L’intestin joue un rôle clé dans le contrôle de l’homéostasie énergétique. Les entérocytes sont des cellules polarisées qui permettent les échanges entre la lumière intestinale (membrane apicale) et le compartiment lymphatique et sanguin (membrane baso-latérale). Dans cette thèse, nous nous sommes particulièrement intéressés au contrôle par les entérocytes de deux processus liés au métabolisme des lipides et du cholestérol : l’excrétion trans-intestinale de cholestérol (TICE) et l’absorption des lipides alimentaires.Très récemment, il a été montré que l’intestin contribue à 20-30% de l’excrétion fécale du cholestérol chez la souris. Ce mécanisme, appelé TICE, implique le passage direct du cholestérol provenant de la circulation sanguine à travers les entérocytes vers les fèces. De par son caractère modulable par des substances pharmacologiques comme l’ézétimibe et les statines, le TICE représente une cible thérapeutique potentielle pour corriger les dyslipidémies athérogènes du diabétique. Cependant, les mécanismes moléculaires gouvernant le transport rétrograde du cholestérol (du pôle baso-latéral au pôle apical) dans l’entérocyte lors du TICE, sont complètement inconnus. Dans une première étude, nous avons mis en évidence la lignée entérocytaire humaine Caco-2/TC7 comme un modèle d’étude des processus trans-entérocytaires liés au TICE. Nous avons d’abord montré que suite à l’incubation avec du plasma humain dans le compartiment baso-latéral et des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 miment des caractéristiques du TICE in vivo. De plus, grâce à ce modèle in vitro, nous avons pu identifier les microtubules comme acteurs nécessaires au transport rétrograde du cholestérol dans l’entérocyte. Dans une seconde étude, nous nous sommes intéressés au contrôle par le récepteur nucléaire Rev-erbα de la production des chylomicrons (CM) par les entérocytes. En effet, bien qu’essentiellement vue comme la conséquence d’une clairance retardée, des données émergentes présentent la surproduction de CM par l’intestin comme un contributeur majeur de la dyslipidémie chez l’insulino-résistant. Il existe une balance, au sein de l’entérocyte, entre l’utilisation des lipides absorbés pour un stockage transitoire sous forme de gouttelettes lipidiques (GL) cytosoliques ou pour l’assemblage de lipoprotéines riches en triglycérides (LRT). Le récepteur nucléaire Rev-erbα est un répresseur transcriptionnel impliqué dans le métabolisme énergétique et le rythme circadien. Rev-erbα contrôle particulièrement le métabolisme lipidique au niveau du foie et le catabolisme des LRT. Pour cette seconde étude, une lignée Caco-2/TC7 invalidée pour Rev-erbα (sh Rev-erbα) a donc été développée par infection lentivirale et différenciée sur insert. Les résultats indiquent que suite à l’incubation avec des micelles lipidiques dans le compartiment apical, les cellules Caco-2/TC7 sh Rev-erbα sécrètent plus de LRT dans le milieu baso-latéral et stockent moins de lipides sous la forme de GL cytosoliques. De plus, la lignée Caco-2/TC7 sh Rev-erbα présente une activité lipophagique plus importante et l’inhibition de l’autophagie par la bafilomycine dans cette lignée restaure la sécrétion baso-latérale de LRT et le stockage intracellulaire de GL aux mêmes niveaux que ceux de la lignée sh control. Cette seconde étude montre donc que l’invalidation de Rev-erbα dans l’entérocyte entraîne une augmentation de la mobilisation des lipides des GL via le processus de la lipophagie résultant en une augmentation de la sécrétion de LRT. Notre hypothèse est que Rev-erbα joue un rôle clé dans le contrôle de la balance GL/LRT et donc de la triglycéridémie post-prandiale.Les deux études présentées dans cette thèse permettent une meilleure compréhension des mécanismes liés au contrôle du métabolisme lipidique par l’intestin et mettent ainsi en avant l’intestin comme une cible thérapeutique potentielle pour corriger les dyslipidémies du diabétique. / The intestine plays a key role in the control of energy homeostasis. Enterocytes, which constitute the main cellular type of intestinal epithelium (> 90%), are polarized cells allowing exchanges between intestinal lumen (apical membrane) and lymph/blood compartment (basolateral membrane). In this thesis, cholesterol and lipid metabolism control by enterocytes was studied and particularly, trans intestinal cholesterol excretion (TICE) and dietary lipid absorption.Recently, it has been estimated that intestine contributes 20-30% of fecal neutral sterol excretion in chow-fed mice. This pathway called TICE involves the direct luminal secretion of plasma-derived cholesterol by enterocytes. Moreover, TICE can be pharmacologically modulated, for instance by ezetimibe and statins and so, represents a new therapeutic target in order to