O gênero Passifloraceae possui mais de 400 espécies, sendo uma delas Passiflora incarnata L., detentora de diversas aplicações , principalmente no âmbito farmacêutico como uma planta capaz de atuar no sistema nervoso central para o tratamento de distúrbios da ansiedade. As substâncias mais abundantes existentes nas folhas dessa espécie pertencem à classe dos flavonóides, principalmente os C-glicosilados, que podem estar diretamente relacionados à atividade farmacológica da planta. Assim, o objetivo do presente trabalho , foi avaliar a influência (ou não ) de fatores externos na concentração dos flavonóides, em um cultivo experimental realizado na região de Botucatu .Os flavonóides analisados foram a luteolina, homoorientina, vitexina, isovitexina e orientina , com sua quantificação realizada por HPLC-UV/DAD e expressos em flavonóides totais calculados com base no padrão de rutina, através do método descrito por Pereira (2002). Realizou-se a correlação quimiométrica destes resultados pela análise multivariada , através de PCA (análise de componentes principais ) e HCA (análise hierárquica de agrupamentos), para avaliar se ocorreu a influência direta de determinados elementos do solo (pH, macronutrientes e micronutrientes) e do ambiente (temperatura e umidade) no teor de flavonóides encontrados nas folhas. Concluiu-se que as amostras se apresentaram de forma heterogênea no decorrer do cultivo (quanto à classificação ) , porém que alguns minerais presentes no solo (por exemplo, ferro, boro e cobre ), possuem relação inversa à concentração dos flavonóides totais encontrados nas folhas de P. incarnata. / Passifloraceae genus has more than 400 species and one of them is Passiflora incarnata L., which holds different application, mainly on the pharmaceutical scope as an herb capable to act on the central nervous system to the treatment of anxiety disturbs. The more copious compounds that exist on the leaves of this specie belong to flavonoid category, mainly C-glicosilates that could have direct relationship with pharmacologic properties of the herb. So, the objective of this task was to evaluate concentration in experimental growth made on Botucatu?s place. Analysed flavonoids were luteolin, homoorientin, vitexin, isovitexin and orientin, which were quantified by HPLC-UV/DAD and expressed as total flavonoids as rutin, through described method from Pereira (2002). Results were analysed through chemometric´s correlation, with PCA (principal components analysis) and HCA (hierarchic clusters analysis), to evaluate if some soil members (pH, macro and micronutrients) and ambient members (temperature and humidity) affected or not the flavonoids content on the leaves. It was conclude that all samples appeared heterogeneous in the course of growth, but some members present on the soil (as example iron, cupper and bore), have opposite relationship with total flavonoids content on the leaves of P. incarnata.

O cupuaçu é uma fruta nativa brasileira, com boa palatabilidade e grande potencial agroindustrial, seja na produção de polpa congelada, seja na de um produto análogo ao chocolate, o cupulate®. Polpas frescas de cupuaçu apresentaram composição centesimal e teor de fenólicos similares ao longo do ano, favorecendo sua industrialização. Esta regularidade também foi observada entre as distintas marcas avaliadas de polpa de cupuaçu congelada. Foi verificado, porém, diminuição de fenólicos, capacidade antioxidante e inibitória de α-amilase da polpa congelada em relação à polpa fresca. Esta enzima foi inibida pela polpa deste fruto; no cupulate não foi detectada nenhuma inibição de α-amilase ou de α-glicosidase. O cupulate apresentou, aproximadamente, metade da capacidade antioxidante, do teor de fenólicos e de proantocianidinas totais do chocolate. Apresentou, ainda, menor teor de proteínas e maior teor de lipídios que o chocolate. Os ácidos graxos presentes em maior quantidade na semente de cupuaçu, tanto fresca como processada (líquor), são o oléico e o esteárico. Os efeitos do processamento das sementes de cupuaçu assemelham-se àqueles observados no cacau: grande perda de umidade, aumento de lipídios, redução de carboidratos e do teor de polifenólicos. O decréscimo destes compostos ocorre, possivelmente, devido à sua condensação oxidativa, gerando taninos insolúveis de alto peso molecular. Os flavonóides identificados no cupuaçu foram catequina, epicatequina, theograndinas e glucuronídeos de isorhamnetina, quercetina e luteolina. Foram, ainda, identificados procianidinas até decâmeros na semente fresca de cupuaçu, já no líquor, devido à baixa concentração, somente até tetrâmeros. / Cupuassu is a brazilian native fruit, with good flavor and great industrial potential, either in the production of frozen pulps or in the manufacturing process of a chocolate-like food, the cupulate®. Cupuassu\'s fresh pulp showed similar chemical composition and phenolics content over the year, supporting its industrialization. This regularity was also observed between different brands of cupuassu frozen pulp. It was found, however, that the processed pulp has a decrease on phenolics content, antioxidant capacity and α-amylase inhibitory activity, in relation to the fresh pulp. This enzyme was inhibited by the fruit pulp. Cupulate, on the other hand, did not inhibit either α-amylase or α-glucosidase. This product showed, approximately, half of the antioxidant capacity, of the total phenolics content and of the total proanthocyanidins content found in the regular chocolate. Cupulate showed, also, less proteins content and more lipids content than chocolate. The most abundant fatty acids, either in the cupuassu\'s fresh seed or in the processed seed (liquor), were the oleic acid and stearic acid. The processing effects observed in the cupuassu\'s seeds were similar to those observed in cocoa processing, i.e. high moisture reduction, lipids increase, carbohydrate and phenolics content reduction. The phenolics decrease occurs, possibly, due to oxidative condensation, yielding unsoluble tannins of high molecular mass. The flavonoids identified on cupuassu were catechin, epicatechin, theograndins and glucuronides of isorhamnetin, quercetin and luteolin. Procyanidins up to decamers on the cupuassu\'s fresh seeds were also identified, but only up to tetramers on cupuassu\'s liquor, due to the their low concentration.

