No description available.

No description available.

5‐羟色胺（5-HT）是一种强有力的血管活性神经递质，被广泛的应用在调节血管张力。当5‐HT 被释放后，会被单胺氧化酶（MAOs）催化的酶促反应代谢，从而产生不同的代谢产物，比如5‐HIAA，5‐HTOL 和过氧化氢（H₂O₂）。然而，5‐HT对于内皮细胞活性氧物种（ROS）的产生作用以及5‐HT 转运体，5‐HT 受体，MAOs和ROS 的产生伴随着细胞内钙变化是否参与了其中的信号传导尚未被阐明。所以，这个研究最初的目的是考查外源性加入的5‐HT 对于脐静脉内皮细胞中ROS产生的影响以及其潜在的生物机理。 / 数据清楚的显示在没有L‐NAME（一种抑制一氧化氮（NO）产生的抑制剂）预处理的情况下，5‐HT 并不能在脐静脉内皮细胞内产生显著性的ROS。然而，在L‐NAME 预处理的情况下，NO 的产生被完全抑制，我们观察到明显的显著性的线粒体内的ROS 产生。5‐HT 产生的线粒体ROS 可以被clorgyline（一种MAO‐A 抑制剂），indatraline（一种5‐HT 转运体阻断剂），LY272015（一种5‐HT‐2B 受体拮抗剂），ketanserin（一种5‐HT2A 受体拮抗剂），XeC（一种IP3 受体拮抗剂），Gd³⁺（一种非选择性TRP 通道阻断剂），BAPTA（一种强效钙离子螯合剂），PEG‐Catalase，U73122（一种选择性PLC 抑制剂）以及没有钙离子的培养基所阻止。同时，5‐HT介导的胞内钙离子变化被XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015 以及没有钙离子的培养基所阻止。另外，MAO‐A 基因敲除抑制了5‐HT 导致的线粒体ROS的产生却对5‐HT 介导的胞内钙离子变化没有影响。基于以上所述的结果，我们可以得出结论，通过5‐HT 转运体，5‐HT 被摄取入细胞内，然后通过MAO‐A 介导的酶促代谢反应，产生钙离子依赖性的线粒体内ROS 的产生，这一结论对于解释血小板聚集而引起的内皮细胞功能性障碍起到非常重要的作用。 / 根据前人所述，内皮细胞内产生的ROS 对于内皮细胞通透性变化有着重要的作用，但是5‐HT 诱导的脐静脉内皮细胞ROS 的增加是否会对内皮通透性有所影响并没有被说明。在这项研究中，我们设计了实验旨在测试平面细胞表面积（ PCSA ）， 跨内皮电阻（ TER ）， 细胞高度， 肌球蛋白轻链磷酸化（MLCphosphorylation）和肌动蛋白细胞骨架（F‐actin cytoskeleton）水平的变化。此外，b‐catenin 在ROS 引起的F‐actin cytoskeleton 重组中的作用也在我们的讨论范围之内。 / 数据表明，在L‐NAME 预处理的情况下，5-HT 降低了脐静脉内皮的PCSA，TER 以及细胞高度，却增加了MLCP 和与b‐catenin 表达负相关的F‐actincytoskeleton 的水平。这些作用明显被PEG‐Catalase 预处理和MAO‐A 基因敲除减弱，证明了5‐HT 通过MAO‐A 介导产生的H₂O₂ 可以增加内皮细胞的通透性。 / 据文献报道，不论内源性还是外源性的低浓度的H₂O₂ 都可以激活导致血管生成的信号通路。文献进一步表明5‐HT 可以通过特定的5‐HT 受体亚型促进各种类型的内皮细胞的血管生成。然而，5‐HT 诱导的H₂O₂ 对于脐静脉内皮的血管生成作用并没有被报道。我们通过最初的实验先验证5‐HT 对于内皮细胞增殖和迁移的影响，然后我们才去验证H₂O₂ 在其中的作用及其潜在的机理。 / 实验结果表明，在L‐NAME 预处理的情况下，不论是急性（30 分钟）还是慢性（24 小时）的5‐HT 的处理都可以导致脐静脉内皮细胞的迁移，而这个作用会被5‐HT‐2 受体拮抗剂ketanserin，LY272015，ROS 清除剂PEG‐Catalase 以及PI3K的抑制剂wortmannin 所抑制。同时，在L‐NAME 预处理下，5‐HT 增加了cortactin,p‐Akt 和 p‐eNOS 的蛋白表达量而并没有影响Akt, eNOS 和p‐cortactin 的蛋白表达量。而5‐HT 增加的p‐Akt 和p‐eNOS 的蛋白表达被wortmannin 和PEG‐Catalase所抑制。不论是在Cyuant 细胞增殖检测还是在BrdU 细胞增殖检测中，5‐HT 诱导了一种非显著性的DNA 合成的增加，并且再BrdU 细胞增殖检测中，增加了的DNA 合成被PEG‐Catalase 显著性降低。总结以上实验结果，我们可以得出结论，通过一种5‐HT‐2 受体介导的PI3K 依赖性通路，而不是cortactin 磷酸化依赖性的信号通，路5‐HT 可以引导内皮细胞迁移。 / 除此之外，ROS 也被印证可以加剧内皮细胞的炎症反应和加速内皮细胞的老化。因此，我们也观察了5‐HT 对于粘附蛋白比如ICAM‐1 和VCAM‐1 以及抗老化因子SIRT‐1 的表达是否有影响。数据表明，在L‐NAME 预处理的情况下，30 分钟的5‐HT 处理显著的增加了ICAM‐1，SIRT‐1 而不是VCAM‐1 的表达。同时，这些作用均可以被PEG‐Catalase 所抑制表明了5‐HT 通过诱导H₂O₂ 的产生来形式其促进炎症反应和抗衰老的作用。 / 最后，总结以上，通过抑制NO 的产生，5‐HT 可以通过MAO‐A 介导的酶促代谢反应在人体脐静脉内皮细胞线粒体诱导ROS 的产生。同时，5‐HT 诱导的H₂O₂参与了改变内皮细胞通透性，促进血管生成（内皮迁移）及炎症反应的过程。 / 5-Hydroxytryptamine (5-HT), a potent vasoactive neurotransmitter, is involved in the regulation of vascular tone. After its release, 5-HT is terminated at the nerve terminals via enzymatic metabolism catalyzed by monoamine oxidases (MAOs), resulting in the generation of different metabolites (e.g. 5-HIAA, 5-HTOL and H₂O₂). Our lab demonstrates for the first time that 5-HT-induced ROS production indeed occurs and therefore, the aim of this study is to investigate exogenously added 5-HT on ROS generation in human umbilical vein endothelial cells (HUVECs), in order to understand the mechanisms involved in 5-HT-induced ROS production. / Our results clearly demonstrated that in the absence of L-NAME(a NO production inhibitor), there wasno apparent ROS production induced by 5-HT. However, after the inhibition of NO synthesis by L-NAME, 5-HT caused a significant increase in mitochondrial H₂O₂ production. The 5-HT-induced mitochondrial H₂O₂ generation was sensitive to clorgyline (a MAO-A inhibitor), indatraline (a 5-HT transporter blocker), LY272015 (a 5-HT2B antagonist) and ketanserin (a 5-HT2A antagonist), Xextospongin C（XeC，a IP3 receptor antagonist), Gd³⁺ (a non-selective TRP channel blocker), BAPTA (a potent Ca²⁺ ions chelator), PEG-Catalase, U73122 (a selective PLC inhibitor), and in [Ca²⁺]o-free medium. Concurrently, 5-HT-mediated [Ca²⁺]i changes were sensitive to XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015, and in [Ca²⁺]o-free conditions. In addition, gene knockdown of MAO-A suppressed 5-HT-elicited H₂O₂ production with no effects on [Ca²⁺]i changes. Based on all the results above, we can conclude that 5-HT caused a Ca²⁺-dependent mitochondrial H₂O₂ generation via MAO-A-mediated metabolism with the pre-requisite uptake of 5-HT into HUVECs through 5-HT transporter. / ROS derived from endothelial cells have been implicated in changes in endothelial permeability, but whether 5-HT-induced H₂O₂ generation could alter endothelial cells permeability has as yet not been demonstrated. Here, we measured the planar cell surface area (PCSA), transendothelial electrical resistance (TER), cell height, myosin light chain phosphorylation and F-actin cytoskeleton level in response to 5-HT challenge to investigate the change of endothelial permeability. Moreover, the participation of β-catenin in regulation of F-actin cytoskeleton remodeling in ROS-modulated alteration in endothelial permeability was also investigated. Results indicated that in the presence of L-NAME, 5-HT reduced the PCSA, TER and cell height in HUVECs. In contrast, 5-HT (with L-NAME) increased myosin light chain phosphorylation (MLCP) expression and F-actin cytoskeleton level, which are negatively associated with β-catenin expression. All of these effects were ameliorated by pre-treatment of PEG-Catalase or gene knockdown of MAO-A, implying 5-HT can consistently elicit