Thesis (Ph. D.)--University of Rhode Island, 2003. / Typescript. Includes bibliographical references (leaves 170-182).

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains v, 137, [79] p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 130-137).

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 216-219).

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Advanced glycated end-products (AGEs) are a heterogenous group of compounds formed through the Maillard reaction. During AGE formation, reactive α-dicarbonyls are formed, such as glyoxal (GO) and methylglyoxal (MG). These α-dicarbonyls are present at elevated concentrations in diabetes, and are frequently implicated in the initiation and progression of diabetic complications. Previous research has linked α-dicarbonyls with formation of reactive oxygen species (ROS) and inflammation. However, much of the prior work has been conducted using concentrations of α-dicarbonyls that are substantially higher than can be generated in vivo, and the biochemistry has been investigated under conditions (e.g. pH) outside the physiological range. The aim of the work presented in this thesis was to test the hypothesis that GO and MG are pro-oxidant and pro-inflammatory at (patho)physiological concentrations in both monocytes and pancreatic β-cells. In this work several new and important observations have been made regarding the action of α-dicarbonyls on oxidative stress and inflammation. 1) The amount of oxidative species production by α-dicarbonyls in glycation reactions with amino acids and proteins may be so low as to be negligible in vivo, despite previous evidence to the contrary. 2) α-dicarbonyls did not appear to generate oxidative stress within inflammatory cells nor pancreatic β-cells by depleting the levels of GSH. 3) At least in the β-cell model, the mechanism of action of the α-dicarbonyls did not involve dysregulation of the antioxidant SOD enzymes. 4) Neither α-dicarbonyl significantly affected insulin production by β-cells, except at cytotoxic concentrations. 5) Treatment of inflammatory cells with α-dicarbonyls induced release of the proinflammatory cytokine IL-8. 6) In both immune cells and pancreatic β-cells, α-dicarbonyls were involved in O2.- generation by activation and/or upregulation of NADPH oxidase. 7) Despite the structural similarities of α-dicarbonyls, they have distinct mechanisms of action with respect to oxidative stress.

The immunoglobulin-like, transmembrane Advanced Glycation End product (AGE) Receptor (RAGE) is a pattern recognition receptor implicated in the transduction of pro-inflammatory signalling and processes. Over the past decade a substantial body of evidence has accrued implicating RAGE in the pathogenesis of several chronic inflammatory and vascular diseases such as diabetes, rheumatoid arthritis, amyloidosis, atherosclerosis and renal failure. More recently RAGE has been linked to cancer progression, possibly through its role in the inflammatory process. AGE products have been shown to exert their intracellular effects through ligation of their cognate receptor RAGE and the subsequent transactivation of NFKB signalling in several cellular contexts. Polycystic Ovary Syndrome (PCOS) is a reproductive endocrine disorder characterized by hyperandrogenism, chronic anovulation and insulin resistance, thus increasing the risk of diabetes mellitus in these patients. Non-enzymatically glycated AGEs are formed at an accelerated rate and accumulate in tissues in conditions of high glucose and oxidative stress. Interestingly, young normoglycemic women with PCOS exhibit higher serum AGE levels and increased RAGE expression in poly-cystic ovaries. RAGE is also regulated through the activity of the estrogen receptor (ER). The natural cyclical expression of estrogen throughout the menstrual cycle is perturbed in endometriosis even post menopause, suggesting that RAGE could also be dysregulated. Finally PCOS has been implicated in increased risk to endometrial cancer progression as has uterine exposure to the selective estrogen receptor modulator Tamoxifen (TX) therefore it is plausible that RAGE has a function in this disease. Objectives: The principal aims of this thesis were to characterise RAGE expression for the first time in fertile and infertile endometriotic and PCO human endometrium, and to initiate RAGE characterisation in endometrium obtained from patients with endometrial hyperplasia and cancer. Secondly, this thesis endeavoured to elucidate the transcriptional mechanisms regulating RAGE in vitro response to 17beta-estradiol and AGEs which are elevated in endometriosis and PCOS pathology respectively, and in endometrial cancer. Methodology: This project employed the use of real time Polymerase Chain Reaction (RT-PCR), Chromatin Immunoprecipitation (ChIP), Immunohistochemistry (IHC) and western blotting (WB). Results: Immunohistochemistry and RT-PCR data revealed that basal RAGE expression was significantly greater in PCO and endometriotic endometrium when compared to fertile controls, and significantly elevated in two cancer patients. RAGE was also characterised in endometrial cell models in which it was shown to be modulated at the mRNA and protein level by AGE-HSA, 17beta-estradiol (E2) and its antagonist 4-hydroxytamoxifen. Moreover, we have shown that RAGE is modulated by two distinct pathways through the estrogen receptor (ER) and NFKB. Novel ChIP results confirmed the presence of p65 and ER-alpha on the RAGE promoter at non- classical Spl and Apl sites in response to AGEs, E2 and TX. Conclusions: The results in this thesis may implicate endometrial RAGE expression in the infertility evident in women with PCOS and endometriosis. Furthermore, recent evidence implicates RAGE in mediating inflammation-driven tumourigenesis. Thus, over-expression of endometrial RAGE in PCOS and endometriosis, and in patients receiving tamoxifen for breast cancer treatment may predispose these women to an elevated risk of cancer.

N-linked protein glycosylation involves the transfer of a glycan onto an Asparagine residue (N) of a polypeptide chain. It is common in Eukaryotes and has recently been observed in Prokaryotes, most notably in Campylobacter jejuni. The C. jejuni N-linked glycosylation system is encoded on a single pgl gene locus that also functions when expressed in Escherichia coli. The key enzyme involved in N-linked protein glycosylation is encoded by the pglB gene and transfers lipid-linked glycan onto N residues of glycoproteins in the periplasm. It is clear from accumulating genome sequence data that pglB orthologues are present in all Campylobacter species and in related species such as Wolinella succinogenes, Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Most Helicobacter species, including Helicobacter pylori, lack the pglB gene but three related Helicobacter species Helicobacter pullorum, Helicobacter canadensis and Helicobacter winghamensis have two distinct pglB genes. These and other orthologues of C. jejuni pgl genes are located not within a single locus but rather at five distinct loci. One of the two pglB genes, termed pglB1, is required for in vitro N-glycosylation of peptides (Jervis et al., 2010). In this thesis I present data on the role of further pgl gene orthologues and previously uncharacterized genes in H. pullorum N-glycosylation. Furthermore I have also identified a number of H. pullorum glycoproteins and provide data comparing N-glycosylation processes in C. jejuni and H. pullorum. These data expand our preliminary observations on the first Helicobacter N-linked glycosylation system, and provide important information on the similarities and differences between the well characterised C. jejuni system and these more recently identified pathways.

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