Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

The etiology of abdominal aortic aneurysms (AAA) is characterized by localized extracellular matrix remodeling and vessel dilation. Population-based studies have shown that AAA account for nearly 1% of all deaths. This thesis seeks to identify the earliest molecular and biomechanical determinants of aneurysm formation. Our initial motivator was the lack of information defining the underlying mechanisms of AAA formation. We used isolated vessel testing and histological analysis to study the mechanical and morphological evolution of AAA. These factors were measured in murine models of reproducible AAA formation. From this study, we determined 1) that molecular events precede mechanical events in AAA progression and 2) aortic circumferential mechanics are well conserved during AAA pathogenesis. Next we sought to explore the mechanistic link between oxidative stress and AAA development. To determine this relationship we used isolated vessel testing as well as measurement of aortic residual circumferential strain. To isolate the role of oxidative stress in these studies we used a line of transgenic mice with vascular smooth muscle cell-specific overexpression of the antioxidant catalase. The results of this study suggest that oxidative stress-mediated elastin degeneration within the aortic media is etiologic of altered aortic mechanics. Lastly, we sought to determine the independent mechanical contribution of the aortic adventitia and media tunica to overall aortic behavior. To accomplish this goal we compared the circumferential and axial mechanical behavior of aortas with and without collagenase treatment. The data demonstrated that the adventitia regulates the circumferential behavior of the aorta by preventing overstretch and the media regulates the axial behavior by maintaining tensile loading. This thesis demonstrates 1) that detecting early aneurysm progression in the form of mechanical or geometric changes may miss the window in which aneurysm pathology may be potentially reversed, 2) that mitigating oxidative stress within the aortic wall may provide protection against AAA, and 3) the adventitia is an important load bearing constituent of the arterial wall and plays a role in vascular adaptation to altered mechanical states. Overall our results impact understanding of early aneurysmal pathogenesis and may facilitate the development of preventative therapies for AAA progression and rupture.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 165 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 131-163).

Lorsque les cellules normales évoluent vers un état néoplasique, elles acquièrent de nombreuses caractéristiques. Par exemple, ces cellules exhibent des voies métaboliques anormales et possèdent la capacité d’échapper à la destruction par les cellules de l’immunité, notamment en exploitant des points de contrôles immunitaires ou « immune checkpoints ». La molécule PD-L1 (Programmed Death-Ligand 1) appartient à la famille de protéines immuno-régulatrices B7 et a tout d’abord été décrite comme impliquée dans l’immuno-échappement tumoral suite à son interaction avec PD-1, récepteur exprimé à la surface des lymphocytes T. Associée à un mauvais pronostic, une expression aberrante de PD-L1 est retrouvée dans les hémopathies malignes ainsi que dans de multiples tumeurs solides. De manière intéressante, il a été montré que PD-L1 possède également des fonctions pro-tumorales intrinsèques. En effet, cette protéine joue un rôle dans la prolifération des cellules cancéreuses et leur résistance aux chimiothérapies, sans interagir avec PD-1. Toutefois, les mécanismes moléculaires modulés par PD-L1 et impliqués dans ces fonctions sont encore inconnus. Des voies métaboliques anormales ont été décrites comme pouvant contribuer à la croissance tumorale et la résistance aux thérapies. Ainsi, les objectifs de ma thèse ont été d’explorer le potentiel rôle de la protéine PD-L1 dans le métabolisme des cellules tumorales. En utilisant la méthode d’édition du génome avec les Zinc Finger Nucleases, nous avons invalidé le gène CD274 codant la protéine PD-L1 dans les cellules cancéreuses de sein MDA-MB-231 et investigué les fonctions métaboliques de cette molécule après surexpression dans ces mêmes cellules. Nous avons observé que PD-L1 induit un shift de la phosphorylation oxydative vers la glycolyse, correspondant à l’effet