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<p> A variety of important cell functions rely on O-GlcNAcylation of proteins, a kind of post-translational glycosylation that modifies nuclear and cytoplasmic proteins on serine or threonine residues by the addition of the single sugar moiety β-N-acetylglucosamine (O-GlcNAc). Generally, two enzymes can catalyze the formation and cleavage of the O-GlcNAc O-glycosidic linkage. The O-GlcNAc transferase (OGT) catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to the hydroxyl group of specific Thr and Ser residues. On the other hand, hydrolysis of the sugar moiety from proteins is achieved via the bi-functional nuclear cytoplasmic O-GlcNAcase and acetyltransferase (NCOAT). Since O-GlcNAcylation plays very important roles in many cell processes such as transcription, translation and protein-protein interaction, my research mainly focused on preparing compounds and developing methods to study the O-GlcNAcylation process and discover the structural information of OGT and NCOAT.</p> <p> In this thesis, the important enzyme substrates (YSDSPSTST for OGT and YSDSP(O-GlcNAc-Ser)TST for NCOAT) were prepared by solid phase synthesis and purified by preparative reverse phase HPLC. Their structure and sequence were confirmed by ESI-MS and tandem MS (MS/MS). The building block S-GlcNAc-Ac3-Ser-COOBn for preparing S-GlcNAcylated peptides was prepared by a multistep procedure. Two potent photoaffinity labeled probes for mapping the active site of sOGT were designed and synthesized, and their IC50 values for sOGT were measured by CapLC and a beta-elimination method. Also, many analytical methods were developed for studying the O-glcNAcylation, including discovering the O-Glycosylation site of peptides by MS/MS and beta-elimination methods, sequencing peptides by MS/MS, analyzing the protein digests by CapLC-MALDI MS and studying the ion mobility of peptides and glycopeptides using ion mobility spectrometry.</p> / Thesis / Master of Science (MSc)

Insects and their natural enemies are engaged in a never-ending battle called the ‘co-evolutionary arms race.’ As a part of these contentious interactions, vulnerable insects evolve natural barriers that prevent successful attacks by their natural enemies. In response, natural enemies evolve strategies that overcome these barriers. Occasionally, microbial symbionts will also participate in these relationships by assisting their insect host in defense against natural enemies or by assisting the natural enemy in subduing its prey. Alternatively, microbial symbionts may become contenders themselves in the co-evolutionary arms race by becoming reproductive parasites of their hosts. To mediate successful outcomes in these relationships, microbial symbionts will often employ diverse protein toxins capable of manipulating and/or harming eukaryotic targets. In this dissertation, I study vertically transmitted Spiroplasma symbionts to address pressing questions about the evolution of symbiont protein toxins involved in insect manipulation and defense. In chapter II, I explore the genome of the first strain of Spiroplasma capable of inducing cytoplasmic incompatibility (CI) - a form of reproductive parasitism. I use bioinformatic techniques to look for potential protein effectors of CI and demonstrate that Spiroplasma evolved this intricate form of reproduction manipulation independent of other symbionts. In chapter III, I use bioinformatic approaches to characterize the expansion and diversification of multiple protein toxin families present in Spiroplasma. I identify dynamic evolutionary processes responsible for expanding and diversifying these toxin families and uncover a striking genus-wide association between protein toxin-associated domains in Spiroplasma and Spiroplasma transmission method. In chapter IV, I explore how protein expansion and diversification have influenced toxin function. Through molecular experiments with diverse Spiroplasma ribosome-inactivating protein (RIP) toxins, I implicate neofunctionalization as a common outcome in RIP toxin expansion. Lastly, in chapter V, I focus on the interactions between host and parasite by describing the first parasitoid wasp known to attack the adult stage of Drosophila hosts. This work introduces a new Drosophila-wasp study model for future novel studies into parasitoid-host interactions. Overall, this dissertation addresses broad questions about the evolution and origins of host, symbiont, and natural enemy interactions, and provides new tools and methods for future investigations.

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/07/24). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.