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Yuen Yee Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 108-125). / Abstracts in English and Chinese. / TITLE PAGE --- p.1 / ACKNOWLEGDEMENTS --- p.2 / ABSTRACT --- p.3 / 摘要 --- p.5 / table of contents --- p.7 / list of figures and tables --- p.13 / CHAPTER 1 GENERAL INTRODUCTION --- p.16 / Chapter 1.1 --- role of PHYTOESTROGENS in soy and red WINE the PREVENTION OF CARDIOVASCULAR DISEASES (CVD) --- p.17 / Chapter 1.1.1 --- INTRoduction and Classification of Phytoestrogens --- p.17 / Chapter 1.1.2 --- estrogenic1ty of phytoestrogens and theIr abundancesin Plasma --- p.18 / Chapter 1.1.3 --- phytoestrogens as one of the active components In cvd Protection --- p.21 / Chapter 1.1.4 --- effects of Phytoestrogens on LDL Receptor and Apolipoprotein A-1 --- p.22 / Chapter 1.2 --- role of estrogen receptors (ers) in gene regulation --- p.24 / Chapter 1.2.1 --- "structure, Classification and tissue distribution of ERS" --- p.24 / Chapter 1.2.2 --- ligands for ERS --- p.25 / Chapter 1.2.3 --- mechaniSMS OF LIgands-ERS complex in GENE regulation --- p.27 / Chapter 1.2.4 --- ligand-independent ER activation --- p.28 / Chapter 1.3 --- aims and scopes of investigation --- p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- chemicals and materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Plasmids --- p.30 / Chapter 2.2 --- mammalian cell culture maintainence --- p.30 / Chapter 2.2.1 --- Maintenance of Cells --- p.31 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.31 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.31 / Chapter 2.3 --- manipulation of dna --- p.31 / Chapter 2.3.1 --- isolation of HEPG2 cells genonmic DNA --- p.31 / Chapter 2.3.2 --- separation and purification of dna from agarose gel --- p.31 / Chapter 2.3.3 --- Restriction digestionof DNA --- p.32 / Chapter 2.3.4 --- Ligation of DNA Fragments --- p.32 / Chapter 2.3.5 --- Transformation of --- p.32 / Chapter 2.3.6 --- Small Scale Plasmids Purification from DH5a --- p.32 / Chapter 2.4 --- construction of expression and reporter plasmids --- p.33 / Chapter 2.4.1 --- Construction of Estrogen Receptorα (Erα) Expression Vectors --- p.33 / Chapter 2.4.2 --- construction of reporter vectors of LDLR promoter and the Respective Mutants --- p.33 / Chapter 2.4.3 --- Construction of Reporter Vectors of APOAI Promoter and the Respective Mutants --- p.33 / Chapter 2.5 --- determination of promoter transcrtiption activities --- p.34 / Chapter 2.5.1 --- Transient Transfection of Cell with ERa Expression Vector and Promoter Reporter using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.34 / Chapter 2.6 --- semi-quantitative and quantitative rt-pcr assay --- p.34 / Chapter 2.6.1 --- Transient transfection of Cell with ERa Expression Vector Using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.6.2 --- "Isolation of RNA using TRIzol® Reagent (Life Technology, USA)" --- p.35 / Chapter 2.6.3 --- Quantitation of RNA --- p.35 / Chapter 2.6.4 --- First Strand cDNA Synthesis --- p.35 / Chapter 2.6.5 --- Sem卜Quantitative PCR Reactions --- p.35 / Chapter 2.6.6 --- Quantitative PCR Reactions --- p.36 / Chapter 2.7 --- western blotting analysis --- p.36 / Chapter 2.8 --- statistical methods --- p.36 / Chapter CHAPTER 3 --- REGULATION BY PHYSIOLOGICAL LEVEL OF 17B-ESTRADIOL ON APOLIPOPROTEIN A-I AND LOW-DENSITY- LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.37 / Chapter 3.1 --- introduction --- p.37 / Chapter 3.2 --- results --- p.39 / Chapter 3.2.1 --- Determination of transient transfection functionality of estrogen receptors in hepg2 cells --- p.39 / Chapter 3.2.2 --- Effect of 17β-Estradiolon LDLR promoter transcription activity --- p.39 / Chapter 3.2.3 --- Effect of 17β-Estradiol on apoai promoter transcription activity --- p.40 / Chapter 3.2 --- discussion --- p.47 / Chapter CHAPTER 4 --- SOY ISOFLAVONES AND RESVERATROL DISPLAY DIFFERENT MECHANISM IN THE UP-REGULATION OF LOVV-DENSITY-LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.49 / Chapter 4.1 --- introduction --- p.49 / Chapter 4.2 --- results --- p.54 / Chapter 4.2.1 --- Association of ERα and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.2 --- Association of ERβ and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol induced LDLR promoter transcription" --- p.55 / Chapter 4.2.4 --- Identification of promoter regions responsible for induction of LDLR transcription by isoflavones in the presence OF ERα --- p.55 / Chapter 4.2.5 --- Identification of promoter regions responsible for induction of LDLR TRANSCRIPTION BY resveratrol IN THE ABSENCE OF ERα --- p.56 / Chapter 4.3 --- DISCUSSION --- p.75 / Chapter CHAPTER 5 --- SOY ISOFLAVONES AND RESVERATROL UP-REGULATE APOLIPOPROTEIN A-I SIMILAR TO 17B-ESTRADIOL IN HEPG2 CELLS --- p.80 / Chapter 5.1 --- INTRODUCTION --- p.80 / Chapter 5.2 --- RESULTS --- p.84 / Chapter 5.2.1 --- Association of ERα phytoestrogens on APCAI gene expression --- p.84 / Chapter 5.2.2 --- Association of ERβ and isoflavones or resveratrol on APOAI promoter transcription activity --- p.85 / Chapter 5.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol in APOAI promoter transcription in the presence of ERα" --- p.85 / Chapter 5.2.4 --- Identification of promoter regions responsible for induction of APOAI transcription by isoflavones and resveratrol in the presence of ERα --- p.85 / Chapter 5.3 --- DISCUSSION --- p.100 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.103 / Chapter CHAPTER 7 --- SUMMARY --- p.106 / BIBLIOGRAPHY --- p.108 / APPENDIX 1 ABBREVIATIONS --- p.126 / APPENDIX 2 MATERIALS AND METHODS --- p.129 / APPENDIX 3 PRIMER LISTS --- p.145 / APPENDIX 4 REAGENTS AND BUFFERS --- p.147

