A leptospirose é uma zoonose mundial causada por espiroquetas patogênicas do gênero Leptospira, que colonizam os túbulos renais de animais domésticos e silvestres e são liberadas ao ambiente externo pela urina. Neste estudo avaliou-se a interação de seis prováveis lipoproteínas de membrana externa de leptospira com as proteínas do hospedeiro: colágeno I, colágeno IV, elastina, fibrinogênio, fibronectina celular e plasmática, laminina e plasminogênio. Os experimentos de adesão demonstraram que as proteínas recombinantes Lp21, Lp22 e Lsa30 apresentaram interação com os componentes do hospedeiro de maneira dose-dependente. Estas aderiram à fibronectina plasmática e laminina, além destes, a Lp21 e a Lp22 interagiram com plasminogênio, a Lp22 e a Lsa30 interagiram com colágeno IV. A Lp22 aderiu à elastina e ao fibrinogênio. No estudo de conservação gênica, os genes que codificam estas proteínas foram observados somente nas Leptospiras patogênicas. Portanto estas proteínas devem contribuir na adesão aos tecidos do hospedeiro na patogênese da Leptospira. / Leptospirosis is a worldwide zoonosis caused by pathogenic spirochetes of the genus Leptospira that colonize the renal tubules of wild and domestic animals and are excreted in the environment by their urine. The aim of this work was to study the interaction of six leptospiral probable outer-membrane lipoproteins with host proteins: collagen I, collagen IV, elastin, fibrinogen, cellular fibronectin, plasma fibronectin, laminin, and plasminogen. The binding experiments demonstrated that the recombinant proteins showed interaction with host components in a dose-dependent manner were Lp21, Lsa30 and Lp22. These proteins adhered to plasma fibronectin and laminin, in addition to these components, Lp21 and Lp22 interacted with plasminogen, Lp22 and Lsa30 interacted with collagen IV. The Lp22 adhered to elastin and fibrinogen. The genes encoding the probable lipoproteins were found only in pathogenic Leptospira. These results demonstrated that these proteins may contribute in the adhesion to host tissues, in the pathogenesis of Leptospira.

Em um delineamento em Quadrado Latino 4X4 balanceado, foram utilizados quatro potros, filhos do mesmo garanhão, com idade entre 10 e 12 meses e peso médio de 270 kg (dp ± 9,80 Kg). Foram analisados os efeitos, por regressão simples polinomial, da inclusão dos níveis de 5, 10, 15 e 20 % de óleo de soja, no concentrado, sobre aceitabilidade, coeficiente de digestibilidade aparente da matéria seca (CDAMS), matéria orgânica (CDAMO), proteína bruta (CDAPB), extrato etéreo (CDAEE), fibra insolúvel em detergente neutro (CDAFDN), em detergente ácido (CDAFDA) e sobre a concentração plasmática de colesterol total (COL) e suas frações nas lipoproteína de densidade muito baixa (VLDL-C), lipoproteína de densidade baixa (LDL-C), lipoproteína de densidade alta (HDL-C) e triglicérides totais (TRG). O aumento do nível de inclusão de óleo afetou (p<0,05) o CDAMO, CDAFDN e CDAFDA, apresentando uma resposta quadrática, com diminuição da digestibilidade após o valor esperado de 10,7%, 9,5% e 10,5% EE na dieta, respectivamente. Observou-se resposta linear (p<0,05) dos tratamentos sobre a concentração plasmática de colesterol e LDL-C, apresentando diminuição 0,65 mg/dL de colesterol e 0,58 mg/dL de LDL-C para cada 1% de aumento no EE no concentrado. A inclusão de óleo de soja afetou a digestibilidade da dieta, principalmente na fração parede celular e diminuiu a concentração plasmática de colesterol e HDL-C. / In a balanced 4x4 Latin Square design, 04 foals from the same stallion were used. They aged between 10 and 12 months and their average weight was 270±9.80 kg. The effects of soybean oil inclusion at the concentrated on acceptability, coefficient of apparent digestibility to dry matter (CADAMS), organic matter (CADOM), crude protein (CADCP), ethereal extract (CADEE), neutral detergent fiber (CADNDF), acid detergent (CADADF) and the plasma concentrations of total cholesterol (COL) and the fractions in Very Low Density Lipoprotein (VLDL - C), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C) and total triglycerides (TRG), at the following levels of 5, 10, 15 and 20%, were analyzed by simple polynomial regression. Increase in the level of oil inclusion affected (P<0.05) CADOM, CADNDF and CADADF, showing a quadratic response. For those parameters, digestibility was decreased after inclusion of 10.7%, 9.5% and 10.5 of EE% in the diet, respectively. There was a linear response (P<0.05) to the treatments on the cholesterol plasma concentration and LDL-C; each 1% of increase in EE on the diet caused a decreased of 0.65 mg/dL on cholesterol and 0.58 mg / dL on LDL-C. The inclusion of soybean oil affected the digestibility of the diet, mainly on cell wall fraction, and decreased the concentration of plasma cholesterol and HDL-C.

