Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

The archaeal flagellum is a unique motility structure. Although functionally similar to the bacterial flagellum, the archaeal flagellum shares more similarities to the bacterial type IV pilus. Using the methanogenic archaea Methanococcus voltae and Methanococcus maripaludis as model organisms, the structural and post-translational requirements for flagellation have been investigated. Known to contain glycosylated flagellin proteins, the N-glycosylation pathway was studied in M. voltae. A number of possible glycosylation component genes, including glycosyl transferases, flippases and an oligosaccharyl transferase were inactivated or deleted in M. voltae and their resulting phenotypes were characterized. Four glycosyl transferases were identified as involved in the assembly of the M. voltae glycan structure, with three of these enzymes, AglA, AglC1 and AglC2, experimentally verified. As well, the oligosaccharyl transferase, AglB, has also been experimentally confirmed and was found to be the homolog of the bacterial and eukaryotic equivalents, PglB and Stt3p, respectively. Disruption of glycan synthesis or attachment resulted in very poorly or non-flagellated cells, implicating for the first time that the glycan structure on archaeal flagellins is necessary for proper flagella assembly and/or stability. These findings also represent the first proven N-glycosylation genes within the domain Archaea. New markerless, in-frame deletion methodology has allowed for advanced studies of a demonstrated and putative set of flagella-related genes in M. maripaludis. This collection of 11 co-transcribed genes, consisting of three flagellin genes (flaB1-flaB3), six genes of unknown function (flaC-flaH) and two genes implicated in flagellin subunit export (flaI and flaJ), make up the fla operon in this organism. Each gene from flaB1-flaI was systematically targeted for deletion and complementation to determine its necessity for flagellation. The analysis showed that both major flagellins, FlaB1 and FlaB2 are required for flagellation, while the minor flagellin, FlaB3, was required for the hook-like region of the flagella filament. FlaC, FlaF, FlaG and FlaH were shown for the first time to be essential for flagellation, while a naturally-occurring, truncated version of FlaD was found not to be required. These results continue to develop our understanding of the archaeal flagellum and the components necessary for its assembly and/or structure. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2007-07-11 16:50:37.119

A large-scale procedure was used to screen for deletions affecting growth of Saccharomyces cerevisiae when combined to ALG6 , ALG88, ALG10, CWH41, ROT2 or CNE1 deletions. 40 genes, grouped in 8 functional categories, were found to interact with the 6 query genes. The resulting network of 61 synthetic interactions was composed of 3 subnetworks, the N-glucosyltransferase (ALG6, ALG8, ALG10), the glucosidase (CWH41, ROT2) and CNE1 interaction sets, respectively. Deletion in 34 interacting genes conferred calcofluor white hypersensitivity, strengthening the relationship between N-glycan glucosylation/processing and cell wall physiology. In addition, a genetic interaction was found between ALG6 and SEC53, the yeast homologues of human ALG6 and PMM2 genes involved in congenital disorders of glycosylation. The alg6sec53 double mutant shows a synthetic growth defect and a CPY underglycosylation. Since this synthetic interaction is conserved from yeast to mammals, this work proposes the use of SGA analysis as a tool to uncover digenic effects that may underlie complex human genetic disorders.

The PMT (Protein O-mannosyl transferase) family members are involved in the initial step of protein-O-mannosylation. A large scale procedure (Systematic Genetic Array) was performed using pmt1, pmt2, pmt3, pmt5, and pmt6 as query deletions and a set of 4700 non-essential array gene deletions, to screen for query/array double deletion mutant combinations affecting growth of Saccharomyces cerevisiae. This procedure revealed a genetic interaction network consisting of 53 interacting genes. Functional grouping of these 53 genes revealed 9 functional categories that were analyzed according to gene function to elucidate how they might buffer defects in protein-O-mannosylation. Synthetic genetic interactions were also identified between PMT family members demonstrating redundancies among them. Protein-O-mannosylation is a protein modification conserved from yeast to human. POMT1 and POMT2 (Human PMTs counterparts) catalyze mannosyl residue transfer in mammals, with mutations identified to be involved in Walker-Warburg Syndrome (WWS). ∼72 % of the genes in the yeast PMT genetic network have human homologs, and ∼55 % of these are associated with human disease. Using the yeast genetic interaction network as a model for human genetic interactions may help in the understanding of complex inherited human disease.

