A ATP-binding cassette transporter A1 (ABCA1) é uma proteína transmembrana responsável pelo efluxo celular de colesterol e fosfolipídeos, que é um passo essencial para o transporte reverso do colesterol e para a biogênese da HDL. Polimorfismos do gene ABCA1 foram associados com risco de doença arterial coronariana, variações no perfil lipídico e diferenças na resposta a fármacos hipolipemiantes. Com a finalidade de avaliar os efeitos de polimorfismos do ABCA1 sobre a expressão gênica e a resposta a vastatinas, foram selecionados indivíduos normolipidemicos (NL, n=143) e hipercolesterolêmicos (HC, n=224). A resposta a atorvastatina (10 mg/dia/4 semanas) foi avaliada pelo perfil lipídico sérico em 141 indivíduos do grupo HC (ATORVA). DNA e RNA total foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G70943A (R219K), C-14T e C-105T, uma variante nova do ABCA1, foram detectados por PCR-RFLP e confirmados por seqüenciamento de DNA. A expressão de RNAm do ABCA1 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR-duplex e PCR em tempo real, utilizando o gene GAPD como referência endógena. A freqüência do alelo -105T foi 1,4% em NL e 2,0% em HC. O alelo 70943A (genótipos GA+AA) foi associado com maior concentração sérica basal de apoAI (NL), de HDL-c (ATORVA) e com menores concentrações basais de triglicerídeos e VLDL-c e menor índice TG/HDL-c (HC e ATORVA) em comparação com o genótipo 70943GG (p<0,05). O polimorfismo C-105T está em desequilíbrio de ligação com o SNP C-14T (p=0,006). Portadores do alelo -105T (genótipos CT+TT), quando comparados aos portadores do genótipo -105CC, tiveram menores valores basais de triglicerídeos e VLDL-c, maior concentração de HDL-c e menor índice TG/HDL-c nos grupos HC e ATORVA e também maiores concentrações de apoAI e menor índice apoB/apoAI no grupo ATORVA (p<0,05). Nos grupos HC e ATORVA, os portadores do haplótipo -14CT+TT/-105CT+TT tiveram menores valores de triglicerídeos e VLDL-c basais, maiores concentrações de HDL-c e menor índice TG/HDL-c quando comparados aos portadores dos outros haplótipos (p<0,05). A expressão basal do ABCA1 foi menor nos HC que nos NL independentemente da taxa de expressão alta (GM1) ou baixa (GM2). Este efeito foi associado com os SNPs C-14T e G70943A SNPs. Após o tratamento com atorvastatina, a expressão de RNAm foi reduzida nos HC portadores do alelo - 14T em comparação com os portadores de alelo -14C. Esses resultados são sugestivos de que ABCA1 SNPs estão envolvidos na variação do perfil lipídico sérico e na expressão de RNAm em resposta a atorvastatina. / The ATP-binding cassette transporter A1 (ABCA1) is a transmembrane protein involved on cholesterol and phospholipid cellular efflux, which is an essential step for the reverse cholesterol transport and HDL biogenesis. Single nucleotide polymorphisms (SNPs) in the ABCA1 gene have been associated with increased risk of coronary heart disease, differences on serum lipid profile and response to lowering-cholesterol drugs. We have evaluated the influence of ABCA1 SNPs on mRNA expression and lipid-lowering response to atorvastatin. Normolipidemic (NL, n=143) hypercholesterolemic (HC, n=224) individuals were enrolled in this study and the response to atorvastatin (10 mg/day/4 weeks) was evaluated in HC individuals (ATORVA, n=141). Blood samples were collected for biochemical analyses, genomic DNA and total RNA extraction. SNPs G70943A (R219K), C-14T and C-105T, a novel variant of ABCA1, were detected by PCR-RFLP and confirmed for DNA sequencing. ABCA1 mRNA expression in peripheral blood mononuclear cells (PBMC) was analysed by PCR-duplex and Real Time PCR, using the GAPD as the endogenous reference. In HC and NL, the frequency of -105T allele was 2.0% and 1.4%, respectively. The 70943A allele (genotypes GA+AA) was associated with higher basal concentrations of apoAI (NL) and HDL-c (ATORVA) and lower triglyceride and VLDL-c and TG/HDL-c ratio (HC and ATORVA) than the 70943GG genotype (p<0.05). We found a linkage disequilibrium between C-14T and C-105T SNPs in HC group (p=0.006). Individuals carrying -105T allele (CT/TT genotypes), when compared with -105CC carriers, had lower basal concentrations of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio in HC and ATORVA groups and also higher concentration of apoAI and lower apoB/apoAI ratio in ATORVA group (p<0.05). In HC and ATORVA, individuals with -14CT+TT/-105CT+TT haplotype had lower basal values of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio than carries of others haplotypes (p<0,05). ABCA1 mRNA basal expression was lower in HC when compared to NL independently of high (GM1) or low (GM2) basal expression rate. This effect was associated with C-14T and G70943A SNPs. After atorvastatin treatment, mRNA expression was reduced in HC individuals carrying -14T allele in comparison with the -14C allele carriers. These results are sugestive that ABCA1 SNPs are involved on variation of serum lipid profile and mRNA expression in response to atorvastatin.

Lipoproteínas do plasma trocam lipídeos e apolipoproteínas constantemente. Além da ação de proteínas de transferência, a habilidade das lipoproteínas em receber ou doar lipídeos depende também de diversos outros fatores. A estrutura e a composição de lipídeos e de proteína das lipoproteínas podem influenciar a fluidez da partícula e, desse modo, esta habilidade da lipoproteína. No plasma, a classe de lipoproteína que é a mais afetada pela transferência de lipídeos é a HDL. O presente estudo foi projetado para estabelecer um método . simples para quantificar a habilidade desta lipoproteína em receber simultaneamente suas principais classes de lipídeos constituintes, fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo. O método é baseado na troca lipídica ocorrida entre uma nanoemulsão artificial (LDE) que se assemelha à estrutura lipídica da LDL, usada como doador de lipídeos radioativos, e as lipoproteínas plasmáticas. Após precipitação da LDE e das demais lipoproteínas, a capacidade da HDL de receber lipídeos é quantificada pela medida da radioatividade presente na lipoproteína. No presente estudo, foi realizada a padronização deste método, assim como analisadas possíveis interferências no método. No mesmo estudo, foi analisada a transferência de lipídeos da LDE para a partícula de HDL em indivíduos controles. A elevação da temperatura (4 a 37°C), do tempo de incubação (5min a 2h) e de HDL-Colesterol (33 a 244 mg/dL) resultaram em progressivo aumento na transferência dos quatro lipídeos da LDE para a HDL. Por outro lado, o aumento do pH (6,5 a 8,5) e da concentração de albumina (3,50 a 7,00 g/dL) não alteraram os valores de transferência. A amostra de plasma mostrou ser inalterada para este ensaio por período de 12 meses (p>0,05), enquanto que a LDE foi inalterada por até 15 dias (p>0.2). Os resultados da análise intra-ensaio apresentaram imprecisão (C.V.) para · a transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo de 0,83, 0,56, 1,49 e 0,51 %, respectivamente. A análise inter-ensaio mostrou imprecisão para os resultados de transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo 0,78, 0,59, 1,32 e 0,58%, respectivamente. A média da transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo da LDE para a HDL nos 53 voluntários foi de 25,5±2,6, 9,9±1 ,6, 4,8±1,3 e 6,9±1 ,1%, respectivamente. As transferências de éster de colesterol, triglicerídeo e colesterol livre se correlacionaram positivamente entre estes lipídeos. Foram encontradas correlações positivas também entre as transferências de fosfolipídeo e de triglicerídeo e entre a transferência de colesterol livre e a concentração de colesterol de HDL. O método de transferência de lipídeos para a fração HDL mostrou ser prático e preciso, .podendo, dentro das condições ideais estabelecidas neste trabalho, determinar a capacidade receptora de lipídeos da HDL. / Plasma lipoproteins constantly exchange lipids and apolipoproteins. Besides the action of transfer proteins, the ability of lipoproteins to receive or donate lipids also depends on several other factors. The structure, lipid and protein composition of the lipoproteins may influence the fluidity of the particle and thereby this ability of lipoprotein. In the plasma, the lipoprotein class that is the most affected by lipid transfers is HDL. The current study was designed to establish a practical method to quantify the ability of this lipoprotein to simultaneously receive its main constituent lipid classes, namely phospholipid, free cholesterol, cholesteryl ester and triglycerides. The method is based on the lipids exchange between an artificial nanoemulsion (LDE) that resembles the LDL, used as a donor of radioactive lipids, and plasma lipoproteins. After precipitation of the LDE and the others plasma lipoproteins, the accept capacity of the HDL\' is quantified by the measure of radioactivity in the lipoprotein. In the present study, the validation of this method was carried through. Moreover, possible interferences in the method had been analyzed. In the same study, the transfer of lipids of LDE for the particle of HDL from control subjets was analyzed. The rise of temperature (4 to 37°C), time of incubation (5min to 2h) and of HDL-Cholesterol (33 to 244 mg/dL) resulted in gradual increase in the transfer of the four lipids from LDE to HDL. On the other hand, the increase of pH (6.5 to 8.5) and albumin concentration (3.50 to 7.00 g/dL) had not modified the values of transfers. The plasma sample showed to be unchanged for this assay for period of 12 months (p>0.05), whereas the LDE was unchanged for 15-days (p>0.2). The intra-assay results showed imprecision (C.V) of 0.83, 0.56, 1.49 and 0.51 % for phospholipid, free cholesterol, cholesteryl ester and triglycerides transfer, respectively. The inter-assay showed imprecision for results of phospholipid, free cholesterol, cholesteryl ester and triglycerides transfer of 0.78, 0.59, 1.32 and 0.58%, respectively. The average of phospholipid, free cholesterol, cholesteryl ester and triglycerides transfers from LDE to HDL in the 53 volunteers was of 25.5±2.6, 9.9±1.6; 4.8±1 .3 and 6.9±1 .1 %, respectively. Cholesterol ester, triglyceride and free cholesterol transfers positively correlated among each other. A positive correlation was also found between phospholipid and triglyceride transfers and free cholesterol transfer and HDL cholesterol concentration. The method of lipids transfer to HDL showed to be practical and reproducible, being able, inside of the ideal conditions established in this work, to determine the aceptora capacity of lipids of the HDL.

Contrairement aux HDL de sujets sains, les HDL de patients diabétiques ont perdu leur capacité à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Les mécanismes en cause ne sont pas connus. Or la glycation et l’oxydation sont deux phénomènes majeurs au cours du diabète. Nous avons donc étudié in vitro, le rôle de la glycation (associée ou non à une oxydation spontanée), et de l’oxydation d’HDL issues de sujets sains, sur leurs capacités à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Chaque condition a conduit au même constat: les HDL modifiées perdent leur pouvoir vasorelaxant en présence de LDL oxydées. En revanche, en l’absence de LDL oxydées, elles n’altèrent pas la vasorelaxation induite par l’acétylcholine. Ainsi les modifications structurelles des HDL (glycation, oxydation, ou les deux) induisent une perte de leur capacité à protéger l’endothélium du stress oxydatif, plutôt qu’un effet délétère direct sur l’endothélium. Un des mécanismes majeur impliqué dans ce phénomène est probablement l’absence de fixation de ces HDL modifiées à leur récepteur SR-BI. Elles ne pourraient plus alors s’opposer au niveau des cavéoles aux effets délétères des LDL oxydées, et ne favoriseraient plus la production de NO. Mais si aussi bien la glycation que l’oxydation des HDL entraînent ces effets néfastes, il semblerait qu’en condition physiopathologique (oxydation spontanée des HDL glyquées), l’oxydation ne majore pas cette perte de capacité des HDL à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation. / Contrary to HDL from normolipidaemic and normoglycaemic subjects, HDL from diabetic patients have lost their capacity to reverse the inhibition of vasorelaxation induced by oxidized LDL. Mechanisms involved are unknown. The glycation and oxidation of HDL are two major phenomena in diabetes mellitus. The aim of this work was to study in vitro the role of glycation (with or without spontaneous oxidation) and oxidation of HDL, on their capacity to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation. Each state showed the same result, modified HDL lost their vasorelaxing power in stress conditions (with oxidized LDL). Nevertheless, modified HDL alone (without oxidized LDL) did not alter vasorelaxation induced by acetylcholine, after noradrenaline-induced vasoconstriction. Thus, modifications of HDL induce a loss of the ability to protect vessels from oxidative stress rather than have a direct deleterious effect on the vessel. One of the major mechanisms involved in this phenomenon is probably the loss of SR-BI binding of these modified HDL, that could lead to the inability of HDL to protect caveolae from deleterious effects induced by oxidized LDL and could not preserve NO production. However, though glycation, like oxidation of HDL, leads to these deleterious effects, it would seem that during physiopathological conditions, with the spontaneous oxidation of glycated HDL, oxidation does not aggravate the loss of the capacity of diabetic HDL to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation.

Made available in DSpace on 2016-05-02T13:54:45Z (GMT). No. of bitstreams: 1 LuisaBarbosaMessora-dissertacao-completa.pdf: 574964 bytes, checksum: f3b5918f00bf09a802e755b11db471d0 (MD5) Previous issue date: 2010-11-21 / LDLr-/ - mice are spontaneously hyperlipidemic and resistant to the development of neointimal lesions. This study determined the influence of plasma levels of high density lipoprotein on cardiovascular inflammation, insulin resistance, and blood cell count in LDL (LDLr-/ -) receptor gene knockout mice. Three groups of 3-month-old male mice were used: Group WT, wild-type mice, Group S, LDLr-/ - mice fed a standard diet, Group HL, LDLr-/ - mice fed a hyperlipidic diet. After 15 days, blood was collected for analysis of plasma lipids, glucose, insulin, and hematological assays. The HOMA index was calculated to determine insulin resistance. The heart and aorta were removed and histologically processed. Heart sections were immunohistochemically processed with the anti-CD40L antibody to evaluate the inflammatory process. Artery sections were stained with hematoxylin-eosin and picrosirius red to assess morphological and morphometric changes. The S mice were resistant to inflammatory, had a low immunoreactivity to CD40L, high HDL plasma levels, and showed no insulin resistance, even with moderate hyperlipidemia in relation to WT. HL mice exhibited severe hyperlipidemia, increased immunoreactivity to CD40L, marked morphological alterations in the aorta wall, and insulin resistance, all associated with a decrease in HDL plasma levels in relation to S. The results showed a negative association between the plasma levels of high density lipoprotein and the total and differential leukocyte and platelet counts in the LDL receptor gene knockout mice. This ratio showed the important influence of the high density lipoprotein on the modulation of the immune and inflammatory response in dyslipidemias. Therefore, the evaluation of the blood cell count results, routinely correlated with the lipid plasma levels, can be promising in the prevention and prognosis of the severity of pathological conditions involving immune responses in dyslipidemias. The high HDL plasma level is a protective factor against the development of cardiovascular inflammation and insulin resistance in LDLr-/- mice, thus preventing the incidence of neointimal lesions. / Os camundongos LDLr-/- são hiperlipidêmicos espontâneos e resistentes ao desenvolvimento de lesões neointimais. O presente estudo teve como objetivo determinar a influência dos níveis plasmáticos de lipoproteína de alta densidade na inflamação cardiovascular, na resistência insulínica e no hemograma de camundongos knockout para gene do receptor de LDL (LDLr-/-). Foram utilizados 3 grupos experimentais de camundongos machos com 3 meses de idade: Grupo WT, camundongos selvagens; Grupo S, camundongos LDLr-/- que receberam ração padrão; Grupo HL, camundongos LDLr-/- que receberam ração hiperlipídica. Após 15 dias, o sangue foi coletado para análises plasmáticas dos lipídeos, glicose, insulina e para análises hematológicas. O índice de Homa foi calculado para determinar a resistência insulínica. O coração e aorta foram removidos e processados histologicamente. Cortes histológicos do coração foram processados imunoistoquimicamente com anticorpo anti-CD40L para avaliar processo inflamatório. Cortes histológicos das artérias foram corados com hematoxilina/eosina e picrosírius red para avaliar alterações morfológicas e morfométricas. Os camundongos S foram resistentes ao processo inflamatório, caracterizado por baixa imunorreatividade para o CD40L, com níveis plasmáticos de HDL elevados, e não desenvolveram resistência insulínica, mesmo com hiperlipidemia moderada em relação aos WT. Os camundongos HL apresentaram uma hiperlipidemia severa, aumento na imunorreatividade cardíaca para o CD40L, pronunciadas alterações morfológicas na parede da aorta e resistência insulínica, associadas a um decréscimo nos níveis plasmáticos do HDL em relação aos S. Os resultados mostraram uma associação negativa entre os níveis plasmáticos de lipoproteína de alta densidade e as contagens total e diferencial de leucócitos e plaquetas nos camundongos knockout para o gene do receptor de lipoproteína de baixa densidade. Essa relação demonstrou importante influência da lipoproteína de alta densidade na modulação da resposta imunológica e inflamatória na dislipidemia. Portanto, a avaliação dos resultados do hemograma correlacionada com os níveis plasmáticos de lipídeos, rotineiramente, pode ser promissora na prevenção e no prognóstico da severidade de quadros patológicos que envolvam respostas imunológicas nas dislipidemias. O nível plasmático elevado de HDL é o fator protetor contra o desenvolvimento de processos inflamatórios cardiovasculares e resistência insulínica nos camundongos LDLr-/-, impedindo o desenvolvimento das lesões neointimais.

Lipoproteínas do plasma trocam lipídeos e apolipoproteínas constantemente. Além da ação de proteínas de transferência, a habilidade das lipoproteínas em receber ou doar lipídeos depende também de diversos outros fatores. A estrutura e a composição de lipídeos e de proteína das lipoproteínas podem influenciar a fluidez da partícula e, desse modo, esta habilidade da lipoproteína. No plasma, a classe de lipoproteína que é a mais afetada pela transferência de lipídeos é a HDL. O presente estudo foi projetado para estabelecer um método . simples para quantificar a habilidade desta lipoproteína em receber simultaneamente suas principais classes de lipídeos constituintes, fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo. O método é baseado na troca lipídica ocorrida entre uma nanoemulsão artificial (LDE) que se assemelha à estrutura lipídica da LDL, usada como doador de lipídeos radioativos, e as lipoproteínas plasmáticas. Após precipitação da LDE e das demais lipoproteínas, a capacidade da HDL de receber lipídeos é quantificada pela medida da radioatividade presente na lipoproteína. No presente estudo, foi realizada a padronização deste método, assim como analisadas possíveis interferências no método. No mesmo estudo, foi analisada a transferência de lipídeos da LDE para a partícula de HDL em indivíduos controles. A elevação da temperatura (4 a 37°C), do tempo de incubação (5min a 2h) e de HDL-Colesterol (33 a 244 mg/dL) resultaram em progressivo aumento na transferência dos quatro lipídeos da LDE para a HDL. Por outro lado, o aumento do pH (6,5 a 8,5) e da concentração de albumina (3,50 a 7,00 g/dL) não alteraram os valores de transferência. A amostra de plasma mostrou ser inalterada para este ensaio por período de 12 meses (p>0,05), enquanto que a LDE foi inalterada por até 15 dias (p>0.2). Os resultados da análise intra-ensaio apresentaram imprecisão (C.V.) para · a transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo de 0,83, 0,56, 1,49 e 0,51 %, respectivamente. A análise inter-ensaio mostrou imprecisão para os resultados de transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo 0,78, 0,59, 1,32 e 0,58%, respectivamente. A média da transferência de fosfolipídeo, colesterol livre, éster de colesterol e triglicerídeo da LDE para a HDL nos 53 voluntários foi de 25,5±2,6, 9,9±1 ,6, 4,8±1,3 e 6,9±1 ,1%, respectivamente. As transferências de éster de colesterol, triglicerídeo e colesterol livre se correlacionaram positivamente entre estes lipídeos. Foram encontradas correlações positivas também entre as transferências de fosfolipídeo e de triglicerídeo e entre a transferência de colesterol livre e a concentração de colesterol de HDL. O método de transferência de lipídeos para a fração HDL mostrou ser prático e preciso, .podendo, dentro das condições ideais estabelecidas neste trabalho, determinar a capacidade receptora de lipídeos da HDL. / Plasma lipoproteins constantly exchange lipids and apolipoproteins. Besides the action of transfer proteins, the ability of lipoproteins to receive or donate lipids also depends on several other factors. The structure, lipid and protein composition of the lipoproteins may influence the fluidity of the particle and thereby this ability of lipoprotein. In the plasma, the lipoprotein class that is the most affected by lipid transfers is HDL. The current study was designed to establish a practical method to quantify the ability of this lipoprotein to simultaneously receive its main constituent lipid classes, namely phospholipid, free cholesterol, cholesteryl ester and triglycerides. The method is based on the lipids exchange between an artificial nanoemulsion (LDE) that resembles the LDL, used as a donor of radioactive lipids, and plasma lipoproteins. After precipitation of the LDE and the others plasma lipoproteins, the accept capacity of the HDL\' is quantified by the measure of radioactivity in the lipoprotein. In the present study, the validation of this method was carried through. Moreover, possible interferences in the method had been analyzed. In the same study, the transfer of lipids of LDE for the particle of HDL from control subjets was analyzed. The rise of temperature (4 to 37°C), time of incubation (5min to 2h) and of HDL-Cholesterol (33 to 244 mg/dL) resulted in gradual increase in the transfer of the four lipids from LDE to HDL. On the other hand, the increase of pH (6.5 to 8.5) and albumin concentration (3.50 to 7.00 g/dL) had not modified the values of transfers. The plasma sample showed to be unchanged for this assay for period of 12 months (p>0.05), whereas the LDE was unchanged for 15-days (p>0.2). The intra-assay results showed imprecision (C.V) of 0.83, 0.56, 1.49 and 0.51 % for phospholipid, free cholesterol, cholesteryl ester and triglycerides transfer, respectively. The inter-assay showed imprecision for results of phospholipid, free cholesterol, cholesteryl ester and triglycerides transfer of 0.78, 0.59, 1.32 and 0.58%, respectively. The average of phospholipid, free cholesterol, cholesteryl ester and triglycerides transfers from LDE to HDL in the 53 volunteers was of 25.5±2.6, 9.9±1.6; 4.8±1 .3 and 6.9±1 .1 %, respectively. Cholesterol ester, triglyceride and free cholesterol transfers positively correlated among each other. A positive correlation was also found between phospholipid and triglyceride transfers and free cholesterol transfer and HDL cholesterol concentration. The method of lipids transfer to HDL showed to be practical and reproducible, being able, inside of the ideal conditions established in this work, to determine the aceptora capacity of lipids of the HDL.

Untersuchung von MPO sowie anti-inflammatorischer HDL Funktionalität bei Patienten mit Vorhofflimmern

L'effet protecteur des HDL sur les pathologies cardio-vasculaires est principalement attribué à leur rôle central dans le Transport Retour du Cholestérol (TRC). Ce processus assure l'efflux du cholestérol excédentaire des cellules périphériques vers le foie, au niveau duquel il est éliminé dans les sécrétions biliaires. Dans ce contexte, notre équipe a identifié à la surface des cellules hépatiques la présence d’un complexe enzymatique, très proche de l’ATP synthase mitochondriale, comme étant un récepteur de haute affinité pour l’apoA-I (protéine majoritaire des HDL). Cette ATP synthase de surface, également appelée ecto-F1-ATPase, joue un rôle clé dans l’endocytose hépatique des HDL. En effet, la liaison de l’apoA-I stimule l’activité ATPasique de l’enzyme, entrainant la production d’ADP extracellulaire puis l’activation spécifique du récepteur nucléotidique P2Y13, aboutissant in fine à l’endocytose des HDL. Ainsi, l’équipe a montré le rôle clé de la voie ecto-F1-ATPase/P2Y13 dans l’endocytose hépatique des HDL et par conséquent dans les effets protecteurs de ces derniers dans l’athérosclérose.Les travaux de thèse présentés ici visent à déterminer les mécanismes de régulation de cette ecto-F1-ATPase. Compte tenu de l’importance de la régulation des taux d’ADP et d’ATP extracellulaires dans l’endocytose des HDL, nous nous sommes intéressés dans un premier temps aux acteurs moléculaires qui pourraient réguler le métabolisme nucléotidique à la surface cellulaire. Nous avons mis en évidence la présence, à la surface des cellules HepG2, de l’adénine nucléotide translocase (ANT), une autre protéine classiquement localisée à la mitochondrie. Nous avons montré que l’ecto-ANT est impliquée dans la régulation des taux des nucléotides adényliques ADP et ATP extracellulaires et que son fonctionnement est lui-même dépendant du taux de ces derniers dans le milieu extracellulaire. / The cardioprotective effect of high-density lipoprotein cholesterol (HDL-C) is mostly attributed to their metabolic functions in reverse cholesterol transport (RCT), a process whereby excess cell cholesterol is taken up from peripheral cells and processed in HDL particles, and later delivered to the liver for further metabolism and bile excretion. ATP synthase, classically known to be located in the mitochondrial inner membrane, has been unexpectedly found expressed at the plasma membrane of hepatocytes, as a receptor for apoA-I, playing a role in HDL-cholesterol uptake. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. The strict dependence of HDL endocytosis on extracellular ADP level led us to study first, whether other plasma membrane proteins than ecto-F1-ATPase could regulate extracellular ADP level. We highlighted the presence on hepatocytes cell surface of Adenine Nucleotide Translocase (ANT), another transmembrane protein of the inner mitochondrial membrane. We showed that ecto-ANT activity could increase or reduce extracellular ADP level, depending on the extracellular ADP/ATP ratio. Furthermore, we demonstrated that pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis in human hepatocytes. We then sought to explore the molecular mechanisms involved in targeting ecto-F1-ATPase to the plasma membrane. Indeed, F1-ATPase ectopic expression at the plasma membrane has been described on several cell types and has been related to several physiological and pathophysiological processes however, the pathway involved in its transport to the cell surface remains unknown.

La prevalencia de riesgo coronario se viene incrementando con una alta incidencia en las ciudades cosmopolitas y de gran auge económico; es por ello que el objetivo de la presente investigación, fue determinar el riesgo coronario en pacientes adultos que acudieron al Servicio Académico Asistencial de Análisis Clínicos (SAAAC) de Enero 2011 a Septiembre 2013; el estudio es de tipo descriptivo, retrospectivo, transversal y no experimental. La muestra estuvo constituida por 624 pacientes, a quienes se evaluó su perfil lipìdico, para determinar el riesgo coronario I y II . Se encontraron los siguientes resultados: de los que tienen colesterol total de alto riesgo el 63,2 % son mujeres, el 36,8 % son hombres y el 52,6 % tiene de 50 a 65 años, del total de pacientes con valores HDL de alto riesgo el 51,4 % son mujeres, el 48,6 % son hombres y el 40,6 % tienen de 50 a 65 años; se aprecia que del total de pacientes con VLDL de riesgo el 53,9 % son mujeres, el 46,1 % son hombres y el 48,5 % tienen de 50 a 65 años; los pacientes con LDL de alto riesgo el 66,8 % son mujeres, el 33,2 % son hombres y el 48,4 % tienen de 50 a 65 años; del total de pacientes con triglicéridos de alto riesgo el 53,1 % son mujeres, el 46,9 % son hombres y el 49,5 % tienen de 50 a 65 años. Se concluye que de los pacientes con riesgo coronario tipo I el 57,4 % son mujeres, el 42,6 % son hombres y el 45,1 % tienen de 50 a 65 años; de los pacientes con riesgo coronario tipo II el 61,6 % son mujeres, el 38,4 % son hombres y el 48,3 % tienen de 50 a 65 años, esto nos indica que el riesgo coronario I y II, se asocia màs con los adultos mayores de sexo femenino Se encontró relación estadísticamente significativa entre el riesgo coronario tipo I para la edad y no significativa para el género, también se encontró relación estadística entre el riesgo coronario tipo II y la edad .

La maladie d’Alzheimer (MA) touche environ 35 millions de personnes à travers le monde ce qui en fait la première forme de démence. Le principal facteur de risque de cette maladie est l’âge et donc, avec l’augmentation de l’espérance de vie, on pense que ce nombre va doubler d’ici 2025. C’est donc un problème de santé publique. Le mécanisme à l’origine de la MA n’est pas connu, et il n’existe aucun traitement permettant de guérir ou de ralentir efficacement l’évolution des symptômes. Il est donc urgent de mieux comprendre cette maladie afin de proposer aux patients des traitements efficaces. Certaines études épidémiologiques ont montré que l’hypercholestérolémie est un facteur de risque pour la MA. A l’inverse, la prise de statines diminuerait les risques de développer cette maladie. De plus, des études réalisées in vitro et in vivo ont montré que le cholestérol peut favoriser la production des peptides amyloïdes qui sont à l’origine de la formation des plaques, caractéristiques de la MA. Enfin, certaines études ont montré qu’une baisse des niveaux de HDL peut être un facteur de risque pour la MA. Tous ces résultats nous amènent à penser que le métabolisme du cholestérol puisse être altéré dans la MA. La PON1 est une enzyme associée aux HDL. Elle possède des propriétés anti-inflammatoires et anti-oxydantes. Certaines études ont montré que son activité est réduite dans la MA, et que certains de ses polymorphismes sont associés à cette maladie. La PON1 pourrait donc jouer un rôle dans la MA. Mon projet de thèse a pour but d’investiguer si l’efflux du cholestérol est altéré dans la MA, et de comprendre le mécanisme à l’origine de cette éventuelle altération. Il vise également à étudier la PON1 dans la MA. Nos résultats montrent que l’efflux du cholestérol est diminué dans la MA. Cette baisse est due à une perturbation de la fonctionnalité des HDL et non, à une baisse de l’expression du transporteur ABCA1 qui est impliqué dans la première étape de l’efflux du cholestérol. Nos résultats suggèrent que la baisse de fonctionnalité des HDL soit due à leur oxydation, et à une altération de leur structure. Notre étude montre que l’activité paraoxonase est réduite de manière non significative dans la MA supposant donc, une légère baisse de la fonctionnalité de la PON1. De plus, les polymorphismes 192Q/R et 55L/M de la PON1 ne sont pas associés à cette maladie. Enfin, nos résultats montrent que les HDL des personnes Alzheimer sont plus sensibles à l’oxydation probablement du fait d’une baisse de la fonctionnalité de la PON1. En conclusion, dans la MA, les HDL sont oxydés et leur structure est altérée ce qui entraîne une baisse de l’efflux du cholestérol. Une altération de la fonctionnalité de la PON1 pourrait expliquer, en partie, la sensibilité des HDL à l’oxydation.

Ce travail décrit la synthèse et le comportement de nouveaux photocatalyseurs à base de dioxyde de titane utilisés pour des applications environnementales. Dans la première partie, la pertinence des composés modèles testés, le colorant acide orange 7 (AO7) et le 4-Chlorophénol (4-CP), pour des traitements photocatalytiques à base de TiO2 a été étudiée et validée. Cette étude a été centrée sur l’effet de la concentration initiale en composé et sur la vitesse d’agitation pendant la réaction photocatalytique. La deuxième partie est consacrée principalement à la synthèse et la caractérisation des suspensions de particules colloïdales de TiO2. Leur séparation puis leur récupération après le traitement étant pratiquement impossible l’immobilisation de ces particules sur des supports de type hydroxydes doubles lamellaires (HDL) a été étudiée afin de préparer un matériau composite TiO2/HDL présentant une activité photocatalytique comparable à celle du dioxyde de titane pur. Un deuxième type de composite basé sur des mélanges de TiO2/SiO2 a également été envisagé et utilisé dans la préparation de fines couches efficaces pour la photodégradation de l’hexane. Enfin, le comportement photocatalytique d’HDL pur à base de zinc et de chrome, sans addition de TiO2, a également été étudié et est présenté dans la partie finale de cette thèse. Les matériaux préparés ont été caractérisés par différentes analyses chimiques, diffraction et fluorescence des rayons X, microscopie électronique à transmission et à balayage, spectroscopie IR à transformé de Fourier, analyse thermogravimétrique, mesure du potentiel Zeta, diffusion de la lumière, mesure d’adsorption N2. Les différents matériaux ont été testés photocatalytiquement via la photo-Oxydation en solution aqueuse de l’acide orange 7 (AO7), du 4-Chlorophénol (4-CP) ou du bleu de méthylène à différents pH. L’activité photocatalytique du matériau composite à base de TiO2/SiO2 sous forme de film fin a été évaluée en phase gaz en présence d’hexane. / This work describes the behaviour and fabrication of new photocatalysts based on titaniumdioxide for the purpose of environmental applications. It consists of five closely connectedparts. In the first part the suitability of chosen model compounds, azo dye Acid Orange 7(AO7) and 4-Chlorophenol (4-CP), for photocatalytic activity assessment of TiO2 was studied.This study was focused on the effect of different initial concentrations of model compoundand different rates of stirring during photocatalytic reaction. The second part then focusedmainly on the synthesis and characterization of aqueous colloidal suspensions of TiO2. Theseparation of TiO2 particles in the form of colloidal suspensions and their regeneration afterthe reaction, while keeping the same photocatalytic properties, is almost not possible.Therefore, it was necessary to find an appropriate method how to immobilize these particleson the support or in the form of composite. The layered double hydroxides (LDH) werechosen as one of suitable supports for TiO2 photocatalyst. The focus was kept on thepreparation of TiO2/LDH composites with the same or higher photocatalytic activity as purecolloidal titanium dioxide. The second chosen type of composite was based on twocomponentTiO2/SiO2 material and these composites were used for the preparation of thinlayers. Photocatalytic behaviour of pure LDHs and their possible use as photocatalyst withoutTiO2 addition was also studied and described in a final part of this work. Prepared materials were characterized by chemical analysis, X-Ray diffraction andflorescence, transmition electron microscopy, scanning electron microscopy, Fouriertransform infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, zetapotential measurement and N2 adsorption. As another step, materials were tested asphotocatalyst by the photooxidation of Acid Orange 7, 4-Chlorophenol and Methylene Blue indifferent pH in aqueous medium. Photocatalytic activity of TiO2/SiO2 composites in the formof thin films was tested in gaseous phase using hexane as a model pollutant. It was found that quantum yields of 4-CP degradation for all prepared alkaline colloidalsuspensions of TiO2 were lower than those obtained for acidic TiO2 colloidal suspensions. Inthe contrary to the quantum yield of acidic TiO2, the quantum yield of alkaline suspensionsdecreased during the aging. Prepared TiO2/Mg2Al1.5 nanocomposites exhibited higherphotocatalytic activity than the original TiO2 in basic conditions and also it was much easierto recover the photocatalyst after reaction by simple sedimentation. In the case of TiO2/SiO2composites, it was found that composite prepared with TiO2:SiO2 ratio 1:1 has higherphotocatalytic activity in aqueous media than starting pure TiO2 but with increasing SiO2content reaction rate of AO7 degradation decreases. Thin layers of TiO2:SiO2 compositeprepared from simultaneously co-Precipitated particles (they have improved crystallinity inrelation to pure TiO2) are able to photocatalyticaly degrade hexane. In the case of pure LDH,it was proved that even noncalcined Zn2CrCO3 LDH can produce HO• radicals. However,mixed oxides (containing ZnO) prepared by LDH calcination at temperatures higher than500°C, showed higher efficiency.