Estudos recentes têm demonstrado uma participação importante do receptor toll-like 4 (TLR4) na evolução de doenças envolvendo desordens metabólicas, como a doença do fígado gorduroso não-alcoólico (NAFLD). No entanto, as alterações do metabolismo lipídico que poderiam ser influenciadas pela ativação do TLR4 são desconhecidas. Neste estudo propomos caracterizar o papel do receptor TLR4 no metabolismo de lipídios no fígado de camundongos deficientes para o receptor de LDL, um modelo que desenvolve NAFLD quando submetido a uma dieta rica em gordura saturada e colesterol. Camundongos controle (C57 black6), deficientes para o receptor de LDL (LDLrKO), deficientes para o receptor TLR4 (TLR4KO) ou deficientes para ambos (duplo KO) receberam dieta controle ou hiperlipídica por quatro, oito ou doze semanas. Após o tratamento e sacrifício dos animais, avaliamos o perfil de lipídios plasmáticos, o conteúdo de lipídios do fígado e a expressão gênica de enzimas relacionadas à síntese e degradação de triglicerídeos (TG) e colesterol no fígado. O perfil inflamatório no fígado também foi avaliado. A dieta hiperlipídica induziu uma hipertrigliceridemia e hipercolesterolemia nos animais LDLr KO e duplo KO, sendo que o grupo duplo KO apresentou níveis séricos inferiores de triglicérides (TG) e ácidos graxos livres a partir de oito semanas de tratamento em comparação aos animais LDLrKO. A dieta hiperlipídica também induziu um aumento significativo no conteúdo de TG e de colesterol no fígado de todos os grupos. Na análise da expressão gênica não foram encontradas diferenças na expressão de proteínas relacionadas à síntese de triglicérides e colesterol (ApoB100, MTTP, GPAT1 e GPAT4) entre os grupos. Porém houve aumento significativo na expressão de proteínas relacionadas à oxidação de ácidos graxos (CPT1, MTP, ACOX, PBE, tiolase) e à síntese de ácidos biliares (CYP7a1) no grupo duplo KO em comparação ao grupo LDLr KO. No perfil inflamatório, a expressão de F4/80 demonstrou infiltração de macrófagos significativamente elevada no grupo LDLrKO tratado com a dieta hiperlipídica comparada a todos os outros grupos. No entanto, houve maior expressão de IL-6, IL-1beta e TNF-alfa no grupo duplo KO em comparação ao grupo LDLr KO. Nossos dados sugerem que a ativação do TLR4 no fígado de animais alimentados com uma dieta hiperlipídica pode contribuir para o acúmulo de lipídios e início da esteatose hepática. Estratégias para a inativação hepática do TLR4 podem diminuir a NAFLD não somente devido a diminuição da inflamação, mas por aumentar a oxidação de ácidos graxos no fígado / Recent studies have shown an important role of toll-like receptor 4 (TLR4) in the evolution of diseases involving metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD). However, changes in lipid metabolism regulated by TLR4 activation are still unknown. In this study, we characterized the role of TLR4 receptor in hepatic lipid metabolism of mice deficient for the LDL receptor, a model that develops NAFLD when exposed to a diet rich in saturated fat and cholesterol. We investigated the role of TLR4 activation in the pathogenesis of diet-induced NAFLD by crossing LDLr KO mice with the TLR4 knockout mice (double KO). Animals were fed for 4, 8 or 12 weeks with high-fat diet (HFD) containing 18% saturated fat and 1.25% cholesterol. We evaluated plasma lipid profile, hepatic lipid content and gene expression of enzymes related to the synthesis and degradation of triglycerides and cholesterol in the liver. Liver inflammatory status was also investigated. We observed that HFD induced hypertriglyceri-demia and hypercholesterolemia in LDLr KO and double KO mice, but double KO animals presented lower serum levels of triglycerides and free fatty acids after eight weeks of treatment. HFD also induced a significant increase in liver contents of triglycerides (TG) and of cholesterol in all groups. We did not find differences in the expression of proteins related to triglycerides and cholesterol synthesis (ApoB100, MTTP, GPAT1, GPAT4) between the groups. However, we observed a significant increase in the expression of proteins related to fatty acid oxidation (CPT1, MTP, ACOX, PBE, tiolase ) and bile acid synthesis (CYP7a1) in double KO group in comparison to LDLr KO. Regarding the inflammatory process, F4/80 expression was elevated in LDLr KO mice fed HFD when compared to all groups. On the other hand, IL-6, IL-1beta e TNF-alfa expression was induced by HFD only in double KO mice. Taken together, our results show that TLR4 activation in liver from mice fed on a high-fat diet may contribute to lipid accumulation and steatosis onset. Strategies regarding localized TLR4 inactivation may increase the oxidation of fatty acids and improve NAFLD not only due to decreased inflammation