A Lesão Renal Aguda (LRA) é a complicação mais grave da rabdomiólise. Nessa síndrome, a liberação do pigmento heme desencadeia uma lesão que se caracteriza por vasoconstrição glomerular e toxicidade celular direta com componente oxidativo. A lesão oxidativa desencadeada é uma das linhas fisiopatológicas mais intrigantes. A renoproteção com antioxidantes tem demonstrado efeito satisfatório. As proantocianidinas são antioxidantes naturais encontrados no extrato da semente da uva. O objetivo deste estudo foi avaliar o efeito antioxidante da Vitis vinifera (extrato da semente de uva) sobre a função renal de ratos submetidos à lesão por rabdomiólise. Foram utilizados ratos Wistar, adultos machos, pesando entre 250-300 gramas. A LRA foi induzida pela administração de glicerol 50% i.m (intramuscular). Os animais foram distribuídos em 4 grupos: grupo Salina (6ml/Kg de NaCl 0,9% via intraperitoneal (i.p) 1 vez ao dia), grupo Glicerol (6ml/Kg de glicerol i.m, cada região femoral recebeu 3ml/Kg de glicerol, 1 vez ao dia), grupo Vitis vinifera (3mg/Kg v.o por 5 dias) e grupo Glicerol+Vitis vinifera que recebeu Vitis vinifera por 5 dias antes do glicerol. Foram avaliados o marcador de lesão muscular (CK), a função renal (FR), a função tubular (FENa e FEK), o perfil oxidativo (peróxidos urinários-FOX-2 e MDA-TBARS) , a histologia e morfometria renal. O grupo Glicerol tratado com Vitis vinifera apresentou melhora da FR e tubular, redução dos níveis da peroxidação lipídica e melhora da histologia renal. Os resultados deste estudo confirmaram a proteção antioxidante, com repercussão histológica, da Vitis vinifera na LRA induzida por glicerol. / The Acute Kidney Injury (AKI) is the worst complication of rhabdomyolysis. In this syndrome, the delivery of heme pigment induces an injury that distinguishes itself by glomerular vasoconstriction and direct cellular toxicity with oxidative component. The oxidative injury is an intriguing one of the pathophysiological mechanism. The renoprotection with antioxidants has demonstrated satisfactory effect. The proanthocyanidins are natural antioxidants found in grape seed extract. The aim of this study was to evaluate the antioxidant effect of Vitis vinifera (grape seed extract) on the renal function of rats submitted to the injury by rhabdomyolysis. Wistar rats, male, adults, weight ranging from 250-300 g were used. The AKI was induced intramuscular (i.m.) administration of glycerol 50%. The animals were distributed in 4 groups: Saline group (6ml/Kg of NaCl 0,9% intraperitoneal (i.p) once a day), Glycerol group (6ml/Kg of glycerol i.m, each femoral region received 3ml/Kg of glycerol, once a day), Vitis vinifera group (3mg/Kg v.o by 5 days) and Glycerol+Vitis vinifera group that has received Vitis vinifera by 5 days before glycerol. Marker of muscular injury (CK), renal function (RF), the tubular function (FENa and FEK), the oxidative profile (urinary peroxides -FOX-2 and MDA-TBARS), the histological and kidney morphometric were evaluated. The Glycerol group treated with Vitis vinifera has shown improvements in RF and tubular, reduction of levels of lipid peroxidation and amelioration of kidney histology. The results of this study have confirmed the antioxidant protection, with histological repercussion of Vitis vinifera in AKI induced by glycerol.

As plantas pertencentes à família das leguminosas apresentam um grande interesse econômico, sendo que o Brasil é um país com vasta utilização de plantas desta família. Neste contexto, destaca-se a leguminosa Phaseolus vulgaris L. popularmente conhecida como feijão, que contém uma boa fonte de constituintes bioativos. Espécie nativa da América Central e dos Andes peruanos possui grande importância econômica para o Brasil. Em sua composição química podemos encontrar os flavonóides. Inúmeros artigos científicos descrevem as atividades dos flavonóides como antioxidantes, antimutagêncio, anticarcinogênico e na captação de nitrogênio durante o desenvolvimento da planta. Sendo esta leguminosa um dos alimentos mais consumidos no mundo e de larga importância econômica, o presente trabalho visou avaliar o perfil cromatográfico do extrato metanólico de 47 cultivares de feijão através da técnica de Cromatografia Líquida de Alta Eficiência (CLAE) e também a validação do seu método. O material vegetal, sementes, teve seu teor de umidade estabelecido pelo método gravimétrico. Através do perfil cromatográfico das sementes dos cultivares de feijão, foi possível identificar a presença de flavonóides como a astragalina, um heterosídeo do canferol. O método foi devidamente validado por Cromatografia Líquida de Alta Eficiência (CLAE) de acordo com os métodos preconizados na literatura. Os cultivares TB 0224, TB0226, TB 0309, Rosinha Precoce e Amarelo Iolanda, foram os que apresentaram teor maior de astragalina, demonstrando que os feijões de cor possuem maior composição de deste composto. / Plants belonging to the legume family have a big economic interest, being Brazil is a country with extensive use of plants in this family. In this context, we highlight the legume Phaseolus vulgaris L. popularly known as beans, which are a good source of bioactive compounds. This species is native of southern Brazil and the main food consumed by the population. In its chemical composition can meet the flavonoids. Numerous scientific articles describing the activities of flavonoids as antioxidants, antimutagênic, anticarcinogenic and nitrogen uptake during plant development. Since this is a legume consumed more food in the world and of great economic importance, this study sought to evaluate the chromatographic profile of the methanol extract of 47 cultivars of beans through the technique of High Performance Liquid Chromatography (HPLC) and also the validation of their method. The plant material, seeds, water content was established by gravimetric method. Through the chromatographic profile of the seeds of bean cultivars, it was possible to identify the presence of flavonoids and astragalin, a kaempferol heteroside. The method was validated for High Performance Liquid Chromatography (HPLC) according to the methods suggested in the literature. The cultivars with colored TB 0224, TB0226, TB 0309, Rosinha Precoce and Amarelo Iolanda, were those with a higher content of astragalin, demonstrating that colored beans have a higher composition of astragalin.