prevent atherosclerosis in type 2 diabetic patients. However, at present, the molecular mechanisms behind the trans-enterocytic process of TICE are still unknown, especially the steps sustaining cholesterol entry, trafficking and efflux in enterocytes. In the first study of this thesis, we highlighted the human enterocytic Caco-2/TC7 cell line as a suitable model to study the enterocyte-related processes of TICE. We have first shown that upon basolateral incubation with human plasma and apical incubation with lipid micelles, differentiated Caco-2/TC7 cells mimic some of the in vivo TICE features. Moreover, using this model, we have identified a key role of the microtubule network in the process.In the second study of this thesis, chylomicron secretion by enterocytes and its control by the nuclear receptor Rev-erbα were investigated. Indeed, although chylomicron remnant accumulation has been associated to a delayed clearance by the liver, some recent studies show that chylomicron overproduction by the intestine is a major contributor to dyslipidemia in insulin resistant patients. Dietary lipid absorption results from a balance between transient storage in enterocytes as cytosolic lipid droplets (LD) and secretion as triglyceride-rich lipoproteins (TRL). The nuclear receptor Rev-erbα is a transcriptional repressor involved in the energy metabolism and the circadian rhythm. Particularly, Rev-erbα controls lipid metabolism in the liver and thus the catabolism of TRL. The aim of this second study was to investigate the role of Rev-erbα in intestinal lipid metabolism and particularly in TRL secretion. To study that, Caco-2/TC7 cells infected with lentivirus encoding or not a shRNA targeting Rev-erbα (sh Rev-erbα) were grown on transwells. Compared to sh control, sh Rev-erbα Caco-2/TC7 cells secrete higher amounts of micelle-derived LRT in the basolateral medium and exhibit lower quantity of neutral lipids stored as cytosolic LD, whereas the apical uptake is not different. Activation of lipophagy in sh Rev-erbα compared to sh control cells was evidenced by a higher autophagic flux and an increased colocalization of the autophagy marker LC3 with LD. Finally, autophagy inhibition with bafilomycin in sh Rev-erbα cells restores lipid secretion to the same level as in sh control cells. This second study show that Rev-erbα knock-down in enterocytes leads to a higher lipophagy-mediated remobilization of intracellular lipids and an increased TRL secretion. Our hypothesis is that Rev-erbα may be a molecular gear in the control of chylomicron secretion and a major regulator of post-prandial triglyceridemia.In conclusion, these two studies allow to better understand lipid metabolism control by the intestine: the first one by identifying the contribution of the microtubule network in enterocytes for trans-enterocytic retrograde cholesterol transport; the second one by highlighting the nuclear receptor Rev-erbα as a regulator of TRL secretion by enterocytes. These two studies point out the intestine as a potential therapeutic target to treat dyslipidemia in type 2 diabetic patients.

Na fase ativa da leishmaniose visceral (LV) ocorrem alterações no metabolismo de lipoproteínas com redução dos níveis de HDL e aumento de triglicérides. A partir desses dados, focamos neste projeto essas alterações na progressão da infecção e apontamos alguns elementos como seus possíveis desencadeantes. Como essas alterações poderiam resultar de redução de atividade e expressão da lipoproteína lipase (LPL), do receptor alfa do proliferador ativado de peroxissoma (PPAR?) e da proteína transferidora de ésteres de colesteril (CETP), a sua expressão foi avaliada durante a progressão da LV em hamster. Em hamsteres infectados com 2 x 107 amastigotas de L. (L.) infantum observamos aumento de triglicérides nos hamsteres com 55 dias (mediana = 294,0 mg/dL) e 90 dias (303,0 mg/dL ) de infecção comparados aos controles de 55 dias (119,0 mg/dL) e de 90 dias (117,0 mg/dL) (p <= 0,05). Os níveis de colesterol total e de HDL não apresentaram diferença significante entre controles e infectados com 30, 55 e 90 dias de infecção. A expressão de mRNA de PPAR? no fígado com 55 e 90 dias de infecção apresentou tendência de redução nos infectados. Já de CETP no fígado dos hamsteres com 55 dias de infecção, a expressão relativa (CT) estava reduzida nos infectados (0,08) comparados aos controles (1,69) (p <= 0,05) e de LPL no coração dos hamsteres com 90 dias de infecção também estava reduzida (1,43) com relação aos controles (2,61) (p <= 0,05). Há dados na literatura sugerindo a importância de lipídios para o desenvolvimento de amastigotas no hospedeiro vertebrado e é possível que as alterações dos níveis de lipoproteínas contribuam na progressão da infecção. Assim, avaliamos neste estudo o efeito da droga hipolipemiante ciprofibrato no controle do parasitismo na LV em hamster, sabendo-se que ciprofibratos atuam aumentando a expressão de PPAR? e a produção e atividade de LPL. O tratamento com ciprofibrato nos hamsteres com 55 dias de infecção gerou redução de triglicérides (123,0 mg/dL) em relação aos infectados não tratados (294,0 g/dL) (p <= 0,05), além dos níveis de triglicérides nos animais infectados não tratados terem aumentado quando comparados aos controles não tratados (119,0 mg/dL) (p <= 0,05). Houve também, redução de triglicérides nos animais não infectados tratados com ciprofibrato (89,0 mg/dL) comparando-se aos infectados não tratados (p <= 0,05). Os níveis de colesterol nos hamsteres não infectados tratados com ciprofibrato reduziram (53,5 mg/dL) em comparação aos infectados não tratados (93,0 mg/dL) (p <= 0,05). Já naqueles que foram infectados e tratados com ciprofibrato, constatamos redução de colesterol (53,5 mg/dL) quando comparados aos infectados não tratados (p <= 0,05). Os níveis de HDL não aumentaram com ciprofibrato e foram similares entre os hamsteres infectados não tratados e os controles não tratados. A carga parasitária no baço e no fígado não foi reduzida com ciprofibrato. Na leishmaniose visceral em hamster ocorrem alterações do metabolismo lipídico com aumento de triglicérides e redução da expressão da mRNA de LPL e CETP. O tratamento com ciprofibrato foi eficaz no controle das alterações de níveis de lipoproteínas. / In the active phase of visceral leishmaniasis (VL) changes occur in lipoprotein me-tabolism with reduction in HDL and increase in triglyceride (TG) levels. From these data, in this project we focused these changes during the progression of the infection and we approached some elements as their underlying factors. Since these changes may result from the reduction of the activity and the expression of the lipoprotein lipase (LPL), of the peroxisome proliferator-activated receptor alpha (PPAR?) and of the cholesteryl ester transfer protein (CETP), their expression were evaluated during VL progression in hamster. In 2 x 107 L. (L.) infantum amastigote-infected hamsters we observed an increase in the triglycerides in hamsters with 55 days (median = 294.0 mg/dL) and 90 days (303.0 mg/dL) of infection compared with controls of 55 days (119.0 mg/dL) and of 90 days (117.0 mg/dL) (p <= 0.05). The total cholesterol and the HDL levels did not present significant differences between control and in-fected groups at 30, 55 and 90 days of infection. The expression of mRNA of the PPAR in the liver with 55 and 90 days of infection tended to be reduced in infected animals. However the relative expression (CT) of CETP in the liver of hamsters with 55 days of infection was signicantly reduced in infected (0.08) compared with control animals (1.69) (p <= 0.05). The relative expression (CT) of LPL in the heart of hamsters with 90 days of infection was also reduced (1.43) in relation to controls (2.61) (p <= 0.05). There are data in the literature suggesting the importance of lipids for the development of amastigotes in vertebrate host and it is possible that the changes in the lipoprotein levels contribute for the infection progression. Therefore, we evaluated in this study the effect of the lipid-lowering drug ciprofibrate in the control of parasitism in VL in the hamster, knowing that ciprofibrate acts increasing the expression of the PPAR? and of the LPL production and activity. The treatment with ciprofibrate in infected hamsters at 55 days lead to the reduction of triglyceride level (123.0 mg/dL) in relation to non-treated infected animals (294.0 g/dL) (p <= 0.05). Further the triglyceride levels in the non-treated infected animals were in-creased when compared with untreated controls (119.0 mg/dL) (p <= 0.05). There was also reduction of triglyceride in ciprofibrate treated-non infected animals (89.0 mg/dL) compared with non-treated infected animals (p <= 0.05). The cholesterol lev-els were reduced in the ciprofibrate-treated non-infected hamsters (53.5 mg/dL) in comparison to the non-treated infected ones (93.0 mg/dL) (p <= 0.05). In the ciprofibrate-treated infected ones we found a reduction of cholesterol level (53.5 mg/dL) when compared with non treated infected animals (p <= 0.05). The HDL lev-els did not increase with ciprofibrate and they were similar between the non-treated infected hamsters and non-treated controls. The parasite load in the spleen and liver were not reduced with ciprofibrate. In the visceral leishmaniasis in hamster changes occur in the lipid metabolism with increase in the triglyceride level and the reduction of expression of mRNA of LPL and CETP. The treatment with ciprofibrate was ef-fective in the control of changes in the lipoprotein levels.

Após o primeiro ano de transplante cardíaco (TC) o desenvolvimento da doença coronária do transplante se torna a principal causa de morbidade e mortalidade desses pacientes. Neste período, aproximadamente 40% dos pacientes com (TC) desenvolvem hiperlipidemias que contribuem para a gênese da doença coronária do transplante. Alterações no metabolismo lipídico, entre elas, no metabolismo dos quilomícrons e da lipoproteína de baixa densidade (LDL) já foram reportadas no pós transplante. Por outro lado, a concentração da lipoproteína de alta densidade (HDL) nesses pacientes é ainda controversa. Tem sido reportado que a avaliação somente da concentração da HDL não é o suficiente para avaliar todo o papel protetor, portanto aspectos funcionais da HDL devem ser testados. Neste estudo, a propriedade fundamental da HDL de receber lipídeos das outras lipoproteínas foi avaliada em pacientes com TC, através da lipoproteína artificial (LDE). Foi também avaliado o diâmetro da HDL e a sua enzima antioxidante, a paraxonase 1 (PON1). Foram estudados 20 pacientes com TC e 20 indivíduos normolipidêmicos pareados por sexo, idade e índice de massa corpórea. Amostras de sangue foram coletadas, após jejum de 12hs, para determinação do perfil lipídico, glicose, atividade da PON1, diâmetro da HDL e transferência de lipídeos da LDE para a HDL. A concentração de colesterol total e LDL-colesterol não foram diferentes entre os grupos, enquanto a concentração de HDL_colesterol foi menor no grupo TC (p=0.01). A concentração de triglicérides no TC foi aproximadamente 40% (p=0.001). A concentração de apo A-I e apo B foram similares entre os grupos. A glicose plasmática está aumentada nos pacientes transplantados (p=0.008). O diâmetro da HDL é menor nos pacientes do grupo TC quando comparados ao do grupo controle (p=0.047), enquanto a atividade da PON1 não diferiu entre os grupos. A transferência de colesterol e éster de colesterol da LDE para a HDL foi menor em pacientes com TC quando comparados aos controles (p= 0,045 and 0,003 respectivamente). Por outro lado, não encontramos diferenças entre os grupos na transferência de triglicérides e fosfolípides. Os resultados nos mostram que a transferência de colesterol e éster de colesterol está diminuída no TC. Como o éster de colesterol é o principal constituinte do núcleo da HDL, a menor transferência de colesterol para a HDL pode ter contribuído para o menor diâmetro da HDL observado neste grupo. Estas alterações no metabolismo da HDL podem potencialmente desestabilizar o pool de colesterol plasmático e o transporte reverso de colesterol. Este achado pode contribuir para o acelerado processo aterosclerótico que frequentemente ocorre nestes pacientes. / After the first year from the transplantation procedure transplant coronary heart disease becomes a major complication and the leading cause of late morbity and mortality of those patients. After the first year, roughly 40% of heart transplantation (HT) patients develop hyperlipidemias what is implicated in the genesis of transplant coronary heart disease. Alterations in plasma lipid metabolism such as disturbed chylomicron and low-density lipoprotein (LDL) metabolism were also reported. On the other hand, levels of high-densitylipoprotein (HDL) cholesterol are controversy in those patients. It has been perceived that the estimation of the lipoprotein concentration does not suffice to evaluate the overall HDL protective role and that the functional aspects of the lipoprotein should be tested. In this study, the fundamental property of HDL to receive lipids from other lipoproteins modeled by a artificial lipoprotein (LDE) was tested in HT patients, together with size and the HDL-associated antioxidant paraxonase 1 (PON 1). We studied 20 heart transplantation patients and 20 healthy normolipidemic subjects paired for sex, age and body mass index. Blood samples were collected after 12h fasting, for determination plasma lipids, glucose, paraxonase 1 activity, HDL size and transfer of lipids from LDE to HDL. The total cholesterol and LDL cholesterol concentration did not differ in the two groups, whereas HDL cholesterol was smaller in HT (p=0.01). Triglycerides were roughly 40% greater than those of the controls (p=0.001). Apo A-I e apo B concentration values were similar comparing HT patients with controls. Plasma glucose was greater in HT than in controls (p=0.008). HDL particle diameter was smaller in HT patients then in controls (p=0.047), whereas the activity of PON 1 is not different in both groups. The transfer of cholesterol and cholesteryl ester from LDE to HDL were smaller in HT patients than in controls (p= 0.045 and 0.003, respectively). On the other hand, there was no difference in the transfer of triglycerides and phospholipids between HT patients and controls. The results showed that the acceptance of cholesterol and cholesteryl esters by the HDL fraction is diminished in HT. Since cholesteryl ester constitute most of the HDL core and cholesterol is transformed in cholesteryl ester, decreased acceptance of both cholesterol from other lipoprotein particles may account for the smaller HDL particle diameter found in the HT patient group. These alterations in HDL metabolism can potentially destabilizing the plasma cholesterol pool and the reverse cholesterol transport. This finding can contribute for the accelerated atherosclerotic process that commonly occurs in those patients.

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

Po Lai See. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 142-152). / Abstracts in English and Chinese. / TITLE PAGE --- p.p.1 / ACKNOWLEGDEMENTS --- p.p.2 / ABSTRACT --- p.p.3 / 摘要 --- p.p.6 / TABLE OF CONTENTS --- p.p.9 / LIST OF FIGURES AND TABLES --- p.p.16 / Chapter CHAPTER 1 --- GENERAL INTRODUCTION / Chapter 1.1 --- Estrogen and Estrogen Receptors and its Action --- p.p.18 / Chapter 1.1.1 --- Estrogen --- p.p.19 / Chapter 1.1.2 --- Estrogen Receptors --- p.p.19 / Chapter 1.1.3 --- Structural Differences between ERa and ERp --- p.p.21 / Chapter 1.1.4 --- Functional Differences --- p.p.22 / Chapter 1.1.5 --- Effects of Selective Estrogen Receptor Modulators --- p.p.22 / Chapter 1.1.6 --- Estrogen works --- p.p.23 / Chapter 1.1.7 --- Estrogen Receptors and Breast Cancer --- p.p.24 / Chapter 1.2 --- Flavonoids： Properties and Biological Activities --- p.p.25 / Chapter 1.2.1 --- Chemical Structure and Classification of flavonoids --- p.p.25 / Chapter 1.2.2 --- Biological Properties and Action Mechanism of Flavonoids… --- p.p.27 / Chapter 1.2.3 --- Flavonoids and breast cancer prevention --- p.p.27 / Chapter 1.3 --- Aims and Scopes of Investigation --- p.p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.p.30 / Chapter 2.1.1 --- Flavonoids --- p.p.30 / Chapter 2.1.2 --- Plasmids --- p.p.30 / Chapter 2.2 --- Mammalian cell culture --- p.p.31 / Chapter 2.2.1 --- Maintenance of cells --- p.p.31 / Chapter 2.2.2 --- Preparation of cell stock --- p.p.32 / Chapter 2.2.3 --- Cell recovery from liquid nitrogen stock --- p.p.32 / Chapter 2.3 --- Identification of estrogenic activity in flavonoids --- p.p.33 / Chapter 2.3.1 --- Steady Glo Luciferase Assay --- p.p.33 / Chapter 2.3.2 --- The Biorad Protein Assay kit (a modified Bradford method). --- p.p.33 / Chapter 2.4 --- Viability Assay --- p.p.34 / Chapter 2.5 --- ERE Luciferase reporter gene assay --- p.p.35 / Chapter 2.5.1 --- Transient transfect ion of cell using lipofectamine PLUS reagent --- p.p.36 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.p.37 / Chapter 2.6 --- ERα competitive binding ASSAY --- p.p.37 / Chapter 2.7 --- Apoptotic death assay --- p.p.38 / Chapter 2.8 --- Semi-quantitative RT-PCR Assay --- p.p.40 / Chapter 2.8.1 --- "Isolation of RNA using TRIzol® Reagent (Life Technology,USA) " --- p.p.40 / Chapter 2.8.2 --- Quantitation of RNA --- p.p.41 / Chapter 2.8.3 --- First strand cDNA synthesis --- p.p.41 / Chapter 2.8.4 --- PCR reactions --- p.p.43 / Chapter 2.9 --- Flow Cytometry Analysis --- p.p.43 / Chapter 2.10 --- Total triglyceride and cholesterol measurement --- p.p.44 / Chapter 2.10.1 --- Determination of the total cholesterol --- p.p.45 / Chapter 2.10.2 --- Determination of the total triglyceride --- p.p.46 / Chapter 2.11 --- Manipulation of DNA and RNA --- p.p.46 / Chapter 2.11.1 --- Transformation of DH5α --- p.p.46 / Chapter 2.11.2 --- Mini preparation of plasmid DNA --- p.p.47 / Chapter 2.11.3 --- Preparation of plasmid DNA using QIAGEN-tip 100 midi-prep kit --- p.p.48 / Chapter 2.11.4 --- Preparation of plasmid DNA using QIAGEN-tip 10000 Giga-prep kit --- p.p.49 / Chapter 2.11.5 --- Ethanol preparation of DNA and RNA --- p.p.50 / Chapter 2.11.6 --- Agarose gel electrophoresis of DNA --- p.p.51 / Chapter 2.12 --- Statistical methods --- p.p.52 / Chapter CHAPTER 3 --- Estrogenic and antiproliferative activities on MCF-7 breast cancer cells by flavonoids / Chapter 3.1 --- Introduction --- p.p.53 / Chapter 3.2 --- Results --- p.p.56 / Screening of phytoestrogens for estrogenic activities on MELN cells --- p.p.56 / Cell proliferation activity of phytoestrogens on MCF-7 and MDA-MA231 cells --- p.p.59 / Estrogenic and antiestrogenic activity of phytoestrogens on ERα or erβ transfected hepg2 cells --- p.p.64 / Chapter 3.3 --- Discussion --- p.p.73 / Chapter Chapter 4 --- interaction of baicalein with estrogen receptors / Chapter 4.1 --- Introduction --- p.p.76 / Chapter 4.2 --- Results --- p.p.78 / Estrogen receptor competition assay --- p.p.78 / ERE-Luciferase gene reporter assay --- p.p.82 / Chapter 4.3 --- Discussion --- p.p.88 / Chapter Chapter 5 --- baicalein and genistein display differential actions on er transactivation / Chapter 5.1 --- Introduction --- p.p.90 / Chapter 5.2 --- Results --- p.p.92 / Estrogenic and antiestrogenic activities of genistein and baicalein on ER transactivation --- p.p.92 / Chapter 5.3 --- Discussion --- p.p.105 / Chapter CHAPTER 6 --- APOPTOTIC EFFECTS OF BAICALEIN ON MCF-7 AND MDA-MB-231 CELL LINES / Chapter 6.1 --- Introduction --- p.p.107 / Chapter 6.2 --- Results --- p.p.111 / ER POSITIVE MCF-7 AND ER NEGATIVE MDA-MB-231 cell death assay --- p.p.111 / "Bcl-2, Bax and PS2 mRNA expression " --- p.p.116 / Arrest at sub G1 phase of MCF-7 by baicalein --- p.p.124 / Chapter 6.3 --- Discussion --- p.p.127 / Chapter CHAPTER 7 --- BAICALEIN CAN REDUCE INTRACELLULAR cholesterol and triglceride / Chapter 5.1 --- Introduction --- p.p.129 / Chapter 5.2 --- Results --- p.p.130 / Baicalein has beneficial effect on lipid metabolism --- p.p.130 / Chapter 5.3 --- Discussion --- p.p.139 / Chapter chapter 8 --- Summary --- p.p.140 / BIBLIOGRAPHY --- p.p.142 / APPENDIX 1 ABBREVIATIONS --- p.p.153 / APPENDIX 2 PRIMER LISTS --- p.p.156 / APPENDIX 3 REAGENTS AND BUFFERS --- p.p.157

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.

Objetivo: O objetivo deste estudo foi avaliar, em pacientes portadores de diabete melito tipo 2 (DM 2) e em indivíduos saudáveis (C), o efeito agudo e crônico do exercício físico aeróbio intenso (TFA) sobre o insulto oxidativo e defesas antioxidantes plasmáticas, bem como o reflexo sobre a habilidade das HDL2 e HDL3 em inibir a oxidação das LDL in vitro. Métodos: O consumo máximo de oxigênio no pico de exercício (VO2 pico) foi medido respiração a respiração, durante teste de esforço máximo, realizado antes e após as 18 sem de TFA supervisionado. Colesterol total (CT), triglicérides (TG), glicose plasmática e insulina foram determinados antes e após TFA. Também foram determinadas a concentração de substâncias reativas ao ácido tiobarbitúrico (TBARS), o perfil antioxidante total e a atividade sérica da paraoxonase-1. As HDL2 e HDL3 foram isoladas do plasma por ultracentrifugação em gradiente descontínuo de densidade. Para determinar o lag time de oxidação de LDL (LAG) e a razão máxima de formação de dienos conjugados (RDC), as HDL2 e HDL3, isoladas nos diferentes períodos experimentais, foram incubadas com pool de LDL de doadores saudáveis, na presença de CuSO4 (10µmol/mL) a 37°C, com leitura a 234 nm, durante 4 h. CT, TG, fosfolípides e apolipoproteína A-I foram determinados nas subfrações de HDL. Resultados: O VO2 pico aumentou em ambos os grupos após TFA. Não se observou variação significativa de peso, TG, HDL colesterol (HDLc), insulina e índice HOMA entre os grupos, tampouco após o treinamento físico. Antes do período de TFA, o CT e o LDL colesterol plasmáticos do grupo C foram maiores que o do grupo DM 2, diferença que não se manteve após o período de treinamento físico. HbA1c e glicemia foram maiores no grupo DM 2, antes e após TFA. O TFA não alterou a HbA1c no grupo DM 2. Na presença de HDL3, o LAG foi semelhante entre os grupos antes do TFA, porém apenas no grupo DM 2 houve aumento do LAG e redução na RDC, mediante incubação com HDL3 isolada após TFA. Na presença de HDL2, o LAG foi menor no grupo DM 2 quando comparado ao grupo C, antes do TFA. Após TFA esta diferença desapareceu. Não houve alteração na RDC em ambos os grupos após TFA. Não houve diferença entre os grupos na composição da HDL3 antes e após TFA. No período basal, a HDL2 do grupo DM 2 apresentou concentrações menores de CT e livre, desaparecendo a diferença após o TFA. Após TFA, observou-se redução no TBARS apenas no DM 2. O TFA não alterou a atividade da paraoxonase-1 e o perfil antioxidante total no plasma em ambos os grupos. Conclusão: O TFA reduziu a peroxidação lipídica no plasma, corrigiu o efeito antioxidante da HDL2 e melhorou o da HDL3 em indivíduos portadores de DM 2. Estes eventos foram independentes de alteração na sensibilidade à insulina e da concentração e composição de HDL no plasma / Objective: The objective of this study was to analyze in type 2 diabetes mellitus (DM 2) and in healthy controls subjects (C) the role of acute and aerobic exercise training (AET) on plasma oxidative stress and antioxidant defenses as well as the HDL2 and HDL3 ability to inhibit the in vitro LDL oxidation. Methods: Peak oxygen uptake (VO2 peak) was measured breath to breath during a maximal cardiopulmonary exercise test before (basal period) and after a 18-wk supervised AET. HDL2 and HDL3 isolated in both periods by discontinuous density gradient ultracentrifugation were incubated with a healthy donor\'s plasma LDL pool for measuring at 234 nm both the lag time for LDL oxidation (LAG) and the maximal rate of conjugated diene formation (MCD) on CuSO4 (10 µmol/mL) at 37°C, for 4 h. Total cholesterol, triglycerides, apolipoprotein A-I and phospholipids were measured in HDL2 and HDL3. TBARS, total antioxidant status and paraoxonase-1 activity were determined in plasma or serum. Results: VO2 peak increased similarly in C and in DM 2 after AET. In the basal period and after AET, both DM 2 and C did not differ according to TG, HDL-c, insulin and HOMA index. HbA1c and glycemia were higher in DM 2 before and after AET. Before AET TC and LDL-c was lower in DM 2 than in C. After AET, TC and LDL-c was similar between C and DM 2. In the presence of HDL3 lag time and MCD was similar in C and DM 2, but only in DM 2 AET improved lag time and reduce MCD. HDL3 composition was similar in DM 2 and C after AET. In the presence HDL2 lag time was lower in DM 2 than in C in the basal period, but did not differ after AET. MCD was lower in DM 2 after AET. HDL2 had less total and free cholesterol in DM 2 than in C but differences vanished after AET. After training plasma TBARS concentration was reduced in DM 2 alone. Training did not modify the total antioxidant status and serum paraoxonase-1 activity in both groups. AET reduces lipid peroxides in plasma, corrects the HDL2 and improves the HDL3 antioxidant effects in DM 2 subjects. These events were independent of changes in insulin resistance and plasma HDL concentration and composition

A hipercolesterolemia é o maior fator de risco para doença arterial coronária e é responsável por um número significante de doenças e mortes. Há evidências que o exercício físico diminui o risco cardiovascular exercendo efeitos benéficos sobre os fatores de risco, incluindo o metabolismo lipídico. Mudanças que ocorrem no metabolismo da LDL podem não ser detectadas através das dosagens rotineiras de lípides plasmáticos. Portanto, avaliamos os efeitos do exercício físico no metabolismo de uma nanoemulsão lipídica artificial com comportamento metabólico semelhante ao da LDL. Foram avaliados 12 indivíduos hipercolesterolêmicos sedentários (H) e 12 indivíduos normolipidêmicos sedentários (N) que foram submetidos a treinamento durante 4 meses. Nos grupos controle, foram estudados 8 indivíduos hipercolesterolêmicos sedentários controle (HC) e 8 indivíduos normolipidêmicos sedentários controle (NC) que não realizaram exercício físico. A emulsão marcada com éster de colesterol -14C (EC-14C) foi injetada endovenosamente. Amostras de sangue foram coletadas em tempos prédeterminados (5 min, 1, 2, 4, 6, 8, 24 horas) após a injeção, para determinação da radioatividade, das curvas de decaimento plasmático e cálculo da taxa fracional de remoção (TFR) dos lípides marcados, por análise compartimental. As avaliações foram feitas antes e após o protocolo de treinamento físico e nos grupos controle foram realizadas 2 avaliações, sendo a segunda 4 meses após a primeira. No grupo H, as concentrações plasmáticas de colesterol total e LDL-c diminuíram (5%, p= 0,0334 e 14%, p= 0,0058), respectivamente, enquanto que, HDL-c, TFR-EC-14C e lag time aumentaram (13%, p= 0,0142; 36%, p= 0,0187; 37%, p= 0,0039), respectivamente após o treinamento físico. No grupo N, a concentração plasmática da HDL foi maior (15%, p= 0,0243), após o treinamento. Nos grupos HC e NC os parâmetros avaliados foram semelhantes. Portanto, o exercício físico acelera a remoção plasmática da LDL em indivíduos hipercolesterolêmicos, indicado pela maior TFR-EC-14C. Este efeito pode ser um dos mecanismos pelos quais o exercício previne a doença arterial coronária. / Hypercholesterolemia has become one of the major risk factors for arterial coronary disease. As such, it is also responsible for a significant number of diseases and deaths. Evidence suggests that physical exercise can, in fact, decrease the risk of cardiovascular diseases by exerting beneficial effects upon the risk factors, including lipid metabolism. The changes that do occur in LDL metabolism are generally not detected by routine clinical laboratory plasma lipid exams. In the present study, the effects of physical exercise on the metabolism of an artificial lipidic nanoemoulsion with similar LDL metabolic behavior were analyzed. 12 hypercholesterolemic sedentary individuals (H) and 12 normolipidemic sedentary individuals (N) were studied. These 24 participants were submitted to a routine training program during a 4-month period. The control group was divided into two groups: one of 8 hypercholesterolemic sedentary individuals (CH) and the other with 8 normolipidemic sedentary individuals (CN) which did not partake in any exercise program. An emulsion labeled with 14Ccholesteryl ester (14C-CE) was endovenously injected into all 4 groups. Blood samples were collected at pre-determined periods (5 min, 1, 2, 4, 6, 8 and 24 hours) after the injection of the emulsion, in order to determine the radioactivity of the plasma decay curves and calculate the fractional clearance rate (FCR) of the labeled lipids for compartimental analysis. Evaluations were made before and after the exercise training protocol. The control groups under went 2 evaluations, the second one 4 months after the first evaluation. In the H group, total cholesterol and LDL-c plasma concentrations decreased (5%, p=0.0334 and 14%, p=0.0058), respectively. HDL-c, 14C-CE-FCR and lag time, on the other hand, increased (13%, p=0.0142; 36%, p=0.0187; 37%, p=0.0039) after exercise training. HDL plasma concentration for the N group was higher (15%, p=0.0243), after exercise training. In groups CH and CN the parameters evaluated were similar. Therefore, exercise accelerates the removal of LDL plasma in hypercholesterolemic individuals as indicated by a higher 14C-CE-FCR. This effect can thus be one of the mechanisms by which exercise can prevent arterial coronary disease.

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

INTRODUÇÃO: Estudos mostraram que, após a injeção de LDE na corrente sangüínea de mulheres portadoras de câncer de mama, ela encontra-se mais concentrada em tecido neoplásico que no tecido normal. Recentemente, estudos pré-clínicos comprovaram que a associação LDE-oleato de paclitaxel é estável, menos tóxica e com mais atividade terapêutica quando comparada ao uso de paclitaxel comercial em animais. O presente estudo teve como objetivo verificar a estabilidade dessa associação na circulação, a sua capacidade em se concentrar no tecido neoplásico e determinar os parâmetros farmacocinéticos em relação ao paclitaxel isolado. MÉTODOS: Para determinar os parâmetros farmacocinéticos foram administrados, por via intravenosa, [3H]-oleato de paclitaxel associado a [14C]- oleato de colesterol-LDE em três pacientes e [3H]-paclitaxel comercial em duas pacientes 24 horas antes do procedimento cirúrgico. Todas as pacientes eram portadoras de neoplasia maligna da mama. Amostras de sangue foram colhidas durante 24 horas. A radioatividade foi medida por cintilação líquida e os parâmetros farmacocinéticos foram calculados usando um modelo multicompartimental. Fragmentos de tecido neoplásico e de tecido normal mamário foram coletados durante a cirurgia e submetidos à contagem radioativa. RESULTADOS: As taxas fracionais de remoção da LDE e do oleato de paclitaxel foram semelhantes (0,0296 ± 0,0264 e 0,0182 ± 0,0186, respectivamente, p = 0,5742). A captação tanto da LDE quanto do oleato de paclitaxel mostrou concentração 2,5 a 3 vezes maior no tecido tumoral do que no tecido mamário normal. O tempo de meia vida do oleato de paclitaxel foi maior do que o da formulação comercial (18,97 ± 7,7 horas e 7,34 ± 0,40 horas) e, a depuração plasmática, menor (1,51 ± 0,18 (L/h) e 7,95 ± 4,32 (L/h)). CONCLUSÃO: A maior parte do fármaco ficou retido na microemulsão até sua remoção da circulação e captação pelas células. O oleato de paclitaxel associado à LDE mostrou-se estável na circulação sangüínea e apresentou tempo de meia vida maior e a depuração plasmática menor do que a formulação comercial, além de se concentrar mais no tecido neoplásico da mama. Os resultados permitem sugerir que essa associação pode se constituir em uma estratégia útil no tratamento de mulheres portadoras de câncer da mama. / INTRODUCTION: Studies had shown that, after the injection of LDE in the circulation of women with breast cancer, it was more concentrate in neoplastic tissue that in the normal tissue. Recently, studies had proven that the LDE-paclitaxel oleate association is steady, less toxic and with more therapeutical activity when compared with the commercial paclitaxel in animals. The present study was designed to verify the stability of this association in the circulation, its capacity in concentrating in the neoplastic tissue and to determine the plasma kinetics of the association compared to that of paclitaxel isolated. METHODS: To determine the pharmacokinetic parameters, [3H]-paclitaxel oleate associated to LDE labeled with [14C]-cholesterol oleate was intravenously injected into three patients and [3H]-commercial paclitaxel into two patients 24 hours before the surgical procedure. All the patients had breast cancer. Blood samples were collected during 24 hours. Radioactivity was quantified in a scintillation solution and the pharmacokinetic parameters were calculated by compartmental analysis. Specimens of tumoral and normal breast were excised during the surgery and submitted to a radioactive counting. RESULTS: Fractional clearance rate of LDE and of the paclitaxel oleate were similar (0,0296 ± 0,0264 and 0,0182 ± 0,0186, respectively, p = 0,5742). The uptake of both [14C]-LDE and [3H]-paclitaxel oleate by breast malignant tissue was two and three fold greater than that of the normal breast tissue. The paclitaxel oleate plasma half-life (h) was greater than the commercial paclitaxel (T1/2 = 18,97 ± 7,7 and 7,34 ± 0,40) and the total plasma clearence (L/h) of paclitaxel oleate was lesser than the commercial (CL = 1,51 ± 0,18 and 7,95 ± 4,32). CONCLUSION: Most of the drug was restrained in the microemulsion until its removal from the circulation and captation by the cells. The paclitaxel oleate associated to LDE is stable in the bloodstream and has greater plasma half-life and lesser clearence than those for commercial paclitaxel. In addition, the association could be concentrated more in malignant breast tissue. The results allow to suggest that this association can consist in a useful strategy in the treatment of women with breast cancer.