The oncogene c-MYC has guanine-rich and complementary cytosine-rich sequences in its P1 promoter region. The P1 promoter is responsible for over 90% of the c-MYC expression. Downregulation of c-MYC expression represents a novel therapeutic approach to more than 50% of all cancers. A stable i-motif formed by the c-MYC C-rich sequence would be an attractive target for cancer treatment. We have previously shown that c-MYC promoter sequences can form stable i-motifs in acidic solution (pH 4.5-5.5). The question is whether c-MYC promoter sequence i-motif will be stable at physiological pH. In this work, we have investigated the stability of mutant c-MYC i-motif in solutions having pH values from 4 to 7 and containing co-solutes or molecular crowding agents. The crowded nuclear environment was modeled by the addition of polyethylene glycol (PEG, having molecular weights from 200 to 12000 g/mol) at concentrations of 10% to 40% w/w. Circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC) were used to establish the presence and stability of c-MYC i-motifs in buffer solutions having pH values of 4 to 7. The results of these studies are: 1) the addition of up to 20% w/w glycerol does not increase i-motif stability, 2) the addition of 30% PEG results in an increase in i-motif stability to pH values as high as 6.7, 3) i-motif stability is increased with increased PEG concentration and increased PEG molecular weight, and 4) the effects of PEG size and concentration are not linear, with larger PEGs forming DNA/PEG complexes, which destabilize the i-motif. In summary, we have shown that the c-MYC i-motif can exist as a stable structure at pH as high as 6.7 in a crowded environment. Molecular crowding, largely an excluded volume effect, drives the formation of the more compact i-motif, even at higher pH values where the cytosine imino-nitrogen is deprotonated and neutral C-C pairs can form only two H-bonds. Based on this research, it seems possible that a stable c-MYC promoter sequence i-motif could form at physiological pH and would be a reasonable drug target for new cancer therapies.

One of the primary goals of active research in molecular biology is to better understand the process of transcription regulation. An important objective in understanding transcription is identifying transcription factors that directly regulate target genes. Identifying these transcription factors is a key step toward eliminating genetic diseases or disease susceptibilities that are encoded inside deoxyribonucleic acid (DNA). There is much uncertainty and variation associated with transcription factor binding sites, requiring these sites to be represented stochastically. Although typically each transcription factor prefers to bind to a specific DNA word, it can bind to different variations of that DNA word. In order to model these uncertainties, we use a Bayesian approach that allows the binding probabilities associated with the motif to vary. This project presents a new method for motif searching that uses expert prior information to scan DNA sequences for multiple known motif binding sites as well as new motifs. The method uses a mixture model to model the motifs of interest where each motif is represented by a Multinomial distribution, and Dirichlet prior distributions are placed on each motif of interest. Expert prior information is given to search for known motifs and diffuse priors are used to search for new motifs. The posterior distribution of each motif is then sampled using Markov Chain Monte Carlo (MCMC) techniques and Gibbs sampling.

Understanding the possible mechanisms of gene transcription regulation is a primary challenge for current molecular biologists. Identifying transcription factor binding sites (TFBSs), also called DNA motifs, is an important step in understanding these mechanisms. Furthermore, many human diseases are attributed to mutations in TFBSs, which makes identifying those DNA motifs significant for disease treatment. Uncertainty and variations in specific nucleotides of TFBSs present difficulties for DNA motif searching. In this project, we present an algorithm, XPRIME-EM (Eliciting EXpert PRior Information for Motif Exploration using the Expectation-Maximization Algorithm), which can discover known and de novo (unknown) DNA motifs simultaneously from a collection of DNA sequences using a modified EM algorithm and describe the variation nature of DNA motifs using position specific weight matrix (PWM). XPRIME improves the efficiency of locating and describing motifs by prevent the overlap of multiple motifs, a phenomenon termed a phase shift, and generates stronger motifs by considering the correlations between nucleotides at different positions within each motif. Moreover, a Bayesian formulation of the XPRIME algorithm allows for the elicitation of prior information for motifs of interest from literature and experiments into motif searching. We are the first research team to incorporate human genome-wide nucleosome occupancy information into the PWM based DNA motif searching.

On January 1, 2007, the Ministry of Agriculture and Marine Resources in the Department of Marine Resources of the Bahamas promulgated significant revisions to their sports-fishing regulations. While the new laws were designed to benefit the Bahamian fisheries, they caused a vociferous uproar among non-resident anglers who frequent the islands of the Bahamas to sports-fish. Of particular concern are the new regulations that limit the maximum weight and number of fish which non-resident anglers may keep on their boat. My research examines the claims-making activities made by American anglers on four different sports-fishermen/cruising forums. The analysis focuses on the motifs, rhetorical idioms, counterrhetorical strategies, and styles among sports-fishermen who fish the Bahamas.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

RNAs play essential roles in various biological processes. Mounting evidence has demonstrated that RNA subcellular localization and intercellular trafficking govern their functions in coordinating plant growth at the organismal level. Beyond that, plants constantly encounter foreign RNAs (i.e., RNAs from pathogens including viruses and viroids). The subcellular localizations of RNAs are crucial for their function. While numerous types of RNAs (i.e., mRNAs, small RNAs, rRNAs, tRNAs, and long noncoding RNAs) have been found to traffic in a non-cell-autonomous fashion within plants, the underlying regulatory mechanism remains unclear. Viroids are single-stranded circular noncoding RNAs, which entirely rely on their RNA motifs to exploit cellular machinery for organelle entry and exit, cell-to-cell movement through plasmodesmata, and systemic trafficking. Viroids represent an excellent model to dissect the role of RNA 3-dimensional (3D) structural motifs in regulating RNA movement. Using nuclear-replicating viroids as a model, we showed that cellular Importin alpha-4 is likely involved in viroid RNA nuclear import, empirically supporting the involvement of Importin-based cellular pathway in RNA nuclear import. We also confirmed the involvement of a cellular protein (Virp1) that binds both Importin alpha-4 and viroids. Moreover, a conserved C-loop in nuclear-replicating viroids serves as a key signal for nuclear import. Disrupting C-loop impairs Virp1 binding, viroid nuclear accumulation and infectivity. Further, C-loop exists in a subviral satellite noncoding RNA that relies on Virp1 for nuclear import. On the other hand, no viroid can systemically infect the model plant Arabidopsis thaliana, suggesting the existence of non-host resistance yet to be understood. Here, we attempted to test whether a gene involved in RNA silencing, RNA-dependent RNA polymerase 6 (RDR6), plays a role in non-host resistance in Arabidopsis. I will discuss the data below in detail.

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