Made available in DSpace on 2017-05-12T14:36:24Z (GMT). No. of bitstreams: 1 EFEITO DA QUERCETINA OVARIECTOMIZADAS.pdf: 2103814 bytes, checksum: ae46af8ec18f6634ae18e23c19f6e393 (MD5) Previous issue date: 2015-12-07 / Tamoxifen was discovered in 1970 and classified as selective estrogen receptor modulator (SERM) being used as therapy in the treatment of breast cancer. Although benefits has been proven, side effects are intense, and are related to different mechanisms of toxic action, such as oxidative stress and changes in the glycolytic pathway that induce a hyperglycemia and liver damage. The administration of quercetin, a flavonoid with antioxidant potential, that has inhibitory effect on enzyme butyrylcholinesterase, an enzyme possibly associated with hepatotoxicity and hyperglycemia, has benefits on the adverse effects caused by tamoxifen. Thus, first to determine the effect of quercetin on the hyperglycemia caused by tamoxifen, ovariectomized rats were treated orally, with tamoxifen and quercetin; concluding that effects of quercetin are dependent on ratio quercetin/tamoxifen coadministered and the 4.5 ratio is more effective. After, trying to elucidate the mechanisms involved in changes in the glycolytic pathway, oxidative stress and liver damage perfusion was performed in rat liver, studying especially glycogenolysis and gluconeogenesis, using as an indicator of liver damage of butyrylcholinesterase activity and monitoring the oxidative stress. The results showed that quercetin has inhibitory effect on glucose production and enhances the effects generated by tamoxifen in gluconeogenesis, concluding that although quercetin is known for antioxidant activity, in certain concentrations (400 μM) intensifies the damage initially caused by tamoxifen, this effect doesn't occur with lower concentrations of quercetin (200 μM). Therefore, although quercetin has shown to be effective in reducing hyperglycemia caused by tamoxifen, the interaction of quercetin with tamoxifen should be viewed with caution since the effect of this interaction in carbohydrate metabolism is intense, can alter the intracellular redox environment, compromising its integrity and causing serious damage to liver tissue. / O tamoxifeno, descoberto em 1970 foi classificado como modulador seletivo de receptor de estrógeno (SERM) sendo utilizado no tramento e prevenção do câncer de mama e embora seja terapia de escolha no período pós-menopausa seus efeitos colaterais são intensos, e estão relacionados a diferentes mecanismos de ação tóxica, como o estresse oxidativo e alterações na via glicolítica que induzem a hiperglicemia e causam danos hepáticos. A coadministração de quercetina, um flavonoide com potencial antioxidante e que possui efeito inibitório na enzima butirilcolinesterase, enzima essa possivelmente associada a hepatotoxicidade e a hiperglicemia, traria benefícios diante dos efeitos adversos gerados pelo tamoxifeno. Sendo assim, primeiramente, para determinar o efeito da quercetina diante da hiperglicemia causada pelo tamoxifeno, ratas ovariectomizadas foram tratadas via oral com quercetina e tamoxifeno; concluindo que os efeitos da quercetina são dependentes da razão Quercetina/Tamoxifeno coadministrado, e na razão 4,5 a quercetina mostra-se altamentente eficaz. Após, buscando elucidar os mecanismos envolvidos nas alterações na via glicolítica, os danos hepáticos e o estresse oxidativo, foram realizados perfusão em fígado isolado de ratas, estudando em especial a glicogenólise e a gliconeogênese, utilizando como indicador dos danos hepáticos a atividade da butirilcolinesterase e monitorando o estresse oxidativo. Os resultados mostraram que a quercetina possui efeito inibitório (200 μM) sobre a produção de glicose e intensifica os efeitos gerados pelo tamoxifeno na gliconeogênese, concluindo que embora a quercetina seja reconhecida pela sua atividade antioxidante, em determinadas concentrações (400 μM) ela acentua os danos inicialmente causados pelo tamoxifeno, não ocorrendo esse efeito com concentrações mais baixas de quercetina (200 μM). Sendo assim, embora a quercetina tenha se mostrado eficaz, reduzindo a hiperglicemia causada pelo tamoxifeno via oral, a interação da quercetina com tamoxifeno deve ser vista com cautela, pois os resultados mostram que o efeito dessa interação no metabolismo de carboidratos é intenso, podendo alterar o ambiente redox intracelular, comprometer a integridade celular e causar danos graves no tecido hepático.

Made available in DSpace on 2017-07-10T13:59:28Z (GMT). No. of bitstreams: 1 EFEITO DA QUERCETINA OVARIECTOMIZADAS.pdf: 2103814 bytes, checksum: ae46af8ec18f6634ae18e23c19f6e393 (MD5) Previous issue date: 2015-12-07 / Tamoxifen was discovered in 1970 and classified as selective estrogen receptor modulator (SERM) being used as therapy in the treatment of breast cancer. Although benefits has been proven, side effects are intense, and are related to different mechanisms of toxic action, such as oxidative stress and changes in the glycolytic pathway that induce a hyperglycemia and liver damage. The administration of quercetin, a flavonoid with antioxidant potential, that has inhibitory effect on enzyme butyrylcholinesterase, an enzyme possibly associated with hepatotoxicity and hyperglycemia, has benefits on the adverse effects caused by tamoxifen. Thus, first to determine the effect of quercetin on the hyperglycemia caused by tamoxifen, ovariectomized rats were treated orally, with tamoxifen and quercetin; concluding that effects of quercetin are dependent on ratio quercetin/tamoxifen coadministered and the 4.5 ratio is more effective. After, trying to elucidate the mechanisms involved in changes in the glycolytic pathway, oxidative stress and liver damage perfusion was performed in rat liver, studying especially glycogenolysis and gluconeogenesis, using as an indicator of liver damage of butyrylcholinesterase activity and monitoring the oxidative stress. The results showed that quercetin has inhibitory effect on glucose production and enhances the effects generated by tamoxifen in gluconeogenesis, concluding that although quercetin is known for antioxidant activity, in certain concentrations (400 μM) intensifies the damage initially caused by tamoxifen, this effect doesn't occur with lower concentrations of quercetin (200 μM). Therefore, although quercetin has shown to be effective in reducing hyperglycemia caused by tamoxifen, the interaction of quercetin with tamoxifen should be viewed with caution since the effect of this interaction in carbohydrate metabolism is intense, can alter the intracellular redox environment, compromising its integrity and causing serious damage to liver tissue. / O tamoxifeno, descoberto em 1970 foi classificado como modulador seletivo de receptor de estrógeno (SERM) sendo utilizado no tramento e prevenção do câncer de mama e embora seja terapia de escolha no período pós-menopausa seus efeitos colaterais são intensos, e estão relacionados a diferentes mecanismos de ação tóxica, como o estresse oxidativo e alterações na via glicolítica que induzem a hiperglicemia e causam danos hepáticos. A coadministração de quercetina, um flavonoide com potencial antioxidante e que possui efeito inibitório na enzima butirilcolinesterase, enzima essa possivelmente associada a hepatotoxicidade e a hiperglicemia, traria benefícios diante dos efeitos adversos gerados pelo tamoxifeno. Sendo assim, primeiramente, para determinar o efeito da quercetina diante da hiperglicemia causada pelo tamoxifeno, ratas ovariectomizadas foram tratadas via oral com quercetina e tamoxifeno; concluindo que os efeitos da quercetina são dependentes da razão Quercetina/Tamoxifeno coadministrado, e na razão 4,5 a quercetina mostra-se altamentente eficaz. Após, buscando elucidar os mecanismos envolvidos nas alterações na via glicolítica, os danos hepáticos e o estresse oxidativo, foram realizados perfusão em fígado isolado de ratas, estudando em especial a glicogenólise e a gliconeogênese, utilizando como indicador dos danos hepáticos a atividade da butirilcolinesterase e monitorando o estresse oxidativo. Os resultados mostraram que a quercetina possui efeito inibitório (200 μM) sobre a produção de glicose e intensifica os efeitos gerados pelo tamoxifeno na gliconeogênese, concluindo que embora a quercetina seja reconhecida pela sua atividade antioxidante, em determinadas concentrações (400 μM) ela acentua os danos inicialmente causados pelo tamoxifeno, não ocorrendo esse efeito com concentrações mais baixas de quercetina (200 μM). Sendo assim, embora a quercetina tenha se mostrado eficaz, reduzindo a hiperglicemia causada pelo tamoxifeno via oral, a interação da quercetina com tamoxifeno deve ser vista com cautela, pois os resultados mostram que o efeito dessa interação no metabolismo de carboidratos é intenso, podendo alterar o ambiente redox intracelular, comprometer a integridade celular e causar danos graves no tecido hepático.

