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

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Applying metagenomics analysis towards a better understanding of freshwater microbial communities

Date created: 
2017-06-15
Abstract: 

Microbial communities may now be studied in more detail using culture-independent methods such as metagenomics (directly analyzing genomes from an environmental sample). One of the many potential applications of metagenomics is in the assessment of water quality. Current methods for detection of fecal pollution in water rely on culture-based microbial testing which is slow and can lack sensitivity and specificity. For the WatershedDiscovery.ca project, it was hypothesized that a molecular-based test, developed based on metagenomics analysis, may be more rapid, and accurate for characterizing fecal pollution. Many bioinformatics methods for metagenomics sequence classification have been developed, but when initiating this research, no comprehensive evaluation of method accuracy had yet been published. Thus, using both in silico and in vitro simulated communities, a comprehensive evaluation of metagenomics taxonomic sequence classification methods was performed. Utilizing knowledge gained from this comparative evaluation, a study was undertaken of microbial community dynamics in monthly water samples from sites in urban, protected, and agricultural watersheds collected over a one-year period. Freshwater samples collected from sites affected by agricultural activity showed distinct microbial profiles versus samples collected from unaffected sites, and a notable presence of Legionella was discovered in all watershed sampling sites (the largest study of Legionella in watersheds to date). Furthermore, biomarkers were developed that could distinguish agriculturally affected samples from pristine samples collected in our watershed study. Finally, there is a lack of methods for the prediction of subcellular localization (SCL) from metagenomics sequences—of interest for the identification of cell surface/secreted proteins for development of ELISA-based diagnostics and other applications. Thus, PSORTb, a precise bacterial and archaeal SCL program, was modified to enable the classification of metagenomics sequences, and applied to the analysis of the watershed samples. A database of protein SCL associated with PSORTb was expanded to make it suitable for a wider diversity of microbes, particularly those with atypical cell envelopes. Collectively this work expands our understanding of metagenomics software accuracy, and available analysis tools, and provides insight into freshwater microbial community dynamics, with potential application in water quality test development.

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

Dissecting the sensory roles of motility-associated ciliary genes in Caenorhabditis elegans

Date created: 
2016-08-30
Abstract: 

Cilia are microtubule-based organelles that emanate from the surface of most mammalian cell types. Motile cilia have well known roles in producing flow, while non-motile cilia play important sensory/signalling roles. Both forms are based on a similar axonemal structure, but ciliary motility requires additional components that conform to a regular arrangement along microtubules thought to be dictated by the protofilament ribbon (pf-ribbon). While pf-ribbon proteins have been implicated in ciliary motility, sensory/signalling functions in non-motile cilia have been less apparent. Although the ciliated organism Caenorhabditis elegans lacks motile cilia, orthologues of several ciliary pf-ribbon-associated proteins are present, including PACRG (Parkin co-regulated gene) and EFHC1 (EF-hand containing 1). In addition to their localisation to motile cilia, the pf-ribbon proteins show expression in neuronal cells of the brain where they may play important sensory roles. In particular, EFHC1 is mutated in the most common form of inherited epilepsy in humans and has been shown to be important for proper neuronal communication. This work investigates these motility-associated genes in C. elegans to dissect their sensory/signalling roles. We find that both PCRG-1 and EFHC-1 localise to a small subset of non-motile cilia in C. elegans, suggesting that they have been adapted to mediate specific sensory/signalling functions. We show that PCRG-1 influences a learning behaviour known as gustatory plasticity, where it is functionally coupled to heterotrimeric G-protein signalling. We also demonstrate that PCRG-1 promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signalling, and that EFHC-1 also promotes longevity, suggesting shared signalling functions for these proteins. In addition, EFHC-1 modulates dopamine signalling where it is required for ciliary mechanosensation and regulating synaptic release of dopamine in cooperation with a voltage-gated calcium channel. Our findings establish previously unrecognised sensory/signalling functions for both PACRG and EFHC1 that may be important for neuronal communication in the human brain, where both proteins are known to be present. Furthermore, our work provides important clues for understanding and ultimately providing novel avenues for intervention of disorders such as epilepsy.