Introdução: A associação de uma estatina com ezetimiba é tão eficaz quanto altas doses da mesma estatina na redução do LDL-colesterol. Os efeitos que não dependem dessa redução são chamados de pleiotrópicos, entre os quais podemos citar: melhora da função endotelial, efeitos anti-oxidantes, efeitos anti- inflamatórios, entre outros. Objetivo: comparar a ação de dois esquemas de tratamento que obtêm reduções equivalentes de LDL-colesterol (sinvastatina 80 mg ao dia e associação sinvastatina 10mg/ezetimiba 10 mg ao dia), sobre os efeitos pleiotrópicos: inflamação, função endotelial e oxidação da LDL. Métodos: estudamos 23 pacientes randomizados e na forma de cross-over 2x2. A inflamação foi mensurada através da PCR-us, a função endotelial por meio de ultra-sonografia e a oxidação de LDL pelas dosagens de LDL eletronegativa (LDL-) e do anticorpo anti-LDL-. Resultados: A redução do LDL-colesterol foi similar nos dois grupos (45,27% no grupo sinvastatina/ezetimiba (p<0,001) e 49,05% no grupo sinvastatina (p<0,001), sem diferença entre os tratamentos (p=0,968)). Os dois grupos apresentaram melhora da função endotelial (3,61% no grupo sinvastatina/ezetimiba (p=0,003) e 5,08% no grupo sinvastatina (p<0,001), não houve diferença entre os tratamentos (p=0,291)). Houve melhora nos níveis da PCR-us (redução de -22,8% no grupo sinvastatina/ezetimiba (p=0,004) e de 29,69% no grupo sinvastatina (p=0,01), sem diferenças entre os tratamentos (p=0,380)). Não houve redução significativa da LDL-. Ocorreu aumento na concentração do anticorpo anti-LDL eletronegativa apenas no grupo sinvastatina (p=0,045). Conclusões: as duas formas de tratamento são eficazes na melhora da função endotelial e dos níveis de PCR-us. Somente com o uso da sinvastatina em alta dose houve aumento nos níveis de anticorpos anti-LDL-. / Introduction: The co-administration of a statin with ezetimibe is as effective as high doses of the same statin in the reduction of the LDL-cholesterol. The effects which don´t depend of this reduction are called pleiotropic effects, some among them can be cited: endothelial function improvement, antioxidative and anti-inflammatory effects. Objective: compare the effectiveness of these two different treatments that obtain equivalent reductions of LDLcholesterol (simvastatin 80 mg once a day and co-administration of simvastatin 10 mg once a day and ezetimibe 10 mg once a day), about pleiotropic effects: inflammation, endothelial function and LDL oxidation. Methods: we have studied 23 randomized patients in a 2x2 cross-over study. Inflammation was measured by high-sensitive C reactive protein, endothelial function by echocardiography and LDL oxidation by electronegative LDL and electronegative anti-LDL antibodies levels. Results: the LDL-cholesterol was similar between the two groups (45,27% reduction in the simvastatin/ezetimibe group (p<0,001) and 49,05% reduction in the simvastatin group (p<0,001); no difference between treatments was found (p=0,968). The two groups had improvement in endothelial function (3,61% in the simvastatin/ezetimibe group (p=0,003) and 5,08% in the simvastatin group (p<0,001)), no differences was found between the two groups (p=0,291). High-sensitive C reactive protein had a 22,8% reduction in the simvastatin/ezetimiba group (p=0,004) and 29,69% reduction in the simvastatin group (p=0,01), with no significative difference in any of the two treatments (p=0,380). There was no significative difference in LDL- levels. The anti-LDL- antibodies concentration was increased only in the simvastatin group (p=0,045). Conclusion: the two forms of treatments presented some similar pleiotropic effects - improvement in endothelial function and decreased hs-CRP levels. Only with a high simvastatim dose the anti-LDL- antibodies concentration was increased.

O uso da LDE como veículo para quimioterápicos tem mostrado ser uma boa estratégia para aumentar a eficácia terapêutica dos mesmos. Nesse estudo, a LDE foi empregada como veículo para um derivado lipofílico do metotrexato (MTX), o di-dodecil metotrexato, que foi obtido com rendimento elevado através de reação de esterificação do MTX. O aumento na lipofilicidade do derivado possibilitou incorporação na LDE com rendimento e estabilidade elevados. O IC50 de LDE-di-dodecil MTX foi cerca de 100 vezes menor em relação ao MTX comercial, sua captação celular mais elevada nas linhagens leucêmicas estudadas e sua toxicidade animal reduzida, mostrando que a LDE é um veículo promissor para este fármaco. / The use of LDE as vehicle to drugs is a great strategy to improve the therapeutic index and reduce the side effects. In this study LDE was used as vehicle to di-dodecyl methotrexate, a lipophilic derivative of MTX, obtained through an esterification reaction with a high yield. The increased lipophilicity of the derivative allowed a high association to LDE and good stability. The IC50 of LDE-di-dodecyl MTX was lower than that of the MTX and the uptake was higher in leukemic cells. The MTX toxicity in mice was reduced after association to LDE, showing that LDE is a promising vehicle to this drug.

