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

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Characterization of FHL2 expression and function in breast cancer cell lines

Author: 
Date created: 
2012-05-11
Abstract: 

Autophagy is a lysosome-mediated catabolic process that is induced by cell stress and functions primarily in cell survival. Previous gene expression studies indicated that the transcription cofactor FHL2 is associated with both cell survival and autophagy in breast cancer cells, but the function of FHL2 in these processes was unknown. My hypothesis was that FHL2 is a component of the molecular machinery regulating survival and/or autophagy in breast cancer cells. To test this, FHL2 expression was examined in breast cancer cell lines following treatment with the autophagy-inducing breast cancer drugs, tamoxifen or epirubicin. These treatments resulted in no substantial change in levels of FHL2 transcripts or FHL2 protein. To assess FHL2 function, siRNA mediated knockdown and FHL2 over-expression approaches were employed in MCF7 breast cancer cells. FHL2 knockdown led to a significant decrease in cell viability, indicating that FHL2 may promote cell survival. Overexpression of FHL2 did not have a significant effect on viable cell numbers but resulted in increased levels of the autophagy protein LC3II, suggesting that elevated FHL2 may alter autophagy.

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

Molecular mechanism of CTXΦ phage uptake into Vibrio cholerae

Date created: 
2012-04-11
Abstract: 

Vibrio cholerae colonize the intestinal lumen and secrete cholera toxin, inducing a massive efflux of water and electrolytes, causing the diarrheal disease cholera. Non-pathogenic serogroups of V. cholerae are made pathogenic by infection with the filamentous cholera toxin bacteriophage Φ (CTXΦ). CTXΦ binding and uptake may occur via a similar mechanism to M13 phage infection of E. coli. By this model the minor coat protein of CTX, pIII, first binds to the toxin coregulated pilus (TCP) on the surface of V. cholerae followed by TolA in the periplasm of V. cholerae. To understand CTXΦ uptake we expressed and purified the N-terminal domain of CTXΦ pIII (N-pIII) and the C-terminal domain of TolA (TolA-C) and demonstrated an interaction between these protein domains using pull down assays. We solved the de novo crystal structure of N-pIII to 2.9 Å resolution and the structure of N-pIII in complex with TolA-C to 1.44 Å. CTXΦ N-pIII has a structure similar to its corresponding domain in M13 pIII proteins despite only 15% sequence identity, but surprisingly binds to TolA at a site distinct from that of M13 N-pIII. These crystal structures provide valuable insights for understanding the mechanism by which CTXΦ infects V. cholerae.

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

Homeodomain-interacting protein kinase regulates Yorkie activity to promote Drosophila tissue growth

Date created: 
2012-04-25
Abstract: 

The Hippo (Hpo) tumour suppressor pathway regulates tissue size by inhibiting cell proliferation and promoting apoptosis. The core components of the pathway, Hpo, Salvador, Warts (Wts) and Mob-as-tumor-suppressor (Mats), form a kinase cascade to inhibit the activity of the transcriptional regulator Yorkie (Yki). Homeodomain-interacting protein kinases (Hipks) are a family of conserved serine/threonine kinases that regulate various transcriptional factors to control developmental processes including proliferation, differentiation and apoptosis. Hipk can induce tissue overgrowth in Drosophila. Genetic interaction studies reveal that Hipk is required to promote Yki activity, overriding the negative regulation induced by the Hpo kinase cascade. Hipk neither affects Yki stability nor its subcellular localization. Moreover, hipk knockdown suppresses the overgrowth and target gene expression caused by hyperactive Yki. Hipk interacts with and phosphorylates Yki and in vivo analyses show that Hipk’s regulation of Yki is kinase-dependent. To the best of our knowledge, this is the first kinase identified to positively regulate Yki.