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

Localization and assembly of the Vibrio cholerae type IV pilus secretin channel

Date created: 
2017-04-12
Abstract: 

Type 4 pili (T4P) are filamentous structures found on the surfaces of many Gram-negative bacteria, including Vibrio cholerae. The V. cholerae T4P are the toxin-coregulated pili (TCP), which mediate bacterial aggregation and exoprotein secretion, critical functions in colonization of the human intestine to cause the diarrheal disease cholera. TCP assemble at the inner membrane (IM), grow through a multiprotein conduit in the periplasm and through a secretin channel in the outer membrane (OM). The multimeric secretin channel is formed by secretin subunits, which are translocated across the IM by the Sec apparatus, and in most T4P systems, are transported to the OM with the help of a lipoprotein co-chaperone. In the V. cholerae TCP the secretin subunit itself, TcpC, is a lipoprotein, and its putative co-chaperone, TcpQ, is non-lipidated. Here we use mutagenesis, cellular fractionation and functional assays to investigate secretin channel assembly in V. cholerae. TcpC must be co-expressed with TcpQ to complement a ΔtcpC mutant in an assay of pilus functions, but the reciprocal is not true. TcpQ is necessary for pilus assembly but not for localization of TcpC to the outer membrane, demonstrating that TcpQ is not a co-chaperone for TcpC. The periplasmic domain of TcpC can be expressed on its own, localizes to the outer membrane, and localizes TcpQ at the outer membrane as well, provided TcpC is lipidated. When the periplasmic domain of TcpC is unlipidated it gets degraded and TcpQ accumulates in the periplasm, suggesting that the periplasmic domain of TcpC interacts with TcpQ and localizes it to the outer membrane via its lipid moiety. Our results lead to a model whereby TcpC Cys1 is lipidated at the IM by the Lgt machinery and transported to the OM in complex with TcpQ. TcpC inserts into the OM at two points: via its C-terminal portion, which forms a β-barrel channel with TcpC C-terminal domains of other secretin subunits, and via lipid moiety in its N-terminal domain, which interacts with TcpQ to link the OM channel to periplasmic pilus conduit.

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

Candidate protein interactors of sexually dimorphic on the Y chromosome (SdY) in Atlantic salmon

Author: 
Date created: 
2017-01-20
Abstract: 

Atlantic salmon depend on genetic cues to determine whether an individual is male or female. A novel sex-determining gene, sexually dimorphic on the Y chromosome (sdY), is found exclusively in all salmonids. Unlike other sex-determining genes, sdY lacks a DNA-binding domain. Instead, it is a divergent, truncated form of interferon regulatory factor 9. As a recently discovered gene, little is known about sdY; how it is involved in sex-determination and what proteins interact with it. Identification of protein interactors was done through a variety of techniques including yeast two-hybridization, co-immunoprecipitation and histidine-tagged pull down assays. These assays identified several proteins: SdY itself, 40S ribosomal protein S16 and SA, isocitrate dehydrogenase, heat shock protein HSP 90-beta, and ras GTPase-activating-like protein IQGAP1, as well as creatine kinase, GDP-mannose-4,6-dehydratase, sodium/potassium-transporting ATPase subunit alpha-1, AP-1 complex subunit beta-1, and hydroxysteroid dehydrogenase (17-beta) 4. The yeast two-hybrid assay also identified 3’ UTR of annexin A7-like and transmembrane protein 91-like, most likely false positives. This broad range of candidates has led me to believe that SdY is involved either in the biosynthesis of testosterone or in the testosterone signalling pathway.

Document type: 
Thesis
Senior supervisor: 
William Davidson
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Myosin Light Chain Phosphatase regulates the transcription of Wingless target genes in Drosophila

Date created: 
2017-03-28
Abstract: 

Canonical Wnt, or Wingless (Wg) in Drosophila, is an evolutionarily well-conserved signalling pathway that is important for a wide range of processes, including cell fate determination, axis formation and stem cell renewal. Wg signalling primarily functions to regulate the cytosolic stability of the key effector β-catenin (Armadillo, Arm, in Drosophila). Arm promotes the transcription of Wg target genes but also is required for the formation of stable adherens junctions. Previously, the Verheyen lab identified the non-muscle myosin II regulator Myosin Light Chain Phosphatase (MLCP) as a putative regulator of Wg signalling. Here we find that reducing the expression MLCP components leads to the attenuation of Wg target gene expression. I present our evidence that MLCP knock down directly regulates Wg signal transduction and that this regulation is through Arm localization. Thus, our work supports mounting evidence of a regulatory relationship between the adherens junctions and the Wg signalling pathway.