Warburg. Afin de valider cette reprogrammation métabolique, nous avons analysé le profil métabolique de ces cellules et mis en évidence une élévation des niveaux des intermédiaires de la glycolyse tels que le F-6-P, le F-1,6-P, le GAP, le DHAP, le PEP et le pyruvate dans la lignée surexprimant PD-L1, confirmant nos précédents résultats. D’autre part, et en accord avec nos observations quant à une augmentation de la production de ROS (Reactive Oxygen Species), nos données transcriptomiques suggèrent une répression de la voie de réponse au stress oxydatif NRF2 suite à l’expression de PD-L1 et notamment de ses gènes cibles tels que NQO2, GSTM3 et ABCC2. En outre, l’analyse in silico de bases de données de cohortes de patients atteints de cancer du sein a révélé une corrélation entre l’expression du gène PD-L1/CD274 et l’expression des gènes de la voie du stress oxydant (GSTM3 ; CYBB) ou des gènes codant les transporteurs de glucose (SLC2A1/GLUT1 ; SLC2A3/GLUT3), ces données supportant nos résultats obtenus in vitro. Par ailleurs, le glucose étant principalement utilisé par les cellules cancéreuses pour favoriser la biosynthèse de diverses biomolécules nécessaires à la prolifération cellulaire, ces résultats pourraient expliquer la tumorigénicité augmentée dans la lignée surexprimant PD-L1 lors des expériences de xénogreffe de cellules de cancer du sein humain chez des souris Nude. Ainsi, les travaux présentés dans cette thèse mettent en évidence de nouvelles fonctions intrinsèques de PD-L1 promouvant le développement tumoral, suggérant l’utilisation d’agents thérapeutiques inhibant ces mécanismes seraient prometteurs pour le traitement du cancer du sein. / Evolving to a neoplastic state, normal cells acquire many characteristics; indeed, tumor cells follow abnormal metabolic pathways and exhibit the ability to avoid immune destruction, partly by exploiting immune checkpoints. Many of these are currently under clinical investigation for new cancer treatments, notably the PD-1/PD-L1 axis.Programmed Death-Ligand 1 (PD-L1) molecule belongs to the B7 immunoregulatory proteins family and was originally described as mediating tumor immuno-escape through interaction with its receptor PD-1 on T cells. Associated with poor cancer outcome, aberrant PD-L1 expression has been observed in hematologic malignancies and in multiple solid tumor types. Actually, this protein has been shown to regulate tumor cell proliferation and resistance to chemotherapy through apoptosis inhibition, without interacting with PD-1. However, cellular mechanisms modulated by PD-L1 and involved in these functions are still unclear. Abnormal metabolic pathways are known for contributing to tumor growth and therapy resistance; therefore, the objective of my PhD thesis was to investigate the impact of PD-L1 in breast cancer cell metabolic reprogramming.Using genome editing, we knocked-out the CD274 gene encoding PD-L1 in breast cancer cell line MDA-MB-231 and investigated metabolic functions after PD-L1 overexpression in the same cells. We observed that PD-L1 induces a shift from oxidative phosphorylation to glycolysis, indicating this molecule promotes the Warburg effect in these tumor cells. To validate PD-L1 metabolic reprogramming, we performed metabolomic profiling that highlighted significantly increased levels of glycolysis intermediated such as F6P, F1,6P, GAP, DHAP, PEP and pyruvate in PD-L1-expressing cells, confirming our latter results. Moreover, in agreement with an increasing mitochondrial reactive oxygen species (ROS) production, transcriptomic study suggested that PD-L1 represses NRF2-mediated oxidative stress response pathway, especially NQO2, GSTM3 and ABCC2 genes. Furthermore, in silico analysis of breast cancer patients databases highlighted a correlation between PD-L1/CD274 gene and oxidative stress gene signature (GSTM3; CYBB) or glucose transporters genes (SLC2A1; SLC2A3) expressions, supporting our results. Besides, glucose is mostly used by cancer cells to favor biosynthesis of diverse biomolecules required for cellular proliferation; the above results could explain our human breast cancer cells xenograft experiments in Nude mice demonstrating that PD-L1 increases tumoreginicity.Thus, the work presented in this thesis evidences novel PD-L1 intrinsic tumor-promoting functions, suggesting that therapeutic agents inhibiting these mechanisms would be promising for breast cancer treatment.