Durante 60 dias foram utilizados seis garanhões de raças variadas, com peso médio inicial de 472,67±90,48 kg, alimentados duas vezes ao dia com dietas compostas por feno de Tifton (Cynodon dactylon) e concentrado comercial, suplementadas com 300 mL de óleo vegetal (óleo de arroz ou óleo de soja), além de sal mineralizado e água ad libitum. Foi avaliado o efeito da suplementação com óleo de arroz semi-refinado com alto teor de gama-orizanol sobre a aceitabilidade da dieta, ganho de peso, escore corporal, lipídeos plasmáticos, digestibilidade aparente dos nutrientes da dieta (Matéria Seca MS, Matéria Orgânica MO, Proteína Bruta PB, Fibra em Detergente Neutro FDN e Fibra em Detergente Ácido FDA), além da qualidade espermática e testosterona plasmática. Para tal observação, foram colhidas amostras sangüíneas com 0, 15, 30, 45 e 60 dias após o início do tratamento para mensuração das concentrações plasmáticas de testosterona, triglicérides, colesterol total e suas frações, tais como lipoproteína de densidade muito baixa (VLDL-C), lipoproteína de densidade baixa (LDL-C) e lipoproteína de densidade alta (HDL-C). Para avaliação da digestibilidade aparente dos nutrientes da dieta, os animais passaram por três dias de colheita total de fezes. Para avaliação da qualidade espermática, os garanhões passaram por colheita seminal com 0, 15, 30, 45 e 60 dias do tratamento. O delineamento experimental foi inteiramente casualisado com medidas repetidas no tempo e as médias foram comparadas considerando-se o nível de 5% de significância. Os valores médios obtidos para a digestibilidade aparente da MS, MO, EE, FDN e FDA foram respectivamente 64,34; 68,03; 71,95; 83,37; 62,15 e 55,05% para o tratamento com óleo de soja, e 58,97; 62,61; 66,96; 81,94; 54,85 e 45,87% para o tratamento com óleo de arroz. Não houve diferença (p < 0,05) em relação à digestibilidade aparente dos nutrientes da dieta para os tratamentos propostos. Os valores médios para testosterona, colesterol total, HDL-C, LDL-C, VLDL-C e triglicérides foram respectivamente 75,93 ng/dL; 92,73; 61,47; 26,99 e 4,28 mg/dL para o tratamento com óleo de soja; e de 62,13 ng/dL; 110,20; 66,73; 38,44 e 5,02 mg/dL para o tratamento com óleo de arroz. Em relação à qualidade espermática, também não se observou (p < 0,05), e os valores médios para volume, motilidade, vigor, concentração, defeitos maiores, defeitos menores e defeitos totais, foram respectivamente, de 71,87 mL; 69 %; 2,63; 123 x 106 espermatozóides/mL; 17,73%; 4,60% e 22,33 % para o tratamento com óleo de soja, e de 78,67 mL; 70,67%; 2,93; 115,67 x 106 espermatozóides/mL; 17,96%; 6,03% e 22,63% para o tratamento com óleo de arroz. Podemos concluir que a suplementação da dieta com óleo de arroz semi-refinado, com alto teor de gama-orizanol, proporciona melhora do ganho de peso e do escore corporal, não afeta a qualidade espermática ou a concentração plasmática de testosterona, VLDL-C, HDL-C e triglicérides, porém, eleva as concentrações plasmáticas de colesterol total e de LDL-C. / Using up six stallions of various breeds during 60 days, with initial average weight of 472,67 ¬± 90,48 kg, fed twice a day with a diet consisting of Tifton hay (Cynodon dactylon) and commercial concentrate, supplemented with 300 mL. of a vegetable oil (rice bran or soybean), moreover mineralized salt and ad libitum water. The experiment evaluated the effect of the supplementation of diet with rice bran oil, with high level of gamma-oryzanol, about acceptability of the diets, weight gain, body score, levels of plasmatic lipids, apparent digestibility of nutrients of the diets (dry matter - DM, organic matter - OM, crude protein - CP, ether extract - EE, neutral detergent fiber - NDF and acid detergent fiber ADF), and spermatic quality and plasmatic testosterone. Blood samples were also held with 0, 15, 30, 45 and 60 days after starting treatment, for analysis of the values of testosterone, triglycerides, total cholesterol and its fractions (HDL-C, LDL-C and VLDL-C). To evaluation of the apparent digestibility of the nutrients of the diet, the animals had passed for three days of total fecal collection. To evaluation of the sperm quality, the sires had passed for seminal collection with 0, 15, 30, 45 and 60 days of the treatment. It was used completely randomized design for repeated measures design with repeated measures over time and the means were compared under 5 % significance level. The gotten average values for the apparent digestibility of the DM, OM, CP, EE, NDF and ADF were respectively 64,34; 68,03; 71,95; 83,37; 62,15 and 55.05 % for the treatment with soybean oil, and 58,97; 62,61; 66,96; 81,94; 54,85 and 45.87 % for the treatment with rice bran oil. There was no statistical difference (p < 0,05) from the apparent digestibility of nutrients of the diet. The average values for testosterone, total cholesterol, HDL-C, LDL-C, VLDL-C and triglycerides had been respectively 75,93 ng/dL; 92,73; 61,47; 26,99 and 4,28 mg/dL for the treatment with soy oil; and of 62,13 ng/dL; 110,20; 66,73; 38,44 and 5,02 mg/dL for the treatment with rice bran oil. In relation to the spermatic quality, also did not have difference (p < 0,05), and the average values for volume, motility, vigor, concentration, defects, lesser defects and total defects, had been respectively, of 71,87 mL; 69%; 2,63; 123 x 106 sptz/mL; 17.73%; 4.60% and 22.33% for the treatment with soybean oil, and 78,67 mL; 70.67%; 2,93; 115,67 x 106sptz/mL; 17.96%; 6.03% and 22,63% for the treatment with rice bran oil. The supplementation of diet with semi-refined rice bran oil, with high level of gamma-oryzanol, provided better weight gain and improves the body score, do not affect the sperm quality or plasmatic levels of testosterone, VLDL-C, HDL-C and triglycerides, however, it increase plasmatic levels of total cholesterol and LDL-C.

A leptospirose é a zoonose mais disseminada no mundo e uma das principais causas de perda econômica no agronegócio. O estudo de novos antígenos de superfície de Leptospira interrogans, é intrigante e pode fornecer conhecimento na interação inicial patógeno-hospedeiro. Os genes LIC13059 e LIC10879, escolhidos por bioinformática, com predição de localização na superfície celular, foram clonados e as proteínas recombinantes expressas em E. coli, para avaliar a interação com componentes do hospedeiro. Após purificação, as proteínas encontravam-se estruturadas e foram reconhecidas por soro de indivíduos infectados. As proteínas recombinantes interagem com plasminogênio, fibrinogênio e laminina. rLIC13059, nomeada Lsa25.6, quando ligada ao fibrinogênio é capaz de inibir a formação de coágulo de fibrina e rLIC10879, nomeada Lsa16, interage com e-caderina, sugerido envolvimento na cascata de coagulação e ligação com o hospedeiro, respectivamente. O plasminogênio ligado às proteínas é convertido em plasmina, o que poderia ajudar a penetração bacteriana no hospedeiro. / Leptospirosis is the most widespread zoonosis and also a major cause of economic loss in animal production worldwide. The study of new surface antigens of Leptospira interrogans is intriguing and may shed light into the initial pathogen-host interactions. We set out to study two novel coding sequences LIC13059 and LIC10879 predicted to be located at the cell surface. The genes were cloned and the recombinant proteins were expressed in E. coli. The purified recombinant proteins presented secondary structures, and interacted with plasminogen, fibrinogen and laminin human components. rLIC13059, named Lsa25.6, when bound to fibrinogen was capable of inhibiting the formation of fibrin clot, while rLIC10879, named Lsa16, interacted with e-cadherin, a mammalian cell receptor, suggesting participation in coagulation pathway and host-cell binding, respectively. The plasminogen captured by both recombinant proteins could be converted into plasmin, a mechanism that could help bacterial penetration in the host.

