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The synthesis of the tertbutyldimethylsilyl derivatives of adenosine is described. The 2',5'-protected nucleosides were rapidly and easily prepared in relatively good yields. / The silylated nucleosides are easily incorporated into ribonucleotides using the fast and efficient dichlorophosphite method. The ribonucleotide GpCpApApCpCpA corresponding to the 3'-terminus of / (DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI) / was successfully synthesized in a stepwise fashion. The stepwise and block condensation pathways were compared in the synthesis of CpCpCpCpC. The syntheses of A2'p5'A2'p5'A and other 2',5'- and symmetrically-linked ribonucleotides were readily accomplished. ('31)P NMR was very useful in elucidating the structure of the diribonucleoside monophosphates. A study of the effect of temperature and solvents on yield of final product is undertaken. Various phosphate protecting groups are also evaluated. / A suitable deprotection procedure is investigated and the identity of the phosphate linkages confirmed by enzyme degradations.

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RNA interference (RNAi) is a gene silencing mechanism induced by double-stranded RNA (dsRNA). RNAi has been used extensively to create loss-of-function mutants in many species to identify the functions of genes, but it also has the potential to be used as a species-specific pesticide if the dsRNA can silence essential genes in pests. The mosquito Aedes aegypti is a vector of numerous viruses including Dengue and West Nile virus, and is frequently controlled by chemical insecticides. With growing concerns about the extensive use of broad-spectrum pesticides, new control methods are eagerly sought. In this study, I examined the efficacy of feeding pesticidal dsRNAs to mosquito larvae. A dose-dependent RNAi response and mortality was observed when larvae were fed dsRNA targeting several different genes. Unlike RNAi in the related dipteran Drosophila melanogaster, RNAi in A. aegypti also appeared to be systemic, spreading beyond the gut to other tissues. A degree of species-specificity was also observed, as dsRNA specific to the D. melanogaster β-tubulin gene killed D. melanogaster larvae but did not kill mosquito larvae. RNAi was also used to determine the function of a newly-identified A. aegypti cytochrome P450 (CYP) gene, Aacyp. This gene showed male-biased expression in the mosquitoes, and was expressed primarily in the male abdomen and/or thorax, but unlike some other insect male-biased CYPs, Aacyp was not highly expressed in the reproductive structures. While dsRNA injections successfully knocked down expression of Aacyp, no discernable change in reproductive or male-specific behaviours were noted. Nevertheless, RNAi is still considered a highly versatile tool for both gene function studies and has promising potential to be developed into a novel class of pesticides.

In this thesis I extend a class of grammars called conjunctive grammars to a stochastic form called stochastic conjunctive grammars. This extension allows the grammars to predict pseudoknotted RNA secondary structure. Since observing sec- ondary structure is hard and expensive to do with today's technology, there is a need for computational solutions to this problem. A conjunctive grammar can handle pseudoknotted structure because of the way one sequence is generated by combining multiple parse trees. I create several grammars that are designed to predict pseudoknotted RNA sec- ondary structure. One grammar is designed to predict all types of pseudoknots and the others are made to only predict a pseudoknot called H-type. These grammars are trained and tested and the results are collected. I am able to obtain a sensitivity of over 75% and a speci city of over 89% on H-type pseudoknots

Ebola virus (EBOV) is an enveloped, negative-sense single-stranded RNA virus that causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes multiple transcripts due to RNA editing at a conserved editing site (ES) (a hepta-uridine stretche). The majority of GP gene transcript is unedited and encodes for a soluble glycoprotein (sGP); a defined function has not been assigned for sGP. In contrast, the transmembrane glycoprotein (GP1,2) dictates viral tropism and is expressed through RNA editing by insertion of a nontemplate adenosine (A) residue. Hypothetically, the insertion/deletion of a different number of A residues through RNA editing would result in another yet unidentified GP gene product, the small soluble glycoprotein (ssGP). I have shown that ssGP specific transcripts were indeed produced during EBOV infection. Detection of ssGP during infection was challenging due to the abundance of sGP over ssGP and the absence of distinguishing antibodies for ssGP. Optimized two- dimensional (2-D) gel electrophoresis verified the expression of ssGP during infection. Biophysical characterization revealed ssGP is a disulfide-linked homodimer that is exclusively N-glycosylated. Although ssGP appears to share similar structural properties with sGP, it does not have the same anti-inflammatory function. Using a new rapid transcript quantification assay (RTQA), I was able to demonstrate that RNA editing is an inherent feature of the genus Ebolavirus and all species of EBOV produce multiple GP gene products. A newly developed dual-reporter minigenome system was utilized to characterize EBOV RNA editing and determined the conserved ES sequence and cis-acting sequences as primary and secondary requirements for RNA editing, respectively. Viral protein (VP) 30, a transcription activator, was identified as a contributing factor of RNA editing— a proposed novel function for this largely uncharacterized viral protein. Finally, I could show that EBOV RNA editing is GP gene-specific because a similar sequence located in L gene did not serve as an ES, most likely due to the lack of the necessary cis-acting sequences. In conclusion, I identified a novel soluble protein of EBOV whose function needs further characterization. I also shed light into the mechanism of EBOV RNA editing, a potential novel target for intervention.