The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.

Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in this malignancy. Several sialyltransferase genes have been shown to be up-regulated at both mRNA and and protein levels in a number of cancers, including that of the ovary. In the present study, we have analyzed the glycosylation patterns of kallikrein 6 in the context of ovarian cancer. We have discovered that the carbohydrate structures found at the single N-glycosylation site of kallikrein 6 derived from ovarian cancer cells found in the ascites fluid of ovarian cancer patients is enriched in sialic acid moieties and has an increased branching pattern when compared to controls. We have also developed a reliable anion-exchange HPLC-based methodology capable of quantifying different glycoform subpopulations of kallikrein 6 in serum and other biological fluids, which was capable of differentiating between samples from ovarian cancer patients and healthy controls. A variety of classic molecular biology and mass spectrometry based techniques were utilized in these experiments. Based on the results of the analysis of kallikrein 6 glycosylation and other literature reports showing upregulated sialylation of proteins in ovarian cancer, we have also identified sialylated glycoproteins from ovarian cancer proximal fluids and conditioned media of ovarian cancer cell lines. Sialylated proteins were enriched utilizing lectin affinity or hydrazide chemistry. In total, 333 sialylated glycoproteins and 579 glycosylation sites were identified. A list of 21 potential candidate ovarian cancer biomarkers was produced from proteins that were identified solely in ovarian cancer proximal fluids, which could form the basis for any future studies.

The amino acid L-proline plays a critical role in many biological processes. Therefore, efforts have been made to understand and control its influence. Since glycosylation is a common post-translational modification known to affect the characteristics of peptides and proteins, in a series of experiments, the effects of glycosylation on the properties of L-proline in peptides have been investigated. A conformationally constrained C-glucosyl proline hybrid is introduced, which has the capacity to vary the N-terminal amide equilibrium in model peptides through derivatization of the carbohydrate scaffold. For the first time, a comprehensive study of the effects of O-glycosylation on the kinetics and thermodynamics of prolyl amide isomerization is reported. The O-glycosylation of 4-hydroxy-L-proline has different effects on amide isomerization depending on the stereochemistry of the 4-hydroxyl group, which alters the orientation of the glycan with respect to the prolyl side chain. 4S-Galactosylation of 4-hydroxy-L-proline affects both the amide isomer equilibrium and the rate of amide isomerization, whereas 4R-galactosylation does not measurably influence either parameter. However, close contacts between the carbohydrate and prolyl rings lead to changes in the conformation and stability of longer peptides. As an expansion on these initial model studies, the effects of prolyl O-glycosylation on the properties of model peptides of two extremely important structural proteins are investigated. O-Galactosylation of 4R-hydroxy-L-proline residues in collagen model peptides does not preclude formation of the collagen triple helix, where the anomeric linkage of the Hyp O-glycan has slightly different influences on the conformational stability of the peptides. Also, the O-galactosylation of Hyp residues in polyproline model peptides causes a large increase in conformational stability. In both cases, interactions between the glycan and the peptide backbone and changes in hydration are implicated in contributing to the conformational stabilization of the peptides. These studies demonstrate that both natural and unnatural glycosylation of L-proline can be used as a means to control amide isomerization, and can increase the conformational stability of peptides, properties that will likely contribute to the development of new biomaterials. Also, these experiments provide further insight into the broad role glycosylation plays in affecting peptide and protein structure.

Thesis (M.S.)--University of Toledo, 2007. / Typescript. "Submitted as partial fulfillments of the requirements for the Master of Science Degree in Chemistry." "A thesis entitled"--at head of title. Bibliography: leaves 56-65.

Thesis (Ph.D.)--York University, 2006. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 113-125). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR29510

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1998. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.