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

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Quantifying trends in bacterial virulence and pathogen-associated genes through large scale bioinformatics analysis

Author: 
Date created: 
2007
Abstract: 

With an increasing number of bacterial genomes becoming available, we are now able to investigate and quantify selected general trends in pathogenicity shared across diverse pathogens which have been previously anecdotally reported but have not yet been quantified on a larger scale. In addition, we can perform more high-throughput approaches for the identification of virulence-associated genes that represent possible therapeutic or prophylactic targets. In this study, I systematically examined up to 267 pathogen and non-pathogen genomes from diverse genera, and identified trends associated with a curated data set of known bacterial virulence factors (VFs). I show, in support of previous anecdotal statements, that genomic islands (clusters of genes of probable horizontal origin) disproportionately do contain more VFs than the rest of a given genome (p < 2.20E-16), supporting their important role in pathogen evolution. To gain insights into the types of genes that may play a more virulence-specific role in pathogens, I also performed an analysis to identify pathogen-associated genes (genes found predominately in pathogens across multiple genera, but not found in non-pathogens). I found that disproportionately high numbers of pathogen-associated VFs are “offensive” (involved in active invasion of the host), such as certain types of toxins, as well as Type III and Type IV secretion systems. Some of the pathogen-specific genes identified have apparently not yet been examined for their potential as vaccine components or drug targets and merit further study. As the first step in the initiation of more sophisticated analyses of trends in virulence, I also developed a Virulence Gene Experiment Database (VGEDB) that incorporates contextual information about virulence. This database is unique in that entries are centered around describing a particular virulence gene experiment, rather than a virulence gene. I used this database in part to investigate a common BLAST-based approach for computationally identifying VFs in genomic sequences. My analysis suggests that this common VF-prediction method is very inaccurate. This work in general provides the first large-scale, multi-genera, quantitative data describing selected trends in bacterial virulence and provides global insights regarding pathogen evolution and pathogen-associated traits of primary importance in a pathogenic lifestyle.

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

Mechanisms of cyclic amp-dependent modulation in pacemaker channels

Author: 
Date created: 
2007
Abstract: 

Pacemaker (HCN) channels are hyperpolarization-activated and their activity is enhanced by cyclic AMP (cAMP), but whether cAMP-gating is coupled to voltage-gating remains unresolved. With long hyperpolarizations, HCN channels form a secondary open state after the initial opening; this briefly sustains the activation of open channels after a return to a resting voltage. CAMP effects on the formation and decay of the secondary open state remain unclear. We studied mouse HCN4 channels in excised oocyte membrane patches, and found cAMP increases sustained activation. We also studied a charge-reversing S4 mutation (K381E) and found it causes surprisingly little change in voltage-dependence. Notably, K381E dramatically increases the cAMP-dependence of sustained activation. Our results suggest cAMP and K381E synergistically enhance the formation of a secondary open state and that this state decays during deactivation through a voltage- and cAMP-dependent step. These results suggest a physical coupling of voltage-sensing and cAMP-modulatory machinery in HCN channels.

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

Strategies for the design of epitope targeting vaccines

Date created: 
2007
Abstract: 

