Título: Estresse oxidativo e hormônios esteróides na associação entre Distúrbios Respiratórios do Sono e Doença Aterosclerótica Coronariana Introdução: Estudos epidemiológicos mostram a existência de associação entre a Doença Aterosclerótica Coronariana (DAC) e os Distúrbios Respiratórios do Sono (DRS). Evidencias sugerem que o estresse oxidativo gerado pela hipóxia intermitente sofrida pelos pacientes com DRS pode estar relacionado à progressão da DAC. Os hormônios esteróides testosterona, progesterona e estradiol estão relacionados ao estresse oxidativo, e podem ter papel em ambas as doenças. A enzima glutationa S-tranferase utiliza a molécula antioxidante glutationa na detoxificação de compostos que podem ser formados neste processo. A enzima paraoxonase-1 hidrolisa peróxidos lipídicos, atuando sobre as lipoproteínas de baixa densidade oxidadas (ox-LDL). Ox-LDL são marcadores de peroxidação lipídica, e são importantes na formação da placa aterosclerótica. O vaso dilatador óxido nítrico (NO●) é considerado ateroprotetor e pode estar reduzido, agravando a DAC. Objetivos: Estudar o estresse oxidativo e as alterações fisiopatológicas decorrentes da associação entre DRS e DAC, e avaliar a participação dos hormônios esteroides neste processo. Material e Métodos: 56 pacientes com prévio diagnóstico para Doença Aterosclerótica Coronariana (DAC) e avaliação do Índice de Apneias-hipopneias (IAH) para diagnóstico de Distúrbio Respiratório do sono (DRS) foram divididos em dois grupos, 29 pacientes controles e 27 pacientes com DAC, definidos por apresentarem obstrução coronariana >30%. Foram quantificadas as concentrações séricas dos triglicerídeos, HDL, LDL, ferritina, tranferrina e ferro disponível, assim como dos níveis séricos dos hormônios testosterona, estradiol e progesterona, das enzimas paraoxonase-1 e glutationa S-transferase, e das ox-LDL. Foram quantificadas as concentrações de glutationa total, glutationa reduzida, glutationa oxidada e nitritos e nitratos (medida indireta de NO●) em eritrócitos. A concentração do marcador de dano oxidativo em DNA 8-oxo-7,8-dihidro-2’-desoxiguanosina foi obtida em leucócitos. Resultados: Pacientes com DAC possuem reduzida concentração de nitritos e nitratos. A concentração de 8-OHdG, a atividade da GsT, os níveis de glutationa total, glutationa reduzida e glutationa oxidada, assim com o estradiol e a progesterona, não apresentaram relação com DAC ou DRS. Além do IAH, a redução da testosterona e do ferro disponível estão relacionados a DAC. A redução da atividade da paraoxonase-1 e a maior concentração de ox-LDL são preditores de DAC. A testosterona está relacionada à concentração de ferritina, transferrina e ferro disponível nestes pacientes. A ferritina correlacionou-se positivamente ao dano oxidativo em proteínas e com o IAH, negativamente aos níveis de nitritos e nitratos, e é maior nos pacientes com DAC. Conclusão: Baixos níveis de testosterona e ferro disponível, assim com o aumento da ferritina podem estar relacionados à fisiopatologia da associação entre DRS e DAC. Paraoxonase-1 e ox-LDL são importantes preditores de DAC, mas parecem não estar diretamente relacionados ao IAH nestes pacientes. / Title: Oxidative stress and steroid hormones in the association between Sleep Disordered Breathing and Coronary Artery Disease Introduction: Epidemiological studies have shown a possible association between Coronary Artery Disease (CAD) and Sleep Disordered Breathing (SDB). Evidences suggest that oxidative stress generated by the intermittent hypoxia experienced by patients with sleep disorders may be related to progression of CAD. The steroid hormones testosterone, progesterone and estradiol are related to oxidative stress, and may have a role in both diseases. Glutathione S-transferase uses the antioxidant molecule glutathione in the detoxification of compounds that can be formed in this process. The enzyme paraoxonase-1 hydrolyzes lipid peroxides, acting on oxidized low-density lipoproteins (ox-LDL). Ox-LDL are lipid peroxidation markers, being important for the atherosclerotic plaque formation. The vasodilator nitric oxide (NO●) is considered atheroprotective and can be reduced, aggravating DAC. Objective: Evaluate the oxidative stress and the pathophysiological changes arising from the association between SDB and CAD, and the role of steroid hormones in this process. Material and Methods: 56 patients with prior Coronary Artery Disease (CAD) diagnosis and