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

HCN channel gating models: a re-evaluation based on how the voltage-sensing and cAMP-sensing domains regulate kinetics

Date created: 
2017-04-03
Abstract: 

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to rhythmic oscillations in the heart and brain. Upon membrane hyperpolarization, HCN channel pore opening is coupled to inward movement of the S4 helix within the transmembrane voltage sensing domain (VSD, helices S1-S4). The gating pathway is proposed to include an initial voltage-dependent VSD movement step followed by a voltage-independent pore movement step (a cyclic allosteric mechanism). Various other mechanisms influence open state stability: A cytosolic cyclic nucleotide-binding (CNB) fold destabilizes the open state when unliganded (an autoinhibition mechanism), whereas binding of the phospholipid PIP2 to the transmembrane domain stabilizes the open state. After pore opening, the channel undergoes a mode-shift, presumed to include lateral movement of S4 towards S2, forming a more stable open state. Despite the knowledge of open state stabilization mechanisms, it remains unclear how these mechanisms affect the kinetics of the gating pathway. Do these mechanisms apply equally strongly to channel thermodynamics and kinetics? Do they apply under a variety of cellular conditions? And do they regulate the VSD movement step, the pore movement step, or both? In this work I examined both the thermodynamics and kinetics of the activation and deactivation pathways in a variety of HCN channel derivatives. I used two-electrode voltage clamp to determine that while channel thermodynamics follow the predictions of the autoinhibition model, a channel with an unliganded CNB fold has faster activation than a channel with autoinhibition relieved by CNB fold deletion. I propose this fast activation is promoted by a “quickening conformation” of the intact CNB fold. The quickening conformation is independent of PIP2 in both autoinhibited and autoinhibition-free channels. I used voltage clamp fluorometry to determine the speed of a VSD movement during channel deactivation in relation to pore closure. The speed of this VSD movement did not limit the rate of the deactivation pathway at strong depolarizations and showed stronger voltage dependence than pore closure. The speed of this VSD movement was independent of both cAMP binding and mode shift. Together my results clarify the HCN gating mechanisms of cyclic allostery, autoinhibition, PIP2 potentiation and mode shift, and produce novel models of both HCN channel activation and deactivation.

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

Genomic identification and functional characterization of essential genes in Caenorhabditis elegans

Author: 
Date created: 
2016-10-18
Abstract: 

For over 30 years, researchers have taken advantage of genetic balancers and forward genetic screens to isolate lethal mutations, which have been studied to identify essential genes in C. elegans. Using traditional genetic methods, such as genetic mapping, complementation tests, and transgenic rescue assays, many essential genes have been successfully identified. However, to pinpoint a specific essential gene the involved experiments are usually labor intensive and time consuming. Nowadays, genetic methods combined with whole genome sequencing (WGS) and bioinformatics analysis provide an effective approach for the molecular identification of essential genes. In my thesis I successfully identified 64 new essential genes with 107 lethal mutations in genomic regions of C. elegans of around 14 Mb from Chromosome III(mid) and Chromosome V(left), by combining genetic mapping, Illumina sequencing, bioinformatics analyses, and experimental validation. Most of these genes have multiple recovered mutant alleles. Of these 64 genes 5 have new alleles identified, which had not been previously studied by RNA interference depletion. Furthermore, by investigating the locations of lethal missense mutations in essential genes, I have identified five novel protein functional domains. Functional characterization of the identified essential genes shows that most of them are enzymes, including helicases, tRNA synthetase, and kinases. There are also ribosomal proteins. Gene Ontology functional annotation also indicates that essential genes tend to execute enzyme and nucleic acid binding activities during fundamental processes, such as intracellular protein synthesis. Essential gene analysis shows that compared to non-essential genes, essential genes have fewer paralogs, and encode proteins that are in protein interaction hubs. Essential genes are also more abundantly and consistently expressed over all developmental stages than non-essential genes. All these essential genes traits in C. elegans are consistent with those of human disease genes. Unsurprisingly, most (90%) human orthologs of essential genes in this study are related to human diseases. Therefore, functional characterization of essential genes underlines their importance as proxies for understanding the biological functions of human disease genes.