Lorsqu’elle est comparée à la pression artérielle (PA) brachiale, la PA centrale est un meilleur reflet du stress hémodynamique infligé aux organes cibles. Les objectifs de ce mémoire sont de clarifier le rôle de la PA centrale pour la prédiction du risque cardiovasculaire (CV), de définir un seuil permettant le diagnostic d’une hypertension centrale et de définir l’effet sur la PA centrale de certains paramètres, tels que les statines et le cholestérol LDL. La base de données CARTaGENE comportant 20 004 sujets a été utilisée. La PA centrale était connue (mesurée à l’aide de l’appareil SphygmoCor Px qui utilise une calibration de type I), ainsi que les autres facteurs de risques CV. Des données prospectives permettant de connaître l’incidence d’événements CV majeurs étaient disponibles. Dans un premier temps, des modèles de régression de Cox et des mesures de discrimination et de reclassification ont permis de comparer la PA systolique centrale et brachiale pour la prédiction du risque CV. Un seuil permettant le diagnostic d’une hypertension centrale a aussi été déterminé à l’aide de l’index de Youden. L’association entre la PA centrale, le cholestérol LDL et les statines a été déterminée à l’aide de régressions linéaires et d’ANOVA. Une analyse de médiation a permis d’éclairer l’effet des statines sur la PA centrale, à savoir si celui-ci est médié par une baisse concomitante du cholestérol LDL. Ces analyses ont permis de déterminer que la PA centrale ne procure qu’une amélioration marginale dans la prédiction du risque cardiovasculaire lorsque comparé à celle de la PA brachiale. Une PA systolique centrale de 120 mmHg a été identifiée comme le meilleur seuil pour poser le diagnostic d’une hypertension centrale lorsqu’évaluée avec un appareil avec calibration de type I. De plus, la PA centrale est influencée par la prise de statines et son effet n’est que partiellement médié par une baisse concomitante du cholestérol LDL. / Compared to brachial blood pressure (BP), central BP is thought to be a better reflection of the hemodynamic strain on target organs. It is unclear though whether this translates into improved cardiovascular (CV) risk stratification when central BP is compared to brachial BP. The objectives of this thesis are to clarify the role of central BP in CV risk stratification, to define a threshold for the diagnosis of central hypertension and to determine what are the impacts of parameters, such as statins and LDL cholesterol, on central BP. The CARTaGENE database, which is comprised of 20,004 individuals, was used for these studies. The central BP (measured with the SphygmoCor Px device and type I calibration) as well as other CV risk factors were known. Prospective data for the incidence of major adverse cardiovascular events (MACE) was available. Cox proportional hazard models and measures of discrimination and reclassification were used to compare central systolic BP and brachial systolic BP in their ability to predict CV risk. A central hypertension threshold was determined using Youden’s index. The association between central BP, LDL cholesterol and statins were assessed using linear regression models and ANOVA. The interactions between central BP, statins and cholesterol LDL were assessed with a mediation analysis. Central BP was only marginally superior to brachial BP in CV risk prediction. A central hypertension threshold of 120 mmHg was identified. Statins reduced both brachial and central BP in a similar fashion, and cholesterol LDL was only partially responsible for the effect of statins on BP. To conclude, central BP, when assessed using type I calibration, is not superior to brachial BP in CV risk prediction. These studies also clarified the central hypertension threshold and the impact of statins and cholesterol LDL on central BP.

Inledning: Övervikt och fetma är förknippat med flera ämnesomsättningssjukdomar. Kostens betydelse har blivit central i det förebyggande arbetet av övervikt och fetma. Periodisk fasta har under de senaste åren blivit ett populärt verktyg för viktnedgång och förbättring av den allmänna hälsan. Tidsbegränsat ätande (TRF) är en form av periodisk fasta där fastandet sker mellan 14–16 timmar per dygn och resterande timmarna är s.k ätfönster. Preliminära data från studier antyder att TRF som kosthållning för med sig en rad hälsofördelar såsom att förbättra den metabola hälsan och kan därmed förebygga en rad ämnesomsättningssjukdomar. Syfte: Syfte med detta litteraturarbete var att analysera ifall tidsbegränsat ätande (TRF) kan främja viktminskning och förbättra de metabola parametrarna hos människor som har övervikt eller lider av fetma. Metod: Sex experimentella studier analyserades. Studierna söktes i databasen PubMed med sökorden: ”time restricted feeding” OR ”time restricted eating”. De inkluderade studierna valdes på basis av fastställda inklusions- och exklusionskriterier. Resultat: Största delen av de inkluderade studierna visade att TRF har en positiv påverkan på viktminskning. De flesta studierna förbättrade inte fasteglukos hos TRF-gruppen i jämförelse med kontrollgruppen. Två studier visade en signifikant förbättring av några av glukosparametrarna. Två av studierna visade att fasteinsulin och HOMA-IR minskade signifikant. Endast en studie visade att faste-triglyceridvärdet minskade.  