Pan, Tak Yin. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.iv / Content --- p.v / Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's Disease --- p.1 / Chapter 1.1.1 --- Cholinergic hypothesis --- p.2 / Chapter 1.1.2 --- p-amyloid hypothesis --- p.2 / Chapter 1.1.3 --- Taupathy hypothesis --- p.3 / Chapter 1.1.4 --- Current therapies --- p.4 / Chapter 1.2 --- Proteins Involved in Alzhemer's Disease --- p.5 / Chapter 1.2.1 --- Acetylcholinesterase (AChE) --- p.5 / Chapter 1.2.2 --- p-amyloid --- p.6 / Chapter 1.2.3 --- Paired helical filaments (PHF) --- p.7 / Chapter 1.2.4 --- Protein kinases --- p.8 / Chapter 1.2.4.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.9 / Chapter 1.2.4.2 --- Cyclin-dependent kinase-5 (CDK-5) --- p.9 / Chapter 1.2.5 --- Protein phosphatase (PP) --- p.10 / Chapter 1.2.5.1 --- Protein phosphatase 1 (PP-1) --- p.11 / Chapter 1.2.5.2 --- Protein phosphatise 2A (PP-2A) --- p.12 / Chapter 1.2.5.3 --- Protein phosphatise 2B (PP-2B) --- p.13 / Chapter 1.2.6 --- Apoptotic and Anti-apoptotic proteins --- p.14 / Chapter 1.2.6.1 --- Caspase-3 --- p.15 / Chapter 1.2.6.2 --- Bcl-2 --- p.15 / Chapter 1.3 --- Flavonoids --- p.16 / Chapter 1.3.1 --- Biosynthesis of flavonoids --- p.17 / Chapter 1.3.2 --- Biological functions of flavonoids in plants --- p.18 / Chapter 1.3.3 --- Beneficial effects of flavonoids on human health --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- Differentiation of SHSY-5Y cells --- p.20 / Chapter 2.1.1 --- SHSY-5Y cell culture --- p.20 / Chapter 2.1.2 --- Counting cells --- p.20 / Chapter 2.1.3 --- Retinoic acid differentiation --- p.21 / Chapter 2.2 --- Western blot analysis --- p.21 / Chapter 2.2.1 --- Extraction of proteins from mammalian cells --- p.21 / Chapter 2.2.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.2.3 --- Semi-dry protein transfer to nitrocellulose membrane --- p.23 / Chapter 2.2.4. --- Membrane blocking and immunostaining --- p.24 / Chapter 2.3 --- MTT assay --- p.25 / Chapter 2.4 --- Hoechst 33342 Nuclei staining --- p.25 / Chapter 2.5 --- Cell cycle analysis --- p.25 / Chapter 2.5.1 --- Ethanol fixation --- p.25 / Chapter 2.5.2 --- Propidium iodide staining --- p.26 / Chapter 2.6 --- Annexin V-FITC & Propidium iodide staining --- p.26 / Chapter 2.7 --- DNA fragmentation analysis --- p.26 / Chapter 2.7.1 --- Phenol/Chloroform extraction of DNA --- p.26 / Chapter 2.7.2 --- Ethanol precipitation of DNA --- p.27 / Chapter 2.7.3 --- Agarose gel electrophoresis of DNA --- p.27 / Chapter 2.8 --- Proteomic analysis --- p.28 / Chapter 2.8.1 --- First dimension: isoelectric focusing --- p.28 / Chapter 2.8.2 --- Second dimension: SDS PAGE --- p.29 / Chapter 2.8.3 --- Gel staining --- p.30 / Chapter 2.8.3.1 --- Silver staining --- p.30 / Chapter 2.8.3.2 --- SYBRO Ruby staining --- p.31 / Chapter 2.8.4 --- Gel scanning and image analysis --- p.31 / Chapter 2.8.5 --- ln-gel digestion --- p.32 / Chapter 2.8.6 --- Zip Tip for desalting the digested sample --- p.33 / Chapter 2.8.7 --- Protein identification with mass spectrometry and database search --- p.33 / Chapter Chapter 3: --- Characterization of Okadaic acid-induced tail hyperphosphorylation in SHSY-5Y cells --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Objectives --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Differentiation of SH-SY5Y cell --- p.38 / Chapter 3.3.2 --- Changes of protein expression after okadaic acid treatment --- p.40 / Chapter 3.3.3 --- Neurite Retraction Induced by okadaic acid --- p.42 / Chapter 3.3.4 --- Okadaic acid-induced Cell Death measured by MTT assay --- p.44 / Chapter 3.3.5 --- Hoechst 33342 Nuclei Staining --- p.44 / Chapter 3.3.6 --- Cell cycle analysis by propidium iodide staining --- p.47 / Chapter 3.3.7 --- Early Apoptotic cells detection by Annexin V/PI staini --- p.49 / Chapter 3.3.8 --- DNA fragmentation --- p.51 / Chapter 3.4 --- Discussion --- p.53 / Chapter Chapter 4: --- Flavonoids screening for protecting neuronal death by preventing tau hyperphosphorylation --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Objectives --- p.58 / Chapter 4.3 --- Tested flavonoids --- p.59 / Chapter 4.4 --- Results --- p.60 / Chapter 4.4.1 --- Toxicity of flavonoids --- p.60 / Chapter 4.4.2 --- Effects of flavonoid pre-treatment on OA-induced neu retractions and cell death --- p.62 / Chapter 4.4.3 --- Western blot analysis --- p.65 / Chapter 4.4.4 --- The effect of different concentrations of hesperidin or OA treatment --- p.70 / Chapter 4.4.5 --- Proteomic analysis --- p.74 / Chapter 4.5 --- Discussion --- p.78 / Chapter Chapter 5: --- General Discussion --- p.82 / References

Yeung, Tak Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 181-197). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / List of Publications --- p.vii / Presentation --- p.vii / List of Abbreviations --- p.viii / Abbreviations in Figures --- p.xiii / Abbreviations in Symbols --- p.xiv / List of Cell Lines Used in this Study --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cellular Redox State and Cancer Biology --- p.1 / Chapter 1.2 --- Reactive Oxygen Species (ROS) --- p.1 / Chapter 1.3 --- Regulation of Cellular Redox State by Intrinsic and Extrinsic Antioxidant Systems --- p.5 / Chapter 1.3.1 --- Intrinsic Antioxidant System --- p.6 / Chapter 1.3.2 --- Extrinsic Antioxidant System --- p.8 / Chapter 1.4 --- Glutathione --- p.9 / Chapter 1.4.1 --- General Information of Glutathione --- p.9 / Chapter 1.4.2 --- Functions of Glutathione --- p.12 / Chapter 1.4.2.1 --- As an Antioxidant and Free Radical Scavenger --- p.12 / Chapter 1.4.2.2 --- As a Detoxifier --- p.13 / Chapter 1.4.2.3 --- As a Regulator of Cell Signaling --- p.14 / Chapter 1.4.3 --- Synthesis of Glutathione --- p.15 / Chapter 1.4.4 --- Catabolism of Glutathione --- p.15 / Chapter 1.4.5 --- Transport and Uptake of Glutathione --- p.16 / Chapter 1.4.6 --- Glutathione in Cancer