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).

No description available.

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.

No description available.

No description available.

Introduction: Alpha-2-macroglobulin (α2m) is a plasma glycoprotein serine protease inhibitor. Previous studies have shown that coagulation factor concentrations are highly variable with age and α2m levels have been found to be twice as high in newborns compared to adults. This may contribute to a resistance towards thrombotic events observed in young populations. Protein glycosylation is known to affect protein activity and the glycosylation profile of adult α2m has previously been analyzed. Information regarding glycosylation of α2m in other age groups has yet to be elucidated. Therefore, the purpose of this study is to examine the differences in the glycosylation profiles between newborn and adult α2m. Methods: To evaluate glycan macroheterogeneity, plasma samples were enzymatically deglycosylated by PNGaseF, followed by SDS PAGE and western blotting (WB) to detect α2m. To evaluate microheterogeneity, plasma samples were incubated with Neuraminidase (Clostridium perfringens) followed by native PAGE and WB to determine sialic acid content. To detect non-sialylated terminal galactose residues, plasma samples were incubated with immobilized RCA120, and lectin-bound molecules were separated from unbound molecules. Additionally, the affinity of α2m for ricin was evaluated by eluting bound proteins with increasing concentrations of galactose. All fractions were subjected to SDS-PAGE and WB to detect α2m. 2D gel electrophoresis was completed to examine differences in pI and molecular weight of α2m in both age groups. Purification by immunoprecipitation was also performed and eluted α2m was analyzed by fluorescence-assisted carbohydrate electrophoresis (FACE) to determine the glycan fingerprint in the two populations. Results: Deglycosylation of both newborn and adult α2m with PNGaseF resulted in a change in migration and apparent molecular weight, however no statistically significant difference was found between newborn and adult. On native PAGE following treatment with neuraminidase, newborn α2m exhibited a statistically significant change in migration compared to adult. Additionally, newborn α2m exhibited a higher percentage of molecules bound to RCA120 than adult (no statistical difference) and elution of α2m from RCA120 with a galactose step gradient produced similar profiles for newborn and adult molecules. 2D electrophoresis and WB revealed a difference in pI of α2m in newborns as compared to adults. Finally, purified newborn and adult α2m were analyzed by FACE and quantification of prominent fluorescent bands revealed a higher secondary:primary band ratio in newborns when compared to adults. Conclusions: To our knowledge, this is the first study investigating glycosylation differences between newborn and adult α2m molecules. The results from PNGaseF analyses indicate no significant difference in total N-glycan content. Neuraminidase results suggest significantly greater sialic acid presence on newborn α2m, however there was no significant difference in galactose content. 2D electrophoresis revealed a difference in pI as well as the way in which newborn and adult α2m degrade when exposed to experimental conditions. A2m was successfully purified from both newborn and adult plasma, and FACE results indicate that the proportion of more branched glycans present in the two major fluorescent bands are of higher quantity in newborns than adults. / Thesis / Master of Science (MSc)