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

No description available.

Orientadores: Eliana Cotta de Faria, Helena Coutinho Franco de Oliveira, Daniel Zanetti Scherrer / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T20:49:38Z (GMT). No. of bitstreams: 1 Zago_VanessaHelenadeSouza_D.pdf: 5059555 bytes, checksum: 854d9d1d1674a14d2ebcf5798acc31b3 (MD5) Previous issue date: 2015 / Resumo: Dado o importante papel desempenhado pelos transportadores ATP binding cassete A1 (ABCA1), G1 (ABCG1), G5 (ABCG5), G8 (ABCG8) e pelo scavenger receptor class B type I (SR-BI) para a homeostase corpórea de colesterol e desenvolvimento da aterosclerose, este trabalho se propôs a: (i) investigar a relação dos polimorfismos rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8) e rs5888 (SCARB1) com gênero, idade e índice de massa corpórea (IMC) e suas interações sobre variáveis clínicas e bioquímicas (n=654); (ii) determinar a repercussão destes polimorfismos sobre os parâmetros estudados na população total e de forma gênero-específica (n=590) e (iii) avaliar se os miRNAs hsa-miR-33a e hsa-miR-128a são diferencialmente expressos em um subgrupo da população (n=51) e averiguar sua associação com as concentrações plasmáticas do colesterol da lipoproteína de alta densidade (HDL-C), aterosclerose subclínica e expressão de ABCA1, ABCG1 e SCARB1. Para tanto, foram selecionados voluntários normolipidêmicos e assintomáticos, de ambos os gêneros, com idade entre 20 e 75 anos. Dados clínicos e antropométricos foram obtidos, assim como sangue venoso periférico para as determinações bioquímicas e extração de DNA e RNA. O subgrupo de 51 voluntários foi classificado de acordo com HDL-C (mg/dL) em hipoalfalipoproteinêmicos (hipo, HDL-C?39), hiperalfalipoproteinêmicos (hiper, HDL-C?68) e controles (CTL, HDL-C?40<68) e determinadas a espessura íntimo-medial das artérias carótidas e proteínas relacionadas ao metabolismo de HDL. Determinamos que o rs1893590 interage com a idade e o IMC, modulando as concentrações de HDL-C, bem como o tamanho e volume da partícula, sugerindo que este pode modificar seu metabolismo e composição. Nas análises comparativas o rs2275543 apresentou efeitos diferentes, porém benéficos para ambos os gêneros; adicionalmente, o rs6720173 determinou um fenótipo lipoproteico proaterogênico no gênero masculino, enquanto as variantes rs5888 e rs6544718 repercutiram sobre marcadores de adiposidade no gênero feminino. A análise dos cinco polimorfismos nesta população fornece evidências de que estes atuam em diferentes vias do metabolismo lipoproteico, e tem na maioria dos casos características gênero-específicas. Adicionalmente, a avaliação da expressão de hsa-miR-33a, hsa-miR-128a, ABCA1, ABCG1 e SCARB1 revelou que os indivíduos hiper apresentam um aumento da expressão de ABCA1 e ABCG1 em relação ao grupo CTL, somado a uma redução de 72% na expressão do hsa-miR-33a; em conjunto, estes resultados indicam um potencial papel regulatório deste miRNA em indivíduos assintomáticos, possivelmente contribuindo para o aumento do efluxo e do transporte reverso de colesterol / Abstract: Given the important role played by ATP binding cassete transporters A1 (ABCA1), G1 (ABCG1), G5 (ABCG5), G8 (ABCG8) and by scavenger receptor class B type I (SR-BI) on body cholesterol homeostasis and atherosclerosis development, this study proposes to: (i) investigate the relationship of polymorphisms rs2275543 (ABCA1), rs1893590 (ABCG1), rs6720173 (ABCG5), rs6544718 (ABCG8) e rs5888 (SCARB1) with gender, age and body mass index (BMI) and its interactions with clinical and biochemical variables (n=654); (ii) determine the effects of these polymorphisms on the studied parameters in the total population and in a gender-specific manner (n=590) and (iii) evaluate if miRNAs hsa-miR-33a e hsa-miR-128a are differentially expressed in a subgroup of the population (n=51) and verify its association with plasma levels of high-density lipoprotein cholesterol (HDL-C), subclinical atherosclerosis plus ABCA1, ABCG1 and SCARB1 expression. Thus, normolipidemic and asymptomatic volunteers from both genders, with ages ranging from 20 to 75 years were selected. Clinical and anthropometric data were