Biology --- p.18 / Chapter 1.4.6.1 --- "Role of Glutathione in the Regulation of Carcinogenesis, Growth and Apoptosis of Cancer Cells" --- p.18 / Chapter 1.4.6.1.1 --- Role of Glutathione in Carcinogenesis --- p.18 / Chapter 1.4.6.1.2 --- Role of Glutathione in the Growth of Cancer Cells --- p.20 / Chapter 1.4.6.1.3 --- Role of Glutathione in Apoptosis of Cancer Cells --- p.21 / Chapter 1.4.6.2 --- Role of Glutathione in the Regulation of Metastasis --- p.23 / Chapter 1.4.6.3 --- Role of Glutathione in Cancer Resistance and Therapy --- p.24 / Chapter 1.4.6.3.1 --- Role of Glutathione in Cancer Resistance --- p.24 / Chapter 1.4.6.3.2 --- Role of Glutathione in Cancer Therapy --- p.24 / Chapter 1.5 --- Aims of the Present Study --- p.25 / Chapter Chapter 2 --- In Vitro Study of Bαicαlein and Baicalin on Glutathione Depletion --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Scutellaria bαicαlensis Georgi --- p.28 / Chapter 2.1.1.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.28 / Chapter 2.1.1.2 --- As an Antioxidant and Free Radical Scavenger --- p.29 / Chapter 2.1.1.3 --- Long History for Treatment of Cancers with the Obscure Mechanism --- p.30 / Chapter 2.1.1.4 --- Major Components --- p.31 / Chapter 2.1.2 --- Baicalein and Baicalin --- p.32 / Chapter 2.1.2.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.32 / Chapter 2.1.2.2 --- As an Antioxidant and Free Radical Scavenger --- p.33 / Chapter 2.1.3 --- Hypothesis: Baicalein and Baicalin Induce Cancer Cell Death Via Glutathione Depletion --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Chemicals --- p.36 / Chapter 2.2.2 --- Buffers and Solutions --- p.36 / Chapter 2.2.3 --- Animals --- p.37 / Chapter 2.2.4 --- Preparation of Rat Brain Microsomes --- p.37 / Chapter 2.2.5 --- Glutathione Depletion Assay In Vitro and Thiol Depletion Assay in Rat Brain Microsomes --- p.38 / Chapter 2.2.6 --- Statistical Analysis --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Glutathione --- p.42 / Chapter 2.3.2 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Rat Brain Microsomes --- p.42 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Effects of Baicalein and Baicalin on Proliferation of Different Human Cancer and Normal Cells --- p.45 / Chapter 3.1 --- Introduction-Importance of Developing A Novel Compound Inducing Cancer Cells to Cell Death with the Least Side Effects on Normal Cells --- p.45 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Instruments --- p.46 / Chapter 3.2.2 --- Chemicals and Cell Culture Reagents --- p.46 / Chapter 3.2.3 --- Buffers --- p.46 / Chapter 3.2.4 --- Cell Lines --- p.47 / Chapter 3.2.5 --- Cell Culture --- p.48 / Chapter 3.2.6 --- Determination of Cell Proliferation by MTT Assay --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.51 / Chapter 3.3.2 --- Effects of Baicalein on Different Normal Cell Lines --- p.56 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.58 / Chapter 3.4.2 --- Effects of Baicalein on Cell Proliferation on Different Human Normal Cell Lines --- p.60 / Chapter Chapter 4 --- Glutathione-Depleting Effects of Baicalein on Cell Proliferation of Different Cell Lines --- p.61 / Chapter 4.1 --- Introduction-Brain Tumors --- p.61 / Chapter 4.1.1 --- Types and Classifications of Brain Tumors --- p.61 / Chapter 4.1.2 --- "Incidence Time, Patient Survival Time and Rate for" --- p.65 / Chapter 4.1.3 --- Symptoms and Diagnostic Methods for Brain Tumors --- p.66 / Chapter 4.1.4 --- "Treatments, Side Effects and Difficulties of Treatments for Brain Tumors" --- p.67 / Chapter 4.1.5 --- Glutathione Levels in Brain Normal and Cancer Cells --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Instruments --- p.70 / Chapter 4.2.2 --- Chemicals --- p.70 / Chapter 4.2.3 --- Buffers --- p.70 / Chapter 4.2.4 --- Determination of Cell Proliferation by MTT Assay --- p.70 / Chapter 4.2.5 --- Determination of Intracellular Glutathione Depletion by Fluorescent Dye CMAC --- p.71 / Chapter 4.2.6 --- Determination of Cellular Reduced Glutathione Levels by DTNB-Coupled Glutathione Reductase Recycling Assay --- p.73 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of Baicalein on Intracellular GSH Levels and Cell Proliferation for Different Cell Lines --- p.75 / Chapter 4.3.2 --- Basal Intracellular Glutathione in Different Cell Lines --- p.81 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Intracellular Glutathione Depletion and Cell Death Induction Effects of Baicalein on Different Cell Lines --- p.84 / Chapter 4.4.2 --- Relationship between Basal Glutathione Levels and Drug Susceptibilities --- p.85 / Chapter Chapter 5 --- Effects of Baicalein on Apoptosis and Caspase Pathways --- p.88 / Chapter 5.1 --- Introduction-Modes of Cell Death --- p.88 / Chapter 5.1.1 --- Necrosis --- p.88 / Chapter 5.1.2 --- Apoptosis --- p.89 / Chapter 5.2 --- Materials and Methods --- p.92 / Chapter 5.2.1 --- Chemicals --- p.92 / Chapter 5.2.2 --- Buffers --- p.92 / Chapter 5.2.3 --- Determination of Change of Mitochondrial Membrane Potential by JC-1 --- p.93 / Chapter 5.2.4 --- Determination of Apoptosis by Annexin V-Propidium Iodide Staining --- p.94 / Chapter 5.2.5 --- Determination of Cell Cycle Arrest by Propidium Iodide Staining --- p.95 / Chapter 5.2.6 --- "Determination of Caspase-3, -8 and -9 Activities by Fluorescent-Labeled Peptides" --- p.96 / Chapter 5.2.7 --- Determination of DNA Fragmentation --- p.97 / Chapter 5.2.8 --- Terminal Deoxynucleotidyl Transferase Mediated dUTP End Labeling (TUNEL) Assay --- p.99 / Chapter 5.2.9 --- Flow Cytometry --- p.101 / Chapter 5.3 --- Results --- p.102 / Chapter 5.3.1 --- Effects of Baicalein on Mitochondrial Membrane Potential by JC-1 Staining --- p.102 / Chapter 5.3.2 --- Effects of Baicalein on Apoptosis and Necrosis by Annexin V-Propidium Iodide Staining --- p.104 / Chapter 5.3.3 --- Effects of Baicalein on Cell Cycle Arrest by Propidium Iodide Staining --- p.108 / Chapter 5.3.4 --- "Effects of Baicalein on Caspase-3, -8 and -9 Activities" --- p.110 / Chapter 5.3.5 --- Effeets of Baiealein on DNA Fragmentation --- p.115 / Chapter 5.3.6 --- Effects of Baicalein on TUNEL Assay --- p.117 / Chapter 5.4 --- Discussion --- p.120 / Chapter Chapter 6 --- Pro-Oxidant Role of Baicalein on Reactive Oxygen Species Generation --- p.122 / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.122 / Chapter 6.2.1 --- Chemicals --- p.122 / Chapter 6.2.2 --- Determination of Cellular Reactive Oxygen Species Generation by Fluorescent Dye cDCFDA --- p.123 / Chapter 6.2.3 --- Determination of Mitochondrial Reactive Oxygen Species Generation by Fluorescent Dye Rhl23 --- p.124 / Chapter 6.3 --- Results --- p.125 / Chapter 6.3.1 --- Effects of Baicalein on Cellular ROS Generation by Fluorescent Dye cDCFDA --- p.125 / Chapter 6.3.2 --- Effects of Baicalein on Mitochondrial ROS Generation by Fluorescent Dye Rhl23 --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- The Anticancer Mechanistic Study of Baicalein --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Materials and Methods --- p.134 / Chapter 7.2.1 --- Chemicals --- p.134 / Chapter 7.2.2 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.134 / Chapter 7.2.3 --- Reversibility of Baicalein-Induced Cellular ROS Generation --- p.136 / Chapter 7.2.4 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.137 / Chapter 7.2.5 --- "Reversibility of Baicalein-Induced Caspase-3, -8 and -9 Activation by Co-Treatment of Different Antioxidants" --- p.138 / Chapter 7.3 --- Results --- p.139 / Chapter 7.3.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.139 / Chapter 7.3.1.1 --- Pre-treatments --- p.139 / Chapter 7.3.1.2 --- Co-treatments --- p.141 / Chapter 7.3.1.3 --- Post-treatments --- p.144 / Chapter 7.3.2 --- Reversibility of Baicalein-Induced Cellular ROS Generation by Co-Treatment of Different Antioxidants --- p.147 / Chapter 7.3.3 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.152 / Chapter 7.3.4 --- Reversibility of Baicalein-Induced Caspase-3 Activation by Co-Treatment of Different Antioxidants --- p.156 / Chapter 7.3.5 --- Reversibility of Baicalein-Induced Caspase-8 and -9 Activation by Co-Treatment of Different Antioxidants --- p.160 / Chapter 7.4 --- Discussion --- p.164 / Chapter 7.4.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death --- p.164 / Chapter 7.4.2 --- "Reversibility of Baicalein-Induced ROS Generation," --- p.167 / Chapter 7.5 --- Concluding Remarks --- p.168 / Chapter Chapter 8 --- General Discussion --- p.169 / Chapter 8.1 --- Drug Delivery to Brain --- p.169 / Chapter 8.2 --- Protective Roles of Baicalein on Brain Cells --- p.170 / Chapter 8.2.1 --- Actions Against Oxidative Stress --- p.170 / Chapter 8.2.2 --- Actions Against Other Neurotoxic Damages --- p.171 / Chapter 8.2.3 --- Actions Against Neuronal Diseases --- p.172 / Chapter 8.3 --- Anticancer Roles of Baicalein on Astrocytoma --- p.173 / Chapter 8.4 --- Implications on the Dual Roles of Baicalein: Antioxidant and Pro-oxidant --- p.175 / Chapter 8.5 --- Future Perspectives --- p.175 / Chapter 8.5.1 --- Effects of Baicalein on Antioxidant System --- p.175 / Chapter 8.5.2 --- Effects of Baicalein on GSH Synthesis --- p.176 / Chapter 8.5.3 --- In Vivo Studies on Cytotoxic Effects of Baicalein --- p.177 / Chapter 8.5.4 --- In Vivo Studies on Anti-Tumor Effects and In Vitro Studies on Anti-Metastasis Effects of Baicalein --- p.178 / Reference List --- p.181

Lam, Cheuk Kai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 148-173). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.x / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Introduction to Cholesterol and Its Related Diseases --- p.1 / Chapter 1.1.1 --- Chemistry of cholesterol --- p.1 / Chapter 1.1.2 --- Physiological importance of cholesterol --- p.1 / Chapter 1.1.3 --- Pathological effects of cholesterol --- p.3 / Chapter 1.1.3.1 --- Mechanism of atherosclerosis --- p.3 / Chapter 1.2 --- Cholesterol Homeostasis --- p.6 / Chapter 1.2.1 --- Liver as the main organ for cholesterol metabolism --- p.6 / Chapter 1.2.2 --- Regulatory sites of cholesterol metabolism --- p.6 / Chapter 1.2.2.1 --- Regulation of cholesterol absorption by acyl coenzyme A: cholesterol acyltransferase (ACAT) --- p.6 / Chapter 1.2.2.2 --- Sterol regulatory element-binding protein 2 (SREBP-2) as a transcription factor for 3 -hydroxy-3 -methylglutaryl coenzyme A reductase (HMGR) and low-density lipoprotein receptor (LDLR) --- p.10 / Chapter 1.2.2.3 --- Roles ofLDLR --- p.11 / Chapter 1.2.2.4 --- Rate limiting role of HMGR in cholesterol de novo synthesis --- p.14 / Chapter 1.2.2.5 --- Roles of liver-X-receptor-a (LXR-a) in cholesterol catabolism --- p.16 / Chapter 1.2.2.6 --- Roles of CYP7A1 in catabolism of cholesterol into bile acids --- p.19 / Chapter 1.2.2.7 --- Roles of cholesterol ester transfer protein (CETP) in maintaining cholesterol distribution in blood --- p.22 / Chapter CHAPTER 2 --- EFFECT OF OCTADECAENOIC ACIDS ON BLOOD CHOLESTEROL IN HAMSTERS / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.1.1 --- Effects of polyunsaturated fatty acids (PUFAs) on blood cholesterol --- p.25 / Chapter 2.1.2 --- Differential effects of 18-C PUFAs on lowering blood cholesterol in vivo --- p.25 / Chapter 2.1.3 --- "Structures, metabolism and conjugation of octadecaenoic acids (ODA)" --- p.26 / Chapter 2.1.4 --- Objectives --- p.26 / Chapter 2.2 --- Experiment 1 --- p.28 / Chapter 2.2.1 --- Materials and methods --- p.28 / Chapter 2.2.1.1 --- Experimental fatty acids --- p.28 / Chapter 2.2.1.1.1 --- Isolation of LN from flaxseed --- p.28 / Chapter 2.2.1.1.2 --- Isolation of CLN from tung seed --- p.28 / Chapter 2.2.1.2 --- Animals --- p.29 / Chapter 2.2.1.3 --- Diets --- p.30 / Chapter 2.2.1.4 --- Plasma lipid measurements --- p.30 / Chapter 2.2.1.5 --- Plasma CETP activity measurement --- p.30 / Chapter 2.2.1.6 --- "Measurement of liver SREBP-2, LDLR, HMGR and CYP7A1 protein abundance by Western blotting" --- p.34 / Chapter 2.2.1.7 --- "Measurement of hepatic SREBP-2, LDLR, HMGR, LXR, CYP7A1, CETP, SR-B1 and LCAT mRNA by real time PCR" --- p.35 / Chapter 2.2.1.7.1 --- Extraction of mRNA --- p.35 / Chapter 2.2.1.1.2 --- Complementary DNA synthesis --- p.36 / Chapter 2.2.1.7.3 --- Real-time polymerase chain reaction (PCR) anaylsis --- p.36 / Chapter 2.2.1.8 --- Determination of cholesterol in liver --- p.37 / Chapter 2.2.1.9 --- Determination of fecal neutral and acidic sterols --- p.38 / Chapter 2.2.1.9.1 --- Determination of fecal neutral sterols --- p.39 / Chapter 2.2.1.9.2 --- Determination of fecal acidic sterols --- p.41 / Chapter 2.2.1.10 --- Statistics --- p.43 / Chapter 2.2.2 --- Results --- p.44 / Chapter 2.2.2.1 --- Growth and food intake --- p.44 / Chapter 2.2.2.2 --- Organ weights --- p.44 / Chapter 2.2.2.3 --- "Effects of ODA on serum TC, TG and HDL-C" --- p.44 / Chapter 2.2.2.4 --- Effect of ODA on liver cholesterol --- p.48 / Chapter 2.2.2.5 --- Effect of ODA on fecal neutral sterol output --- p.48 / Chapter 2.2.2.6 --- Effect of ODA on fecal acidic sterol output --- p.48 / Chapter 2.2.2.7 --- Effect of ODA on cholesterol balance in hamsters --- p.52 / Chapter 2.2.2.8 --- Effect of ODA on plasma CETP activity --- p.52 / Chapter 2.2.2.9 --- Correlation between blood TC and liver cholesterol --- p.52 / Chapter 2.2.2.10 --- Correlation between blood HDL-C and liver cholesterol --- p.52 / Chapter 2.2.2.11 --- Correlation between blood nHDL/HDL ratio and liver cholesterol --- p.52 / Chapter 2.2.2.12 --- Effect ofODA on liver SREBP-2 immunoreactive mass --- p.58 / Chapter 2.2.2.13 --- Effect of ODA on liver LDLR immunoreactive mass --- p.58 / Chapter 2.2.2.14 --- Effect of ODA on liver HMGR immunoreactive mass --- p.58 / Chapter 2.2.2.15 --- Effect of ODA on liver LXR immunoreactive mass --- p.58 / Chapter 2.2.2.16 --- Effect of ODA on liver CYP7A1 immunoreactive mass --- p.63 / Chapter 2.2.2.17 --- Effects ofODA on hepatic CETP mRNA --- p.65 / Chapter 2.2.2.18 --- Effects of ODA on hepatic LDLR mRNA --- p.65 / Chapter 2.2.2.19 --- Effects of ODA on hepatic LXR mRNA --- p.65 / Chapter 2.2.2.20 --- Effects of ODA on hepatic CYP7A1 mRNA --- p.65 / Chapter 2.3 --- Experiment 2 --- p.70 / Chapter 2.3.1 --- Materials and Methods --- p.70 / Chapter 2.3.1.1 --- Experimental diets --- p.70 / Chapter 2.3.1.2 --- Animals --- p.70 / Chapter 2.3.1.3 --- Intestinal acyl coenzyme A: cholesterol acyltransferase (ACAT) activity measurement --- p.70 / Chapter 2.3.1.3.1 --- Preparation of intestinal microsome --- p.71 / Chapter 2.3.1.3.2 --- ACAT activity assay --- p.71 / Chapter 2.3.2 --- Results --- p.73 / Chapter 2.3.2.1 --- Growth and food intake --- p.73 / Chapter 2.3.2.2 --- Organ weights --- p.73 / Chapter 2.3.2.3 --- "Effect of ODA on serum TC, TG and HDL-C" --- p.73 / Chapter 2.3.2.4 --- Effect of ODA feeding on fecal neutral sterol content --- p.77 / Chapter 2.3.2.5 --- Effect of ODA feeding on fecal acidic sterol content --- p.77 / Chapter 2.3.2.6 --- Effect of ODA feeding on intestinal acyl coenzyme A: acyl cholesterol transferase (ACAT) activity --- p.77 / Chapter 2.4 --- Discussion --- p.81 / Chapter CHAPTER 3 --- EFFECT OF OCTADECAENOIC ACIDS ON CHOLESTEROL-REGULATING GENES IN HepG2 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HepG2 as a model of cholesterol regulation --- p.86 / Chapter 3.1.2 --- Effect of polyunsaturated fatty acids (PUFAs) on cholesterol regulating genes in cultured cells --- p.87 / Chapter 3.1.3 --- Objectives --- p.89 / Chapter 3.2 --- Materials and Methods --- p.90 / Chapter 3.2.1 --- Cell culture --- p.90 / Chapter 3.2.2 --- "Measurement of SREBP-2, LDLR, HMGR and CYP7A1 protein abundance by Western blotting" --- p.92 / Chapter 3.2.3 --- "Measurement of cellular SREBP-2, LDLR, HMGR, LXR, CYP7A1 and CETP mRNA by real time PCR" --- p.93 / Chapter 3.2.4 --- Statistics --- p.93 / Chapter 3.3 --- Results --- p.95 / Chapter 3.3.1 --- Effect of ODA on HepG2 SREBP-2 immunoreactive mass --- p.95 / Chapter 3.3.2 --- Effect of ODA on HepG2 HMGR immunoreactive mass --- p.95 / Chapter 3.3.3 --- Effect of ODA on HepG2 LDLR immunoreactive mass --- p.95 / Chapter 3.3.4 --- Effect of ODA on HepG2 LXR immunoreactive mass --- p.95 / Chapter 3.3.5 --- Effect of ODA on HepG2 CYP7A1 immunoreactive mass --- p.96 / Chapter 3.3.6 --- Effect of ODA supplementation on HepG2 SREBP-2 mRNA expression --- p.102 / Chapter 3.3.7 --- Effect of ODA supplementation on HepG2 SREBP-2 mRNA expression --- p.102 / Chapter 3.3.8 --- Effect of ODA supplementation on HepG2 LDLR mRNA expression --- p.102 / Chapter 3.3.9 --- Effect of ODA supplementation on HepG2 LXR mRNA expression --- p.106 / Chapter 3.3.10 --- Effect of ODA supplementation on HepG2 CYP7A1 mRNA expression --- p.106 / Chapter 3.3.11 --- Effect of ODA supplementation on HepG2 CETP mRNA expression --- p.106 / Chapter 3.4 --- Discussion --- p.110 / Chapter CHAPTER 4 --- EFFECT OF APPLE POLYPHENOLS ON BLOOD CHOLESTEROL IN HAMSTERS / Chapter 4.1 --- Introduction --- p.114 / Chapter 4.1.1 --- Apple is a commonly consumed fruit worldwide --- p.114 / Chapter 4.1.2 --- Potential health effects of apples --- p.114 / Chapter 4.1.3 --- Abundance of polyphenols in apple --- p.115 / Chapter 4.1.4 --- Fuji variety of apple --- p.116 / Chapter 4.1.5 --- Objectives --- p.116 / Chapter 4.2 --- Materials and Methods --- p.118 / Chapter 4.2.1 --- Isolation of AP --- p.118 / Chapter 4.2.2 --- Characterization of AP extract --- p.118 / Chapter 4.2.3 --- Effect of AP on CETP activity in vitro --- p.118 / Chapter 4.2.4 --- Effect of AP on blood cholesterol in hamsters --- p.119 / Chapter 4.2.4.1 --- Animals --- p.119 / Chapter 4.2.4.2 --- Diets --- p.120 / Chapter 4.2.4.3 --- Plasma lipids measurement --- p.121 / Chapter 4.2.4.4 --- Plasma CETP activity measurement