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

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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): 
Senior supervisor: 
Nancy Hawkins
Department: 
Science:
Thesis type: 
(Thesis) M.Sc.

The organization, regulation and functions of genes in the pericentric heterochromatin of the Drosophila melanogaster third chromosome

Date created: 
2013-11-22
Abstract: 

Alterations to chromatin through the post-translational modification of histone proteins or the binding of non-histone proteins is important for the tight control and specification of nuclear processes (e.g. transcription) occurring at most chromosomal regions. In this thesis, I report on the roles of enzymes responsible for adding methyl groups to histone H3 at lysine 4 (H3K4)—a modification pattern which is found at most active genes. I focus on a functional characterization of dSet1, which I show is responsible for most di- and trimethylation of H3K4. I also present evidence that dSet1 interacts with members of an evolutionarily conserved complex and that complex members are collectively required for H3K4 methylation. This work establishes a model system for better understanding functions of methyl-H3K4 in metazoans. Given that the dSet1 gene was found in pericentric heterochromatin, I became interested in the regulation, functions and organization of other genes in this specialized genomic compartment. Pericentric heterochromatin encompasses approximately 1/3 of the Drosophila melanogaster DNA sequences, yet contains just over 1% of Drosophila genes. Since pericentric heterochromatin has a low gene density, remains condensed throughout the cell cycle, and ‘silences’ most genes ectopically placed within it, it has in the past been mistakenly regarded as transcriptionally inert. However, there are still approximately 200 transcribed genes present in pericentric regions, and many of these genes are essential and/or expressed ubiquitously throughout development. Here I report a broad requirement for structural components of heterochromatin (Su(var) proteins) in the expression of genes in Drosophila pericentric heterochromatin, expanding on previous work reporting that Su(var) proteins and a heterochromatic environment are required for the expression of genes in this region. Due to difficulties arising in sequencing, mapping and genetically manipulating heterochromatic regions, much work remains in order to fully characterize the heterochromatic genome. I have assisted in these efforts by identifying and characterizing essential functions in 3rd chromosome pericentric heterochromatin by sequencing genetic mutants and through gene-targeting using RNA interference (RNAi). Together my data will contribute to collaborative efforts to obtain a basic understanding of the regulation, organization and functions of genes in pericentric heterochromatin.

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

Identification, Evolution, and Expression of the Trace Amine-Associated Receptor (TAAR) Gene Family in Atlantic Salmon (Salmo salar)

Date created: 
2014-09-12
Abstract: 

It is widely hypothesized that Atlantic salmon are imprinted at a young age with olfactory cues, which they use as a guide in order to return to their natal streams to spawn. However, the molecular mechanism(s) behind this biological phenomenon remain unknown. Therefore, in order to better understand imprinting and homing in Atlantic salmon, it is important to characterize the repertoire of olfactory receptors in thisspecies. A search of the first assembly of the Atlantic salmon genome revealed 27 putatively functional trace amine-associated receptor (TAAR) genes and 25 putative TAAR pseudo-genes. Genetic mapping, phylogenetic analysis, binding-site prediction, and quantitative PCR were performed using the Atlantic salmon TAAR genes. The identification of this gene family in Atlantic salmon will facilitate additional studiesinvolving olfaction and homing such as determining the range of allelic variation in olfactory receptors genes of different salmon populations.

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

Enabling systems-level analyses of the host response to infectious diseases in bovine and other mammalian species

Date created: 
2014-09-29
Abstract: 

The innate immune response is a critical branch of immunity, providing a first line of defense against pathogens and shaping subsequent adaptive immune responses. The complexity of this system necessitates the application of systems-level approaches. InnateDB is an integrated web-accessible database and systems biology platform being developed to facilitate the systems level analysis of innate immunity pathways and networks. One of the aims of this thesis was to enhance InnateDB with bovine data, thereby providing a resource for investigation of this agriculturally important model organism. Using an orthology based approach, over 70% of InnateDB’s human protein-protein interactions (PPIs), and a similar fraction of human pathways were reconstructed in cow and integrated into InnateDB. Pathway analysis, the statistical association of observations at the molecular level with processes at a more systems level, plays a crucial role in the interpretation of high-throughput experimental datasets. A widely neglected challenge in pathway analysis relates to the handling of multifunctional genes. I therefore developed SIGORA, a novel pathway analysis method that identifies genes and gene-pairs that are unique signatures of a pathway and examines their over-representation in a given list of genes of interest (e.g. the list of differentially expressed genes in an infectious condition). With several biological datasets, SIGORA outperformed traditional methods, delivering biologically more plausible and relevant results. This was also reflected in significantly lower false positive rates for simulated datasets. An additional challenge in high-throughput dataset interpretation concerns the lack of functional annotation for many genes. The guilt by association (GBA) principle was applied in a conservative manner to a large tissue expression dataset (105 Tissues, 13000 genes) to infer gene functions from co-expression data. Overall, 180 previously un-annotated bovine genes were assigned a putative function by this approach. In 20% of the cases, the inferred function was additionally supported by literature in other species. microRNAs are emerging as important innate immune response regulators and as biomarkers of disease. Determining microRNA functions requires the identification of their targets, yet computational prediction of such targets is challenging. As part of a group investigating microRNA roles in bovine mastitis, I used a combination of prediction tools to compile a list of likely targets. Here, the overall emerging picture (including pathway enrichment) is consistent with our current understanding of this condition. Collectively this work provides new tools and insights that may more broadly be used to improve systems-based analysis of bovine and other mammalian responses.

Document type: 
Thesis
Senior supervisor: 
Fiona Brinkman
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Genomic Analysis of Infectious Agents, Mutations and Immune Cells Associated with Cancer

Date created: 
2014-09-02
Abstract: 

Cancer is primarily a genetic disease that can be actuated by carcinogens, such as infectious agents, and attenuated by the body’s own immune response. This thesis is a multifaceted study of cancer genomics, metagenomics and immunogenomics. In our metagenomic study of colorectal cancer, we used RNA-seq followed by host sequence subtractions and found marked over-representation of Fusobacterium nucleatum sequences in tumours. We verified the overabundance of Fusobacterium sequences by quantitative PCR analysis from a total of 99 subjects, and we observed a positive association with lymph node metastasis. In our cancer genomics study of ovarian cancer, the clonal structure and evolution of tumours were profiled by whole exome sequencing of serial tumour samples in three patients. Tumours from all three patients harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight novel pathways that are mutated in ovarian cancer and shows that recurrent disease arises from multiple clones present in the primary tumour. In our immunogenomics study of ovarian cancer immunity, T and B cell clonality was surveyed by high-throughput sequencing of their antigen receptor. First, we characterized the errors in receptor sequencing and developed filtering strategies to reduce the false discovery rate. Errors were discovered in the form of substitutions and chimeras and additionally, sequence contamination and biases were observed between samples. We applied our error filtering model to survey the T and B cells in serial ovarian tumour samples and found that the tumour-associated immune repertoires diverged over time. Furthermore, we discovered that tumour-responsive lymphocytes can be recognized by in vitro expansions of T cells and by discovering B cells with highly mutated antigen receptor sequences. In conclusion, genomics approaches were employed first to study colorectal cancer, which revealed a tumour-associated bacteria. Secondly, genomics was used to study ovarian cancer tumours, which showed tumour clonal evolution, alterations in novel biological pathways and a dynamic adaptive immune response.

Document type: 
Thesis
File(s): 
Senior supervisor: 
Robert Holt
Department: 
Science:
Thesis type: 
(Thesis) Ph.D.

Structural and kinetic analysis of Escherichia coli signal peptide peptidase A

Date created: 
2013-06-19
Abstract: 

Secretory proteins contain a signal peptide at their N-terminus. The signal peptide functions to guide proteins to the membrane and is cleaved off by signal peptidase. The remnant signal peptides must be removed from the membrane to prevent their accumulation which can lead to membrane destabilization. Escherichia coli signal peptide peptidase A (SppAEC) has been identified as a major enzyme that processes the remnant signal peptide to smaller fragments. SppAEC, however, had remained uncharacterized such that the structure, catalytic mechanism, and substrate preference were unknown. I have cloned, overexpressed, purified and crystallized an active soluble domain of SppAEC. We have determined the structure of SppAEC by X-ray crystallography, which revealed that: 1) SppAEC has two similar alpha-beta domains (despite limited sequence identity), 2) SppAEC forms a tetramer that results in a dome shaped structure with a hydrophobic interior and hydrophilic exterior, 3) the four active sites within the cavity each utilize a Ser-Lys dyad where the general base (Lys209) arrives from the N-terminal domain and the nucleophile (Ser409) from the C-terminal domain. I have further characterized SppAEC using steady-state kinetics and cocrystallization. Using a series of fluorometric peptide substrates, I discovered that leucine is the most preferred residue at the P1 substrate position. I cocrystallized an SppAEC active site mutant (K209A), in complex with the substrate Z-LLL-MCA. The electron density within the active site of the 1.95 Å resolution structure is consistent with the carbonyl carbon of the substrate’s C-terminal leucine being covalently linked via an ester bond to the Ser409 O, thus revealing an acyl-enzyme complex in SppAEC. This is direct evidence that Ser409 O serves as a nucleophile in the SppAEC catalyzed reaction and confirms the identity of the S1 and S3 substrate specificity pockets. Lastly, I measured the pH dependence of both WT and S431A enzymes (Ser431 Ois hydrogen-bonded to Lys209 Nζ). The pH-rate profiles are consistent with S431 playing a role in lowering the pKa of the lysine general base, which would be critical for activity at physiological pH. We propose that the active site architecture of SppAEC may be best described as a Ser-Lys-Ser triad.

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

Refolding Recombinant Escherichia coli BamA: the integral membrane component of the BAM (Beta-barrel Assembly Machinery) complex

Author: 
Date created: 
2014-07-23
Abstract: 

The selective permeability of the outer membrane (OM) of Gram-negative bacteria is mainly determined by the pore-forming proteins within it, which are called outer membrane proteins (OMPs). The proteinaceous apparatus required to fold and assemble the OMPs is known as the beta-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is made up of five proteins. BamA is a beta-barrel protein integrated into the OM, while BamB, BamC, BamD and BamE are lipoproteins attached to the periplasmic side of the OM by a covalently bound lipid. Over-expression of BamA without its signal peptide results in insoluble inclusion body formation. I have investigated refolding strategies for E. coli BamA. The refolded BamA has been analysed by size-exclusive chromatography, heat-modifiability, circular dichroism spectroscopy and limited proteolysis. The experimental results are consistent with both a full length BamA and a truncated BamA construct being refolded properly using my discovered conditions. Both refolded constructs are able to crystallize. These initial crystallization conditions will be helpful in the structural analysis of E. coli BamA.

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

The functional characterization of the gene coding for O-linked β-N-acetylglucosaminidase (OGA) in Drosophila melanogaster

Date created: 
2014-08-15
Abstract: 

O-linked β-N-acetylglucosaminidase (OGA) is the enzyme responsible for removing the O-linked β-N-acetylglucosamine (O-GlcNAc) modification from serine and threonine residues of a variety of proteins, while its addition to protein targets is catalyzed by O-linked β-N-acetylglucosamine transferase (OGT). sxc/Ogt is essential in Drosophila melanogaster; however, it is unknown whether Oga is also essential in flies. I found that, in flies, a significant decrease in Oga transcript induced by RNAi knockdown is not lethal and that a nonsense mutation that putatively results in the translation of a C-terminally truncated version of OGA is viable when crossed to a deficiency known to span the Oga locus in the genome; however, reduced viability was observed when ubiquitously overexpressing two copies of Oga cDNA. Reduced expression of Oga and Ogt in Drosophila insulin-producing cells, via targeted RNAi expression with a dILP2-GAL4 driver, results in a slight increase and decrease, respectively, in male body weight.

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

A search for new players in the signalling pathways regulating dorsal closure of the Drosophila embryo

Author: 
Date created: 
2014-08-14
Abstract: 

Dorsal closure in Drosophila is a widely used model system to study developmental epithelial fusions and wound healing, and had been shown to require coordinated cell morphogenesis and reciprocal communication between different tissue types. In this thesis, I focused on identifying new components of the signalling pathways regulating dorsal closure. First, I extended the characterization of Epidermal Growth Factor Receptor (EGFR) signalling, which is an important regulator of dorsal closure, and established new leads in the search for components upstream and downstream of EGFR during dorsal closure. I have also identified the ligand Folded gastrulation (Fog) as a candidate upstream regulator of signalling during dorsal closure. Fog appears to regulate the transcript levels of decapentaplegic and zipper, two important genes known to participate in dorsal closure. Finally, I initiated studies to explore if regulators of cell adhesion at the neuromuscular junction are conserved regulators of cell adhesion during dorsal closure.

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

Transcriptional regulation of the ciliopathy gene MKS1/mks-1

Author: 
Date created: 
2014-06-12
Abstract: 

Meckel Grubber Syndrome (MKS) is a severe ciliopathy. The first identified causal loci of MKS resided in the gene MKS1. The only known transcription factor of MKS1 is Regulatory Factor X (RFX). C. elegans, an organism with well-characterized ciliated neurons, is ideal for studying ciliary genes. Many ciliary genes including MKS1 are conserved in C. elegans. My study aims to find candidate mutants of transcription factor(s) for mks-1, the C. elegans ortholog of MKS1. In order to track in vivo mks-1 expression in C. elegans, I generated a transgenic strain that expressed Green Fluorescence Protein (GFP) driven by mks-1 promoter. I carried out genetic screens to identify mutations that altered GFP fluorescence intensity and expression profile, as a way to search for potential transcription factor(s) for mks-1 in C. elegans. I successfully found an X-linked recessive mutant, which suppresses mks-1 reporter gene expression in subset of labial neurons. The mutant has normal labial neurons development. This research has set up a solid stage for studying the transcriptional regulation of mks-1 and other ciliary genes.

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