There is need for vaccines that target production of antibodies (Ab) to conserved sites on highly-variable pathogens, such as HIV-1. Strategies targeting Ab production to such sites are called “epitope-targeting vaccines”, and this dissertation explores this theme by: i) using filamentous phage-based carriers for focusing Ab responses to peptides, and ii) developing a protein-prime, peptide-boost immunization strategy for targeting Ab production to a specific epitope on a protein. In Chapter 2, phage are compared as a carrier for a synthetic peptide to the classical carrier protein, ovalbumin (OVA). Both immunogens elicited strong anti-peptide Ab titers, with those elicited by OVA-peptide conjugate exceeding those produced by phage-peptide conjugate. Comparison of the anti-peptide and anti-carrier Ab responses showed that phage-peptide conjugate elicited a more focused anti-peptide Ab response than OVA-peptide conjugate. In Chapter 3, the dominant epitopes on phage are removed or altered. Engineered phage are tested as immunogens to determine if reduction of B-cell epitopes on the virion could further focus an Ab response against a peptide. One engineered phage variant (Δ3) produced lower Ab titers than wild type (WT) phage. A recombinant peptide displayed on the surface of the Δ3 phage immunogen produced a poor anti-peptide Ab responses compared to WT. In contrast, the Δ3 phage did focus an Ab response against a chemically-conjugated synthetic peptide compared to WT. These differences may be due to variation in copy number or inherent peptide immunogenicity. Chapter 4 tests if an Ab-specific peptide can selectively amplify an oligoclonal population of memory B-cells that were primed by an immunization with the polyclonal antigen hen egg lysozyme (HEL). This approach is enhanced by adding a T-cell epitope from OVA to the priming and boosting immunogens. A prime with HEL followed by a boost with phage bearing a HEL-specific peptide, produced a moderate increase in Abs that cross-react with HEL. However, this came at the expense of producing a de novo Ab response to non-targeted epitopes on the boosting immunogen. Taken together, these results suggest that, in some situations, moderate epitope targeting is possible; however, more studies are required to optimize this approach.

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

The cell biology of Nek8, defective in cystic kidney diseases

Author: 
Date created: 
2007
Abstract: 

Ciliopathies, which are caused by defects in cilia and basal bodies include the cystic kidney diseases. Mutations in the NIMA-related kinase, Nek8, are associated with the cystic kidney disease, nephronophthisis, and cause renal cysts in juvenile cystic kidney (jck) mice. In this study, I find that Nek8 localizes to cilia in cell culture and in developing mouse kidney. Specifically, Nek8 is expressed in cilia of epithelial cells lining the tubules of the uretic bud lineage. Cystogenic forms of Nek8 show differential ciliary localization, but do not affect ciliogenesis. In jck mouse kidney, ciliary Nek8 localization is reduced. These data indicate Nek8 functions within the cilium and defects in localization are correlated with renal cystogenesis.

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

Crystallographic analysis of the Sec-dependent secretion chaperone CsaA.

Author: 
Date created: 
2007
Abstract: 

The eubacterial protein CsaA has been proposed to act as a protein secretion chaperone in the Sec-dependent translocation pathway. Two structures of CsaA from Bacillus subtilis were solved by X-ray crystallography and refined to 1.9 and 2.0 Å resolution. Structural analysis revealed two potential substrate binding pockets on the surface of CsaA. These pockets display biochemical properties consistent with the substrate binding preference of CsaA. A structure of CsaA from Agrobacterium tumefaciens in complex with a phage display derived peptide was solved to 1.65Å resolution. The peptide binds to the substrate binding pocket on the surface of CsaA. Three residues of the bound peptide form specific interactions with CsaA: glutamine at position (-6) and small hydrophobic residues at positions (-5) and (-3). A conserved arginine residue in the binding site of CsaA likely acts as a clamp that transiently interacts with and stabilizes the peptides in the binding site.

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

Computational characterization of prokaryotic genomic islands

Date created: 
2007
Abstract: 

