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

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Investigating the role of the small Heat Shock Protein, HSP-12.6, in longevity in Caenorhabditis elegans

Author: 
File(s): 
Date created: 
2012-05-24
Supervisor(s): 
Eve Stringham
Michel Leroux
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
Abstract: 

The insulin-like/IGF-1 signaling (IIS) pathway is evolutionarily conserved from yeast to humans (Barbieri et al., 2003). Mutations in the insulin receptor homologue in worms, daf-2, extend lifespan in Caenorhabditis elegans and this phenotype is dependent on the activity of the forkhead transcription factor, DAF-16/FOXO (forkhead box, subgroup O) (Murphy et al., 2003). The small heat shock protein (smHSP) HSP-12.6 is a target of DAF-16/FOXO and is implicated to influence lifespan (Murphy et al., 2003). To monitor HSP-12.6 expression in vivo, a transgenic strain carrying a translation fusion of phsp-12.6::HSP-12.6::DSRED2 was constructed. Using this transgenic mutant, longevity assays were performed and we found that overexpression of HSP-12.6 extends lifespan of the animal while reduction of function of hsp-12.6 by RNAi decreases lifespan. Longevity assay data also suggest that HSP-12.6 requires the transcription factor DAF-16 but not necessarily HSF-1 for its function.

Document type: 
Thesis

The protein-protein interactions involved in the periplasmic components of the β-barrel assembly machinery (BAM) complex of Escherichia coli

File(s): 
Date created: 
2012-06-06
Supervisor(s): 
Mark Paetzel
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
Abstract: 

The β-barrel assembly machinery (BAM) complex plays the essential role of folding and inserting outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. In Escherichia coli, the BAM complex is comprised of five proteins: BamA, BamB, BamC, BamD, and BamE. This thesis project investigates the interactions between the periplasmic components of the BAM complex by analyzing complex formation using gel-filtration chromatography. Results from the interaction studies have identified the unstructured N-termini of BamC and BamE as requirements for BamCDE subcomplex formation. Furthermore, BamAPOTRA was shown to form stable BamAPOTRA-BamB and BamAPOTRA-BamD-BamE complexes, but was unable to form the latter complex in the presence of BamC. Together these results provide a model for how the proteins assemble and suggest that the complex is dynamic. By understanding how the BAM components come together brings us one step closer to determining their individual roles and how the BAM complex may function overall.

Document type: 
Thesis

Characterization of FHL2 expression and function in breast cancer cell lines

Author: 
File(s): 
Date created: 
2012-05-11
Supervisor(s): 
Sharon Gorski
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
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

Molecular mechanism of CTXΦ phage uptake into Vibrio cholerae

File(s): 
Date created: 
2012-04-11
Supervisor(s): 
Lisa Craig
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
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

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

File(s): 
Date created: 
2012-04-25
Supervisor(s): 
Dr. Esther Verheyen
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
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

Structural characterization of Vibrio cholerae toxin-coregulated pilus

Author: 
File(s): 
Date created: 
2012-04-16
Supervisor(s): 
Lisa Craig
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.
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

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

Author: 
File(s): 
Date created: 
2012-04-10
Supervisor(s): 
David Baillie
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.
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

Probing non-coding RNA structural dynamics with 2-aminopurine

File(s): 
Date created: 
2012-02-24
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.
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

Characterization of the p21-activated kinase Pak during Drosophila oogenesis

Author: 
File(s): 
Date created: 
2012-01-06
Supervisor(s): 
Nicholas Harden
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
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

Prokaryotic protein subcellular localization prediction and genome-scale comparative analysis

Author: 
Date created: 
2011-12-14
Supervisor(s): 
Fiona Brinkman
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
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