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

Hip Hop: Hipk promotes tumourigenesis in Drosophila through JAK/STAT signaling

Date created: 
2015-11-24
Abstract: 

Signal transduction pathways are crucial for co-ordinated development and growth of multicellular organisms. Dysregulation and mutations of components in these pathways can often lead to tumourigenesis. Evolutionarily conserved Homeodomain-Interacting-Protein-Kinase (Hipk) is a strong growth regulator of many signal transduction pathways, and elevated levels of hipk lead to tumour-like masses. While many known regulators of Hipk exist, we attempted to identify novel phospho-regulators of Hipk activity. Here we present evidence that Hopscotch, a core tyrosine kinase of the JAK/STAT cascade, is a putative phospho-regulator of Hipk activity in multiple contexts. We show that modulation of Hipk expression levels modifies JAK/STAT activity in both wildtype and tumourous tissues. Finally, we show that Hipk interacts with the JAK/STAT transcriptional effector STAT92E. Thus, our work provides a role for Drosophila Hipk in tumourigenesis and regulation of the JAK/STAT cascade.

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

Comparative analysis of ciliary gene regulation in nematodes

Author: 
Date created: 
2016-09-08
Abstract: 

Cilia are highly-conserved organelles ubiquitously present in metazoans and some unicellular eukaryotes. In humans, ciliary defects result in a plethora of serious genetic diseases termed ciliopathies. Despite their diverse morphology and function, cilia are comprised of a core set of proteins, and many ciliary genes share similar but likely not identical regulation mechanisms. Our research aims to understand the variations in cis-regulatory elements in ciliary genes and the impact of such variations on transcriptional regulation. We hypothesize that cis-regulatory elements in different ciliary promoters are unique and that this uniqueness impacts the expression and function of ciliary genes. We focus on a particular cis-regulatory element, the X-box motif, which functions as the binding motif for RFX/DAF-19, a transcription factor that regulates ciliary gene expression. We identify and analyze X-box motifs for a set of 32 well-studied ciliary genes in C. elegans and their orthologs in 25 additional nematodes, including both free-living and parasitic species. My research consists of three modules. First, we curate ciliary gene orthologs using a combined approach, including homology-based gene finding and RNA-seq-based improvement. The primary goal of this step is to ensure that the 5' ends of the genes are accurately defined in order to properly locate ciliary promoters. Second, we search for putative X-box motifs in these promoters using computational tools to identify motifs that resemble the consensus. For the promoters from which consensus X-box motifs are not found, we will search for X-box motifs that may show more differences from the consensus using frequency matrix-based search and regular expressions, which we call "atypical" X-box motifs. Third, we analyze the putative atypical X-box motifs, focusing on their sequence similarities, positions in promoter sequences, and flanking sequences, and compare them against the consensus X-box motifs. In this study, I will highlight progress made and challenges encountered for defining X-box motifs in ciliary genes.

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

Analysis of Genomic Islands and Other Features in Draft Versus Complete Bacterial Genomes

Date created: 
2016-08-19
Abstract: 

Short-read DNA sequencing technologies have revolutionized bacterial genomics, but these technologies have limitations. It is easy to produce a high quality draft genome, but relatively costly and/or time consuming to complete a genome, so most bacterial genomes remain as drafts. Despite this, limitations of draft bacterial genomes for functional analysis have not been well assessed. To characterize the importance of missing and poor quality regions of draft genomes, analyses of COG categories and genes of medical importance were performed using analogous draft and complete genomes. A popular genomic island prediction tool, IslandViewer, was updated to allow draft genomes as input, and its ability to detect genomic islands in draft genomes was assessed. There are limitations to bacterial draft genome analysis, with respect to disproportionately missing certain types of genes. However, valuable information of medical interest, including virulence and antimicrobial resistance genes, can still be obtained from some draft genome datasets.

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