Diskussion: Sammanfattningsvis visar de flesta studierna att TRF ger viktminskning utan kaloribegränsning och därmed skulle TRF kunna fungera som ett bra verktyg för viktnedgång. Vidare verkar längden på ätfönstret (4-12 timmar) och tiden på dagen (morgon och förmiddag) när matintaget sker viktigt både för att ge viktminskning och för att förändra de metabola parametrarna. TRF skulle därför kunna användas som ett möjligt redskap för att förebygga fetma och därmed förbättra olika metabola parametrar som i sin tur kanske kan förhindra/skjuta upp insjuknande i metabola syndromet, diabetes typ 2 samt hjärt- och kärlsjukdomar. / Introduction: Overweight and obesity are associated with several metabolic diseases. The importance of diet has become central in the preventive work of overweight and obesity. Periodic fasting has in recent years become a popular tool for weight loss and improvement of general health. Time-restricted eating (TRF) is a form of periodic fasting where the fasting window is between 14–16 hours per day and the remaining hours are so-called eating windows. Preliminary data from studies suggest that TRF contributes to a number of health benefits, improves metabolic health, and thus can prevent a number of metabolic diseases. Aim: The aim of this review was to analyze whether time-restricted eating (TRF) can promote weight loss and improve the metabolic parameters of overweight and obese people. Method: Six experimental studies were analyzed. The studies were searched in the PubMed database; the keywords were: "time restricted feeding" OR "time restricted eating". The included studies were selected on the basis of established inclusion and exclusion criteria. Results: Most of the included studies showed that TRF has a positive effect on weight loss. Most studies showed no significant improvement in fasting glucose in the TRF group compared to the control group. Two studies showed a significant improvement in some of the glucose parameters. Two of the studies showed a significant reduction in fasting insulin and HOMA-IR. Only one study showed a significant reduction in the fasting triglyceride value. Discussion: In summary, most studies showed that TRF provides weight loss without calorie restriction, and thereby TRF could work as a good tool for weight loss. Furthermore, the length of the eating window (4-8 hours) and the time of day (morning and before noon) when food intake takes place are important both to provide weight loss and to change the metabolic parameters. TRF could therefore be used as a possible tool to prevent obesity and thereby improve various metabolic parameters that may prevent / delay the onset of metabolic syndrome, type 2 diabetes, and cardiovascular disease.

Wong Yuen Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / LIST OF ABBREAIATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- History of Kudingcha --- p.1 / Chapter 1.2 --- Classification of Kudingcha --- p.1 / Chapter 1.3 --- Composition of Kudingcha --- p.3 / Chapter 1.4 --- Introduction to phenylethanoid glycosides --- p.4 / Chapter 1.4.1 --- Isolation and purification of phenylethanoid glycosides --- p.4 / Chapter 1.4.2 --- Taxonomy of phenylethanoid glycosides --- p.5 / Chapter 1.4.3 --- Structure of phenylethanoid glycosides --- p.5 / Chapter 1.4.4 --- Biosynthesis of phenylethanoid glycosides --- p.6 / Chapter 1.4.5 --- Pharmacological effects of phenylethanoid glycosides --- p.9 / Chapter 1.4.5.1 --- Anticarcinogenic activity --- p.10 / Chapter 1.4.5.2 --- Inhibitory activity of protein kinase C --- p.10 / Chapter 1.4.5.3 --- Immunosuppressive activity --- p.11 / Chapter 1.4.5.4 --- DNA repairing activity --- p.11 / Chapter 1.4.5.5 --- Antibacterial and antiviral activities --- p.11 / Chapter 1.4.5.6 --- Antiinflammatory and antinociceptive activities --- p.12 / Chapter 1.4.5.7 --- Hepatoprotective activity --- p.12 / Chapter 1.4.5.8 --- Inhibitory activity of xanthine oxidase --- p.13 / Chapter 1.4.5.9 --- Antioxidative and scavenging activities --- p.13 / Chapter Chapter 2 --- Isolation and purification of phenylethaonid glycosides in Kudingcha / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Objectives --- p.16 / Chapter 2.3 --- Materials and Methods --- p.17 / Chapter 2.3.1 --- Extraction and isolation --- p.17 / Chapter 2.3.2 --- High performance liquid chromatograph (HPLC)analysis --- p.19 / Chapter 2.3.2.1 --- "Acteoside, ligupurpuroside A and osmanthuside B" --- p.19 / Chapter 2.3.2.2 --- cis-Ligupurpuroside B and trans-ligupurpuroside B --- p.19 / Chapter 2.3.3 --- Isolation and purification of isoacteoside --- p.19 / Chapter 2.4 --- Results --- p.24 / Chapter 2.4.1 --- A cteoside --- p.24 / Chapter 2.4.2 --- Osmanthuside B --- p.24 / Chapter 2.4.3 --- Ligupurpuroside A --- p.24 / Chapter 2.4.4 --- trans-Ligupurpuroside B --- p.25 / Chapter 2.4.5 --- cis-Ligupurpuroside B --- p.25 / Chapter 2.4.6 --- Isoacteoside --- p.25 / Chapter 2.4.6.1 --- Thermal stability --- p.25 / Chapter 2.5 --- Discussions --- p.27 / Chapter 2.5.1 --- Acteoside --- p.27 / Chapter 2.5.2 --- Osmanthuside B --- p.27 / Chapter 2.5.3 --- Ligupurpuroside A --- p.28 / Chapter 2.5.4 --- trans-Ligupurpuroside B --- p.29 / Chapter 2.5.5 --- cis-Ligupurpuroside B --- p.29 / Chapter 2.5.6 --- Isoacteoside --- p.30 / Chapter Chapter 3 --- Inhibitory effect of phenylethanoid glycosides isolated from Kudingcha on Cu2+-mediated LDL oxidation in vitro / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Mechanisms of lipoprotein oxidation in vivo --- p.36 / Chapter 3.2.1 --- Oxidants underlying LDL oxidation --- p.36 / Chapter 3.2.2 --- Oxidative modification of LDL --- p.37 / Chapter 3.2.3 --- Role of oxidatively modified LDL in atherogenesis --- p.38 / Chapter 3.2.4 --- Antioxidants and atherosclerotic heart disease --- p.40 / Chapter 3.2.5 --- Measuring the thiobarbituric acid-reactive substances (TBARS) formation as an index to monitor LDL oxidation --- p.41 / Chapter 3.2.6 --- Effect of flavonoids on Cu2+-mediated human LDL oxidation --- p.41 / Chapter 3.3 --- Objectives --- p.43 / Chapter 3.4 --- Materials and methods --- p.44 / Chapter 3.4.1 --- LDL isolation --- p.44 / Chapter 3.4.2 --- LDL oxidation --- p.44 / Chapter 3.4.3 --- Thiobarbituric acid-reactive substances (TBARS) assay --- p.45 / Chapter 3.4.4 --- Interactions of phenylethanoid glycosides isolated from Kudingcha with Cu2+ in human LDL oxidation --- p.45 / Chapter 3.4.5 --- Statistics --- p.46 / Chapter 3.5 --- Results --- p.47 / Chapter 3.5.1 --- Protective effect of the major phenylethanoid glycosides isolated from Kudingcha on LDL oxidation --- p.47 / Chapter 3.5.2 --- Varying protective effect of individual major Kudingcha phenylethanoid glycosides --- p.47 / Chapter 3.5.3 --- Interactions of Kudingcha phenylethanoid glycosides with Cu2+in human LDL oxidation --- p.51 / Chapter 3.5 --- Discussions --- p.55 / Chapter Chapter 4 --- Inhibitory effects of Kudingcha phenylethanoid glycosides on a-tocopherol oxidation in vitro / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- LDL oxidation and atherosclerosis --- p.58 / Chapter 4.1.2 --- Role of vitamin E in LDL lipid peroxidation --- p.59 / Chapter 4.1.3 --- Interaction of tocopherol interactions with other antioxidants and synergists --- p.61 / Chapter 4.2 --- Objectives --- p.62 / Chapter 4.3 --- Materials and Methods --- p.63 / Chapter 4.3.1 --- Depletion of a-tocopherol in LDL --- p.63 / Chapter 4.3.2 --- Regeneration of a-tocopherol in LDL --- p.63 / Chapter 4.3.3 --- HPLC analysis of a-tocopherol in LDL --- p.64 / Chapter 4.3.4 --- Statistics --- p.64 / Chapter 4.4 --- Results --- p.66 / Chapter 4.4.1 --- Protective effects of Kudingcha phenylethanoid glycosides on a-tocopherol depletion --- p.66 / Chapter 4.4.2 --- Regeneration of a-tocopherol by acteoside --- p.70 / Chapter 4.5 --- Discussions --- p.72 / Chapter Chapter 5 --- Relaxing effects of Kudingcha extract and purified acteoside in rat aortic rings / Chapter 5.1 --- Introduction --- p.75 / Chapter 5.1.1 --- Mechanisms of calcium mobilization --- p.76 / Chapter 5.1.1.1 --- Voltage-dependent calcium channel --- p.76 / Chapter 5.1.1.2 --- Thromboxane A2 Receptor-mediated calcium channel --- p.77 / Chapter 5.1.1.3 --- Protein kinase C in signal transudation --- p.77 / Chapter 5.1.2 --- Contractile proteins and regulation of contraction of vascular smooth muscle --- p.78 / Chapter 5.2 --- Objectives --- p.82 / Chapter 5.3 --- Materials and Methods --- p.83 / Chapter 5.3.1 --- Arterial ring preparation --- p.83 / Chapter 5.3.2 --- Vascular action of Kudingcha extract and acteoside --- p.85 / Chapter 5.3.2.1 --- Relaxant responses of Kudingcha extract and acteoside on U46619 -induced contraction --- p.85 / Chapter 5.3.2.2 --- Relaxant responses of Kudingcha extract and acteoside on high K+ and CaCl2-induced contraction --- p.85 / Chapter 5.3.2.3 --- Relaxant responses of