Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

The study aimed to investigate the following: 1. Investigate a putative glutathione peroxidase gene (Glyma17g34110) within Glycine max by an in silico analysis and spatial expression. 2. Determine the effects of exogenously applied nitric oxide on the expression of Glyma17g34110. 3. Investigate the antioxidant mechanism with attention to Glyma17g34110,reactive oxygen species and cell death in the response to salt stress. 4. Establish whether Glyma17g34110 is a glutathione peroxidase or thioredoxindependent peroxidase gene. / Magister Scientiae - MSc

The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress. / >Magister Scientiae - MSc

目錄…………………………………………………………………………I 圖目錄……………………………………………………………………IV 表目錄……………………………………………………………………VI 縮寫表……………………………………………………………………VII 謝誌………………………………………………………………………IX 中文摘要…………………………………………………………………XI 英文摘要…………………………………………………………………XIII 第壹章、前言………………………………………………………………1 第貳章、文獻探討…………………………………………………………3 第2-1節 紫外線…………………………………………………….…..4 2-1-1. 紫外線……………………………………………………….4 2-1-2. 紫外線的分類……………………………………………….5 2-1-3. 紫外線對人體健康的益處………………………………….6 2-1-4. 紫外線對人體的傷害……………………………………….7 第2-2節 自由基…………………………………………………………8 2-2-1. 自由基的介紹……………………………………………….8 2-2-2. 自由基的來源……………………………………………….8 2-2-3. 紫外線與自由基…………………………………………….9 2-2-4. 自由基種類………………………………………………….9 第2-3節 抗氧化酵素訊息的傳遞路徑………………………………..12 2-3-1. Nf2…………………………………………………………..12 2-3-2. Keap1………………………………………………………..12 2-3-3. ARE………………………………………………………….13 2-3-4. Nrf2的活化機制…………………………………………….14 第2-4節 細胞抗氧化的防禦系統……………………………………17 2-4-1. 酵素型抗氧化防禦系統…………………………………..18 2-4-2. 非酵素型抗氧化防禦系統………………………………...24 第參章、研究動機與實驗設計架構圖……………………………………...25 第3-1節 硏究動機……………………………………………………..26 第3-2節 實驗設計架構………………………………………………..27 第肆章、實驗材料與方法……………………… …………………………28 第4-1節 實驗材料……………………………………………………...29 第4-2節 實驗儀器……………………………………………………...32 第4-3節 實驗方法……………………………………………………...33 4-3-1. 細胞培養…………………………………………………...33 4-3-2. 存活率試驗………………………………………………...36 4-3-3. ROS產量測定………………………………………………38 4-3-4. 細胞毒性的測定…………………………………………...40 4-3-5. Comet assay彗星試驗………………………………………41 4-3-6. 細胞GSH含量測定………………………………………..44 4-3-7. 細胞凋亡試驗……………………………………………...46 4-3-8. 細胞總蛋白質萃取………………………………………...48 4-3-9. 細胞核與細胞質之蛋白質萃取…………………………...49 4-3-10. 蛋白質定量……………………………………………….51 4-3-11. 西方墨點分析…………………………………………….52 4-3-12. 免疫螢光染色…………………………………………….56 4-3-13. 轉染作用………………………………………………….58 4-3-14. 冷光酵素報導基因分析………………………………….60 4-3-15. RNA干擾…………………………………………………62 4-3-16. 統計分析………………………………………………….64 第伍章、實驗結果與圖表…………………………………………………65 第5-1節 ERG對HaCaT細胞的抗光氧化能力………………………66 5-1-1. ERG以及UVA對HaCaT細胞的細胞毒性影響…………66 5-1-2. ERG對UVA誘導活性氧物種含量之影響………………..66 5-1-3. ERG對UVA誘導的細胞膜損傷之影響…………………..67 5-1-4. ERG對UVA誘導的DNA損傷之影響……………………67 5-1-5. ERG對UVA所誘導之氧化傷害的影響…………………..68 5-1-6. ERG對UVA引發之細胞凋亡的影響……………………..68 第5-2節 ERG誘導之抗氧化系統的機制探討………………………..79 5-2-1. ERG對抗氧化酵素的影響…………………………………79 5-2-2. ERG活化Nrf2路徑探討……………………………………80 5-2-3. ERG影響Nrf2入核的表現………………………………..80 5-2-4. ERG對抗氧化酵素轉錄的影響……………………………81 5-2-5. 以siNrf2探討ERG的保護機制和Nrf2之間的關係……81 第陸章、討論………………………………………………………………95 第6-1節 ERG對於UVA刺激HaCaT細胞造成的細胞損傷之影響..97 第6-2節 ERG對抗氧化基因的表現之影響………………………….97 第6-3節 ERG與UVA影響Nrf2-Keap1 pathway……………………98 第6-4節 Nrf2對ERG所抑制UVA造成之光氧化傷害的影響……..98 第柒章、結論……………… …………………………………………..100 參考文獻 ………………….……………………………………………..102