Thesis (M.Sc. (Medical Sciences)) -- University of Limpopo, 2020 / Infertility affects nearly 186 million couples globally, with male factors contributing to half of the cases. Oxidative stress is an established cause of declining semen quality. Moringa oleifera has proven antioxidants. This study aimed to investigate in vitro effects of aqueous leaf extract of M. oleifera on human sperm functions. Semen samples from donors (n = 40) and patients (n = 30) were washed with HTF-bovine serum albumin (BSA), and then incubated with various concentrations of M. oleifera (0, 0.625, 6.25, 62.5, and 625 μg/ml) at 37°C for 1 hour. Sperm motility, vitality, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA fragmentation, capacitation, and acrosome reaction were assessed. Sperm motility, vitality, MMP, and capacitation were enhanced, while ROS production, and DNA fragmentation decreased after M. oleifera treatment. Uncapacitated spermatozoa increased significantly with a reduction in acrosome reaction in donors. M. oleifera antioxidant compounds suppressed excessive ROS, preserved mitochondrial membrane, DNA and acrosome integrity, while enhancing sperm motility and viability. / National Research Foundation (NRF)

Poon, Chui Wa Christina. / "August 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-131). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / PUBLICATIONS --- p.x / Abstracts --- p.x / ABBREVIATIONS --- p.xii / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1. --- Diabetes --- p.1 / Chapter 1.1.1. --- Overview --- p.1 / Chapter 1.1.2. --- Diagnostic Criteria of Type-2 Diabetes --- p.2 / Chapter 1.1.3. --- Type-2 Diabetes (T2DM) --- p.3 / Chapter 1.1.3.1. --- Impaired Insulin Synthesis and Insulin Secretory Defects in Type-2 Diabetes --- p.3 / Chapter 1.1.3.2. --- β-Cell Dysfunction --- p.5 / Chapter 1.1.3.3. --- Insulin Resistance --- p.5 / Chapter 1.1.4. --- Glucose Toxicity --- p.6 / Chapter 1.1.4.1. --- Fasting Hyperglycemia --- p.8 / Chapter 1.1.4.2. --- Postprandial Hyperglycemia --- p.8 / Chapter 1.2. --- Oxidative Stress --- p.8 / Chapter 1.2.1. --- ROS and Mitochondria --- p.8 / Chapter 1.2.2. --- ROS Production by Mitochondria --- p.9 / Chapter 1.2.3. --- The Relationship of Glucose Recognition by β-cells and Oxidative Stress --- p.11 / Chapter 1.2.4. --- Important Roles of Glutathione in Pancreatic β-cells and Glutathione Synthesis --- p.14 / Chapter 1.2.5. --- N-acetyl-L-cysteine - A Potential Drug Treatment for Type-2 Diabetes? --- p.17 / Chapter 1.3. --- Role of F-actin Cytoskeleton on Glucose-induced Insulin Secretion --- p.18 / Chapter 1.4. --- Current Clinical Treatments for Type-2 Diabetes Mellitus --- p.21 / Chapter 1.4.1. --- Metformin --- p.22 / Chapter 1.4.2. --- Sulfonylureas --- p.22 / Chapter 1.4.3. --- Thiazolidinediones --- p.23 / Chapter 1.4.4. --- Glinides (Meglitinide Analogues) --- p.23 / Chapter 1.4.5. --- α-Glucosidase (AG) Inhibitors --- p.24 / Chapter 1.4.6. --- Dipeptidyl Peptidase-4 (DPP-4) Inhibitors --- p.24 / Chapter 1.4.7. --- (Clinical) Antioxidant Treatment --- p.24 / Chapter 1.5. --- Animal Models Used in Type-2 Diabetes Research --- p.25 / Chapter 1.6. --- Aims of Study --- p.27 / Chapter 2. --- RESEARCH DESIGN & METHODS --- p.28 / Chapter 2.1. --- Materials --- p.28 / Table 1. Sources and concentrations of drugs tested in this study: --- p.28 / Culture Medium - --- p.29 / General Reagents --- p.29 / Chapter 2.2. --- Isolation of Islets of Langerhans and Single Pancreatic β-Cells --- p.31 / Chapter 2.3. --- Measurement of Mitochondrial ROS Levels --- p.32 / Chapter 2.4. --- Measurement of Islets Insulin Release and Insulin Content --- p.34 / Chapter 2.4.1. --- Preparation of Samples --- p.34 / Chapter 2.4.2. --- Enzyme-Link Immunosorbent Assay (ELISA) --- p.35 / Chapter 2.5. --- Immunocytochemistry --- p.35 / Chapter 2.6. --- Data and Statistical Analysis --- p.37 / Chapter 3. --- RESULTS --- p.38 / Chapter 3.1. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Releasable Insulin Levels and Insulin Contents in Response to Low Glucose (5 mM) and High Glucose (15 mM) of Isolated Pancreatic Islets of (db+/m+) and (db+/db+) Mice" --- p.38 / Chapter 3.1.1. --- Effect of L-NAC on Insulin Secretion and Insulin Contents --- p.38 / Chapter 3.1.2. --- Effect of Cytochalasin B on Insulin Secretion and Insulin Contents --- p.39 / Chapter 3.1.3. --- Effect of 4-Phenyl Butyric Acid on Insulin Secretion and Insulin Contents --- p.43 / Chapter 3.1.4. --- Effect of Ursodeoxycholic Acid on Insulin Secretion and Insulin Contents --- p.46 / Chapter 3.1.5. --- Effect of Hydrogen Peroxide on Insulin Secretion and Insulin Contents --- p.49 / Chapter 3.1.6. --- Effect of Jasplakinolide on Insulin Secretion and Insulin Contents --- p.53 / Chapter 3.1.7. --- Effect of Thapsigargin on Insulin Secretion and Insulin Contents --- p.57 / Chapter 3.1.8. --- Effect of BSO on Insulin Secretion and Insulin Contents --- p.61 / Chapter 3.2. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on Mitochondrial ROS Levels in Response to High Glucose (15 mM) Challenge in Isolated Single Pancreatic β-Cells of (db +/m+) and (db +/db +) Mice" --- p.65 / Chapter 3.2.1. --- "Effects of L-NAC (20 mM), 4-Phenyl Butyric Acid (4-PBA) (1 mM), Ursodeoxycholic Acid (UA) (500 μg/ml), H202 (200 μM), Thapsigargin (0.5 μM) and DL-Buthionine-[S,R]-Sulfoximine (BSO) (0.1 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge" --- p.65 / Chapter 3.2.2. --- "Effects of L-NAC (20 mM), Cytochalasin B (10 μM) and Jasplakinolide (5 μM) Pre-treatments on Mitochondrial ROS Level in Response to High Glucose (15 mM) Challenge_" --- p.76 / Chapter 3.3. --- "Effects of L-NAC, Various Oxidative Stress Inducers/Reducers and Actin Polymerisation/Depolymerisation Inducers on F-actin Cytoskeleton Levels Incubated in Low Glucose (5 mM) and High Glucose (15 mM) Medium in Single Pancreatic β-Cells of Non-Diabetic (db +/m+) and Diabetic (db +/db +) Mice" --- p.81 / Chapter 4. --- DISCUSSION --- p.100 / Chapter 4.1. --- General Discussion --- p.100 / Chapter 5. --- SUMMARY --- p.120 / Chapter 6. --- FUTURE PERSPECTIVES --- p.121 / Chapter 7. --- REFERENCES --- p.123

In the aerobic cell, oxygen can be converted into a series of reactive metabolites, together called as "reactive oxygen species" (ROS). This large group include both radical and non-radical species such as superoxide anion (02"), hydroxyl radical ("0H), H202, nitric oxide (N0') and lipid hydroperoxides (LOOH). ROS are generated in very small amounts at all stages of aerobic life, and probably have a role in cellular regulation. However, their formation in excess leads to toxicity and damage to tissues. This situation, called 'oxidative stress', is responsible, atleast in part, to the pathophysioiogy of a number of disease states such as inflammation, arthritis, cancer, ageing, ischemia-reperfusion and several neurodegenerative disorders. Compared to other organs in the animal body, brain tissue is more vulnerable to oxidative stress. This is due to three major reasons; (1) brain has a high oxygen consumption (2) high content of polyunsaturated fatty acids and iron, that can promote lipid peroxidation, and (3) low levels of antioxidant enzymes such as catalase and glutathione peroxidase. The inability of neurons to regenerate also contributes to exacerbate an oxidant damage in the brain. The main objective of this investigation was to identify biochemical systems in the brain that are susceptible to ROS, on the following two issues: 1. What are the targets for the action of H2O2 and NO in the glycolytic cycle, the major route for the oxidation of glucose in brain? 2. What are the targets for the action of polyunsaturated fatty acids and their oxidative metabolites among the enzymes of phosphoinositide cycle (PI cycle), the ubiquitous signal transduction event in the brain? Using sheep brain cytosol , it was found that among the various glycolytic enzymes, only glyceraldehyde 3-phosphate dehydrogenase (GAPD) was inhibited by H2O2. The enzyme was purified to homogeneity from sheep brain and its inactivation with H202 was studied in detail. Commercial preparations of rabbit skeletal muscle GAPD was also used in this study. An unusual requirement of glutathione for the complete inactivtion of the enzyme by H2O2 was observed. The H2O2-inactivated GAPD was partially reactivated by prolonged treatment with thiol compounds. Using CD-spectral analysis, a significant change was found in the secondary structure in H2O2-treated GAPD. GAPD was inactivated by NO only in presence of high concentrations of DTT and after prolonged incubation. The N0-inactivated GAPD was partially reactivated by treatment