apnea-hypopnea index (AHI) evaluation for diagnosis of sleep-disordered breathing (SDB) were divided into two groups, 29 control patients and 27 patients with CAD, defined by present a coronary obstruction > 30%. The serum concentration of triglycerides, HDL, LDL, ferritin, transferrin and available iron was obtained, as well as the serum levels of the hormones testosterone, estradiol and progesterone, enzymes paraoxonase-1 and glutathione S-transferase, and ox-LDL. Were measured concentrations of total glutathione, reduced glutathione, glutathione disulfide and nitrites and nitrates (NO● indirect measure) in erythrocytes. The concentration of the 8-oxo-7,8-dihydro-2'-deoxyguanosine, oxidative DNA damage marker, was obtained from leukocytes. Results: CAD patients have reduced concentrations of nitrates and nitrites. The concentration of 8-OHdG, GST activity, levels of total glutathione, reduced glutathione and glutathione disulfide, and estradiol and progesterone, showed no relationship with CAD or SDB. In addition to AHI, the reduction of testosterone and iron available are related to CAD. The reduced activity of paraoxonase-1 and the highest concentration of ox-LDL are CAD predictors. Testosterone is related to the concentration of ferritin, transferrin and iron available in these patients. Ferritin was positively correlated to oxidative damage in protein and with the AHI, and negatively to the levels of nitrites and nitrates, and is higher in CAD patients. Conclusion: Low testosterone levels and iron available, as well as the increase ferritin may be related to the pathophysiology of the association between SDB and CAD. Paraoxonase-1 and ox-LDL are important CAD predictors, but do not seem to be directly related to AHI in these patients.

A família paraoxonase (PON1, PON2 e PON3) tem sido objeto de grande interesse por prevenir o estresse oxidativo e o processo inflamatório, condições importantes na carcinogênese. O Linfoma Difuso de Grandes Células B (LDGCB) consiste no subtipo histológico mais comum dentre os linfomas, doenças que se originam a partir das células do tecido linfoide e exibem distintos comportamentos clínicos, fatores patológicos e características epidemiológicas. Há escassez de dados sobre a atuação das paraoxonases na susceptibilidade diferencial ao risco de linfomas. Deste modo, o objetivo do presente estudo foi investigar a frequência alélica e genotípica dos polimorfismos 192QR e 55LM, no gene da PON1, e 148AG e 311SC, no gene da PON2 e o efeito desses polimorfismos sobre as atividades da enzima PON e perfil lipídico em 182 indivíduos (78 pacientes com LDGCB e 104 indivíduos saudáveis). O sangue foi coletado, em 4 momentos, para a determinação do perfil lipídico e das atividades arilesterase e paraoxonase da PON. O DNA foi extraído de leucócitos do sangue periférico pelo método de extração salina. A análise dos polimorfismos foi realizada por PCR/RFLP. Não houve diferença estatística na distribuição de genótipos e frequência de alelos dos polimorfismos nos genes da PON1 e PON2. A atividade sérica da arilesterase apresentou valores significativamente maiores apenas entre os indivíduos saudáveis (p=0,001). As variantes 55MM e 192QQ, do gene da PON1, influenciaram as atividades arilesterase (p=0,011) e paraoxonase (0,001). O polimorfismo PON2 311SS associou-se a atividade arilesterase (p=0,021). A concentração de autoanticorpos oxLDL foi alterada, pela presença do genótipo 55LM (p=0,037) nos indivíduos com LDGCB / The paraoxonase family (PON1, PON2 and PON3) have been the subject of great interest, since they are responsible for preventing oxidative stress and inflammation, conditions important in carcinogenesis. The Diffuse Large B Cell Lymphoma (DLBCL) is the most common histological subtype among lymphomas, diseases that originate from cells of the lymphoid tissue and exhibit clinically distinct behaviors and pathological and epidemiological factors. There are paucity of data on the activity of paraoxonase in the differential susceptibility to the risk of lymphoma. Thus, the objective of this study was to investigate the genotypic and allelic frequency of polymorphisms 192QR and 55LM, in the PON1 gene and 148AG and 311SC, in the PON2 gene and the effect of these polymorphisms on PON enzyme activities and lipid profile in 182 subjects (78 patients with DLBCL and 104 healthy subjects). Blood was collected in four moments for the determination of lipid profile and paraoxonase and arylesterase activities of PON. The DNA was extracted from peripheral blood leukocytes by salt extraction method. The analysis of polymorphisms was performed by PCR/RFLP. There was no statistical difference in the distribution of genotypes and allele frequencies of polymorphisms in the PON1 and PON2 genes. The serum arylesterase activity was significantly higher only among healthy subjects (p=0.001). 192QQ and 55MM variants of the PON1 gene, influenced arylesterase (p=0.011) and paraoxonase (0.001) activities. The PON2 polymorphism was associated with 311SS arylesterase activity (p = 0.021). The concentration of oxLDL autoantibodies was altered by the presence of 55LM genotype (p = 0.037) in patients with DLBCL

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2015-03-20T21:43:39Z No. of bitstreams: 2 Dissertação Cristiane Pereira.pdf: 1114775 bytes, checksum: 2a455906de543e8e5836442a98096bc7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2015-03-21T00:04:47Z (GMT) No. of bitstreams: 2 Dissertação Cristiane Pereira.pdf: 1114775 bytes, checksum: 2a455906de543e8e5836442a98096bc7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2015-03-21T00:05:04Z (GMT) No. of bitstreams: 2 Dissertação Cristiane Pereira.pdf: 1114775 bytes, checksum: 2a455906de543e8e5836442a98096bc7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-03-21T00:05:04Z (GMT). No. of bitstreams: 2 Dissertação Cristiane Pereira.pdf: 1114775 bytes, checksum: 2a455906de543e8e5836442a98096bc7 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-03-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A Restrição Calórica (RC) tem sido associada a inúmeros benefícios à saúde, como redução do peso e gordura corporal, melhora no perfil lipídico e aumento de enzimas antioxidantes que resultam na prevenção de diversas doenças, como as cardiovasculares (DCV) e na prevenção e atenuação de doenças neurodegenerativas relacionadas à idade em diversas espécies animais. No entanto, há poucos estudos que avaliam o impacto da RC iniciada em ratos jovens comparada com a iniciada em ratos adultos e sobre diferentes períodos de tratamento. O presente estudo avaliou o estado nutricional e metabólico, risco cardiovascular (CV) associado ao perfil lipídico sérico, parâmetros comportamentais e oxidativos em ratos Wistar submetidos à intervenção nas fases de vida jovem e adulta. Ratos com 40 ou 70 dias de vida foram alimentados ad libitum ou submetidos à RC por 12 ou 16 semanas. Nossos resultados mostraram que o peso corporal (PC) dos ratos submetidos à RC foi inferior – 21% nos jovens e 16% nos adultos, comparados aos controles (p<0,05). Os parâmetros bioquímicos séricos avaliados indicaram bom estado de saúde. Após 12 semanas, a RC diminui o colesterol total (CT), HDL, o colesterol não-HDL e a razão LDL/HDL nos ratos jovens e triacilglicerois (TG) e razão TG/LDL nos adultos – neste último com aumento de LDL e da razão LDL/HDL (p<0,05). O prolongamento da intervenção não resultou em mudança do risco CV associado ao perfil lipídico sérico. A atividade da paraoxonase 1 (PON1) foi mantida com a RC e a maior relação PON1/HDL foi encontrada nos jovens submetidos à RC por 12 semanas (p<0,05). Após 12 semanas, no hipocampo, a RC aumentou os níveis de glutationa (GSH) nos jovens e diminuiu a produção de espécies reativas de oxigênio (ERO) e peroxidação lipídica nos adultos, comparados aos controles (p<0,05). A GSH e a atividade da glutationa peroxidase (GPx) parecem aumentar com a idade independente do tratamento. Nenhuma alteração foi observada na memória, na atividade da superóxido dismutase (SOD) e na produção de óxido nítrico (ON). A idade per se não ocasionou diferenças nos parâmetros de estresse oxidativo avaliados. Este estudo indica que a RC iniciada precocemente e mantida por 12 semanas proporcionou redução do risco CV associado ao perfil lipídico sérico quando comparada a iniciada na fase adulta. Já seu prolongamento não manteve esse efeito benéfico. A atividade da PON1 foi mantida com a RC independentemente da idade de início e do tempo de tratamento. Ainda, a RC resultou em melhora significativa de parâmetros celulares de autodefesa do hipocampo, tanto iniciada em jovens, quanto na fase adulta. Contudo, a RC iniciada em ratos jovens alterou de forma positiva e expressiva um importante tampão redox do hipocampo. / Caloric restriction (CR) has been associated to numerous health benefits such as weight and body fat reduction, lipid profile improvement and increased antioxidant enzymes that result in prevention of various diseases such as cardiovascular disease (CVD) and prevention and attenuation of age-related neurodegenerative diseases in several animal species. However, there are few studies that assess the impact of CR initiated in young rats compared with adult rats and started in on different treatment periods. The present study evaluated the nutritional and metabolic status, cardiovascular (CV) risk associated with serum lipid profile, behavioral and oxidative parameters in Wistar rats subjected to intervention in young and adult phases of life. Rats with 40 or 70 days old were fed ad libitum or subjected to CR for 12 or 16 weeks. Our results showed that CR-fed rats had lower body weight (BW) - 21% in young rats and 16% in adults rats compared to controls (p<0.05). The serum biochemical parameters indicated good health. After 12 weeks, in hippocampus, the CR decreases total cholesterol (TC), HDL, non-HDL cholesterol and LDL/HDL ratio in young rats and triacylglycerols (TG) and TG/LDL ratio in adults - the latter with increased LDL and LDL/HDL ratio (p<0.05). The extension of the intervention resulted in no change in CV risk associated with serum lipid profile. Paraoxonase 1 (PON1) activity was maintained with CR and PON1/HDL highest ratio was found in young rats underwent CR for 12 weeks (p<0.05). After 12 weeks, the CR increased glutathione (GSH) levels in young rats and decreased reactive oxygen species (ROS) production and lipid peroxidation in adult rats compared to controls (p<0.05) GSH and glutathione peroxidase (GPx) activity seem to increase with age regardless of treatment. No change was observed in memory, superoxide dismutase (SOD) activity and nitric oxide (NO) production. Age per se did not cause differences in oxidative stress parameters evaluated. This study indicates that CR maintained for 12 weeks resulted in an reduction CV risk associated with serum lipid profile when initiated in young rats compared to initiated in adulthood. The extension has not kept this beneficial effects. PON1 activity was maintained with CR regardless the age at onset and treatment time. Still, CR resulted in a significant improvement in hippocampus cellular self-defense parameters, both begun in youth, as in adulthood. However, the CR initiated in young rats changed positively and significantly an important hippocampus redox buffer.

Abstract Atherosclerosis is a multifactorial disease with numerous genes and environmental factors affecting its intiation and progression. During the past years many candidate genes for atherosclerosis have been suggested, and it has also become evident that the immune system plays a part in atherogenesis. Early atherosclerotic changes can be effectively detected by measuring carotid artery intima-media thickness (IMT). In the present study the associations between IMT and polymorphisms of three candidate genes for atherosclerosis were studied: endothelial nitric oxide synthase (eNOS), apolipoprotein E (apoE) and paraoxonase-1 (PON1). To assess the role of immunological factors determining carotid atherosclerosis, CRP and circulating autoantibodies to oxidised LDL were studied in relation to IMT. The study population consisted of 519 hypertensive and 526 control subjects from a middle-aged population in Oulu, Finland. The results showed that the investigated polymorphisms of eNOS and PON1 genes were not associated with IMT, suggesting that these polymorphisms are not major risk factors for atherosclerosis in the general Caucasian population. A significant interaction between the apoE polymorphism and smoking in relation to IMT was observed among men, indicating that carriers of the ε4 allele may be particularly prone to the atherogenic effects of smoking. This interaction was especially clear in hypertensive subjects. CRP was strongly associated with IMT before adjusting for confounding factors. After the adjustment, this association diasppeared. The finding suggests that instead of early atherosclerosis CRP may be related to the later phases of the disease. This may partly explain the strong correlation between CRP and future cardiovascular events. IgM type of autoantibodies binding to oxidised LDL were inversely associated with IMT, and this finding remained after adjusting for previously known risk factors for atherosclerosis, implying a possible protective role for these antibodies in atherogenesis.

Le stress oxydant joue un rôle majeur dans le développement et l’évolution des maladies inflammatoires de l’intestin. Le corps humain est doté d’une panoplie d’enzymes antioxydantes ayant pour fonction de protéger l’intégrité cellulaire. De nouvelles enzymes au fort potentiel antioxydant, les paraoxonases (PON) 1, 2 et 3, ont récemment été identifiées tout au long du tube digestif, mais leurs rôles y restent inconnus. Les cellules intestinales Caco-2/15, qui ont la capacité de se différencier et d’acquérir les caractéristiques physiologiques de l'intestin grêle, ont été utilisées dans le présent travail pour étudier la régulation des PON. Les cellules ont été traitées avec différents effecteurs physiologiques (cytokines, LPS, stress oxydant) et pharmacologiques (fibrates, thiazolidinédiones) et l’expression des leurs gènes et protéines a été évaluée. Les résultats ont mis en lumière la modulation distincte de l’expression des PON par le stress oxydant et l’inflammation. Ceci suggère que chaque PON peut jouer un rôle différent au niveau intestinal et être impliquée dans le maintien de l’homéostasie. La régulation de l’expression des PON a également été largement explorée dans un article de revue. Pour définir le rôle de PON2, celle-ci étant potentiellement la plus importante pour l’homéostasie intestinale, les cellules Caco-2/15 ont été infectées à l’aide de lentivirus contenant des ARN d’interférence, ce qui a fortement réduit l’expression de PON2. En l’absence de PON2, les cellules Caco-2/15 étaient plus susceptibles face à un stress oxydant, la réponse inflammatoire était exacerbée et la perméabilité cellulaire paraissait altérée. Toutes ces composantes sont majeures dans le développement des maladies inflammatoires de l’intestin chez l’humain. De plus, des cellules Caco-2/15 de la PON2, ce qui a renforcé la force de la défense antioxydante cellulaire. Les résultats suggèrent que les PON jouent un rôle dans le maintien de l’homéostasie intestinale et pourraient être impliquées dans l’étiologie et la pathogenèse des maladies inflammatoires de l’intestin. / Oxidative stress is a major part of the pathogenesis of inflammatory bowel disease (IBD). The endogenous antioxidative defence is formed of multiple enzymes that have to protect cellular integrity. Paraoxonases (PON1, 2 and 3) are antioxidant enzymes that have recently been identified throughout the digestive tract, but their roles remain unclear in the intestine. Intestinal Caco-2/15 cells, which have the capacity to differentiate and exhibit the functionality of the small intestine, were used to study PON’s regulation. Cells were treated with various physiological effectors (cytokines, lipopolysaccharides, oxidative stress) and pharmacological molecules (fibrates, thiazolidinediones) and gene and protein expression were determined. Results obtained showed that PONs are distinctly modulated especially by inflammation and oxidative stress and suggests that they could play different roles in the maintenance of intestinal homeostasis. PONs regulation has also been the main topic of a review article. Our results and the literature pointed to PON2 as the most important PON for intestinal health. To better define PON2 functions in the intestine, PON2 expression was knocked-down using lentiviral infection and plasmids containing anti-PON2 shRNA. In the relative absence of PON2, Caco-2/15 cells were more susceptible towards induction of oxidative stress, the inflammatory response was exacerbated and cell permeability seemed altered. All of these components are major players involved in the development of human IBD. Moreover, Caco-2/15 cells were treated with purified PON2, which increased their antioxidative defence. All of these results suggest that PONs are implicated in the antioxidative and anti-inflammatory response in intestinal epithelial cells and makes them potentially important players for the aetiology and pathogenesis of IBD.

La present tesi avalua els efectes de certs factors ambientals sobre la paraoxonasa1 (PON1), enzim antioxidant, possiblement protector enfront les malalties cardiovasculars, a través de dos estudis d'intervenció i un de transversal. En primer lloc, el tractament amb simvastatina dels pacients amb hipercolesterolèmia familiar, que presentaven una activitat paraoxonasa baixa, s'associava a un increment de l'activitat fins a valors similars als d'individus normolipèmics, independentment dels polimorfismes PON1-55 o PON1-192. En segon lloc, l'entrenament físic s'associava a un augment de l'activitat paraoxonasa en els individus QQ i una disminució de la mateixa en els portadors de l'al·lel R pel polimorfisme PON1-192. L'increment de l'activitat paraoxonasa immediatament després de l'exercici físic agut era seguit per una disminució subseqüent de l'activitat. La recuperació dels nivells basals d'activitat paraoxonasa a les 24h de l'exercici físic agut es donava en els individus QQ independentment del seu estat d'entrenament, i en els individus portadors de l'al·lel R només quan estan entrenats. En tercer lloc, el consum elevat d'àcid oleic comportava un augment de la concentració de c-HDL i de l'activitat paraoxonasa en els homes portadors dels genotips QR i RR del polimorfisme PON1-192, respectivament.Paraules claus: paraoxonasa, PON1, genotips, simvastatina, hipercolesterolèmia familiar, interacció gen-dieta, lipoproteïna d'alta densitat (HDL), exercici físic agut, entrenament físic, estrès oxidatiu, àcid oleic, oli d'oliva, peròxids lipídics, malaltia cardiovascular. / The present thesis evaluates some environmental factor effects on paraoxonase1 (PON1), an possibly protective against cardiovascular disease antioxidant enzyme, through two intervention studies and a cross-sectional one. First, treatment with simvastatin of the familial hypercholesterolemic patients, which had low paraoxonase activity, was associated with an increase in the activity to values similar to the normolipemic ones, regardless of the PON1-55 or PON1-192 polymorphisms. Second, Regular exercise was associated with an increase in PON1 activity in QQ subjects and with a decrease in R carriers. Increased PON1 activity immediately after a bout of exercise was subsequently followed by a decrease of activity. The recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. Third, high oleic acid intake was associated with increased HDL cholesterol and PON1 activity levels only in men who were QR and RR of the PON1-192 polymorphism, respectively.