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

Structural characterization of Vibrio cholerae toxin-coregulated pilus

Author: 
Date created: 
2012-04-16
Abstract: 

Vibrio cholerae are Gram-negative bacteria responsible for cholera, a severe and fatal gastrointestinal disease. The ability of V. cholerae and many other bacterial pathogens to cause disease is dependent on type IV pili. V. cholerae use toxin-coregulated pili (TCP) to colonize the human intestine. TCP are long, thin, flexible polymers of the TcpA subunit that self-associate to hold cells together in microcolonies, serve as the receptor for the cholera toxin bacteriophage CTXφ and secrete colonization factor proteins. To better understand TCP’s roles in pathogenesis, its structure was characterized using hydrogen/deuterium exchange mass spectrometry, computational modeling, electron microscopy (EM) and three-dimensional image reconstruction. The V. cholerae TcpA pilin crystal structure was docked into the pilus EM reconstruction to generate a pseudo-atomic resolution TCP structure. Tight packing of the hydrophobic N-terminal α-helices holds the pilin subunits together, but loose packing of the globular domains leaves gaps on the filament surface. These findings explain filament flexibility, suggest a molecular basis for pilus:pilus interactions and reveal a potential therapeutic target. TCP are members of the type IVb pilus subclass, which is distinguished from the type IVa subclass by differences in amino acid sequence, length and topology of the pilin globular domains. To understand the biological significance of the distinct pilin folds, circular dichroism spectroscopy was used to compare the stability of the V. cholerae type IVb TCP with that of the Neisseria gonorrhoeae type IVa gonococcal (GC) pilus together with their pilin counterparts. We show that TcpA pilin monomers are more stable than GC pilins but surprisingly GC pili are more stable than TCP filaments. Thus, while the type IVb pilin fold appears to be more stable than the type IVa fold, differences in quaternary structures, including tighter packing and stacking of aromatic side chains appear to contribute to the extreme stability of the GC pili. The robustness of GC pili may be necessary to withstand high stress forces in the urogenital tract. This may also be a common feature of type IVa pili as an adaptation to the niches occupied by the bacteria, biological demands and functions of these filaments.

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

Distinct cell guidance pathways control the extension of the excretory canals in C. elegans

Author: 
Date created: 
2012-04-10
Abstract: 

The excretory cell is the functional equivalent to the kidney in the nematode Caenorhabditis elegans, and provides an excellent in vivo model to study directional cell migration and extension. The leading edges of the growing canals resemble neuronal growth cones in that they must be able to sense and integrate directional cues in both the dorsoventral and anteroposterior axes. The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons, as well as the excretory cell. UNC-53 and the Abelson kinase interactor (ABI-1) appear to function cell autonomously in the excretory canals to promote outgrowth, have overlapping expression patterns, and display similar cell migration phenotypes in the canals and mechanosensory neurons. Moreover, proteins known to function with abi-1 in actin dynamics, including members of the ARP2/3 complex exhibit similar excretory cell and neuronal outgrowth defects by RNAi, suggesting that UNC-53 may act as a scaffold that links ABI-1 to the ARP2/3 complex to regulate actin cytoskeleton remodelling. Genetic analysis of putative interactors of UNC-53 suggests that UNC-53 appears to function together with UNC-71/ADAM, while the kinesin-like motor VAB-8 appears to act in a separate pathway to control the posteriorly directed excretory canal outgrowth. Analysis of putative interactors of VAB-8 suggests that VAB-8, SAX-3/ROBO, SLT-1/Slit and EVA-1 are functioning together in the outgrowth of the excretory canals. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double mutant combination with either unc-53 or vab-8. Rescue experiments suggest that a cell autonomous pathway is mediated by the Rho specific GEF domain of the UNC-73E isoform in conjunction with UNC-53, UNC-71, and ABI-1, and a cell non-autonomous pathway is mediated by the Rac specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1. Taken together, my studies indicate that the migration of the excretory canals requires two or more signaling pathways, and the guidance cues involved are also essential migration molecules functioning to guide neuronal cells and axon growth cones.

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

Probing non-coding RNA structural dynamics with 2-aminopurine

Date created: 
2012-02-24
Abstract: 

This thesis investigates sequence-dependent structural dynamics of non-coding (nc) RNAs utilizing the fluorescent base 2-Aminopurine (2Ap). We conclude that the highly homologous adenine (ARNA) and guanine (GRNA) riboswitches exhibit distinct structural dynamics in the ligand-bound state. Relative to ARNA, GRNA is more preorganized towards the closed/native conformer (CC), displays enhanced thermostability, and higher magnesium (Mg2+) binding affinity. We then focus on the role of nucleoporin 50-kilodalton (Nup50) Alu double-stranded (ds) RNAs in adenosine-to-inosine (A-to-I) editing. Here, we deduce the folding pathways of three constructs containing an internally substituted 2Ap, which are representative of Nup50-Alu dsRNAs in human, chimp, and rhesus species, respectively. Our fluorescence-based data do not fit well to a two state (folded/unfolded) model, but are well modeled by a four-state (two intermediate) model. Despite the high interspecies homology, the human sequence is most heavily edited in vivo. Interestingly, we find that the 2Ap at position five in the human Nup50-Alu dsRNA has the most thermodynamically stable intermediate, which is an optimal substrate for the A-to-I editing enzyme. The investigations of ncRNA folding dynamics underscore how Nature utilizes subtle sequence variations to achieve remarkable diversity.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Dr. B.M. Pinto (for Dr. M.A. O'Neill)
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Characterization of the p21-activated kinase Pak during Drosophila oogenesis

Author: 
Date created: 
2012-01-06
Abstract: 

Understanding the mechanisms involved in tissue reorganization and organ formation are fundamental questions that are of particular interest to those studying epithelial morphogenesis. Model organisms such as Drosophila melanogaster have been instrumental in identifying key components required for cellular processes that are critical for tissue morphogenesis as a whole. These include cell fate specification, cell shape change, cell growth and cell migration. The Rho subfamily of small GTPases Rho, Rac and Cdc42, are master regulators of actin cytoskeletal dynamics and the well-characterized Rac/Cdc42 effector kinase Pak has been implicated in morphogenesis of epithelia both in mammalian cell culture as well as Drosophila development. Drosophila oogenesis is a highly favourable system for studying epithelial morphogenesis and differentiation of epithelia from a stem cell. Newly formed germline cysts become encapsulated by follicle cells that arise from follicle stem cells (FSCs) to form egg chambers. This study on Pak function during oogenesis, combined with the work of others, has opened the door to our understanding of how the FSC and its niche produce a simple yet very organized epithelium. Pak appears to be required during early stages of oogenesis at or around the time point at which FSCs and/or their niches are specified. Loss of Pak during early stages of oogenesis leads to a novel side-by-side egg chamber phenotype with pak mutant germaria having duplicated FSC niches, implicating Pak in FSC niche formation. This work has led to a model for stem cell niche formation that may be broadly applicable. Later in oogenesis Pak is required for the polarized organization of the basal F-actin in follicle cells, which drives egg chamber elongation. Further investigation of Pak’s role has demonstrated that it acts antagonistically to the Rho1-activated actomyosin contractility pathway in regulation of this F-actin. The basal F-actin of the follicle cells is similarly organized as the stress fibres of mammalian cells and insights gained from this work are likely to be relevant to understanding regulation of the mammalian cytoskeleton. Overall this work has revealed new roles for Pak in epithelial development.

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

Prokaryotic protein subcellular localization prediction and genome-scale comparative analysis

Author: 
Date created: 
2011-12-14
Abstract: 

Identifying protein subcellular localization (SCL) is important for deducing protein function, annotating newly sequenced genomes, and guiding experimental designs. Identification of cell surface-bound and secreted proteins from pathogenic bacteria may lead to the discovery of biomarkers, novel vaccine components and therapeutic targets. Characterizing such proteins for non-pathogenic bacteria and archaea can have industrial uses, or play a role in environmental detection. Previously, the Brinkman lab has developed PSORTb, the most precise SCL prediction software tool for bacteria. However, as we increasingly appreciate the diversity of prokaryotic species and their cellular structures, it became clear that there was a need to more accurately make predictions for more diverse microbes. For my thesis research, I developed a new version of PSORTb that now provides SCL prediction capability for more prokaryotes, including Archaea and Bacteria with atypical cell wall and membrane structures. The new PSORTb also has significantly increased proteome prediction coverage for all bacterial species. The software is the first of its kind to predict subcategory localizations for bacterial organelles such as the flagellum as well as host cell destinations. Using both computational validations and a new proteomic dataset I produced, I established that PSORTb 3.0 outperforms all other published prokaryotic SCL prediction tools in terms of both precision and recall. Furthermore, I have developed a semi-automated version of a comprehensive prokaryotic SCL database (PSORTdb) that provides access to experimentally verified and pre-computed SCL predictions for all sequenced prokaryotic genomes. I developed an ‘outer membrane prediction method’ which allows auto-detection of bacterial structure, distinguishing bacteria with one vs. two membranes. This method allows the database to be automatically updated as newly sequenced genomes are released. In addition, the method can aid more general analysis of a bacterial genome for which the bacteria’s associated cellular structure is not initially clear. Finally, I performed a global analysis of SCL proportions for over 1000 sequenced bacterial and archaeal genomes. This is the most comprehensive SCL analysis of prokaryotes to date. My findings provide insights into prokaryotic protein network evolution, elucidate relationships between SCL proportions and genome size, and provide directions for future SCL prediction research.

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

Comparative analysis of Caenorhabditis genomes

Date created: 
2011-08-04
Abstract: 

Comparative genomic studies between Caenorhabditis species as well as within C. elegans strains have proved useful for unveiling the genetic basis of differences in biological processes and phenotypical variation. An important step for such studies is the accurate detection and characterization of genomic conservation and divergence. Using newly developed tools OrthoCluster and OrthoClusterDB, perfect and imperfect conserved synteny between the two chromosomal assemblies of C. elegans and its sister species C. briggsae is estimated, showing that syntenic information can be used for improving hundreds of gene models and for detecting new ones. Additionally, a large segmental duplication within the C. elegans genome involving 216 kb is detected using OrthoCluster. Genotyping of 76 N2 strains reveals that this duplication is polymorphic and occurred very recently in the C. elegans genome. In particular, this large segmental duplication was found to be absent in a wild isolate from Hawaii (CB4856) which has a high degree of polymorphism and a number of trait differences with N2, some of which have an explained genetic basis. This study provides the first genome-wide detection of breakpoint-resolution single nucleotide variants, small InDels and large genomic variations (GVs) between the genomes of N2 and CB4856 based on complementary next-generation sequencing technologies. Assessment of the co-occurring GVs on the protein-coding genes with a newly developed tool, Variant-Analyzer, shows that hundreds of single-copy genes with lethal and sterile phenotypes are impacted by GVs, many of them homologs to human genes with associated diseases. This study confirms most of the known GVs associated with trait differences and serves as a source of candidates that could explain the genetic basis for others. Overall, this thesis provides a deeper understanding of the conservation and divergence between Caenorhabditis species as well as within C. elegans strains.

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

Kinetic studies of O-GlcNAc processing enzymes: implications for enzyme regulation

Author: 
Date created: 
2011-04-15
Abstract: 

The modification of nuclear and cytoplasmic protein with O-linked N-acetylglucosamine (O-GlcNAc) residues is a unique cellular process in higher eukaryotes. Levels of this modification are regulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine: polypeptidyl transferase (OGT) and O-GlcNAcase (OGA). Based on structural information obtained for an OGT homolog from Xanthomonas campestris, we characterized the active center of human OGT and proposed histidine 558 to be the catalytic general base. We also described a method to generate O-GlcNAcylated recombinant proteins in E. coli. This allowed, for the first time, kinetic studues of human OGA to be carried out using protein substrates. In combination with human OGT kinetics on the same substrates, we are able to predicted the relative O-GlcNAc stoichiometry between the protein substrates. Lastly, we explored the substrate specificity of human OGT and discovered uridine diphosphate glucose (UDP-Glc) may be a substrate of OGT within cells.

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