Kudingcha extract and acteoside on protein kinase C- mediated contraction --- p.86 / Chapter 5.3.2.4 --- Effect of acteoside on acetylcholine-induced relaxation --- p.87 / Chapter 5.3.3 --- Statistics --- p.87 / Chapter 5.4 --- Results --- p.88 / Chapter 5.4.1 --- Effects of Kudingcha extract and acteoside on U46619-induced contraction --- p.88 / Chapter 5.4.2 --- Effects of Kudingcha extract and acteoside on high K+-induced contraction --- p.94 / Chapter 5.4.3 --- Effect of Kudingcha extract and acteoside on protein kinase C-mediated contraction --- p.98 / Chapter 5.4.4 --- Effect of acteoside on acetylcholine-induced relaxation --- p.100 / Chapter 5.5 --- Discussions --- p.103 / Chapter Chapter 6 --- Effect of Kudingcha on lipid contents of hamsters and New Zealand Rabbits / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.1.1 --- Factors related to CHD --- p.106 / Chapter 6.1.2 --- Animal model --- p.107 / Chapter 6.2 --- Objectives --- p.108 / Chapter 6.3 --- Materials and Methods --- p.109 / Chapter 6.3.1 --- Rabbit --- p.109 / Chapter 6.3.1.1 --- Measurement of atheroma formation --- p.112 / Chapter 6.3.2 --- Hamster --- p.114 / Chapter 6.3.3 --- Serum lipid determinations --- p.116 / Chapter 6.3.4 --- Determination of hepatic cholesterol content --- p.116 / Chapter 6.3.5 --- Statistics --- p.117 / Chapter 6.4 --- Results --- p.119 / Chapter 6.4.1 --- Growth and Food intake --- p.119 / Chapter 6.4.2 --- "Effect of Kudingcha supplementation on Serum TG, TC and HDL-C" --- p.119 / Chapter 6.4.3 --- Effect of Kudingcha supplementation on hepatic cholesterol contents --- p.124 / Chapter 6.4.4 --- Effect of Kudingcha supplementation on atheroma formation --- p.124 / Chapter 6.5 --- Discussions --- p.129 / Chapter Chapter 7 --- Conclusions --- p.131 / References --- p.134

Yang, Yaling. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 187-207). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Ho Hing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 140-156). / Abstracts in English and Chinese. / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- History of soybean --- p.1 / Chapter 1.2 --- Health benefits of soybean --- p.2 / Chapter 1.3 --- Introduction to flavonoids --- p.2 / Chapter 1.4 --- Bioavailability of flavonoids from foods --- p.3 / Chapter 1.5 --- Pharmacological effects of flavonoids and their glycosides --- p.4 / Chapter 1.5.1 --- Anticarcinogenic activity --- p.4 / Chapter 1.5.2 --- Antioxidative activity --- p.7 / Chapter 1.5.3 --- Cardioprotective activity --- p.9 / Chapter 1.5.4 --- Osteoprotective activity --- p.10 / Chapter 1.5.5 --- Neuroprotective activity --- p.12 / Chapter 1.5.6 --- Antiangiogenic activity --- p.12 / Chapter 1.6 --- Soy leaves --- p.13 / Chapter Chapter 2 --- Isolation and purification of kaempferol glycosides and genistin in soy leaves / Chapter 2.1 --- Introduction --- p.14 / Chapter 2.2 --- Objectives --- p.15 / Chapter 2.3 --- Materials and Methods --- p.16 / Chapter 2.3.1 --- Extraction and isolation --- p.16 / Chapter 2.3.1.1 --- Preparation of soy leaves butanol extract --- p.16 / Chapter 2.3.1.2 --- Preparation of kaempferol glycosides from soy leaves butanol extract --- p.16 / Chapter 2.3.2 --- High performance liquid chromatography (HPLC) analysis --- p.19 / Chapter 2.3.2.1 --- Sample preparation for the HPLC analysis --- p.19 / Chapter 2.3.2.2 --- HPLC analysis --- p.19 / Chapter 2.3.2.3 --- Quantification of the flavonoids and their glycosides --- p.23 / Chapter 2.3.2.4 --- Change in flavonoids and their glycosides in soy leaves --- p.23 / Chapter 2.4 --- Results --- p.24 / Chapter 2.4.1 --- Compound 1 --- p.24 / Chapter 2.4.2 --- Compound 2 --- p.24 / Chapter 2.4.3 --- Compound 3 --- p.25 / Chapter 2.4.4 --- Compound 4 --- p.25 / Chapter 2.4.5 --- Compound 5 --- p.25 / Chapter 2.4.6 --- Compound 6 --- p.26 / Chapter 2.4.7 --- Quantification of flavonoids in soybean and soy leaves --- p.32 / Chapter 2.4.8 --- Age-dependent changes in flavonoids and their glycosides --- p.32 / Chapter 2.5 --- Discussion --- p.35 / Chapter 2.5.1 --- Compound 1 --- p.35 / Chapter 2.5.2 --- Compound 2 --- p.35 / Chapter 2.5.3 --- Compound 3 --- p.37 / Chapter 2.5.4 --- Compound 4 --- p.38 / Chapter 2.5.5 --- Compound 5 --- p.39 / Chapter 2.5.6 --- Compound 6 --- p.40 / Chapter 2.5.7 --- Age-dependent changes in flavonoids and their glycosides --- p.40 / Chapter Chapter 3 --- Hypolipidemic effects of soy leaves in hamsters / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.1.1 --- Different lipoproteins and their functions --- p.41 / Chapter 3.1.2 --- Risk factors of cardiovascular disease --- p.42 / Chapter 3.1.3 --- Animal model --- p.43 / Chapter 3.2 --- Objectives --- p.44 / Chapter 3.3 --- Materials and Methods --- p.45 / Chapter 3.3.1 --- Animals --- p.46 / Chapter 3.3.2 --- Serum lipid and lipoprotein determinations --- p.46 / Chapter 3.3.3 --- Determination of cholesterol in the liver and adipose tissue --- p.46 / Chapter 3.3.4 --- Extraction of neutral and acidic sterols from fecal samples --- p.49 / Chapter 3.3.4.1 --- Determination of neutral sterols --- p.49 / Chapter 3.3.4.2 --- Determination of acidic sterols --- p.50 / Chapter 3.3.4.3 --- GLC analysis of neutral and acidic sterols --- p.51 / Chapter 3.3.5 --- Statistics --- p.51 / Chapter 3.4 --- Results --- p.54 / Chapter 3.4.1 --- Growth and food intake --- p.54 / Chapter 3.4.2 --- "Effects of SLP and SLEE supplementation on serum triacylglycerol (TG), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C)" --- p.54 / Chapter 3.4.3 --- Effects ofSLP and SLEE supplementation on non-HDL-C and ratio of non-HDL-C to HDL-C --- p.55 / Chapter 3.4.4 --- Effects of SLP amd SLEE supplementations on concentration of hepatic cholesterol --- p.58 / Chapter 3.4.5 --- Effects of SLP and SLEE supplementations on perirenal adipose tissue cholesterol --- p.58 / Chapter 3.4.6 --- Effects of SLP and SLEE supplementations on fecal neutral and acidic sterols --- p.61 / Chapter 3.5 --- Discussion --- p.64 / Chapter Chapter 4 --- Effects of soy leaves and its flavonoid glycosides on haemolysis and on LDL oxidation / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.1.1 --- Role of low density lipoprotein oxidation in the development of atherosclerosis --- p.68 / Chapter 4.1.2 --- LDL oxidation --- p.70 / Chapter 4.1.3 --- Thiobarbituric acid reactive substances (TBARS) as an index of LDL oxidation --- p.71 / Chapter 4.1.4 --- Antioxidant and LDL oxidation --- p.74 / Chapter 4.2 --- Objective --- p.75 / Chapter 4.3 --- Materials and methods --- p.76 / Chapter 4.3.1 --- Isolation of LDL from human serum --- p.76 / Chapter 4.3.2 --- LDL oxidation --- p.77 / Chapter 4.3.3 --- Determine the formation of thiobarbituric acid-reactive substances (TBARS) --- p.77 / Chapter 4.3.4 --- Assay for erythrocyte haemolysis --- p.78 / Chapter 4.3.5 --- Statistics --- p.79 / Chapter 4.4 --- Results --- p.80 / Chapter 4.4.1 --- Effects of three different soy leaves extracts and flavonoid glycosides on LDL oxidation --- p.80 / Chapter 4.4.2 --- Effects of three soy leaves extracts and flavonoid glycosides on erythrocyte haemolysis --- p.80 / Chapter 4.5 --- Discussion --- p.85 / Chapter Chapter 5 --- Relaxing effects of soy leaves and its flavonoids / Chapter 5.1 --- Introduction --- p.89 / Chapter 5.1.1 --- Smooth muscle contraction --- p.90 / Chapter 5.1.1.1 --- Sliding filament mechanism --- p.91 / Chapter 5.1.2 --- Intracellular mechanisms involved in the regulation of smooth muscle contraction --- p.92 / Chapter 5.1.2.1 --- Voltage-gated Ca2+ channels --- p.92 / Chapter 5.1.2.2 --- Protein kinase C (PKC) mediated smooth muscle contraction --- p.93 / Chapter 5.1.2.3 --- Thromboxane A2 receptor-mediated calcium channel --- p.94 / Chapter 5.2 --- Objectives --- p.96 / Chapter 5.3 --- Materials and methods --- p.97 / Chapter 5.3.1 --- Drugs preparation --- p.97 / Chapter 5.3.2 --- Vessel preparation --- p.97 / Chapter 5.3.3 --- Contraction experiments --- p.99 / Chapter 5.3.3.1 --- Relaxant responses of soy leaves butanol extract on the contraction induced by different constrictors --- p.99 / Chapter 5.3.3.2 --- Relaxant responses of soy leaves butanol extract on U46619 and PGF2a- induced contraction --- p.99 / Chapter 5.3.3.3 --- "Relaxant responses of genistein, genistin and the kaempferol glycosides on U46619-induced contraction" --- p.100 / Chapter 5.3.4 --- Statistics --- p.100 / Chapter 5.4 --- Results --- p.102 / Chapter 5.4.1 --- Effect of soy leaves butanol extract --- p.102 / Chapter 5.4.2 --- Role of endothelium in extract-induced relaxation --- p.102 / Chapter 5.4.3 --- Effect of the soy leaves butanol extract on contractile response to prostaglandins --- p.103 / Chapter 5.4.4 --- Effects of kaempferol glycosides and kaempferol --- p.111 / Chapter 5.4.5 --- Effects of genistein and genistin --- p.111 / Chapter 5.5 --- Discussion --- p.118 / Chapter Chapter 6 --- Effect of soy leaves on mammary tumor / Chapter 6.1 --- Introduction --- p.123 / Chapter 6.1.1 --- Carcinogenesis --- p.123 / Chapter 6.1.1.1 --- In itiation --- p.124 / Chapter 6.1.1.2 --- Promotion --- p.124 / Chapter 6.1.1.3 --- Progression --- p.125 / Chapter 6.2 --- Objective --- p.126 / Chapter 6.3 --- Materials and methods --- p.127 / Chapter 6.3.1 --- Animal --- p.127 / Chapter 6.3.2 --- Determination of estrus cycle --- p.128 / Chapter 6.3.3 --- Statistics --- p.129 / Chapter 6.4 --- Results --- p.131 / Chapter 6.4.1 --- Incident rate of tumor induction --- p.131 / Chapter 6.4.2 --- Number of tumor induced --- p.131 / Chapter 6.5 --- Discussion --- p.136 / Chapter Chapter 7 --- Conclusions --- p.136 / References --- p.140