with thiol compounds. A new activity, namely ADP-ribosylation (ADPR) emerged in the NO-treated mammalian, but not in yeast. GAPD, ADPR activity could be generated in GAPD through NO-independent treatments such as incubation with NADPH and aerobic dialysis. During NADPH treatment no loss of dehydrogenase activity occurred. Thus, it was concluded that loss of dehydrogenase activity and emergence of ADPR in NO-treated GAPD were not correlated but coincidental, and that NO treatment yielded small amounts of modified-GAPD that had ADPR activity. In the brain, onset of ischemia is characterized by a significant elevation in free fatty acid (FFA) levels, predominantly, arachidonic acid (AA). It is suggested that AA can be oxidised to its metabolites like prostaglandins and 15-hydroperoxy arachidonic acid (15-HPETE) and some of these might exert toxic effects during reperfusion. Using whole membranes or tissue slices prepared from rat brain, effects of polyunsaturated fatty acids and their oxidative metabolites on five enzymes of PI cycle namely PI synthase, PI and PIP kinases, agonist-stimulated PLC and DG kinase was studied. Hydroperoxides of linoleic- and arachidonic acids inactivated PI synthase selectively among the PI cycle enzymes. Interestingly, AA selectively stimulated DG kinase in neural membranes. Docasahexaenoic acid (DHA) a highly unsaturated fatty acid found in the brain, also stimulated DG kinase activity while saturated, mono-and di-unsaturated fatty acids were ineffective. It was concluded that AA and DHA have a role in modulating neural DG kinase. The data presented in the thesis indicate that ROS have selective targets in cells and the consequent protein modifications can be used to modulate cellular functions under normal and oxidative stress conditions.

Heart failure is the leading cause of death worldwide. In 2013, the American Heart Association estimated that one American will die of cardiovascular disease every 39 seconds. While heart transplantation is the most viable treatment option, the limited availability of donor hearts has necessitated the search for treatment alternatives such as the use of adult stem cells for cardiac repair and regeneration. Following myocardial infarction (MI), the inflammatory cardiac microenvironment, limited survival of stem/progenitor cells, myocardial scarring and fibrosis affect cardiac regeneration. This dissertation examines adult stem cell based approaches for cardiac regeneration by studying the effect of i) H₂O₂- mediated oxidative stress on mesenchymal stem cells, ii) Notch1 activation in cardiac progenitor cells using a self-assembling peptide hydrogel containing the Notch1 ligand mimic RJ in vitro and functional consequences in a rat model of MI. Through these approaches, the central hypothesis that modulation of stem cell response using cues such as oxidative stress and activation of Notch1 signaling can improve functional outcome following myocardial infarction has been studied.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Metals environment aquatic contamination has been a growing problem with serious consequences to life of different species over time, even after interrupted their emission into the environment. Among relevance metals, manganese (Mn) has shown importance to be related to several activities such as oil exploration, coal deposits extraction, fertilizer use in agriculture, among others. In living organisms, Mn is an essential trace element for a number of vital functions, and involves energy regulation by blood clotting. On the other hand, high concentrations of Mn can cause irreversible damage to living organisms primarily affecting central nervous system (CNS). Thus, waterborne Mn toxicity can switch from aquatic species, while metal bioaccumulation in marine fish or freshwater has been found around 0.2 to 19.0 mg/kg dry weight. In this study, silver catfish (Rhamdia quelen) were exposed to different Mn concentrations (4.2; 8.4 e 16.2 mg/L), under two different dissolved oxygen levels, normoxia (7.48 ± 0.28) and hypoxia (3.88 ± 0.41) for 15 days. At the end of protocol, Mn bioaccumulation as well parameters of oxidative stress and mitochondrial viability were evaluated in different tissues. In hypoxic conditions, the highest Mn concentration (16.2 mg/L) showed the lowest silver