obtained, as well as peripheral venous blood for biochemical determinations plus DNA and RNA extraction. The subgroup of 51 individuals was classified according HDL-C (mg/dL) in hypoalphalipoproteinemics (hypo, HDL-C?39), hyperalphalipoproteinemics (hyper, HDL-C?68) and controls (CTL, HDL-C?40<68); then, were determinated the carotid intima-media thickness and proteins related to HDL metabolism. The polymorphism rs1893590 interacts with age and BMI, modulating HDL-C levels as well as the particle size and volume, suggesting its role on HDL metabolism and composition. Comparative analysis demonstrated that rs2275543 has different, but beneficial repercussions in both genders; furthermore, rs6720173 determines a pro-atherogenic lipoprotein profile in males, while the variants rs5888 and rs6544718 affect positively adiposity markers in females. The analyses of the five studied polymorphism in this population provide evidences of its role in several pathways of lipoproteins metabolism, in most cases in a gender-specific manner. Moreover, the ABCA1, ABCG1, SCARB1, hsa-miR-33a and hsa-miR-128a expression analysis revealed that hyper group presents a significant increase of ABCA1 and ABCG1 expression in relation to the control group; additionally, hsa-miR-33a decreased by 72%. Together, these results indicate a potential regulatory role of this miRNA in asymptomatic individuals, probably contributing to increased cholesterol efflux and reverse cholesterol transport / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas

Background: Obstructive sleep apnea (OSA) consists of repeated, involuntary breathing suspension during sleep. These events induce rapid depletion/repletion of blood/tissue oxygen content, a phenomenon known as intermittent hypoxia. Aside from causing daytime sleepiness, the most important health consequence of OSA is a 2-fold increase in cardiovascular (CVD) risk. Animal studies provide evidence that intermittent hypoxia, a simulating model of OSA, causes important rise in plasma TG, especially in the postprandial state. However, the underpinning mechanisms linking intermittent hypoxia to altered postprandial TG levels remain unknown. As such, the objective of this study was to characterize the effects of acute intermittent hypoxia on postprandial TG levels in 2 distinct lipoprotein subtypes in humans: chylomicrons which are secreted by the intestine and carry dietary lipids, and denser TG carriers (mainly VLDL) which are secreted by the liver and carry endogenous lipids. Methods: The research consisted of a randomized crossover design. In collaboration with the Sleep laboratory at Montfort Hospital, 7 individuals diagnosed with moderate sleep apnea were recruited through phone calls as well as 8 healthy individuals without OSA from the University of Ottawa. While lying on a bed, participants were given a meal after which they were exposed for 6 hours to normoxia or intermittent hypoxia corresponding to moderate OSA, e.g. 15 hypoxic events per hour. Blood lipid levels were measured hourly.  Results: Plasma TG levels increased over time in both experimental conditions and tended to be greater under 6-h exposure to intermittent hypoxia (p=0.093, effect size ηp2= 0.383.). This trend toward higher total plasma TG under intermittent hypoxia was attributable to increased levels in denser TG carrying lipoproteins such as VLDL and CM remnants (p= 0.009, ηp2 = 0.173).  Conclusion: Acute intermittent hypoxia, a simulating model of obstructive sleep apnea, tends to negatively affect postprandial TG levels, which is attributable to an increase in denser TG carrying lipoprotein levels such as VLDL and CM remnants. These results lend support to the increase in blood lipid levels in animal studies observing the effect of acute hypoxia in mice.  Contribution to advancement of knowledge: This proposed research will allow a better understanding of the mechanisms by which obstructive sleep apnea may alter blood lipid profile. This information will be beneficial to the treatment of obstructive sleep apnea related dyslipidemia and contribute to reduce CVD risk in the large proportion of obstructive sleep apnea patients who are reluctant to current treatment avenues.