The common risk factors for atherosclerosis, such as obesity, high cholesterol levels, sedentary lifestyle, diabetes and high alcohol intake, only explain approximately 50% of cardiovascular disease events. It is thereby important to identify new mechanisms that can stimulate the process of atherosclerosis. During the past decades, a wide range of investigations have demonstrated connections between infections by the respiratory bacterium Chlamydia pneumoniae and atherosclerosis. Earlier studies have focused on the interaction between C. pneumoniae and monocytes/macrophages, T-lymphocytes, smooth muscle cells and endothelial cells, which are present in the atherosclerotic plaque. However, another important player in atherosclerosis and which is also present in the plaques is the platelet. Activation of platelets can stimulate both initiation and progression of atherosclerosis and thrombosis, which is the ultimate endpoint of the disease. The aim of the present thesis was to investigate the capacity of C. pneumoniae to activate platelets and its role in atherosclerosis. The results show that C. pneumoniae at low concentrations binds to platelets and stimulates platelet aggregation, secretion, reactive oxygen species (ROS) production and oxidation of low-density lipoproteins (LDL), and that these effects are mediated by lipopolysaccharide (LPS). Activation of protein kinase C, nitric oxide synthase and 12-lipoxygenase (12-LOX) was required for platelet ROS production, whereas platelet aggregation was dependent on activation of GpIIb/IIIa. Pharmacological studies showed that the C. pneumoniae-induced platelet activation is prevented by inhibitors against 12-LOX, platelet activating factor (PAF) and the purinergic P2Y1 and P2Y12 receptors, but not against cyclooxygenase (COX). These findings were completely opposite to the effects of these inhibitors on collagen-stimulated platelets. We also present data from a clinical study indicating that percutaneous coronary intervention (PCI or balloon dilatation) leads to release of C. pneumoniae into the circulation, which causes platelet activation and LDL oxidation. In conclusion, these data support a role for C. pneumoniae-induced platelet activation in the process of atherosclerosis. Stimulation of platelets by C. pneumoniae leads to release of growth factors and cytokines, oxidation of LDL and platelet aggregation, which are processes that can stimulate both atherosclerosis and thrombosis. Development of novel drugs that prevent C. pneumoniae-platelet interaction by inhibiting 12-LOX and/or PAF, may be important in the future treatment of cardiovascular disease.

Atherosclerosis is a vascular degenerative disease leading to progressive thickening in the intima of large and medium sized arteries through the formation of plaque that is very rich with cholesterol. The cholesterol is carried by LDL (low density lipoprotein) particles which pass through the endothelium and accumulate in the intima. The passage of LDL is influenced by wall shear stress which activates physiological responses of the endothelium. However, the causal relationship between the physiological responses and their effect on LDL mass transport is not fully understood. To obtain blood flow patterns in human carotid arteries, a fluid structure interaction (FSI) computational approach is employed, based on the in-vivo arterial geometry constructed from black blood magnetic resonance images (BBMRI) and flow rate boundary conditions obtained from phase contrast images (PC). Wall shear stress (WSS) on the luminal surface is computed, and this variable is related to the formation of leaky junctions, which is a major transendothelial pathway for LDL. A model for the fraction of leaky junction at a surface is incorporated into the overall computational scheme for mass transport, along with pore theory. The theoretical model is applied to images from three human carotid arteries in which the degree of disease ranges from mild to moderate. Maximum mass flux is predicted to be in the downstream region of stenoses where WSS is low, and this result is consistent with the clinical observation of plaque progression downstream of the stenosis. The hypothesis that the majority of LDL enters into the intima through leaky junctions is supported by observation of similar distributions between the pattern of volume flux via leaky junctions and mass flux. These studies suggest that mass flux of LDL can be a predictor to indicate areas with potential for plaque formation and progression in human carotid artery disease.