Thesis (M.S.)--Springfield College, 2004. / Includes bibliographical references.

By immumolabeling and electronmicroscopy, the subcellular localization of GmPAP3 has been proved to be mainly localized in mitochondria, a primary site for ROS production. Ectopic expression of GmPAP3 in transgenic tobacco BY-2 cells mimicked the protective effects exhibited by the antioxidant ascorbic acid by: (1) increase the percentage of cells with active mitochondria; (2) reduce the percentage of dead cells; and (3) lower the accumulation of ROS under NaCl and osmotic stress treatments. However, when ectopically express a truncated GmPAP3 with the mitochondria transit peptide removed, such protective effect was not observed. This provides evidences on the significance of mitochondria localization to the physiological function of GmPAP3. In addition, when GmPAP3 transgenic Arabidopsis thaliana seedlings were subjected to NaCl, osmotic stress, and oxidative stress treatments, the growth performance of the transgenic lines was significantly better than the wild type. To summarize, these studies has demonstrate that the mitochondrial localized GmPAP3 may play a role in stress tolerance by enhancing ROS scavenging. / Mitochondrion is one of the major sites for the production of reactive oxygen species (ROS). Abiotic stresses such as salinity and osmotic stress can cause oxidative damage to organelle membranes due to excess accumulation of ROS. The inducibility of GmPAP3 gene expression by salinity and oxidative stresses and the putative mitochondrial localization of GmPAP3 prompt us to further investigate the possible physiological roles of GmPAP3 under abiotic stress-induced oxidative stress. / My Ph.D. study has been focused on the detailed functional analysis of the GmPAP3 gene. The objectives of my research include: (i) to verified the subcellular localization of GmPAP3; (ii) to investigate the physiological functions of GmPAP3 under NaC1 and osmotic stress in both cellular level and in planta level. and (iii) to examine the significance of mitochondria] localization of GmPAP3 in relationship to its protective roles. / Purple acid phosphatases (PAPs) represent a diverse group of acid phosphatases in animals and plants. While the mammalian PAPs were found to be related to Reactive Oxygen Species (ROS) evolution in important physiological functions, the roles of plant PAPs remain largely unknown. / Recently, we have isolated a novel PAP-like gene (GmPAP3) from soybean that is induced by NaC1 and oxidative stresses. Subcellular localization prediction programs suggested that GmPAP3 may be a novel PAP that localized in mitochondria. Most other PAPs are extracellularly located and membrane localization of PAPs was only verified in a few cases. / by Li, Wing Yen Francisca. / "December 2009." / Adviser: Lam Hon-Ming. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 123-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

o,o???-Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection. The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported. An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer???s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer???s and control subjects. Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer???s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.

Bibliography: leaves 162-174.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 150 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xxiii, 236 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 220-236).

When proteins are unable to fold properly in the endoplasmic reticulum (ER), the resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress often have a higher abundance of reactive oxygen species (ROS). Previous studies suggest that ROS could aggravate ER stress by further disrupting the ER protein folding process. More recent studies suggest that the unfolded protein response signaling pathways activated by ER stress could lead to the production of ROS. Such studies lead to the hypothesis that ER stress could be promoted by ROS, and vice versa. The aim of the present study is to test the above hypothesis by studying how ROS could be generated in ER-stressed cells. This is followed by investigating if ROS could increase or decrease the level of ER stress in cells. Finally, the extent of ER stress induced cell death in the presence and absence of ROS is assessed. The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress inducing agent, resulted in the elevation of intracellular ROS that could be detected with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not dihydroethidium which is relatively specific towards superoxide anion. The Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol reductants such as dithiothreitol and N-acetylcysteine but not with the free radical scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be prevented by the over-expression of catalase in HeLa. These data is consistent with the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells. In addition to elevation of ROS level, HeLa cells treated with tunicamycin also resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the presence of cycloheximide to inhibit protein synthesis so as to deplete protein substrates for folding in the ER, tunicamycin-induced ER stress was greatly minimized as was evident by the absence of both the phosphorylation of PERK and splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of DCF-detectable ROS remained unaffected. The cycloheximde-resistant phosphorylation of eIF2α could be prevented when cells were co-treated with thiol reductants, or upon the over-expression of catalase. These data suggest that the production of ROS in Tm-treated cells does not require the presence of ER stress as a prerequisite. Furthermore, the ROS so produced could induce phosphorylation of eIF2α without the need to cause ER stress in the first place. The quenching of ROS through the use of thiol reductants, or the over-expression of catalase, had no effect on inhibition of protein synthesis in cells treated with tunicamycin. However, the extent of cell death was significantly increased. The data obtained in this study is not consistent with the idea that ROS is a downstream product of ER stress, capable of inducing more ER-stress by a feedback mechanism. Therefore, a mutually enhancing effect between ER stress and ROS may not exist. The ROS found in stressed cells may serve to extend cellular survival under the condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Caenorhabditis elegans clk-1(qm30) and isp-1(qm150) mutants exhibit highly pleiotropic phenotypes that include slow development and long lifespan. clk-1(qm30) and isp-1(qm150) correspond to loss of function mutations in genes necessary for ubiquinone biosynthesis and complex III electron transport, respectively. Previous research has lead to the hypothesis that altered levels of cellular reactive oxygen species (ROS) may underlie clk-1(qm30) and isp-1(qm150) mutant phenotypes. To test this hypothesis RNA interference (RNAi) by feeding was used to indirectly alter cellular ROS levels by knocking down genes that encode ROS detoxification enzymes. Specifically, genes that detoxify ROS using glutathione or thioredoxin, both of which are important cellular thiol-redox molecules, were knocked down to examine the role of ROS in determining clk-1(qm30) and isp-1(qm150) lifespan, post-embryonic development, and germline development. In summary, knocking down ROS detoxification genes does not severely appear to affect the phenotypes that were studied. ROS detoxification gene knockdowns consistently induced mild decreases in wild type, clk-1(qm30), and isp-1(qm150) lifespan. However, knocking down NAD+-dependent isocitrate dehydrogenases, which are not closely involved in ROS detoxification, similarly affected lifespan, indicating that decreases are not specific to ROS detoxification. Of note, knocking down gcs-1, which is required for glutathione biosynthesis, induced lethal intestinal abnormalities in wild type, c1k-1(qm30), and isp-1(qm150) worms. Overall, findings do not support that low ROS underlies the clk-1(qm30) and isp-1(qm150) mutant phenotypes.

o,o???-Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection. The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported. An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer???s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer???s and control subjects. Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer???s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 137-158). Also available for download via the World Wide Web; free to University of Oregon users.