La vigne est soumise à la pression de nombreux bioagresseurs dont des parasites obligatoires tels que l’oïdium et le mildiou. La lutte contre les maladies causées par les pathogènes biotrophes nécessite une utilisation souvent intensive de fongicides. Le vignoble consomme à lui seul 16% des fongicides commercialisés chaque année en France. Pour réduire leur impact environnemental qui conduit à l’acquisition de la résistance aux pesticides des pathogène et la présence de résidus dans les vins et dans l’atmosphère, des efforts doivent être entrepris pour développer des stratégies de protection innovante de remplacement ou complémentaire permettant de réduire les intrants pesticides.Les stimulateurs des défenses des plantes permettent de limiter le développement des bioagresseurs en conditions contrôlées. Toutefois, leurs efficacités in natura sont variables et souvent décevantes. Suite au grand nombre de produits potentiellement stimulateurs des défenses des plantes, et à l’intérêt que leur portent les viticulteurs, il est nécessaire de disposer de connaissances et d’outils qui permettent d’évaluer leus efficacités et mieux connaitre leurs potentiels de protection du vignoble. Pour ce faire, une méthode d’évaluation de l’efficacité de produits potentialisateurs ou éliciteurs a été développée au niveau biologique, moléculaire (expression de gènes impliqués dans les défenses) et biochimique (analyses qualitatives et quantitatives des polyphénols), nommée "BioMolChem". Cette méthode a permis d’évaluer l’efficacité de deux phosphonates et d’un analogue de l’acide salicylique, sur différents génotypes et phénotypes de mildiou de la vigne et d’oïdium. Cette approche méthodologique "BioMolChem" a permis d’établir des corrélations entre l’expression de gènes de défense, la présence de certains stilbènes et une efficacité des défenses de Vitis vinifera cv. Cabernet-Sauvignon vis-à-vis de l’oïdium et du mildiou. Les modifications des patrons d’expression des 19 gènes suivis dans les feuilles de vigne et les profils HPLC de polyphénols révèlent des mécanismes de défense multigéniques et complexes. Ainsi, les réactions de défense de la plante sont-elles modulées, en fonction de l’éliciteur considéré, mais aussi en fonction de la diversité phénotypique et génétique des agents pathogènes contre lesquels elle se défend. Ces défenses se caractérisent par une sur-expression d’un ensemble de gènes de défense et une accumulation de composés phénoliques spécifiques.Les marqueurs (gènes et molécules) ainsi identifiés, la méthode "BioMolChem" a été appliquée in natura et a conforté, pour partie, les résultats obtenus au laboratoire. Dans des conditions de fortes pressions parasitaires, il est donc possible de protéger les feuilles et les grappes, à l’aide de SDP et des essais d’association ou d’alternance avec des fongicides conventionnels montrent l’intérêt potentiel de l’emploi des SDP au vignoble. Chemin faisant, dans le cadre d’une viticulture innovante et durable, les SDP et la méthode "BioMolChem" ont été appliqués sur des génotypes hybrides (Vitis vinifera x Muscadinia rotundifolia). Nous révélons que selon le niveau de résistance intrinsèque des génotypes (plus ou moins résistants à l’oïdium et au mildiou), il est possible d’augmenter le niveau de la résistance exprimée par élicitation. Ainsi, les SDP pourraient-ils s’avérer des alliés d’intérêt pour l’utilisation de variétés partiellement résistantes et limiter potentiellement le contournement des QTL de résistance. L’ensemble de ce travail, à but appliqué, a conduit à l’obtention de résultats qui nous permettent de mieux comprendre comment la vigne réagit aux SDP dans son environnement agronomique. Leur exploitation et leur finalisation devraient nous permettre d’exploiter et de mettre en place une utilisation des éliciteurs mieux adaptée, à des stratégies alternatives ou complémentaires de la gestion des bioagresseurs de la vigne. / Powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) are very important grapevine diseases (Vitis vinifera). These two biotrophic pathogens, which are native to the United States, infect green vine tissues and cause significant economic loss as well as environmental damage through the repetitive applications of fungicides. To reduce their environmental impact efforts should be made to develop strategies to protect innovative alternative or