Genomic islands, including pathogenicity islands, are commonly defined as clusters of genes in prokaryotic genomes that have probable horizontal origins. These genetic elements have been associated with rapid adaptations in prokaryotes that are of medical, economical or environmental importance, such as pathogen virulence, antibiotic resistance, symbiotic interactions with plants, and notable secondary metabolic capabilities. As the number of genomic sequences increases, the impact of genomic islands in prokaryotic evolution has become more apparent and detecting these regions using bioinformatics approaches has become an integral part of studying microbial evolution and function. I therefore constructed and analyzed a set of previously reported genomic islands, most of which are pathogenicity islands, to quantify features associated with islands as a prelude to improving their identification and characterization. I integrated some of the features associated with islands into a bioinformatics tool, named IslandPath, which aids visual detection and analysis of genomic islands. Features incorporated included %G+C of genes, dinucleotide bias of gene-clusters, occurrence of mobility genes, and presence of tRNA genes. Using IslandPath, and our knowledge of features of islands, I constructed sets of computationally predicted genomic islands and characterized the classes and functions of the gene products in these regions. I conducted the first large-scale analysis of genomic islands from over 60 prokaryotic genomes and showed that the distribution of genes in the islands versus the rest of a given genome is non-random for a wide range of phyla. Certain protein functional categories, including many virulence factors and novel hypothetical proteins, are significantly more prevalent in genomic islands. This study also led to the proposal that the horizontally-inherited gene pool is characteristically distinct from, and may be larger than, the vertically-inherited prokaryotic gene pool. I also carried out detailed analyses of genomic islands in the Rhodococcus sp. RHA1 genome to shed light on its evolutionary history. The results suggest that RHA1’s unusually large genome is not due to recent horizontal gene transfer, but rather formed through more ancient evolutionary processes. Overall, my studies offer new insights into the features associated with genomic islands and the significance of these regions in prokaryotic evolution.

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

Gfat1/zeppelin is an essential heterochromatic gene involved in cuticle formation in D. melanogaster

Date created: 
2007
Abstract: 

Many essential genes exist in heterochomatin despite the high composition of repeat or ‘junk’ DNA in these regions. The gene zeppelin (zep) was genetically mapped to a distal segment of 3R heterochromatin by the Bejsovec lab. As well as being essential, it is characterised by an abnormal expansion of the embryo cuticle, a ‘blimp’ phenotype. Gfat1 is part of the hexosamine pathway that uses glucose to form chitin, a major component of the exoskeleton and therefore a candidate for causing the ‘blimp’ phenotype. Using genetic and molecular approaches, I have determined that the zep gene coincides with Gfat1 and further alleles have also been identified and characterized. Inter se complementation has revealed an interesting semi-viable Splayed (Spl) phenotype, characterised by weak legs and melanotic deposits at leg joints. Reproduction of the Spl phenotype via RNAi has further confirmed the correspondence of the zeppelin locus with Gfat1

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

The role of p21-activated kinase in Drosophila development

Author: 
Date created: 
2007
Abstract: 

Dept. of Molecular Biology and Biochemistry

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

Characterization of putative effector proteins for the small GTPase, Cdc42, during drosophila development

Author: 
Date created: 
2005
Abstract: 

The Rho subfamily GTPases (Rho, Rac, Cdc42) are small GTP-binding proteins that act as molecular switches, controlling many cellular functions. These GTPases fluctuate between a GTP-bound 'on7 state and a GDP-bound 'off state, and convey signals into the cell. Cdc42 has been implicated in a diverse array of processes, including vesicular trafficking, gene expression, formation of F-actin based membrane protrusions (filopodia), cell polarity, apoptosis, and cell cycle regulation. Model systems such as Drosophila have furthered the understanding of the hnctional roles of Cdc42 in epithelial morphogenesis, establishment of cell polarity, and neuronal path finding and development. The use of model systems allows the study of molecular processes at levels not possible in cell culture. These include genetic approaches and the study of gene function at the level of tissue morphogenesis. Three putative downstream effectors for Cdc42, originally identified in mammals, were studied in Drosophila. DCIP4, a putative cytoskeletal regulator, is expressed in a dynamic pattern throughout development. DCIP4 is required during oogenesis and functions with Cdc42 in crossvein development. The two members of the Drosophila ACK nonreceptor tyrosine kinase family, DACK and DPR2, are both expressed at the leading edge epidennis during the embryonic process of dorsal closure. DACK functions downstream of Cdc42 in dorsal closure, but is not required for JNK signalling during embryonic development. However, DACK may modulate signalling downstream of, or in parallel to, the Decapentaplegic pathway in this process.

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

Bardet-Biedl Syndrome : molecular genetics of a rare recessive disorder

Date created: 
2003
Document type: 
Thesis
File(s): 
Department: 
Theses (Dept. of Molecular Biology and Biochemistry) / Simon Fraser University
Thesis type: 
Thesis (M.Sc.)