analysis of ABC Transporter Genes in Pathogenic and Non-pathogenic Nematodes

Author: 
Date created: 
2015-11-20
Abstract: 

The ATP-binding cassette (ABC) transporter gene superfamily is a large protein family with diverse physiological functions in different organisms. Recent genome sequencing projects have reported expansion of ABC transporter gene family in parasitic nematodes and hypothesized that such expansion may enable the parasites to become pathogenic or have increased virulence. Some of these reported expansions may reflect the completeness of sequenced genomes, use of bioinformatics programs, and parameters and criteria used in these projects. The goal of this thesis research is to develop a robust bioinformatics pipeline for annotating high-quality ABC transporter genes so that we can reduce the contribution of technical errors. Our comparative analysis of 29 nematode genomes suggests that pathogenic nematodes generally contain fewer ABC transporter genes than non-pathogenic nematodes, suggesting that expansion in ABC superfamily may not be a mechanism for pathogenic nematodes to survive in their host environment. However, many pathogenic nematodes have genome-specific ABC transporter genes.

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

Computational and molecular dissection of an X-box cis-Regulatory module

Date created: 
2015-10-06
Abstract: 

Ciliopathies are a class of human diseases marked by dysfunction of the cellular organelle, cilia. While many of the molecular components that make up cilia have been identified and studied, comparatively little is understood about the transcriptional regulation of genes encoding these components. The conserved transcription factor Regulatory Factor X (RFX)/DAF-19, which acts through binding to the cis-regulatory motif known as X-box, has been shown to regulate ciliary genes in many animals from Caenorhabditis elegans to humans. However, accumulating evidence suggests that RFX is unable to initiate transcription on its own. Therefore, other factors and cis-regulatory elements are likely required. One such element, a DNA motif called the C-box, has recently been identified in C. elegans. It is still unclear if the X-box and C-boxes are the only regulatory elements involved and how they interact. To this end, I analyzed the transcriptional regulation of dyf-5, the C. elegans ortholog of the human ciliopathy gene Male-Associated Kinase (MAK). Using computational methods, I was able to confirm the presence of the previously reported X-box and C-boxes as well as identifying an additional C-box. By sequentially mutating each of the identified motifs, I identified the role each potential motif plays in transcriptional regulation of dyf-5. My results showed that only the X-box and the three C-boxes are necessary and are sufficient to drive transcription, with the X-box and the centre C-box being the major contributors and the other two C-boxes enhancing expression. This study advances the knowledge of gene regulation in general and will further our understanding of ciliopathies and the mutations that cause them.

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

Towards a Prebiotically Plausible Mechanism for the Emergence of RNA, Ribozymes and the RNA World

Author: 
Date created: 
2015-08-25
Abstract: 

Available evidence, both in vitro and in vivo, attests to the descent of life from the RNA World; however, the prebiotic genesis of such RNA life remains ambiguous. How did The RNA World emerge from the abiotic chemistry on the Archean Earth? Montmorillonite clays have been shown to catalyze the polymerization of activated nucleotides (eg. adenosine 5′ phosphorimidazolide) into RNA, but polymerization has not previously been demonstrated for a prebiotically plausible nucleotide such as cyclic 2′, 3′-adenosine monophosphate (A>p). I reacted A>p in the presence of montmorillonite clay and could detect RNA polymers up to 5 nucleotides in length using a combination of HPLC, enzymatic labeling and mass spectrometry. This chemistry was found to be sensitive to pH and temperature. Reactions at pH 6 were found to produce more polymerization products than reactions at pH 7 or 8. Similarly A>p was found to be very stable at pH 6 with a hydrolysis rate at 25ºC of 6.2 x 10-9 sec-1 (t1/2 = 3.5 years) and at 4ºC of 4.4 x 10-10 sec-1 (t1/2 = 50 years). Also discussed are the requirements for the transition from abiotic chemistry to life. Important considerations for the emergence of replicating RNA networks from randomly synthesized RNA are outlined. Finally, the progress towards recreating the RNA world in the laboratory is summarized.

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

Functional Divergence of Photolyase and Cryptochrome-DASH: The Role of Loop Dynamics

Date created: 
2015-09-11
Abstract: 

Photolyase (PL) is an enzyme that repairs thymine dimers, a form of DNA damage caused by UV light. To do so, the damaged bases of the cyclobutane pyrimidine dimer (CPD) are displaced from the duplex into an extrahelical position. A “recognition loop” in PL must be displaced to allow it to bind substrate. Cryptochrome-DASH (CRYD) is a structurally homologous protein to PL, with a high sequence identity, which cannot perform the same physiological function as PL. Here I ask how PL is able to bind to its substrate and how CRYD functionally diverged from PL. I hypothesize that there exist optimized conformational dynamics of these recognition loop regions that allow PL to bind CPD. Limited proteolysis experiments determined that the recognition loops of PL and CRYD are the most dynamic regions of each of these proteins. Furthermore, the conformational dynamics of the CRYD recognition loop are greater than that of PL. A difference between the dynamics is consistent with my hypothesis, but does not definitively prove it. The differences in recognition loop conformational dynamics could be due to primary, secondary or tertiary structures. To determine the impact sequence has on these conformational dynamics, end-to-end contact was measured in isolated recognition loop peptides using fluorescence quenching. It was found that the CRYD recognition loop peptide is more dynamic than that of PL. This indicates that sequence, at least in part, is responsible for conformational dynamic differences in these protein loops. To definitively determine the role the recognition loop plays in how PL and CRYD functionally diverged, I outlined a series of directed evolution experiments. To this end random mutagenesis was planned to be carried out only in the PL recognition loop region in search for partially functional PLs. If a correlation exists between conformational dynamics and function, then the recognition loop would be implicated in the functional divergence of these proteins. Technical development, including random mutagenic PCR, a mutation rate detection assay, in vivo functional assays and in vitro functional assay was undertaken to test this hypothesis.

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

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.