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

Das Rett-Syndrom ist eine postnatal progressiv verlaufende neurologische Entwicklungsstörung, die x-chromosomal vererbt. Das klassische Rett-Syndrom entsteht durch eine spontane Mutation des MECP2-Gens, welches für die Kodierung des Transkriptionsfaktors MeCP2 (methyl CpG binding protein 2) verantwortlich ist. Das Krankheitsbild verläuft in vier Stadien und ist vor allem von geistiger Retadierung, motorischer Dysfunktion und Unregelmäßigkeiten der Atmung geprägt. In verschiedenen Untersuchungen wurden bereits verschiedene zelluläre Dysfunktionen und Beeinträchtigungen der Mitochondrien bestätigt, weshalb wir weitere Untersuchungen anstrebten. Ziel dieser Dissertation war es, mögliche Einflüsse verschiedener Enzyme auf die ROS-Produktion zu untersuchen und somit die Erkenntnisse aus vorangegangenen Arbeiten zu erweitern. Im Fokus dieser Untersuchungen lagen zum einen die mitochondriale ROS-Produktion sowie die extramitochondriale ROS-Produktion. Durch unterschiedliche pharmakologische Modulationen wurden beide Systeme beeinflusst, um den jeweiligen Beitrag zur gesamten ROS-Produktion abzuschätzen.

Genetiniai ir funkciniai tyrimai parodė, kad oksidacinį stresą sukeliančios reaktyvios deguonies formos (angl. Reactive oxygen species, ROS) užima svarbų vaidmenį uždegiminių žarnų ligų (UŽL) patogenezėje. NADPH oksidazės šeimos fermentai (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 ir Duox2) - svarbus ROS šaltinis organizme. Šių fermentų gaminamos ROS molekulės veikia kaip antriniai signalų nešikliai, kurie aktyvina bei reguliuoja įvairius biologinius procesus, tokius kaip augimas, ląstelių diferenciacija, apoptozė ar uždegimas. NADPH oksidazės izoformos randamos gaubtinės žarnos ir virškinamojo trakto barjeriniuose audiniuose, turi tiesioginį kontaktą su žarnų simbiotinėmis bei patogeninėmis bakterijomis. Todėl manoma, kad šių fermentų biologinės funkcijos gali būti susijusios su įgimtais apsauginiais, gynybiniais mechanizmais, ląstelės signaliniais keliais bei virškinamojo trakto uždegiminių ligų patogeneze. Darbo tikslas – ištirti epitelio ląstelių NADPH oksidazės reikšmę žarnų uždegimo patogenezei, sergant opiniu kolitu ir DSS sukeltu kolitu. Mūsų darbas atskleidė NADPH oksidazės svarbą žarnų uždegimo patogenezei. Mes pirmieji parodėme, jog NADPH oksidazės fermentų slopinimas pasižymi uždegimą malšinančiu poveikiu pirminėms žmogaus ir pelių žarnų epitelio ląstelėms in vitro. / Genetic and functional studies indicated the importance of reactive oxygen species (ROS) induced oxidative stress in the development of chronic inflammatory bowel disease (IBD). NADPH oxidase enzymes (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2) - are the important source of ROS in the organism. These enzymes - derived ROS act as intracellular second messengers for a variety of cellular receptor signal transduction pathways, and they play pivotal roles in various biological activities, including host defence, cell growth and differentiation, stimulation of pro-inflammatory genes, and cell death. The epithelial NADPH oxidase isoforms are highly expressed in the colon, particularly in the colon epithelial cells. These enzymes come into close contact with normal and pathogenic bacteria and may play an important role in local innate immune, cell signalling pathways and inflammation in the gut. The aim of this study is to investigate the role of NADPH oxidase of colon epithelial cells in the pathogenesis of colon inflammation during ulcerative colitis and DSS-induced colitis. The results of our study revealed the importance of NADPH oxidase in the pathogenesis of colon inflammation. Moreover, we are the first to show that the treatment with NADPH oxidase inhibitors has a protective effect against pro-inflammatory action of LPS in human and mice primary colon epithelial cells in vitro.

Atrial fibrillation (AF), the most common arrhythmia encountered in clinical practice, is a major source of morbidity and mortality, and is highly associated with inflammation and oxidative stress. In the present study, we show that acute exposure of mice atrial tissue to tumor necrosis factor-α (TNF-α) increases susceptibility to AF. We further show that acute exposure to TNF-α led to increased spontaneous sarcoplasmic reticulum (SR) calcium release and generated triggered activities in isolated mice atrial myocytes. This increase in spontaneous SR calcium activity was found to be due to elevated reactive oxygen species production from mitochondria and NADPH oxidase sources triggered by TNF-α. Hence we concluded that acute exposure to TNF-α leads to elevated oxidative stress that increases spontaneous SR Ca2+ release and triggered activity through which it can lead to AF induction and maintenance