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

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DNA Helix-stack Switching as the Basis for the Design of Versatile Deoxyribonsensors

Date created: 
2004
Abstract: 

The charge conduction properties of deoxyribonucleic acids (DNA) can be harnessed for monitoring the binding of a ligand to its receptor. Here, we show how DNA-based sensors (deoxyribosensors) consisting of a photo-activated oxidant tethered to a receptor-containing DNA molecule can be used to monitor the presence of a ligand. Phosphorescence measurements, reflective of changes in charge conduction to a targeted region in these deoxyribosensors are made in the presence and absence of the test ligand. The deoxyribosensors described here, exploit established rules for DNA helical stacking in three-way junctions and use previously selected aptamer sequences as receptors for target analytes. More specifically, a systematic investigation outlining the characteristics of a deoxyribosensor for the detection of the amino acid derivative, argininamide are presented. These results suggest a general design for deoxyribosensors for any molecular ligand for which an aptamer sequence can be obtained. Also, a new methodology for investigating helical stacking of nucleic acids of unknown tertiary structure, such as DNAzymes or RNAzymes is discussed.

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

In-vitro selection of charge conducting DNA sequences

Author: 
Date created: 
2006
Abstract: 

Double helical DNA transports electrical charge, via hole transport, over distances over 2008, (58 base pairs). Because the exact mechanism of hole transport is under debate, an alternative method to the current 'rational' design approach used by investigators may shed new light on the problem. In vitro selection was used to isolate DNA sequences most efficient at hole conduction out of a random pool. Several selections were carried out, each providing valuable information for subsequent selection design. Tests carried out on the Round 7 of the SYB series confirmed that selection superior conductors was indeed occurring. 93 clones sequenced from Round 7 were analyzed against 89 clones from the initial round and found to have statistically elevated levels of specific purine motifs segregated to only one specific strand of the duplex. These sequences, termed 'transport permissive candidate motifs', suggested that Phononassisted Polaron-like hole hopping occurs in duplex DNA.

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

Computational prediction and comparative analysis of protein subcellular localization in bacteria

Date created: 
2006
Abstract: 

Predicting the subcellular localization of a protein is a critical step in processes ranging from genome annotation to drug and vaccine target discovery. Previously developed methods for localization prediction in bacteria exhibit poor predictive performance and are not conducive to the high-throughput analysis required in this era of genome-scale biological analysis. We therefore developed PSORTb, a hig h-precision, hig h-throug hput tool for the prediction of bacterial protein localization. PSORTb implements a multi-component approach to prediction, incorporating the detection of several sequence features known to influence subcellular localization. With a reported overall precision of 96%, it is the most precise method available and one of the most comprehensive methods capable of assigning a query protein to one or more of four Gram-positive or five Gram-negative localization sites. The PSORTb algorithm comprises a series of analytical steps, each step - or module - being an independent piece of software which scans the protein for the presence or absence of a particular sequence feature. Modules include: SCL-BLAST for homology-based detection, the HMMTOP transmembrane helix prediction tool, a signal peptide prediction tool, a series of frequent subsequence-based support vector machines, as well as motif and profile-matching modules. The modules return as output either a predicted localization site or - if the feature is not detected - a result of unknown'. The output is then integrated by a Bayesian network into a final prediction. Development of PSORTb also required the creation of PSORTdb, a database storing both known and predicted localization information for bacterial proteins. This is a valuable resource to both the localization prediction and microbial research communities, providing a source of training data for new predictive algorithms and acting as a discovery space. The release of PSORTb v.2.0 allowed us to carry out a number of analyses related to localization. We performed the first genome-wide computational and laboratory screen for Nterminal signal peptides in the opportunistic pathogen Pseudomonas aeruginosa, used PSORTb as a complement to laboratory-based high-throughput 2D gel studies of individual cellular compartments, and examined protein localization in a global context, revealing trends with implications for adaptive evolution in microbes.

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

Repetitive landscape of the Atlantic salmon genome

Date created: 
2006
Abstract: 