Après administration intraveineuse, un nanovecteur va interagir avec de nombreuses molécules endogènes, notamment celles présentes dans la circulation sanguine. En fonction de la composition chimique du nanovecteur, ces molécules vont conférer à celui-ci une signature spécifique qui va orienter sa biodistribution et sa reconnaissance par certaines cellules de l’organisme. Plusieurs études sur l'identification des protéines adsorbées à la surface des nanovecteurs ont été menées alors que moins d'attention a été consacrée à l'interaction avec les lipoprotéines (LPs). Or, un nombre élevé de récepteurs aux LPs a été observé dans les cellules à croissance rapide et des études ont démontré que certaines cellules cancéreuses surexpriment ces récepteurs. De ce fait, l’utilisation des LPs comme vecteurs de médicaments anticancéreux a été proposée afin de favoriser le ciblage des cellules tumorales. Ce projet de thèse repose sur l’utilisation d’un bioconjugué (SQGem) issu du couplage chimique de la gemcitabine (Gem), une molécule anticancéreuse, au squalène (SQ) (un lipide naturel et précurseur de la biosynthèse du cholestérol), dont l’auto-organisation sous forme de nanoparticules à préalablement été décrite au laboratoire. Nous avons pu mettre en évidence la capture et le transport spontané de la SQGem par les LPs plasmatiques. Les résultats in vitro et in vivo que nous avons obtenus démontrent parfaitement que l’association préférentielle de la SQGem aux LPs corrèle fortement avec la quantité de cholestérol présente dans ces derniers. De plus, les simulations in silico effectuées ont révélé l’incorporation de SQGem dans le noyau hydrophobe des LPs. Par la suite, cette interaction spontanée a été mise à contribution pour effectuer le ciblage indirect des cellules cancéreuses ayant une expression élevée de récepteurs aux LPs, ce qui a été confirmé in vitro sur cellules ainsi qu’in vivo sur un modèle de tumeur expérimentale chez la souris. L’ensemble de ces résultats suggère l’originalité de notre approche, basée sur le ciblage des tumeurs de manière indirecte via les LPs qui constituent ainsi des « vecteurs » endogènes de SQGem. La « squalénisation » évite ainsi la préparation fastidieuse de vecteurs à base de LPs reconstitués et représente, par l’utilisation des LPs endogènes, une stratégie innovante et potentiellement révolutionnaire dans le traitement expérimental du cancer. / The in vivo fate of intravenously injected nanoparticles is strongly affected by their interactions with the blood components. Several studies have focused on the identification of proteins adsorbed at the surface of nanoparticles whereas less attention has been devoted to the interaction with lipoproteins (LPs). Interestingly, LPs have been previously described as excellent carriers for delivery of anticancer drugs due to their particularly high receptor-mediated uptake on several cancer cell lines. In this PhD project, we focused on a bioconjugate obtained by covalent linkage of the anticancer drug gemcitabine (Gem) to squalene (SQ) (a natural lipid and precursor of the cholesterol’s biosynthesis) whose ability to spontaneously self-assemble in the form of nanoparticles has been previously described in our laboratory. We have demonstrated that this conjugation enables the spontaneous capture and transport of the SQGem by circulating lipoproteins. In vitro and in vivo experiments revealed a preeminent affinity of SQGem towards cholesterol-rich LP particles and in silico simulations further displayed their incorporation into the hydrophobic core of LPs. Such spontaneous interaction allowed for indirect targeting of cancer cells with high expression of LP receptors, which was confirmed both in vitro and in vivo in an experimental tumor model in mice. To the best of our knowledge, the use of squalene to induce drug insertion into LPs for indirect cancer cell targeting is a novel concept in drug delivery. It represents a flexible, highly versatile platform that would enable efficient drug delivery by simply exploiting endogenous lipoproteins without the need for complex nanoparticles surface functionalization or artificial lipoproteins production.

A lot of attention has been focused on reducing cholesteric problems before individuals reach middle age, but is it not possible to reduce the risk factors for CHD after middle age? This study was initiated to determine id moderate exercise had an effect on the serum lipoproteins in postmenopausal women. Plasma concentrations of glucose, triglyceride (TRIG), TC, HDL, and LDL were measured before and after a monitored regime of moderate exercise.