catfish kidney and brain lipoperoxidation (LPO) levels, while brain reduced glutathione (GSH) levels were increased in lower Mn concentration (4.2 mg/L) and kidney catalase activity was reduced in the same metal concentration, in relation to normoxia. Lowest Mn concentration (4.2 mg/L) in hypoxia showed higher gills mitochondrial viability, compared to normoxia. Hematocrit of silver catfish exposed to two highest Mn concentration (8.4 and 16.2 mg/L) was reduced in normoxia conditions while under hypoxia, these values were similar to control. Plasma except, Mn bioaccumulation in liver, kidney and gills were higher in normoxia than hypoxia. From these results is possible to suggest that hypoxia stimulates the development of adaptive mechanisms and/or hormesis in silver catfish exposed to Mn, mainly because the lower metal bioaccumulation occurred in this oxygen level. Contributing with this, under hypoxia, oxidative damage indicators were lower than those observed in normoxia, which were accompanied by changes in antioxidant system represented by GSH and catalase. In conclusion, our results show in the first time that silver catfish exposed to Mn contamination is able to show a better survival under hypoxia. These findings indicate need for continuing studies in search of molecular mechanisms involved in the adaptation and or hormesis processes, which were suggested here. / A contaminação do meio aquático por metais tem sido um problema crescente para à vida de diferentes espécies, mesmo depois de interrompida sua emissão no ambiente. Entre os metais de relevância, o manganês (Mn) apresenta importância por estar relacionado a uma série de atividades tais como exploração de petróleo, extrativismo em jazidas de carvão mineral, utilização de fertilizantes na agricultura, entre outros. Nos organismos vivos, o Mn constitui um oligoelemento essencial para uma série de funções vitais, e envolve desde a regulação da energia até coagulação sangüínea. Por outro lado, quando em concentrações elevadas, o Mn pode causar danos irreversíveis aos organismos afetando principalmente o sistema nervoso central (SNC). A toxicidade do Mn dissolvido na água pode variar entre as espécies aquáticas, a bioacumulação do metal em peixes marinhos ou de água doce tem sido encontrada em torno de 0,2 até 19,0 mg/kg de peso seco. No presente estudo, jundiás (Rhamdia quelen) foram expostos a diferentes concentrações de Mn (4,2; 8,4 e 16,2 mg/L), sob dois diferentes níveis de oxigênio dissolvido: normóxia (7,48±0,28 mg/L) e hipóxia (3,88±0,41 mg/L), durante 15 dias. Ao final do protocolo, a bioacumulação de Mn bem como, parâmetros de estresse oxidativo e a viabilidade mitocondrial foram avaliados em diferentes tecidos vitais. Em condições de hipóxia, a maior concentração de Mn (16,2 mg/L) mostrou os menores níveis de lipoperoxidação (LPO) em rim e encéfalo dos jundiás, enquanto os níveis de glutationa reduzida (GSH) encefálico foram aumentados na menor concentração de Mn (4,2 mg/L) e a atividade da catalase renal foi reduzida nesta mesma concentração de metal, em relação à normoxia. A menor concentração de Mn (4,2 m/L) em hipóxia proporcionou maior viabilidade mitocondrial das brânquias, em relação à normóxia. O hematócrito dos jundiás expostos às duas maiores concentrações de Mn (8,4 e 16,2 mg/L) foi reduzido em condições de normóxia, enquanto sob hipóxia, estes valores foram semelhantes ao grupo sem a presença do metal. Sob normóxia, a bioacumulação de Mn em fígado, rim e brânquias foi maior que sob hipóxia, excetuando o plasma. A partir desses resultados é possível sugerir que a hipóxia pode ter estimulado o desenvolvimento de mecanismos adaptativos ou de hormesis nos jundiás, em presença de Mn na água, principalmente porque neste nível de oxigênio, a bioacumulação do metal ocorreu em menor grau. Contribuindo com este resultado, sob hipóxia, os indicadores de danos oxidativos foram mais baixos que os observados sob normóxia, os quais foram acompanhados de alterações do sistema antioxidante representado pela GSH e catalase. Neste sentido, nossos resultados apontam para uma melhor sobrevivência da espécie em estudo, sob hipóxia, quando exposta ao Mn. Estes achados indicam a necessidade da continuidade dos estudos, em busca dos mecanismos moleculares envolvidos no processo de adaptação aqui sugeridos.