complementary to reduce pesticide inputs.In this study, the efficacy and the role of Benzothiadiazole (BTH), a salicylic acid analogue, and two phosphonate derivatives strengthen plant defence mechanisms against various isolates of downy and powdery mildews (Plasmopara viticola and Erysiphe necator). These compounds showed differences in their efficacy depending on the variability of mildews and highly dependent on plant genetics, environmental conditions and selection pressure. The plant defense stimulation could be an alternative or additional method to traditional pest management in the grapevine.Tools “BioMolChem” were developed to better assess the defence status of the plant defences in vitro and in natura. Transcript kinetics of selected defence-related genes and polyphenol contents profiles, during Vitis vinifera-biotrophic pathogen interaction, were characterized, and the impact of pathogen diversity was investigated in the absence or presence of elicitation. In vineyard, under strong pathogen pressures, it is thus possible to protect leaves and clusters, with SDP and assays of association or alternation with conventional fungicides show the potential interest of the use of these SDP in the vineyard.The grapevine defense mechanisms are complex, depending on the elicitor, leading to the coordinated accumulation of pathogenesis-related proteins (PR), the production of phytoalexins, and the reinforcement of plant cell walls.On the way, within the framework of an innovative and sustainable viticulture, the SDP was applied to hybrid genotypes (V. vinifera x M. rotundifolia). We reveal that according to the level of intrinsic resistance of the genotypes (more or less resistant to powdery and to downy mildew), it is possible to increase the level of the expressed resistance. The SDP could become allies of interest in the use of partially resistant grapevine varieties.The present findings provide insights into the potential use of transcripts and stilbenes as markers of the defense status of grapevine leaves with or without elicitation or infection, which should allow us to exploit and develop a better use of elicitors in alternative or complementary strategies in grapevine pest management.

No / Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1beta-mediated inflammatory signaling. Resveratrol suppressed IL-1beta-induced activation of NF-kappaB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1beta-induced NF-kappaB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IkappaB kinase, IkappaBalpha phosphorylation, and inhibition of nuclear translocation of NF-kappaB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-kappaB activation. Inhibition of PI3K by wortmannin attenuated IL-1beta-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1beta-induced activation of NF-kappaB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-kappaB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-kappaB.

No / Osteoarthritis is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective on pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Resveratrol is a phytoalexin stilbene produced naturally by plants including red grapes, peanuts and various berries. Recent research in various cell models has demonstrated that resveratrol is safe and has potent anti-inflammatory properties. However, its potential for treating arthritic conditions has not been explored. In this study we provide experimental evidence that resveratrol inhibits the expression of VEGF, MMP-3, MMP-9 and COX-2 in human articular chondrocytes stimulated with the pro-inflammatory cytokine IL-1beta. Since these gene products are regulated by the transcription factor NF-kappaB, we investigated the effects of resveratrol on IL-1beta-induced NF-kappaB signaling pathway. Resveratrol, like N-Ac-Leu-Leu-norleucinal (ALLN) suppressed IL-1beta-induced proteasome function and the degradation of IkappaBalpha (an inhibitor of NF-kappaB) without affecting IkappaBalpha kinase activation, IkappaBalpha-phosphorylation or IkappaBalpha-ubiquitination which suppressed nuclear translocation of the p65 subunit of NF-kappaB and its phosphorylation. Furthermore, we observed that resveratrol as well as ALLN inhibited IL-1beta-induced apoptosis, caspase-3 activation and PARP cleavage in human articular chondrocytes. In summary, our results suggest that resveratrol suppresses apoptosis and inflammatory signaling through its actions on the NF-kappaB pathway in human chondrocytes. We propose that resveratrol should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

No / Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-kappaB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IkappaBalpha kinase and IkappaBalpha phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-kappaB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-kappaB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-kappaB, inhibition of NF-kappaB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating osteoporosis and rheumatoid arthritis-related bone loss.