and immunoreactive mass by Western blotting --- p.123 / Chapter 4.2.4.5 --- "Measurement of liver SREBP-2, LDL-R, HMG-R and CYP7A1 protein abundance by Western blotting" --- p.124 / Chapter 4.2.4.6 --- Statistics --- p.124 / Chapter 4.3 --- Results --- p.125 / Chapter 4.3.1 --- Polyphenol content in AP --- p.125 / Chapter 4.3.2 --- Effect of AP on CETP activity in vitro --- p.125 / Chapter 4.3.3 --- Growth and food intake --- p.128 / Chapter 4.3.4 --- Organ weights --- p.128 / Chapter 4.3.5 --- Effect of AP supplementation on the plasma lipid profile of hamsters --- p.131 / Chapter 4.3.6 --- Effect of AP feeding on plasma CETP activity of the hamsters --- p.131 / Chapter 4.3.7 --- Effect of AP on plasma CETP immunoreactive mass --- p.134 / Chapter 4.3.8 --- Effect of AP on liver SREBP-2 immunoreactive mass --- p.134 / Chapter 4.3.9 --- Effect of AP on liver LDLR immunoreactive mass --- p.134 / Chapter 4.3.10 --- Effect of AP on liver HMGR immunoreactive mass --- p.134 / Chapter 4.3.11 --- Effect of AP on liver CYP7A1 immunoreactive mass --- p.134 / Chapter 4.3.12 --- Effect of AP on liver cholesterol level --- p.140 / Chapter 4.4 --- Discussion --- p.142 / Chapter CHAPTER 5 --- CONCLUSION --- p.145 / REFERENCES --- p.148

Li, Wing Yan Kate. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 130-154). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Abstract (Chinese Version) --- p.vi / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Overview of cancer --- p.1 / Chapter 1.2. --- Apoptosis and cancer --- p.4 / Chapter 1.3. --- Roles and regulation of caspase-dependent apoptosis --- p.7 / Chapter 1.3.1. --- Extrinsic death receptor pathway --- p.8 / Chapter i. --- TNFR1 and TNFa --- p.13 / Chapter ii. --- CD95/Fas and CD95 Ligand/FasL --- p.14 / Chapter iii. --- "TRAIL-R1(DR4), TRAIL-R2 (DR5) and TRAIL" --- p.14 / Chapter 1.3.2. --- Intrinsic mitochondrial pathway --- p.16 / Chapter i. --- Bcl-2 family of proteins --- p.17 / Chapter ii. --- Reactive Oxygen Species (ROS) --- p.19 / Chapter 1.4. --- Phytochemicals from Traditional Chinese Medicine (TCM) as a source of new therapeutics --- p.22 / Chapter 1.5. --- Biological effects of baicalein --- p.25 / Chapter 1.5.1 --- Roles of baicalein as a lipoxygenase inhibitor --- p.28 / Chapter 1.5.2 --- Dual roles of baicalein as an antioxidant and prooxidant --- p.28 / Chapter 1.5.3 --- "Roles of baicalein as an anti-carcinogenic, anti-proliferative and anti-metastatic agent" --- p.29 / Chapter 1.6. --- Aims of current study --- p.30 / Chapter Chapter 2 --- Effects of Baicalein on Growth and Survival of Human Cancer Cells / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Cell culture --- p.35 / Chapter 2.2.2 --- Measurement of growth and survival of various cell lines --- p.36 / Chapter 2.2.3 --- Statistical analysis --- p.37 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Baicalein retards the growth and survival of human melanoma A375 and colorectal carcinoma Caco-2 --- p.37 / Chapter 2.3.2 --- Baicalein reduces the growth and survival of melanoma A375 but not in normal skin fibroblast Hs68 cells --- p.40 / Chapter 2.4 --- Discussion --- p.42 / Chapter Chapter 3 --- Effects of Baicalein on Cell Cycle and the Apoptosis in Human Melanoma A375 Cells / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Determination of cell cycle changes and quantification of apoptosis --- p.51 / Chapter 3.2.2 --- Immunoblotting --- p.52 / Chapter 3.2.3 --- Inhibition of caspase-8 by caspase-8 inhibitor --- p.54 / Chapter 3.2.4 --- Fluorometric measurement of caspase-3 activity --- p.54 / Chapter 3.2.5 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Baicalein induces S-phase arrest in cell cycle and triggers apoptosis --- p.55 / Chapter 3.3.2 --- Baicalein induces proteolytic inactivation of PARP and activation of caspases --- p.59 / Chapter 3.3.3 --- Caspase-8 is the major initiator caspase eliciting the baicalein-induced apoptosis --- p.62 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Immunoblotting --- p.75 / Chapter 4.2.2 --- Determination of sub-lethal dose of exogenous TRAIL --- p.76 / Chapter 4.2.3 --- Determination of the combinatory effect of exogenous TRAIL and baicalein --- p.76 / Chapter 4.2.4 --- Statistical analysis --- p.77 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Baicalein upregulates the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) --- p.77 / Chapter 4.3.2 --- Baicalein sensitizes the melanoma cells to sub-lethal dose of exogenous TRAIL --- p.80 / Chapter 4.4 --- Discussion --- p.84 / Chapter Chapter 5 --- Effects of Baicalein on the Extrinsic Apoptotic Pathways in Human Melanoma A375 Cells Cancer Cells / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Materials and Methods / Chapter 5.2.1 --- Analysis of mitochondrial membrane potential --- p.94 / Chapter 5.2.2 --- Fractionation of cell lysates into cytosolic and mitochondrial fractions for immunoblotting --- p.95 / Chapter 5.2.3 --- Immunoblotting --- p.95 / Chapter 5.2.4 --- Determination of cellular reactive oxygen species (ROS) production --- p.96 / Chapter 5.2.5 --- Verification of ROS generation via the addition of Trolox´ёØ --- p.96 / Chapter 5.2.6 --- Statistical analysis --- p.97 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Baicalein induces mitochondrial membrane depolarization --- p.97 / Chapter 5.3.2 --- Cytochrome c is released in the baicalein-induced mitochondrial membrane depolarization --- p.100 / Chapter 5.3.3 --- Baicalein does not elicit the intrinsic apoptotic pathway via modulation of some better-characterized Bcl-2 family proteins in A375 cells --- p.102 / Chapter 5.3.4 --- Baicalein induces ROS production --- p.105 / Chapter 5.3.5 --- Baicalein induces mitochondrial permeabilization via ROS-mediated mechanisms --- p.108 / Chapter 5.4 --- Discussion --- p.112 / Chapter Chapter 6 --- General Discussion --- p.119 / References --- p.130