The duplication of genes and genomes is considered a major force in evolution. Ohno, in 1970, theorized that with two copies of the genome, one copy would be under normal evolutionary constraints while the other copy could serve as raw material for evolution. Salmonids are a classical example of species containing duplicated genomes, and they offer an opportunity to investigate how such genomes undergo reorganization as they attain a stable diploid state. Repetitive elements play an important role in genome reorganization. Therefore, I investigated the repeat structure and organization of the Atlantic salmon genome. An analysis of the fingerprinted CHON-214 BACs classified the singletons, or those that contained few restriction sites, into three categories of repetitive structures. The first group contains histone genes in a tandemly repeating cluster of H4 - H2B - H1 - H2A - H3. A second group contains the ribosomal DNA (rDNA) cistron. Intriguingly, fluorescent in situ hybridization (FISH) analysis indicated that one of each of the duplicated histone and rDNA clusters was lost or rearranged in the genome. The remaining group of BACs contains novel repetitive sequences and tRNA clusters. The Atlantic salmon EST libraries and BAC-end sequences were data-mined for simple sequence repeats (SSRs) and 2,497 SSRs were recovered. 154 SSR loci gave clean PCR amplicons and 94 produced polymorphic banding patterns with eleven of the EST-SSRs indicating duplicated loci. These loci were mapped on the Atlantic salmon linkage map. Novel repetitive elements were detected in the sequences of Atlantic salmon BACs and ESTs. Using computational tools for data-mining, repetitive elements were identified and classified based on sequence similarity to other known repetitive elements such as SINES, LINES and retrotransposons. A repeat database that can be used to mask repetitive elements, is now available for the Atlantic salmon genome. A website was developed to host the Atlantic salmon linkage and physical maps, correlating them based on marker hybridization. A BAC annotation pipeline analyzes the BAC sequences for ORFs, transcript similarity and repetitive elements. Information generated from sequence annotation, microsatellite development and repetitive element identification provides essential resources for investigating salmonid genomes.

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

Evolution of mitochondrial DNA in the genus Salmo

Author: 
Date created: 
2006
Abstract: 

The mitochondrial genome (mtDNA) is a valuable source of data for evolutionary studies because of its small size, lack of recombination and its higher rate of accepted mutations than nuclear coding sequences. All salmonid rnitochondrial genomes are -16.7 Kb in size and identical in their genome organization. PCR amplification with thirty-three conserved primer pairs and subsequent direct sequencing was used to obtain whole mitochondrial genome sequences from fourteen Atlantic salmon (Salmo snlar) samples. The nucleotide and amino acid sequences were aligned and compared with those of a sister species, brown trout (Salmo trutta) to study the mode and tempo of mtDNA evolution. Varying percent sequence divergence was observed in different parts of the genome suggesting that different constraints operate across the genome. Further, by measuring the amount of variation in the Atlantic salmon from different geographical locations, previous hypotheses regarding the structuring of Atlantic salmon populations were confirmed.

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

Pinguid, the drosophila homolog of huntingtin interacting protein 14, encodes an essential protein involved in TGF beta signalling

Author: 
Date created: 
2006
Abstract: 

To elucidate the cellular function of Huntingtin interacting protein 14 (Hipl4), a genetic approach using Drosophila was initiated. The gene encoding Pinguid was identified as the closest Drosophila homolog to human Hip14 with 44% identity and 59% similarity. A P-element insertion in the 5' UTR of pinguid was used for carrying out a transposase mediated P-element excision screen. Subsequent genetic and molecular experiments confirmed that pinguid encodes an essential gene. Five pinguid alleles were identified: three excision alleles and two EMS-induced alleles. No visible defects were observed in the mutants since the morphology and patterning of the embryos appeared normal, CNS development appeared wild type, and pharate adults had no visible external defects. However, the ectopic expression of pinguid identified genetic interactions with components of the TGFP signalling pathway. Strong genetic interactions were seen with short gastrulation, crossveinless 2, baboon, and the ecdysone receptor.

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

Analysis of antibody reactivities in lysozyme-immunized mice with peptide ligands: A model for epitope-targeted vaccine design

Date created: 
2006
Abstract: 

Many viruses evade antibody (Ab)-mediated clearance through the presentation of immunodominant, highly variable epitopes, and the masking of conserved ones. It has been proposed that vaccines incorporating peptides that cross-react with conserved epitopes could be used to target the production of Abs that protect against a range of viral isolates. For this thesis the concept of epitope-targeted vaccine design is explored with the model protein hen egg lysozyme (HEL). The goals of the work are to develop peptide markers for murine anti-HEL Abs that can also be used as immunogen to amplify Ab production against target HEL epitopes. Three types of peptides are analyzed: (i) a peptide selected from a phagedisplayed random peptide library (RPL) with the anti-HEL monoclonal Ab D1.3, (ii) peptides identified from RPLs with polyclonal (pc) Abs from the sera of HEL-immunized mice, and, (iii) peptides derived from the linear sequence of HEL. A peptide selected with Dl .3 Ab shares critical binding residues in common with its cognate discontinuous epitope but showed no reactivity with anti-HEL sera. A prime-boost immunization study, in which mice were primed with HEL to produce anti-HEL pcAbs, and boosted with the peptide to amplify the production of Dl .3 or Dl .3-like Abs, did not yield crossreactive Abs. Thus, Dl .3 Ab may not have been produced in the priming immunization because it is a rare specificity in the immune response to HEL. A peptide marker for an anti-HEL Ab commonly produced amongst mice was identified in a RPL screening with anti-HEL pcAbs. The commonly reactive peptide was also used in ELISPOT analysis to characterize epitope-specific B cell responses to HEL; the peptide-reactive cells represented less than 1% of IgG-producing B cells. The peptide was not recognized by anti-HEL IgG from rabbit, however, emphasizing the limitations of using animal models for developing epitope-targeted vaccines. Lastly, a set of 10- mer overlapping HEL peptides showed common patterns of reactivity with different murine anti- HEL sera. These findings indicate that common HEL epitopes are targeted amongst mice in the humoral immune response to HEL and that a prime-boost immunization strategy may succeed with a commonly reactive peptide marker.

