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

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The Group-I Paks participate in a regulatory network controlling actomyosin contractility in the Drosophila follicular epithelium

Author: 
Date created: 
2012-11-08
Abstract: 

Epithelial morphogenesis takes place in response to F-actin modulation, which is regulated by signaling cues from the Rho family of small GTPases and their downstream effectors such as the Group-I p21-activated kinases, Pak and Pak3. The basal F-actin fibres of the Drosophila ovarian follicular epithelium (FE) form parallel bundles that contribute to egg chamber elongation. Pak has been demonstrated to modulate the levels of phosphorylated myosin light chain (pMLC) in the FE, but little is known about Pak3. Here, I present evidence that Pak3 is a major MLC kinase and is repressed by Pak in the elongating FE. In addition, actomyosin contractility promoted by Pak and the Rho1 pathway drives Pak3 expression through the MAL-SRF transcriptional regulatory complex, to ensure there is sufficient active myosin to provide contractility. The more Factin in the FE, the greater the activity of MAL-SRF and consequently the higher the levels of Pak3 and pMLC.

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

Structural and computational analysis of the Escherichia coli chaperone protein DmsD

Peer reviewed: 
No, item is not peer reviewed.
Date created: 
2012-09-19
Abstract: 

In Gram-negative bacteria, the secretion of proteins that contain redox cofactors is accomplished using the twin arginine translocase (TAT) system, so named because the cofactor containing secretory proteins contain an N-terminal leader peptide with a twin arginine motif. The redox enzyme maturation proteins (REMPs) are molecular chaperones that prevent TAT substrate translocation until the preprotein is folded and its cofactor is incorporated. REMPs then direct the substrate to the TAT translocase. The work presented here explores, from a structural biology perspective, a model REMP: Escherichia coli DmsD. DmsD was crystallized and the structure determined and refined to 2.0 Å resolution. This is the first structure of E. coli DmsD, and contains clear electron density for all 204 amino acid residues in the protein molecule. This was complemented by NMR analysis that characterized the local backbone dynamics of the protein. The dynamic properties of DmsD were also explored by molecular dynamics simulation. These analyses have identified three flexible regions of DmsD, two of which contribute to the putative leader peptide binding site. The third flexible region is located in a patch of residues that were implicated in GTP binding in the homologue TorD. A method for the purification of active TAT leader peptides was devised and used in the generation of a selectively labeled sample for NMR analysis. Chemical shift perturbation analyses are consistent with a hydrophobic groove on the surface of DmsD interacting with the DmsA leader peptide. DmsA is the cofactor containing catalytic subunit of DMSO reductase and the specific substrate of DmsD. The leader peptide binding groove on DmsD overlaps with the previously identified “hot pocket” which is predicted to interact with the twin-arginine motif of the leader peptide, and encompasses regions of the x-ray structure that form crystal contacts with crystallization reagents. Finally, a new model for TAT leader peptide binding is proposed which combines all of the available data.

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

Profiling antibody responses to engineered HIV-1 gp120 antigens

Date created: 
2012-08-09
Abstract: 

Conserved regions on the HIV-1 envelope spike are important for vaccine design due to their vulnerability to antibodies that can block virus infectivity – termed neutralizing antibodies (NAbs). The CD4-binding site (CD4bs) on the HIV spike subunit gp120 is one such region. Eliciting antibody responses to the CD4bs is difficult because it is surrounded by sequence-variable segments. Here, I conducted immunizations in mice to investigate approaches to focus NAb responses to the CD4bs. I found that formulating gp120 mutants that preferentially expose the CD4bs with the saponin adjuvant QuilA improved CD4bs-directed responses; however neutralizing activity was not enhanced. In subsequent work I found that priming animals with a gp120 mutant fused to a string of pathogen-derived CD4 T-helper epitopes followed by booster injections with alternate gp120 constructs that differentially present the CD4bs elicited higher antibody titers and improved neutralizing activity. Overall, my work provides insight into strategies for focusing antibody responses to desired epitope targets with relevance to HIV vaccine design.

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

Characterization of TCRβ sequence diversity in colorectal carcinoma

Author: 
Date created: 
2012-06-13
Abstract: 

T cells can play a critical role in mediating anti-tumour responses in cancer, but thus far have been characterized at low resolution. Each T cell receptor beta subunit (TCRβ) possesses a hypervariable sequence (CDR3) that is a principal site of contact with peptide-MHC complexes on heterologous cells and can be used to characterize TCRβ diversity. Here, I used a sequencing approach developed for interrogating peripheral blood immune repertoires to explore TCRβ sequence diversity associated with colorectal carcinoma (CRC). TCRβ sequences were amplified from RNA isolated from biopsies of tumour and matched control tissue of 43 CRC patients. Amplicons were indexed and survey sequenced on the Illumina platform. Sequence reads were assembled and filtered revealing a diverse sequence repertoire. Some abundant sequences shared between patients were significantly associated with improved overall survival. These results have great potential utility in the design of immunological approaches targeting tumours, and in the design of specific screening approaches for CRC.

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

Gene identification in Caenorhabditis elegans through next generation sequencing

Author: 
Date created: 
2012-08-14
Abstract: 

The emergence of Next Generation DNA Sequencing (NGS) technologies has made cloning of genes much more efficient by dramatically reducing the cost, labour and time required. In this work, I have applied a NGS and devised a bioinformatics program pipeline to detect genomics alterations in the mutant strains of the nematode Caenorhabditis elegans. In the first project, I have detected a mutation in tba-5, validated through complementation tests, and proposed a model elucidating the role of TBA-5 in cilia structure. In the second project, the variant detection pipeline is used to isolate potential candidate genes corresponding to various dsh-2(or302) suppressors, namely Sup327, Sup245 and Sup305. Additionally, I have applied the bioinformatics pipeline to confirm that the current C. briggsae reference genome harbours thousands of assembly errors, many of which affect the correct prediction of gene models.

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

Structural analysis of the Escherichia coli β-barrel assembly machinery complex

Date created: 
2012-05-31
Abstract: 

The outer membrane (OM) is a unique structural feature of Gram-negative bacteria. Residing within the outer membrane are β-barrel outer membrane proteins (OMPs) that serve many important cellular functions. As proper folding and assembly of these proteins are crucial for cell viability, Gram-negative bacteria possess a specialized proteinaceous machine, known as the BAM (β-barrel assembly machinery) complex, to catalyze the folding and membrane insertion of OMPs. In Escherichia coli, the BAM complex consists of five proteins: one β-barrel membrane protein – BamA, and four lipoproteins – BamB, BamC, BamD, and BamE. The roles of the individual components and how they are arranged into the BAM complex to function together is not yet clearly understood. During the course of this thesis project, I determined the structures of E. coli BamB, BamC, BamE and the BamCD subcomplex. Analysis of the conserved residues and the molecular surface properties of these solved structures helped to identify potential protein-protein interaction sites on each lipoprotein. For example, BamC has two ‘helix-grip’ domains that are ideally shaped to accommodate α-helices. BamB, on the other hand, has a β-propeller fold that could potentially interact with BamA or substrates via β-augmentation, a mode of interaction in which a pre-existing β-sheet is augmented by an addition of a β-strand of another protein. Comparing the solved structures with their structural homologs with known functions has also provided important clues about the functional roles of each lipoprotein. BamD structure, for example, closely resembles the binding pocket of a peroxisomal targeting signal receptor PEX5, suggesting a similar substrate recognition function for BamD. Interestingly, our BamCD complex structure shows that the putative substrate binding pocket of BamD is bound and blocked by the conserved unstructured N-terminal region of BamC. This suggests a possibility that BamC may have a regulatory function.The structural and interaction data acquired from this thesis project contributes to a better understanding of the BAM complex structure and provides a platform for future research driven by structure-based hypotheses.

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

Investigation of DNAzymes catalyzing thymine dimer photoreactivation and phosphodiester cleavage using a new method of iodine-mediated phosphorothioate cross-linking

Author: 
Date created: 
2012-05-25
Abstract: 

In recent years, many artificial and naturally occurring catalytic nucleic acids molecules have been discovered. These enzymes, composed purely of RNA or DNA, are referred to as ribozymes or DNAzymes. With an ever-increasing repertoire of chemical reactions they are able to catalyze, researchers have attempted to discover the mechanisms governing catalysis by identifying the functional groups involved in catalysis. This thesis introduces a novel Iodine-mediated phosphorothioate cross-linking (IMPC) method, which enables structure mapping around the phosphodiester backbone of DNA. Two DNAzymes that I have investigated using this method are the 8-17 RNA-cleaving enzyme and the UV1C cyclobutane thymine dimer-repairing enzyme. The UV1C DNAzyme adopts a G-quadruplex structure that is responsible for enhanced, in comparison to non-G-quadruplex forming DNA, absorption of light energy of >300 nm wavelength to photo-reactivate cyclobutane thymine dimers in a DNA substrate. IMPC results suggest that the topology of this G-quadruplex is an all-parallel propeller orientation. UV1C was originally in vitro selected for repair of a substrate lacking an intra-dimer phosphodiester phosphate. I show that UV1C can catalyze repair of thymine dimer mutations in a “natural” intact single stranded DNA substrate at wavelengths greater than 300 nm. Additionally, evidence is produced indicating that UV1C shifts the photostationary state of thymine dimer formation to favour monomer formation in comparison to single stranded and double stranded DNA at 280 nm wavelength. While other biochemical cross-linking methods explore base-to-base contacts, IMPC identifies base-to-backbone contacts. This provides an advantage at identifying phosphodiester cleaving ribozyme and DNAzyme nucleobases directly involved in phosphodiester cleavage at the enzyme’s active site. Using this approach, not only is the 8-17’s catalytic core base contacts identified, but also a chirally resolved phosphorothioate at the cleavage site provides a stereochemical glimpse of a key cytosine (C13) base. Another important cytosine (C3) is also identified, for the first time. On the basis of the C3’s proximity to the cleavage site and the impact of its mutation on the DNAzyme’s catalytic rate, it is functionally implicated to perform an acid-base role in catalysis.

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

Multiple roles for UNC-53/NAV2 in cell migration, trafficking and innate immunity in Caenorhabditis elegans

Date created: 
2012-06-01
Abstract: 

unc-53 is the Caenorhabditis elegans homolog of Nav2, and a member of the Neuron Navigator protein family, a group of cytoskeletal binding proteins with conserved roles in the guidance and outgrowth of cells and cellular processes. The signalling pathways that employ UNC-53 during development, and the role of UNC-53 after development is complete, are largely unknown. A proteomics screen for interactors of UNC-53 identified that UNC-53 interacts with ABI-1. The Calponin Homology domain unique to the long-isoform of UNC-53 is sufficient to bind ABI-1 in vitro and is required in vivo for longitudinal migration. ABI-1 and UNC-53 are co-expressed, and abi-1 genetic loss causes many of the same migration defects as unc-53 mutants. abi-1 and unc-53 function cell-autonomously and in a common genetic pathway in the posterior migration of the excretory canals, and genetically inactivating abi-1 interactors (nck-1, wve-1, arx-2) resulted in phenotypes similar to unc-53 and abi-1. abi-1 and unc-53 are also required for endocytosis as measured by assaying the in vivo uptake of GFP into coelomocytes and primary oocytes. Lastly, abi-1 mutants have unc-53 independent migration defects, and ABI-1 binds to MIG-10A through its SH3 domain, thereby demonstrating that ABI-1 interacts with two proteins that function cell-autonomously in the longitudinal migration of the excretory canals. To identify a post-developmental role for unc-53, a potential role for unc-53 in innate immunity was assessed. unc-53 mutants are susceptible to the human and nematode pathogen Pseudomonas aeruginosa PA14. unc-53 mutants are hypersensitive to Aldicarb and have increased RNA levels of the daf-16 antagonist ins-7 as well as decreased nuclear DAF-16 localization following recovery from cellular stress, suggesting that unc-53 functions in the daf-16 pathway. Loss of function and null alleles of daf-2 and ins-7 only partially suppress unc-53 in immunity, and a null pmk-1 mutant does not enhance unc-53. Together, this data suggests that unc-53 participates in innate immunity through multiple tissues, isoforms and genetic pathways. These findings expand on the varied functions of unc-53 and the signal transduction pathways that it controls, opening up new prospects for future areas of research.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Nancy Hawkins
Eve Stringham
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis/Dissertation) Ph.D.

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

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

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

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