Résumé<p>L’objectif de ce travail a été d’évaluer la toxicité du butyrate de sodium (NaBu), un inhibiteur des<p>histones désacétylases (HDACs), et ses mécanismes d’action sur les cellules de cancer du sein<p>humain, les cellules MCF-7 déficientes pour la caspase-3, et lignées dérivées :les cellules MCF-<p>7/caspase-3, et les cellules VCREMS résistantes à la vincristine, et dans une moindre mesure à la<p>doxorubicine. La contribution de l’apoptose dans la létalité induite par le NaBu a été recherchée<p>dans les cellules MCF-7wt en estimant l’exposition de la phosphatidylsérine ainsi que le clivage de<p>la PARP. La présence de caspase-3, n’a ni amplifié ni accéléré l’apoptose qui a impliqué le<p>rhéostat Bax/Bcl-2 en faveur d’une induction de Bax. La cytostasie du NaBu dans les cellules<p>MCF-7 s’est manifestée par un blocage des cellules en phase G2/M. L’évaluation du niveau<p>d’expression des régulateurs du cycle cellulaire dans les cellules MCF-7wt et MCF-7/caspase-3 a<p>montré une surexpression de p21, de façon indépendante de p53. L’action cytostatique du NaBu<p>a été associée à une accumulation légère et modeste des formes non-phosphorylées de pRB, un<p>facteur dont la phosphorylation par les complexes cycline D/cdk4,6 et cycline E/cdk2 est<p>nécessaire à la transition G1/S. Dans ces conditions, les niveaux de cdk2 et de Cdc25A, une<p>oncoprotéine activatrice de cdk2, sont restés stables. Le NaBu est une molécule à effet<p>pléïotropique, l’utilisation de la trichostatine A, inhibiteur par excellence des HDACs, a permis<p>d’établir la relation de causalité entre l’inhibition des HDACs et la toxicité du NaBu. La plupart<p>des inhibiteurs des HDACs induisent l’apoptose en perturbant le métabolisme oxydatif de la<p>mitochondrie ce qui pourrait modifier le statut redox cellulaire. Nous avons cherché une<p>implication du métabolisme du glutathion (GSH), le thiol anti-oxydant non-protéique majoritaire<p>de la cellule, dans la toxicité induite par le NaBu. Les résultats montrent que le NaBu induit une<p>déplétion du GSH dans les cellules MCF-7wt et dérivées de façon dose-dépendante, corrélée avec<p>la mortalité cellulaire. Devant l’éventualité d’une consommation accrue de GSH par les enzymes<p>associées à son métabolisme, nous avons évalué le niveau des activités des enzymes glutathion<p>peroxydase, glutathion réductase et glutathion S-transférases. Dans les cellules MCF-7, le NaBu a<p>induit de façon significative ces enzymes anti-oxydantes, à l’exception des GSTs, de même que la<p>catalase, une enzyme indépendante de ce système. Les expériences visant à libérer le pool de<p>GSH lié aux protéines ont montré que la déplétion du GSH intracellulaire est parallèle à celle du<p>GSH lié aux protéines. Par conséquent, la consommation du GSH est réellement la cause de la<p>chute du niveau de GSH générant un stress oxydant. La doxorubicine, un inhibiteur des<p>topoisomérases, a une utilisation clinique limitée en raison de ses effets secondaires irréversibles<p>(cardiotoxicité entre autres). Dans le but d’améliorer son efficacité, nous avons expérimenté des<p>combinaisons NaBu/doxorubicine sur les cellules VCREMS et MCF-7, étant donné la capacité<p>du NaBu à induire l’expression des topoisomérases et favoriser la conformation déployée de la<p>chromatine. L’utilisation de la technique isobologramme nous a permis de déterminer les index<p>de combinaison pour une application simultanée ou séquentielle des drogues. Les résultats<p>indiquent que le NaBu sensibilise les cellules VCREMS et MCF-7 à l’action de la doxorubicine.<p>Dans les cellules VCREMS, cet effet s’est produit en dépit de la stimulation des enzymes de<p>détoxication, GSTs et GPX. L’ensemble de ces résultats indique que l’utilisation du NaBu en<p>combinaison avec certains anticancéreux constitue une stratégie très intéressante en<p>cancérothérapie. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished