Estudos clínicos e epidemiológicos indicam que baixas concentrações plasmáticas da lipoproteína de alta densidade (HDL) estão forte e independentemente associadas a uma maior incidência de doença arterial coronária (DAC). Entretanto, o insucesso dos agentes que são capazes de aumentar a concentração de HDL-C sugere que a funcionalidade da HDL pode representar um alvo terapêutico mais apropriado. Para a avaliação de um dos aspectos funcionais da HDL, o presente trabalho descreve o desenvolvimento de um método de grande praticidade que permite uma visão integrada de uma etapa fundamental do metabolismo que é a transferência de lípides entre as diferentes classes de lipoproteínas. A avaliação deste fenômeno nas subfrações de HDL, aspecto ainda não explorado, poderá fornecer novas informações a respeito da fisiopatologia da DAC. O método descrito no presente trabalho permite a avaliação da transferência simultânea das quatro principais classes lipídicas doadas por uma nanoemulsão semelhante à LDL para a HDL3. Foi realizada análise dos possíveis interferentes neste método. Verificou-se que a elevação da temperatura de 0 a 40 °C resultou em aumento progressivo na transferência de todos os lipídeos para a HDL3. A variação de pH entre 6,5 e 8,5 e o aumento na concentração de albumina não alteraram os valores de transferência. O aumento no tempo de incubação acima de 60 minutos promoveu diminuição na transferência de colesterol esterificado para a HDL3 e aumento na transferência de fosfolipídeos. O método apresentou boa precisão intra e inter-ensaio, sendo o coeficiente de variação menor que 5% para todos os lipídeos. A porcentagem média de transferência de colesterol livre, fosfolipídeos, triacilglicerol e colesterol em 45 invíduos saudáveis foi de respectivamente de 1,1±0,06; 13,5±0,15; 2±0,05 e 0,84±0,04% e em 45 portadores de doença arterial coronária foi respectimente 1,0±0,04; 15,8±0,44; 1,77±0,04 e 1,0±0,06%. Não houve diferença nos valores de idade, IMC, colesterol total, HDL-C, LDL-C, triacilglicerol, apo A-1, apo B, CETP, PLTP e LCAT, mas os indivíduos portadores de doença arterial coronária apresentaram valores maiores de colesterol livre e colesterol total em relação aos indivíduos saudáveis. O método desenvolvido no presente estudo é prático, preciso e de potencial relevância como ferramenta no estudo dos distúrbios de função da HDL.. / Clinical and epidemiological studies show that low concentrations of high density lipoproteins (HDL) are strongly and independently associated to an increased incidence of coronary artery disease (CAD). However, the lack of success of some drugs developed to increase HDL cholesterol concentrations (HDL-C) suggests that the functional aspects of HDL may represent a more appropriate therapeutic target. To study one of the functional aspects of HDL, the present work describes the development of a practical method that provides an integrated view of a fundamental step of lipid metabolism, namely, the lipid transfer among different lipoprotein classes. This phenomenon in the HDL subfractions is yet unexplored, and could provide new insights on the pathophysiology of CAD. The method described here allows the measurement of the ability of HDL3 to receive the major lipid classes from a radioactively labeled nanoparticle that resemble LDL. The possible interfering factors at the lipid transfer to HDL3 were studied. The increase in the assay temperature from 0 to 40 °C results in a progressive increase in the net transfer of all lipids to HDL3. The increase in incubation time above 60 minutes resulted in a reduced transfer of cholesterol esters to HDL3 with a concomitant increase in the transfer of phospholipids to the latter. The method presented adequate intra and inter-assay precision, with a coefficient of variation smaller than 5% for all lipids. The average percentage of free cholesterol, phospholipids, triacilglycerol an cholesterol transfer to HDL3 was respectively of 1,1±0,06; 13,5±0,15; 2±0,05 e 0,84±0,04% in 45 healthy individuals and 1,0±0,04; 15,8±0,44; 1,77±0,04 e 1,0±0,06% in 45 CAD patients. There was no difference in the age, BMI, total cholesterol, HDL-C, LDL-C, triacilglycerol, apo A-1, apo B, CETP, PLTP and LCAT but the CAD patients had higher levels of total cholesterol and free cholesterol. The method described here is practical, precise and potentially relevant as a tool to study HDL function.

Orientadores: Eliana Cotta de Faria, Andrei Carvalho Sposito / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T05:11:23Z (GMT). No. of bitstreams: 1 Vendrame_Felipe_M.pdf: 1091840 bytes, checksum: 4ae9b0626a5c1d06cdf37066842a24fc (MD5) Previous issue date: 2014 / Resumo: Baixas concentrações plasmáticas da lipoproteína de alta densidade (HDL-C) constituem um dos fatores de risco para o desenvolvimento da doença cardiovascular aterosclerótica, a principal causa de morte nos países desenvolvidos e emergentes. A niacina é conhecida por ser o principal fármaco a aumentar as concentrações de HDL-C. A concentração plasmática reduzida das lipoproteínas de alta densidade está associada à vulnerabilidade ao estresse oxidativo e a propensão à disfunção endotelial. A niacina através da ativação de seu receptor específico acoplado à proteína G (GPR109A) promove efeitos anti-inflamatórios e antioxidantes. Com base nestas informações, este estudo investigou o efeito de curto prazo da niacina e da niacina associada ao laropipranto sobre a função endotelial, parâmetros séricos bioquímicos e físico-químicos em indivíduos hipoalfalipoproteinêmicos. O estudo foi realizado em 18 indivíduos assintomáticos, de ambos os gêneros com idade entre 20 e 60 anos apresentando concentrações plasmáticas de HDL-C abaixo de 40 mg/dL. Os indivíduos foram tratados com niacina de liberação prolongada 1g/dia (NLE, Metri, Libbs Farmacêutica, São Paulo, Brasil) e niacina associada ao laropipranto 1g/20mg (NLE/LRPT, Cordaptive, Merck, São Paulo, Brasil), em um estudo crossover e a sequência de tratamentos foi realizada de forma aleatória. As amostras de plasma e a dilatação fluxo mediada da artéria braquial (FMD) foram obtidas no início do estudo, no 7º dia do tratamento 1, no 7º dia após washout e no 7 º dia do tratamento 2. Após terapias com NLE e NLE/LRPT as concentrações de triglicérides diminuíram 4,0% e 3,0% (p <0,05) e o tamanho da HDL 5,8% e 6,2%, (p <0,05) além de aumentarem a glicose 5,0 e 8,0 % (p <0,02) e a bilirrubina direta 62% e 50% (p <0,04); também foi observado aumento médio do FMD de 4,5% e 4,1% nos grupos NLE e NLE/LRPT, respectivamente. Não houve alteração de HDL-C e na atividade da proteína de transferência de colesteril éster (CETP) após ambos os tratamentos. Todas estas modificações foram revertidas após o período de washout. Na análise intergrupos, não houve diferenças em relação à variação de HDL-C, triglicérides, proteína C reativa, bilirrubina direta e FMD. Os resultados revelam que a terapia de niacina de curto prazo pode melhorar a função endotelial em indivíduos com baixas concentrações de HDL-C. A adição do antagonista de PGD2 (laropipranto) não influenciou o efeito da niacina sobre a função endotelial / Abstract: Low plasma levels of high-density lipoprotein-cholesterol (HDL-C) are one of the risk factors involved in the development of atherosclerotic cardiovascular disease, the main cause of death in developed and emerging countries. Niacin is known as the main drug used to increase HDL-C levels. Reduced concentrations of high density lipoproteins are associated to oxidative stress vulnerability and tendency to endothelial dysfunction. Niacin, through the activation of its specific receptor coupled to G protein (GPR109A) promotes anti-inflammatory and antioxidant effects. Therefore, this study investigated the short-term effect of niacin and niacin associated to laropiprant on endothelial function, biochemical and physical-chemical serum parameters, in hypoalphalipoproteinemic subjects. The study was carried out in 18 asymptomatic subjects, male and female, aged between 20 and 60 years, with HDL-C plasma concentrations below 40 mg/dL. Subjects were treated with extended-release niacin 1g/day (NLE, Metri, Libbs Pharmaceutics, Sao Paulo, Brazil) and niacin associated to laropiprant 1g/20mg (NLE/LRPT, Cordaptive, Merck, Sao Paulo, Brazil), in a crossover study. The sequence of treatments was randomly determined. Plasma samples and flow-mediated dilatation of the brachial artery (FMD) were obtained in the beginning of the study, on the 7th day of treatment 1, on the 7th after washout and on the 7th day of treatment 2. After therapies with NLE and NLE/LRPT respectively, triglycerides (TG) concentrations decreased 4,0% and 3,0% (p <0,05) and HDL size 5,8% and 6,2%, (p <0,05); besides, glucose increased 5,0 and 8,0 % (p <0,02) and direct bilirubin 62% and 50% (p <0,04). The average increase of FMD was 4,5% and 4,1% on NLE and NLE/LRPT groups, respectively. No changes in HDL-C and the activity of cholesteryl ester transfer protein (CETP) after both treatments were observed. All these changes were reversed after the washout period. In intergroup analysis, no differences were found regarding variations in HDL-C, triglycerides, C reactive protein (CRP), direct bilirubin and FMD. These results suggest that short-term niacin therapy may improve endothelial function in subjects with low HDL-C concentrations. The addition of the PGD2 antagonist (laropiprant) did not influence niacin¿s effect on endothelial function / Mestrado / Clinica Medica / Mestre em Ciências

Orientadores: Andrei Carvalho Sposito, Eliana Cotta de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T13:48:30Z (GMT). No. of bitstreams: 1 Panzoldo_NataliaBaratella_D.pdf: 2220803 bytes, checksum: b1c1a7df4771466e6a32411bceb02387 (MD5) Previous issue date: 2014 / Resumo: Doenças cardiovasculares constituem a principal causa de mortalidade no Brasil e no mundo. Baixas concentrações de HDL - colesterol são consideradas um fator de risco cardiovascular independente. Esta relação inversa tem sido atribuída às diferentes propriedades protetoras da HDL, dentre as quais podemos destacar seu papel no transporte reverso de colesterol, por meio do efluxo de colesterol, sua habilidade de inibir a agregação plaquetária, e suas atividades antioxidantes e anti-inflamatórias. No entanto, estudos recentes indicam que baixas concentrações de HDL - colesterol constituem um preditor significante de doença aterosclerótica somente em indivíduos assintomáticos e que a capacidade de efluxo de colesterol é um melhor preditor de carga aterosclerótica do que HDL - colesterol. Em conjunto, estes achados sugerem que a função da HDL poderia ser um fator chave na relação entre concentrações de HDL - colesterol e o desenvolvimento de doença aterosclerótica. Se o for, alterações das funções da HDL podem discernir melhor, entre os indivíduos com hipoalfalipoproteinemia, aqueles com maior propensão a desenvolver doença aterosclerótica. Assim, investigamos, em indivíduos sem doença cardiovascular previamente manifesta, se funções da HDL estão associadas a concentrações de HDL - colesterol e carga aterosclerótica. Participantes foram classificados em HDL - colesterol baixo (LH; HDL-C? 32 mg/dL; n=33), intermediário (IH; HDL-C= 40-67 mg/dL; n=33), ou alto (HH; HDL-C?78mg/dL; n=35). Nós avaliamos composição química da HDL, tamanho da partícula, capacidade de efluxo de colesterol, atividade antioxidante, susceptibilidade à oxidação, atividade anti-inflamatória, e habilidade de inibir agregação plaquetária. O grupo LH foi associado à espessura intimo-medial de carótidas (IMT) aumentada (p?0,001), maior conteúdo de triglicérides (4±2% vs. 4±2% em IH e 3±1% em HH, p?0,001), menor conteúdo de fosfolípides (12±4% vs. 14±5% em IH e 13±3% em HH, p=0,035), menor tamanho de partículas (7,33±0,33nm vs. 7,72±0,45nm em IH e 8,49±0,42nm em HH, p?0,001) e menor capacidade de efluxo de colesterol celular (9±3 % vs. 12±3 % em IH e 11±4 % em HH, p?0,001). Indivíduos HH apresentaram menor atividade antioxidante (37(53)% vs. 48(35)% em IH e 55(41)% em LH, p=0,003), maior suscetibilidade à oxidação (57±22% vs. 45±20% em IH e 46±25% em LH, p=0,017) e maior habilidade de inibir agregação plaquetária (45±25% vs. 31±18% em IH e 37±24 % em LH, p=0,0026). Indivíduos com IMT acima de 1 mm apresentaram partículas com menor tamanho (7,55±0,49 nm vs. 7,89±0,64 nm, p?0,001), atividade antioxidante (37(23)% vs. 49(42)%, p=0,018) e capacidade de efluxo de colesterol (31±14% vs. 40±14%, p=0,02). Nenhuma diferença foi encontrada para as outras características ou propriedades funcionais da HDL. Nós concluímos que, em um contexto de prevenção primária, o menor tamanho da partícula, o conteúdo reduzido de fosfolípides, e capacidade de efluxo de colesterol diminuída são relacionados com ambos LH e magnitude da doença aterosclerótica subclínica. Nestes indivíduos, estas características podem explicar a associação entre HDL - colesterol e o desenvolvimento da doença aterosclerótica. / Abstract: Cardiovascular diseases are the main cause of death in Brazil and worldwide. Low HDL-C levels are considered an independent cardiovascular risk factor. This inverse relationship has been attributed to different protective properties described for HDL, such as its role in the reverse cholesterol transport, through cholesterol efflux, its ability to inhibit platelet aggregation, and its antioxidant and anti-inflammatory effects. However, recent studies indicate that low HDL-cholesterol is a significant predictor of atherosclerotic disease in healthy individuals and that cholesterol efflux capacity is a better predictor of carotid atherosclerotic burden as compared to HDL-cholesterol. Altogether these findings have suggested that HDL function would be the key factor for the link between HDL-cholesterol concentration and the subclinical disease in a primary prevention setting. If so, changes in HDL function could help to discriminate, among individuals with hypoalphalipoproteinemia, those who are prone to develop atherosclerotic disease. Hence, in a primary prevention setting, we investigated whether HDL dysfunction is associated with HDL-cholesterol concentration and atherosclerotic burden. Participants were classified as low (LH; HDL-C? 32 mg/dL; n=33), intermediate (IH; HDL-C= 40-67 mg/dL; n=33), or high HDL-cholesterol (HH; HDL-C?78mg/dL; n=35). We measured HDL chemical composition, particle size, cholesterol efflux capacity, antioxidant activity, susceptibility to oxidation, anti-inflammatory activity, and ability to inhibit platelet aggregation. LH was associated to enhanced carotid intima-media thickness (IMT;p?0.001), high HDL triglyceride (4±2% vs. 4±2% in IH and 3±1% in HH, p?0.001), low HDL-phospholipids (12±4% vs. 14±5% in IH and 13±3% in HH, p=0.035), decreased particle size (7.33±0.33nm vs. 7.72±0.45nm in IH and 8.49±0.42nm in HH, p?0.001) and reduced cholesterol efflux capacity (9±3 % vs. 12±3 % in IH and 11±4 % in HH, p?0.001). The HH group presented reduced antioxidant activity (37(53)% vs. 48(35)% in IH and 55(41)% in LH, p=0.003), and increased susceptibility to oxidation (57±22% vs. 45±20% in IH and 46±25% in LH, p=0.017) and ability to inhibit platelet aggregation (45±25% vs. 31±18% in IH and 37±24 % in LH, p=0.0026). Carotid IMT>1mm was associated with reduced HDL size (7.55±0.49 nm vs. 7.89±0.64 nm, p?0.001), antioxidant activity (37(23)% vs. 49(42)%, p=0.018), and cholesterol efflux capacity (31±14% vs. 40±14%, p=0.02). No differences were found for the other HDL characteristics or functional properties. We conclude that in a primary prevention setting, small particle size, reduced HDL-phospholipids content, and diminished cholesterol efflux capacity are related to both LH and carotid IMT. In these individuals, these characteristics may underlie the association between HDL-cholesterol and atherosclerotic burden / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Η παχυσαρκία και οι σχετιζόμενες με αυτήν παθολογικές καταστάσεις συνιστούν μία από τις κυριότερες αιτίες θανάτου παγκοσμίως, με τα επίπεδά της να αυξάνονται σε ανησυχητικό βαθμό. Επιδημιολογικές έρευνες σε ασθενείς με μεταβολικό σύνδρομο έδειξαν ευθεία συσχέτιση μεταξύ παχυσαρκίας και χαμηλών επιπέδων HDL χοληστερόλης πλάσματος, ενώ μελέτες σε πειραματικά μοντέλα ποντικών αποδεικνύουν πως η συσχέτιση αυτή είναι αιτιολογική. Η HDL είναι ένα μίγμα λιποπρωτεϊνικών σωματιδίων που, ανάλογα με τη σύστασή τους σε λιπίδια, μπορούν να είναι δισκοειδή ή σφαιρικά. Η σημαντικότερη αθηροπροστατευτική δράση της HDL οφείλεται στο γεγονός ότι η συγκεκριμένη λιποπρωτεΐνη συλλέγει την περίσσεια χοληστερόλης από τους περιφερικούς ιστούς και τη μεταφέρει στο ήπαρ, όπου καταβολίζεται, μια διαδικασία γνωστή και ως ανάστροφη μεταφορά χοληστερόλης. Κύρια πρωτεΐνη της HDL είναι η απολιποπρωτεΐνη Α-Ι (apoA-I), η οποία αλληλεπιδρώντας με τον μεταφορέα λιπιδίων ABCA1 προάγει την de novo σύνθεση δισκοειδών HDL σωματιδίων, τα οποία μετατρέπονται σε σφαιρικά με τη δράση του ενζύμου LCAT. Μία πρόσφατη μελέτη έδειξε ότι οι απολιποπρωτεΐνες E (apoE) και CIII (apoCIII) είναι ικανές να προάγουν την de novo βιογένεση HDL σωματιδίων ανεξάρτητα από την apoA-I. Η παρατήρηση αυτή ενίσχυσε την ιδέα ότι ο πληθυσμός της HDL είναι ένας συνδυασμός διαφορετικών σωματιδίων που έχουν διακριτή απολιποπρωτεϊνική σύνθεση. Επιπλέον, μεταβολές στις αναλογίες των απολιποπρωτεϊνών της HDL φαίνεται να καθορίζουν την κατανομή και τη λειτουργικότητα των υποπληθυσμών της. Στην παρούσα μελέτη στόχος ήταν ο δομικός και λειτουργικός χαρακτηρισμός της HDL σε ασθενείς με υπερνοσογόνο παχυσαρκία που υποβλήθηκαν σε χολοπαγκρεατική εκτροπή με γαστρική παράκαμψη κατά Roux en Y. Μελετήθηκαν 20 ασθενείς με υπερνοσογόνο παχυσαρκία, πριν και μετά την χειρουργική επέμβαση, και 7 υγιή άτομα ελέγχου. Απομονώθηκε πλάσμα από την ομάδα ελέγχου και από τους 20 ασθενείς πριν την επέμβαση καθώς και έξι μήνες μετά. Ακολούθησε κλασματοποίηση λιποπρωτεινών του πλάσματος με υπερφυγοκέντρηση σε βαθμίδωση πυκνότητας KBr και ανάλυση των κλασμάτων με Western Blot για ανίχνευση και ποσοτικοποίηση των επιπέδων των απολιποπρωτεινών apoA-I, apoE και apoCIII. Ακολούθησαν περαιτέρω βιοχημικές αναλύσεις των κλασμάτων, δομικές αναλύσεις με μη αποδιατακτική ηλεκτροφόρηση δύο διαστάσεων (2D-PAGE) και ηλεκτρονική μικροσκοπία, προκειμένου να υπάρξει μία πιο ξεκάθαρη εικόνα για τα σωματίδια που απαρτίζουν την HDL ενώ μετρήθηκε και η αντιοξειδωτική ικανότητα της HDL και στις τρεις ομάδες. Τέλος, μετρήθηκε η δραστικότητα του ενζύμου LCAT και συσχετίστηκε με την ηπατική εναπόθεση τριγλυκεριδίων των ασθενών. Η σειρά αναλύσεων των λιποπρωτεϊνικών κλασμάτων με Western Blot έδειξε ότι, έξι μήνες μετά την επέμβαση, υπάρχει μείωση των απολιποπρωτεϊνών A-I, E και CIII των ασθενών. Τα αποτελέσματα από την δισδιάστατη ηλεκτροφόρηση και την ηλεκτρονική μικροσκοπία αποδεικνύουν την ύπαρξη διαφορετικών σωματιδίων της HDL με διακριτή απολιποπρωτεϊνική σύνθεση, ενώ η μέτρηση του αντιοξειδωτικού δυναμικού της HDL φανερώνει τη ύπαρξη πιο λειτουργικής HDL μετά την επέμβαση. Η αυξημένη δραστικότητα της LCAT στους ασθενείς σε σχέση με την ομάδα ελέγχου και η μείωσή της μετά την επέμβαση υποδηλώνει ένα πιθανό αντιρροπιστικό μηχανισμό για την αναστολη της συσσώρευσης δισκοειδούς HDL. Έτσι προάγεται ο σχηματισμός περισσότερης ώριμης σφαιρικής HDL, ενώ αποτρέπεται και η εναπόθεση ηπατικών τριγλυκεριδίων που παρατηρείται στο ήπαρ των ασθενών αυτών. Η παρατήρηση ότι η δομή και η λειτουργικότητα της HDL βελτιώνονται στους ασθενείς έξι μήνες μετά την επέμβαση, οδηγεί στο συμπέρασμα ότι η χειρουργική επέμβαση στην οποία υποβάλλονται οι ασθενείς επιδρά θετικά στην ποιότητα της HDL. / Obesity and its related pathologies are one of the leading causes of death worldwide, with levels increasing to an alarming extent. Epidemiological studies in patients with metabolic syndrome showed a direct correlation between obesity and low levels plasma of HDL cholesterol and experimental studies in mice show that this correlation is causative. HDL is a mixture of lipoprotein particles, which, depending on their composition in lipids, may be discoidal or spherical. The most important atheroprotective action of HDL is due to the fact that this lipoprotein collects excess cholesterol from peripheral tissues and transports it to the liver where it is catabolized. The main protein of the HDL is apolipoprotein A-I (apoA-I), which interacts with the lipid carrier ABCA1 and promotes de novo synthesis of discoidal HDL particles, which are converted to spherical with the LCAT enzyme. A recent study showed that apolipoprotein E (apoE) and CIII (apoCIII) areable to promote de novo biogenesis of HDL particles regardless of apoA-I. This observation supported the idea that the population of HDL is a combination of different particles with distinct apolipoprotein composition. Moreover, changes in the apolipoprotein ratiosof HDL appear to determine the distribution and function of its subpopulations. Although HDL cholesterol (HDL-C) has been traditionally associated to atheroprotection, recently it became clear that HDL particle functionality is also a key in reducing cardiovascular mortality. To this date the effects of obesity on HDL structure and functionality remain unclear. In this study, the objective was the structural and functional characterization of HDL frommorbidly obese patients undergoing biliopancreatic diversion by Roux en Y before and six months after the operation, and in lean control subjects. We studied 20 morbidly obese patients before and after surgery, and 7 control healthy individuals. Plasma was isolated from the control group and 20 patients before surgery and six months. Plasma was fractionated by KBr density gradient ultracentrifugation and lipoprotein fractions were isolated and analyzed, by Western Blot, for detection and quantification of the levels of apolipoproteins apoA-I, apoE and apoCIII. Further biochemical analysis of fractions, structural analysis with non denaturing two-dimensional electrophoresis (2D-PAGE) and electron microscopy, in order to have a more clear picture of the particles that compose HDL. In order to determine whether or how the efficiency of HDL is affected by the operation, we performed an assay measuring the antioxidant capacity of HDL in all three groups. Finally, the activity of the LCAT enzyme was measured and was correlated with the triglyceride deposition observed in the liver of these patients. The sequence analyzes of lipoprotein fractions by Western Blot showed that six months after surgery, there was a decrease in the levels of apolipoproteins AI, E and CIII. The results from the two-dimensional electrophoresis and electron microscopy showed the existence of different particles with distinct HDL apolipoprotein composition while the increased antioxidant potential of HDL indicates the existence of a more functional HDL after surgery. The increased activity of LCAT in patients plasma compared with the control group and its reduction after surgery, suggests that perhaps there may be a saving grace that tries to overcome the accumulation of discoidal HDL and promotes the formation of more mature, spherical HDL, so as to prevent the triglyceride deposition observed in the liver of these patients. The observation that the structure and function of HDL improved six months after surgery, leads to the conclusion that the surgery undergone by patients has a positive effect on the quality of HDL.

<p>High density lipoprotein (HDL) is an independent risk factor for thedevelopment of coronary heart disease. HDL mediated reverse cholesterol transport is a key element responsible for the cardioprotective effects of HDL. In addition HDL exerts other atheroprotective effects in vascular cells. The HDL receptor, scavenger receptor class I type B (SR-BI) derives the process of reverse cholesterol transport, mediates HDL signaling in the vasculature and protects against atherosclerosis. However, the exact atheroprotective mechanisms of HDL and SR-BI are not clearly understood.This thesis starts by characterizing a model of occlusive coronary arteryatherosclerosis, the SR-BI/apolipoprotein E double knockout mice and tests the effectsof phenolic rich pomegranate extract on disease progression. Coronary artery disease in these mice starts at three weeks of age and progresses rapidly leading to sudden death within three to five weeks. The administration of pomegranate extract reduced the extent of coronary artery atherosclerosis possibly via mechanisms that involved alterations in lipid metabolism and reduced inflammation and oxidative stress.The next two chapters aimed to gain better understanding of the atheroprotectiveactions of HDL and SR-BI. Increased macrophage apoptosis is a key event in the development of atherosclerotic plaques. HDL signaling via SR-BI reduced macrophage apoptosis while the lack of macrophage SR-BI was associated with increased macrophage apoptosis and necrotic core areas, features of plaque instability. Next HDL and SR-BI effects on macrophage migration, a key event in atherosclerotic plaque regression, are described. HDL stimulated the migration of macrophages in a manner that was dependent on SR-BI, its adaptor protein, PDZK1, and the G-protein coupled receptor, sphingosine-1-phosphate receptor 1. SR-BI mediated macrophage migration may suggest a potential role of SR-BI in atherosclerotic plaque regression.To expand our view of HDL effects on macrophages we have used proteomics as an approach. HDL treatment of macrophages altered the expression of multiple proteins.Validation experiment confirmed changes in interesting and particularly relevant protein targets in HDL mediated protection against macrophage apoptosis and inflammation and in HDL induced macrophage migration. Follow up experiments will determine their involvement in HDL and SR-BI mediated signaling. Overall this work represents a milestone in understanding the atheroprotective effects of HDL and SR-BI in macrophages.</p> / Doctor of Philosophy (PhD)

Introdução: estudos recentes demonstram que o aumento do colesterol na lipoproteína de alta densidade (HDL-C), induzido por medicamentos ou mutações genéticas, não é associado à redução de eventos coronarianos. A lipoproteína de alta densidade (HDL) apresenta aspectos funcionais distintos em relação ao seu papel cardioprotetor. Objetivo: desenvolver um escore de funcionalidade da HDL (EFH) e avaliar a sua associação com algoritmos de predição de risco cardiovascular e aterosclerose subclínica em indivíduos brasileiros. Metodologia: trata-se de um estudo transversal composto por duas etapas. Na 1ª etapa, o EFH preditor de risco cardiovascular (EFH-RCV) foi desenvolvimento e validado a partir de uma subamostra do estudo CARDIONUTRI (n=354). Na 2ª etapa, o EFH preditor de aterosclerose subclínica (EFH-AS) foi desenvolvido e validado com dados de uma subamostra do estudo ELSA-Brasil (n=4549). No estudo CARDIONUTRI foram avaliadas a atividade da paraoxonase 1 (PON1) e da proteína de transferência de ésteres de colesterol (CETP), a concentração da apolipoproteína AI (APOAI), a capacidade antioxidante da HDL (lag time) e as subfrações da HDL pelo método Lipoprint®. O estudo ELSA-Brasil avaliou as subfrações da HDL pelo método Vertical Auto Profile (VAP) e Ressonância Magnética Nuclear (RMN), e a aterosclerose subclínica por tomografia computadorizada, quantificação da calcificação da artéria coronária (CAC) e calculo do escore da CAC. Resultados: no desenvolvimento do EFH-RCV, a HDL grande apresentou maior força de associação com o risco cardiovascular no modelo múltiplo final (OR = 0,797; p <0,001). O EFH-RCV demonstrou bom desempenho em relação ao escore de risco de Framingham (AUC = 0,899; p <0,001), escore de risco de Reynolds (AUC = 0,722; p <0,001) e Adult Treatment Panel III/2013 (AUC = 0,864; p <0,001). Além disso, apresentou boa reprodutibilidade e correlação com aterosclerose subclínica, quando testado na amostra do estudo ELSA-Brasil, utilizando medidas da HDL grande derivadas do método VAP (AUC = 0,864; p <0,001 e r = 0,252 p <0,001) ou do método de RMN (AUC = 0,876; p <0,001 e r = 0,277; p <0,001). O EFH-AS foi desenvolvido a partir do tamanho da HDL (nm), que apresentou a associação mais forte com aterosclerose subclínica no modelo múltiplo final (OR = 0,549; p <0,001) e demonstrou bom desempenho (AUC = 0,769; p <0,001). Conclusão: o EFH apresentou associações mais fortes com o risco cardiovascular e a aterosclerose subclínica, independente do HDL-C, com destaque para a HDL grande. Os resultados controversos entre as subfrações da HDL e o risco cardiovascular parecem manter relação com as metodologias distintas utilizadas nas análises. Portanto, a validação dos métodos e a inclusão do tamanho da HDL como marcador de risco cardiovascular revela um futuro promissor como adjuvante na estimativa do risco cardiovascular, manejo de medicamentos e tomada de decisões na prática clínica. / Introduction: current studies have not presented association between high density-lipoprotein cholesterol (HDL-C) increase, induced by drugs or genetic mutations, and coronary events reduction. HDL plays different functional cardioprotective role. Objective: to develop a HDL functionality score (HFS) and to assessment its association with predictive cardiovascular risk algorithms and subclinical atherosclerosis outcomes in Brazilian subjects. Methods: cross-sectional study based in two steps. In the first step, the HFS predictor of cardiovascular risk disease (HFS-CVR) was developed and validated on CARDIONUTRI study subsample (n=354). In second step the HFS predictor of subclinical atherosclerosis (HFS-SA) was developed and validated on ELSA-Brasil study subsample (n=4549). CARDIONUTRI study evaluated paraoxonase 1(PON1) and cholesterol ester transfer protein (CETP) activity, apolipoprotein AI (APOAI) concentration, HDL antioxidant capacity, and HDL subfractions by standard Lipoprint® method. ELSA-Brasil study evaluated the size of HDL and subfractions by Vertical Auto Profile (VAP) and Nuclear Magnetic Resonance (NMR) method, and the diagnosis of subclinical atherosclerosis by computed tomography, quantifying coronary artery calcification (CAC) and CAC score. Results: in the development of HFS-CVR, the large HDL presented greater strength of association with cardiovascular risk in the multiple final model (OR = 0.797; p <0.001). The HFS-CVR showed satisfactory performance by Framingham risk score (AUC = 0.899; p <0.001), Reynolds risk score (AUC = 0.722; p <0.001) and Adult Treatment Panel III/2013 guidelines (AUC = 0.864; p <0.001). In addition, HFS-CVR presented satisfactory reproducibility and was associated with subclinical atherosclerosis on ELSA-Brasil sample using large HDL measurements derived from the VAP method (AUC = 0.864; p <0.001 and r = 0,252; p <0,001) or the NMR method (AUC = 0.876; p <0.001 and r = 0.277; p <0,001). HFS-AS was developed from the HDL size (nm), because presented greater association with subclinical atherosclerosis in the final multiple model (OR = 0.549; p <0.001). HFS-AS demonstrated satisfactory performance (AUC = 0.769; p <0.001). Conclusion: the HFS demonstrates strong association with cardiovascular risk and subclinical atherosclerosis, independent of HDL-C, with emphasis on large HDL. Controversial results, between HDL subfractions and cardiovascular irsk seem to maintain a relation with the different methodologies used in analysis. Therefore, the validation of the methods and the inclusion of the HDL size as a cardiovascular risk marker reveal a promising future as an adjunct in the estimation of cardiovascular risk, drug management and decision making in clinical practice.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le confinement des antibiotiques dans des matrices inorganiques, constituent une classe de matériaux particulièrement bien adaptée, pour la conservation de leurs activités. Cette thèse à pour objectifs: (1) d’établir une corrélation entre la localisation des antibiotiques dans les matrices HDL et leur activité antibactérienne, (2) de définir la matrice la plus adéquate pour protéger les antibiotiques de la dégradation thermique et/ou photonique. Nous nous sommes intéressés à étudier l’influence de la localisation de l’antibiotique dans le matériau sur son activité antibactérienne, et de vérifier si la phase HDL peuvent protéger les antibiotiques de la dégradation thermique et/ou photonique.Dans ces travaux, nous avons utilisé des hydroxydes doubles lamellaires (HDL) de type Zn2Al, Mg2Al, Ni2Al et Mn2Al comme des supports pour différentes types d’antibiotiques (cycline, polypeptide et enzymatique). Nous avons étudié l’immobilisation de deux antibiotiques de types cyclines tetracycline (TCH) et oxytetracycline (OXY), par deux méthodes (la coprécipitation et l’échange anionique). Nous avons étudié l’impact de l’irradiation du UV ou/et stockage à différentes températures (30, 60 et 120°C) pour évaluer leurs activités antibactériennes. Les résultats ont montré que les antibiotiques sont localisés à la surface des grains de la phase HDL. Les matériaux préparés par coprécipatation présentent un taux de relargage et une activité antibactérienne plus importante que celles préparé par échange anionique. Une baisse significative de l’activité antibactérienne après un stockage à haute température et l’exposition à la lumière UV est observée. Cela permet de conclure que la phase HDL accélère la dégradation des antibiotiques sous l’effet du température et la lumière UV. Nous avons étudié l’immobilisation de la nisine dans des différentes phases HDL, en tenant compte de l’effet du rapport molaire, l’effet de matrice, l’effet de l’anion et l’effet de morphologie. Les résultats ont montré que l’immobilisation de la nisine dans les différentes phases HDL n’affecte pas son activité antibactérienne à 4°C. En revanche, on remarque que l’activité de la nisine est moins sensible à la température quand elle est confinée.. La localisation de la nisine joue donc un rôle très important dans la meilleure protection de l’activité antibactérienne. Enfin nous avons effectué l’immobilisation du lysozyme dans les différentes phases HDL. Nos résultats ont montré que, le lysozyme est localisé à la surface des grains du matériau, on note un abattement plus important pour le lys libre que lYS adsorbé. Cela peut être du à une dénaturation du site enzymatique. / The confinement of antibiotics in inorganic matrices results in a class of materials particularly suitable for the conservation of their activities. This thesis aims to: (1) establish a correlation between the localization of antibiotics in HDL matrices and their antibacterial activity, (2) define the most appropriate matrix to prevent the antibiotics thermal and / or photon degradation. We were interested in studying the influence of the antibiotic localization in the material and the effect on the material antibacterial activity and whether the HDL phase can protect the antibiotics from thermal and / or photon degradation.In this work, we used double lamellar hydroxide (HDL) type Zn2Al, Mg2Al, Ni2Al and Mn2Al as supports for different types of antibiotics (cyclin, polypeptide and enzymatic). We investigated the immobilization of two cyclin types tetracycline (TCH) and oxytetracycline (OXY), by two methods (coprecipitation and anion exchange). We investigated the impact of UV irradiation and / or storage at different temperatures (30, 60 and 120°C) to evaluate their antibacterial activities. The results showed that the antibiotics are localized on the grain surface of the HDL phase. The materials prepared by coprecipitation have a higher release rate and antibacterial activity than those prepared by anion exchange. A significant decrease in the antibacterial activity after storage at high temperature and exposure to UV light was observed. This leads to the conclusion that the HDL phase accelerates the degradation of antibiotics under the effect of temperature and UV light. We investigated the immobilization of nisin in different HDL phases, taking into account the effect of the molar ratio, of the matrix, of the anion and the morphology. The results showed that the immobilization of nisin in the different HDL phases does not affect its antibacterial activity at 4°C. On the other hand, we noteiced that the activity of nisin is almost temperature sensitive under confinement. The localization of nisin plays therefore a very important rolefor the best protection of the antibacterial activity. Finally, we immobilized lysozyme in the various HDL phases. Our results showed that lysozyme is localized on the surface of the material grains, one notes a more important abatement for free lys than adsorbed LYS. This can be due to denaturation of the enzyme site.

Apolipoprotein A-I (apoA-I) and ATP-Binding Cassette A1 (ABCA1) transporter play important roles in nascent high density lipoprotein (nHDL) biogenesis – the first step in the reverse cholesterol transport pathway. Based on the crystal structure of a C-terminally truncated form of apoA-I (apoA-I(1-184)) determined in the laboratory, structurally designed and naturally occurring mutants of apoA-I were conformationally characterized in solution. The function of these mutants in nHDL formation was assessed in ABCA1-transfected HEK293 cells. An apoA-I mutant designed to destabilize the N-terminal helical bundle at the first hinge region, 38/40G, exhibited a locally reduced α-helical content, destabilized overall structure, and increased lipid binding ability in solution, indicating a destabilized N-terminal helical bundle. In the cellular system, 38/40G showed significantly enhanced nHDL forming ability, suggesting that a destabilized N-terminal bundle will facilitate nHDL formation. Other designed N-terminal mutants (Q41A, P66G, G65A, V67P, T68P, 65/67/68P) and the naturally occurring mutants (R153P, L178P, and insertion mutant apoA-INashua) all showed either unchanged or destabilized overall structure, unchanged lipid binding abilities in solution and unchanged nHDL formation and cholesterol efflux promotion from the cells. Mutants designed to progressively extend the C-terminus (1-184, 1-198, 1-209, 1-220, 1-231) yielded progressively increased nHDL formation and cholesterol efflux, suggesting that the C-terminus of apoA-I is critical for these two activities. Central Helix 5 triple glycine mutation (H5 3xG) designed to lock the monomer conformation of apoA-I resulted in reduced nHDL formation but unaffected cholesterol efflux, suggesting that hindering apoA-I monomer to dimer conversion could retard nHDL formation. Remarkably, studies of cholesterol efflux and nHDL particle formation indicated that the two processes might be two uncoupled events. Analysis of the nHDL particles revealed the presence of ganglioside (GM1) in the complexes. Cross-linking data demonstrated binding of apoA-I to ABCA1-expressing cells. The binding level of apoA-I mutants to ABCA1-expressing cells was positively correlated with nHDL forming ability of these mutants. ABCA1 was isolated from FreeStyle™ HEK293-F cells in suspension by detergent solubilization and was shown to have ATPase activity. A direct interaction between apoA-I and amphipol solubilized- ABCA1 in solution was detected for the first time. Furthermore, the successful purification of ABCA1 has laid the foundation of structure determination of this protein in the future.

<p>The need for a more general hardware description language aimed specifically at processors, and vague notions and visions of how that language would be realized, lead to this thesis. The aim was to use the visions and initial ideas to evolve and formalize a language and begin implementing the tools to use it. The language, called NoGAP Common Language, is designed to give the programmer freedom to implement almost any processor design without being encumbered by many of the tedious tasks normally present in the creation process. While evolving the language it was chosen to borrow syntaxes from C++ and verilog to make the code and concepts easy to understand. The main advantages of NoGAP Common Language compared to RTL languages are;</p><p>-the ability to define the data paths of instructions separate from each other and have them merged automatically along with assigned timings to form the pipeline.</p><p>-having control paths automatically routed by activating named clauses of code coupled to control signals.</p><p>-being able to specify a decoder, where the instructions and control structures are defined, that control signals are routed to.</p><p>The implemented compiler was created with C++, Bison, and Flex and utilizes an AST structure, a symbol table, and a connection graph. The AST is traversed by several functions to generate the connection graph where the instructions of the processor can be merged into a pipeline. The compiler is in the early stages of development and much is left to do and solve. It has become clear though that the concepts of NoGAP Common Language can be implemented and are not just visions.</p> / <p>Behovet av ett mer generellt hårdvarubeskrivande språk specialiseret för processorer och visioner om ett sådant gav upphov till detta examensarbete. Målet var att utveckla visionerna, formalisera dem till ett fungerande språk och börja implementera dess verktyg. Språket, som kallas NoGAP Common Language, är designat för att ge programmeraren friheten att implementera nästan vilken processordesign som helst utan att bli nedtyngd av många av de enformiga uppgifter som annars måste utföras. Under utvecklingsprocessen valdes det att låna många syntax från C++ och verilog för att göra språket lätt att förstå och känna igen för många. De största fördelarna med att utveckla i NoGAP Common Language jämfört</p><p>med vanliga RTL språk som verilog är; </p><p>-att kunna specificera datavägar för instruktioner separat från varandra och få dem automatiskt förenade med hjälp av tidsangivelser till en pipeline.</p><p>-att få kontrollvägar automatiskt dragna genom att aktivera namngivna klausuler med kod kopplade till kontrollsignaler. </p><p>-att kunna specifiera en avkodare som kontrollvägarna kan kopplas till där</p><p>kodning för instruktioner kan anges. </p><p>Kompilatorn som implementerats med C++, Bison och Flex använder sig av en AST struktur, en symboltabell och en signalvägsgraf. AST strukturen traverseras av flera funktioner som bygger upp signalvägsgrafen där processorns instruktioner förenas till en pipeline. Utvecklingen av kompilatorn är ännu bara i de första stadierna och mycket är kvar att göra och lösa. Det har dock blivit klart att det är möjligt att implementera koncepten i NoGAP Common Language och att de inte bara är lösa visioner. </p>