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

A serotonin-dependent deoxyribozyme that uses light to repair thymine dimers in DNA

Author: 
Date created: 
2005
Abstract: 

Thymine dimers are the most common lesion formed in DNA as a result of exposure to ultraviolet (UV) light. Naturally occurring protein enzymes are known which repair these lesions in different ways. It was hypothesized that in early Earth it may have been RNA that played the functional role, which proteins play today. Also in early Earth it was suspected that the intensity of UV radiation was strong, which could produce dinier products within RNA. A previous in vitro selection has been performed to determine whether DNA was capable of catalyzing a photochemical reaction to reverse this damage. This selection yielded two different deoxyribozymes able to catalyze thymine dimer repair in DNA. The deoxyribozyme SerolC, the topic of this thesis, uses light and serotonin as a cofactor to repair the thymine dimers. Characterization of SerolC provided hypotheses for structure, the proposed reaction mechanism, and aspects of substrate specificity.

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

Identifying correlates of CTL-mediated control of HIV using computational and biomedical approaches

Author: 
Date created: 
2013-01-08
Abstract: 

Cytotoxic T lymphocytes (CTL) select HIV variants that encode mutations in peptide epitopes presented by HLA. Studying HLA-associated viral polymorphisms may therefore elucidate characteristics of effective CTL responses. Using population-level data, we identified 2100 HLA-associated polymorphisms located throughout the HIV proteome. In 9-mer epitopes, viral mutations at HLA anchor residues occurred two-fold more frequently than expected and reduced predicted peptide-HLA binding affinity by ten-fold. Epitopes presented by more protective HLA displayed greater enrichment of anchor residue mutations. CTL epitope recognition is determined by its T-cell receptor (TCR). To assess the impact of epitope mutations on TCR signaling, we implemented an in vitro reporter cell assay. This method was validated using a TCR specific for the HLA-A*02:01-FK10 peptide (FLGKIWPSYK), and proved sensitive, scalable, and robust. Alanine mutants of the TCR and peptide variants identified an interaction between FK10 position 4 and TCR alpha CDR3 that may be critical for signaling.

Document type: 
Thesis
File(s): 
Supervisor(s): 
Mark Brockman
Zabrina Brumme
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.

Characterization of the Role of Nemo and Homeodomain-interacting Protein Kinase, Two Conserved Regulators of Signalling, in Drosophila Development

Author: 
Date created: 
2012-11-23
Abstract: 

Development of multicellular organisms requires regulated cell-cell communication mediated by a handful of conserved signalling pathways. Precise regulation of signalling is essential in integrating the many inputs that cells receive. In this study, I investigated several different aspects of cell communication and signal transduction. First, I describe a novel mode of cross-talk between the Wingless (Wg) and the Bone Morphogenic Protein (BMP) Pathway where Wg-dependent gene transcription is suppressed by ectopic BMP signaling, and the BMP effector Mad disrupts the interaction between Wg effectors Arm and dTcf. This represents a novel mode of regulation to integrate input from various pathways, and does not involve post-translational modification.Protein phosphorylation by kinases is a post-translational modification reiteratively used in many different pathways and processes to regulate signaling. My further studies have involved two kinases, Nemo (Nmo) and Homeodomain-interacting Protein Kinase (Hipk), and their roles in cell signaling and development. Nmo is an evolutionarily conserved kinase involved in the modulation of several pathways. It was originally implicated to regulate cell movements during Planar Cell Polarity (PCP) signaling. Our genetic and molecular studies show that Nmo binds the core PCP factors, and phosphorylates the E-cadherin-Arm complex, implicating Nmo as the molecular link between these two complexes to promote cell motility and rotation. Additionally, we found that nmo inhibits the BMP pathway and suppresses BMP-regulated gene transcription during wing development. Nemo binds to and phosphorylates Mad at a conserved serine, leading to its nuclear exclusion in a kinase dependent manner. This is the first known kinase to target the MH1 domain of Mad to alter its subcellular localization.Hipk represents another class of conserved kinases important in the regulation of signaling. Hipk was previously implicated in growth, apoptosis and patterning. I describe a novel role for Hipk in the regulation of cell junctions and apicobasal polarity in the adult midgut. Loss of hipk leads to a disruption in junctions, epithelial disorganization and cell death. Hipk genetically and physically interacts with the polarity protein Discs Large (Dlg) and is required for its membrane localization.

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