the increase in endothelial permeability via MAO-A mediated H₂O₂ generation. / Low dose of ROS from exogenous or endogenous source can activate signaling pathway that lead to angiogenesis.5-HT can promote endothelial angiogenesis through specific 5-HT receptor subtype in various endothelial cell types, but the concomitant ROS generation had not previously been indicated to play a role in the process. In this study, we seek to test out the effects of 5-HT on endothelial cells migration, and should there be a functional role for ROS in the process. / Our results revealed that in the presence of L-NAME, both acute (30 min) and chronic (24 hr) treatment of 5-HT caused HUVECs migration, the effects of which were reversed by pre-incubation of 5-HT-2 receptor antagonists, ketanserin, LY272015, ROS scavenger PEG-Catalase or selective PI3K inhibitor wortmannin. With L-NAME, 5-HT consistently increased cortactin, p-Akt and p-eNOS expression without affecting total Akt, eNOS and p-cortactin protein expression whereas the increased p-Akt and p-eNOS expression are suppressed by pre-treatment of wortmanin or PEG-Catalase. Both in Cyuant cell proliferation assay and BrdU assay, 5-HT caused a trend but non-significant increase in DNA synthesis whereas the pre-treatment of PEG-Catalase significantly suppressed cell proliferation in the BrdU assay. Based on these results, we can conclude that 5-HT elicits endothelial migration via 5-HT-2 receptor-mediated H₂O₂ generation in a PI3K-dependent pathway. Under this circumstance, cortactin phosphorylation-dependent pathway was excluded. / Besides, ROS is notorious for effects like aggravation of inflammation and acceleration aging processes. The investigation extends to looking at alterations ofexpression of adhesion protein including ICAM-1 and VCAM-1 in the inflammatory response pathway and also to looking at the major aging parameter SIRT-1 in the presence of 5-HT in endothelium. Our data showed that in the presence of L-NAME, 30 min treatment of 5-HT significantly increased ICAM-1 and SIRT-1 expression without altering VCAM-1 expression and the up-regulation of ICAM-1 and SIRT-1 expression was prevented by PEG-Catalase. / In conclusion, with the eradication of the influence of NO, 5-HT induced mitochondrial H₂O₂ production via MAO-A-mediated metabolism in HUVECs. At the same time, 5-HT-induced H₂O₂ generation was involved in increasing endothelial permeability, inflammation and angiogenesis (cell migration). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Qian. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 326-450). / Abstracts also in Chinese. / Zhang, Qian.

Ho, Ka Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-124). / Abstracts in English and Chinese. / Thesis committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Abbreviations --- p.x / Content --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species (ROS) --- p.1 / Chapter 1.1.1 --- Definition and examples --- p.1 / Chapter 1.1.2 --- Generation of ROS in biological systems --- p.2 / Chapter 1.1.3 --- Features of specif ic ROS --- p.3 / Chapter 1.1.3.1 --- Superoxide anion --- p.3 / Chapter 1.1.3.2 --- Peroxyl radical --- p.4 / Chapter 1.1.3.3 --- Hydrogen peroxide --- p.4 / Chapter 1.1.3.4 --- Hydroxyl radical --- p.5 / Chapter 1.1.4 --- Damaging effects of ROS on biomolecules --- p.5 / Chapter 1.1.4.1 --- Lipid peroxidation --- p.6 / Chapter 1.1.4.2 --- DNA damage --- p.8 / Chapter 1.1.4.3 --- Protein oxidation --- p.9 / Chapter 1.2 --- Antioxidants --- p.11 / Chapter 1.2.1 --- Introduction --- p.11 / Chapter 1.2.2 --- Mode of action --- p.11 / Chapter 1.2.3 --- Endogenous Antioxidants --- p.12 / Chapter 1.2.3.1 --- Antioxidant enzymes --- p.12 / Chapter 1.2.3.2 --- Antioxidant compounds --- p.15 / Chapter 1.2.4 --- Exogenous antioxidants --- p.16 / Chapter 1.3 --- Oxidative stress --- p.17 / Chapter 1.3.1 --- Balance between ROS and antioxidants --- p.17 / Chapter 1.3.2 --- Diseases associated with oxidative stress --- p.18 / Chapter 1.4 --- Previous studies on edible mushroom antioxidants --- p.19 / Chapter 1.4.1 --- Previous studies on Agrocybe aegerita --- p.20 / Chapter 1.5 --- Cell culture models for antioxidant research --- p.21 / Chapter 1.6 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Mushroom fruiting bodies --- p.24 / Chapter 2.1.2 --- Cell lines and their subcultures --- p.24 / Chapter 2.2 --- Principle of Methods and Procedures --- p.26 / Chapter 2.2.1 --- Sample preparation and extraction --- p.26 / Chapter 2.2.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.28 / Chapter 2.2.2.1 --- ABTS + scavenging activity --- p.28 / Chapter 2.2.2.2 --- Hydroxyl radical scavenging activity --- p.30 / Chapter 2.2.2.3 --- Hydrogen peroxide scavenging activity --- p.32 / Chapter 2.2.3 --- Total phenolic content --- p.34 / Chapter 2.2.4 --- Cytotoxicity of hydrogen peroxide --- p.36 / Chapter 2.2.5 --- Cytoprotectivity of mushroom extracts --- p.36 / Chapter 2.2.6 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.37 / Chapter 2.2.7 --- Lactate dehydrogenase (LDH) assay --- p.39 / Chapter 2.2.8 --- Total cellular protein loss --- p.40 / Chapter 2.2.9 --- Comet assay (Single cell gel electrophresis assay) --- p.41 / Chapter 2.2.10 --- Thiobarbituric Acid Reactive Substances (TBARS) assay ..… --- p.44 / Chapter 2.2.11 --- Preparation of cell lysate for evaluating cellular antioxidant defense system --- p.45 / Chapter 2.2.12 --- Total Glutathione level --- p.46 / Chapter 2.2.13 --- Enzyme activity --- p.49 / Chapter 2.2.13.1 --- Catalase (CAT) --- p.49 / Chapter 2.2.13.2 --- Glutathione peroxidases (GPx) --- p.51 / Chapter 2.2.13.3 --- Glutathione Reductase (GR) --- p.53 / Chapter 2.2.13.4 --- Superoxide dismutase (SOD) --- p.54 / Chapter 2.2.14 --- Determination of protein --- p.56 / Chapter 2.2.15 --- Statistical analysis --- p.56 / Chapter Chapter 3 --- Results and discussions --- p.57 / Chapter 3.1 --- Extraction yield --- p.57 / Chapter 3.2 --- Chemical assays for in vitro antioxidative properties of mushroom extracts --- p.60 / Chapter 3.2.1 --- ABTS + scavenging activity --- p.60 / Chapter 3.2.2 --- Hydroxyl radicals scavenging activity --- p.61 / Chapter 3.2.3 --- Hydrogen peroxide scavenging activity --- p.64 / Chapter 3.3 --- Total phenolic content --- p.67 / Chapter 3.4 --- Cytotoxicity of hydrogen peroxide --- p.69 / Chapter 3.4.1 --- "Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay" --- p.71 / Chapter 3.4.2 --- Lactate dehydrogenase (LDH) assay --- p.72 / Chapter 3.4.3 --- Total cellular protein loss --- p.73 / Chapter 3.4.4 --- Residual hydrogen peroxide level --- p.76 / Chapter 3.4.5 --- Lipid peroxidation --- p.77 / Chapter 3.4.6 --- DNA damage --- p.79 / Chapter 3.5 --- Cytotoxicity of extracts --- p.85 / Chapter 3.6 --- Protection of H2()2-induced oxidative damage in HDFa cells --- p.88 / Chapter 3.6.1 --- Protective effect of mushroom water extracts --- p.88 / Chapter 3.6.2 --- Protective effect of CfAa on H2()2-incluced damage to HDFa --- p.93 / Chapter 3.6.3 --- Protective effect of CfAa on DNA damage in HDFa cells --- p.96 / Chapter 3.7 --- Modulation of cellular antioxidant defense system by CfAa --- p.99 / Chapter 3.7.1 --- Intracellular total glutathione --- p.100 / Chapter 3.7.2 --- Enzyme activities --- p.102 / Chapter 3.8 --- Speculation on the possible components in CfAa --- p.108 / Chapter Chapter 4 --- Conclusion and further works --- p.109 / References --- p.111

No description available.

Thesis (M.S.)--University of Michigan, 1991.

Indiana University-Purdue University Indianapolis (IUPUI) / In this thesis, we proposed and investigated the efficacy and safety of dental bleaching as a non-invasive aesthetic treatment option for stained arrested caries lesions (s-ACLs). Chapter 1 reports the suitability of this approach in extracted human teeth, as well as in a selected clinical case. Visual improvement in the color lighteness of the s-ACLs was observed and reported in photographs. In order to systematically study the impact of dental bleaching on the s-ACLs, we developed in vitro models simulating the development of metallic and non-metallic s-ACLs (Chapter 2). Human dental specimens were submitted to incipient caries-like lesion formation, followed by a 5-day cycling protocol based on remineralization and staining episodes. The created lesions were then bleached (simulating in-office/40% hydrogen peroxide). Color change was measured spectrophotometrically at baseline, after lesion creation, staining/remineralization cycling and bleaching; while mineral loss and lesion depth were quantified by transversal microradiography after staining/remineralization cycling. Metallic s-ACLs were darker, more remineralized and more difficult to bleach, compared to the non-metallic ones (p<0.05). In Chapter 3, we tested the efficacy and safety of different dental bleaching systems (simulating at-home/15% carbamide peroxide and in-office/40% hydrogen peroxide) using the in vitro models previously developed. Similar methods and outcomes were used, with the addition of demineralization after bleaching to simulate and test changes in caries susceptibility. At-home bleaching showed greater efficacy in color improvement compared to in-office (p<0.05), but also increased susceptibility to further demineralization (p<0.05), regardless of the type of stain. Overall, bleached non-metallic s-ACLs were more susceptible to demineralization compared to metallic ones (p<0.05). Within the limited laboratory testing conditions, we concluded that dental bleaching can improve the aesthetics of s-ACLs, with efficacy being dependent on the nature of the stain. At-home bleaching presented greater efficacy, but also raised some potential safety concerns, which should be further investigated in clinical conditions. In-office bleaching protocol showed to be an effective and safe procedure for the aesthetic treatment of stained arrested caries lesions.

The entire epidermis of patients with vitiligo exhibits accumulation of up to 10-3M concentrations of hydrogen peroxide (H₂O₂) (Schallreuter, Moore et al. 1999). Over the last decade our group and others have focused on the effect of H₂O₂-mediated oxidative stress on the function of many proteins and peptides due to oxidation of target amino acid residues in their structure including L-methionine, L-tryptophan, L-cysteine and seleno cysteine (Rokos, Beazley et al. 2002; Gillbro, Marles et al. 2004; Hasse, Kothari et al. 2005; Schallreuter, Chavan et al. 2005; Spencer, Chavan et al. 2005; Chavan, Gillbro et al. 2006; Elwary, Chavan et al. 2006; Gibbons, Wood et al. 2006; Schallreuter, Bahadoran et al. 2008; Shalbaf, Gibbons et al. 2008; Wood, Decker et al. 2009). Moreover, it was shown that patients with vitiligo possess up regulated wild type functioning p53 protein in their skin (Schallreuter, Behrens- Williams et al. 2003). The reason behind this up regulation has remained unclear (Schallreuter, Behrens-Williams et al. 2003). Therefore the aim of this thesis was to get a better understanding of these puzzling data. Along this project different techniques have been used including Western blot, dot blot, immuno precipitation, immuno fluorescence, EMSA and computer modelling. In this thesis we confirmed the previous result on up regulation of p53 in vitiligo and we showed that p90MDM2, the master regulator for p53 protein is not different in patients and healthy controls. Therefore we decided to test for expression of p76MDM2 which mediates the inhibition of p90MDM2-p53 binding. Our results show for the first time the presence and over expression of p76MDM2 protein in vitiligo compared to 3 healthy individuals. This result could provide an explanation, why up regulated p53 is not degraded in this disease. Since epidermal H₂O₂ accumulation has been extensively documented in vitiligo, we wanted to know whether other ROS could also contribute to the overall oxidative stress in this scenario. Therefore we turned our interest to nitric oxide (NO) and its possible effects on p53 protein. In order to elucidate this role in more detail, the expression levels of epidermal nitric oxide synthesase (iNOS) and the oxidation product of NO and O2 - i.e peroxynitrite (ONOO-) were investigated. Our data revealed over expression of iNOS and nitrated tyrosine residues, the foot print for ONOO-. Moreover, we show for the first time the presence of abundant nitration of p53 protein in vitiligo. In addition using purified p53 from E. coli strain (BL21/DE3) and mutant p53 protein from HT-29 cells (colon cancer cells), we show that nitration takes place in a dose and time dependent manner. On this basis we investigated the effect of both H₂O₂ and ONOO- on p53-DNA binding capacity employing EMSA, since this is the most acceptable technique to follow the binding between proteins and DNA. Our results revealed that ONOO- abrogated p53-DNA binding capacity at concentrations >300 μM, meanwhile oxidation of p53 protein with H₂O₂ at the same concentrations does not affect binding capacity. Importantly, a much higher p53- DNA binding capacity was observed after exposure to both ONOO- and H₂O₂. Taken together, p53 is regulated by both ROS (H₂O₂) and RNS (ONOO-). Next we identified the presence of phosphorylated and acetylated p53 in vitiligo. Phosphorylation of ser 9 and ser 15 residues of the protein are associated with over expressed ATM protein kinase, while acetylation of lys 373, 382 residues correlates with increased PCAF expression. We show that up regulated p53 is associated with over expressed p21 (cyclin dependent kinase inhibitor 1) and induced PCNA 4 expression. Hence, we can conclude that p53 in patients with vitiligo is up regulated, activated and functional. Finally we show up regulated BCL-2 supporting the long voiced absence of increased apoptosis in vitiligo. Given that patients with vitiligo have no increased risk for solar induced skin cancer and increased photo damage (Calanchini-Postizzi and Frenk 1987; Westerhof and Schallreuter 1997; Schallreuter, Tobin et al. 2002), despite the presence of increased DNA damage as evidenced by increased 8-oxoG levels in the skin and in the plasma, our findings suggest that both p53 and PCNA provide a powerful machinery to mediate DNA repair via hOgg1, APE1 and DNA polymerase ß (Shalbaf 2009). On this basis it is tempting to conclude that DNArepair is the overriding mechanism to combat oxidative stress in this disease.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.

The use of two online sensors, the S::CAN and the FLD, was used to evaluate the treatment of synthetic wastewater by UV/H₂O₂ advanced oxidation. The UV dose and intensity for the flow through reactor was also determined using H₂O₂ actinometry. The total power of the lamps in the reactor was determined to be 139 W which, with the absorbance of the water, produced a UV dose of 39.4 mJ/cm². It was determined that the S::CAN was unable to differentiate between treatment of a particular water, however it could differentiate between different test waters. The FLD was very sensitive and was able to detect minute variations in treatment of the test water.