Le stress oxydant joue un rôle majeur dans le développement et l’évolution des maladies inflammatoires de l’intestin. Le corps humain est doté d’une panoplie d’enzymes antioxydantes ayant pour fonction de protéger l’intégrité cellulaire. De nouvelles enzymes au fort potentiel antioxydant, les paraoxonases (PON) 1, 2 et 3, ont récemment été identifiées tout au long du tube digestif, mais leurs rôles y restent inconnus. Les cellules intestinales Caco-2/15, qui ont la capacité de se différencier et d’acquérir les caractéristiques physiologiques de l'intestin grêle, ont été utilisées dans le présent travail pour étudier la régulation des PON. Les cellules ont été traitées avec différents effecteurs physiologiques (cytokines, LPS, stress oxydant) et pharmacologiques (fibrates, thiazolidinédiones) et l’expression des leurs gènes et protéines a été évaluée. Les résultats ont mis en lumière la modulation distincte de l’expression des PON par le stress oxydant et l’inflammation. Ceci suggère que chaque PON peut jouer un rôle différent au niveau intestinal et être impliquée dans le maintien de l’homéostasie. La régulation de l’expression des PON a également été largement explorée dans un article de revue. Pour définir le rôle de PON2, celle-ci étant potentiellement la plus importante pour l’homéostasie intestinale, les cellules Caco-2/15 ont été infectées à l’aide de lentivirus contenant des ARN d’interférence, ce qui a fortement réduit l’expression de PON2. En l’absence de PON2, les cellules Caco-2/15 étaient plus susceptibles face à un stress oxydant, la réponse inflammatoire était exacerbée et la perméabilité cellulaire paraissait altérée. Toutes ces composantes sont majeures dans le développement des maladies inflammatoires de l’intestin chez l’humain. De plus, des cellules Caco-2/15 de la PON2, ce qui a renforcé la force de la défense antioxydante cellulaire. Les résultats suggèrent que les PON jouent un rôle dans le maintien de l’homéostasie intestinale et pourraient être impliquées dans l’étiologie et la pathogenèse des maladies inflammatoires de l’intestin. / Oxidative stress is a major part of the pathogenesis of inflammatory bowel disease (IBD). The endogenous antioxidative defence is formed of multiple enzymes that have to protect cellular integrity. Paraoxonases (PON1, 2 and 3) are antioxidant enzymes that have recently been identified throughout the digestive tract, but their roles remain unclear in the intestine. Intestinal Caco-2/15 cells, which have the capacity to differentiate and exhibit the functionality of the small intestine, were used to study PON’s regulation. Cells were treated with various physiological effectors (cytokines, lipopolysaccharides, oxidative stress) and pharmacological molecules (fibrates, thiazolidinediones) and gene and protein expression were determined. Results obtained showed that PONs are distinctly modulated especially by inflammation and oxidative stress and suggests that they could play different roles in the maintenance of intestinal homeostasis. PONs regulation has also been the main topic of a review article. Our results and the literature pointed to PON2 as the most important PON for intestinal health. To better define PON2 functions in the intestine, PON2 expression was knocked-down using lentiviral infection and plasmids containing anti-PON2 shRNA. In the relative absence of PON2, Caco-2/15 cells were more susceptible towards induction of oxidative stress, the inflammatory response was exacerbated and cell permeability seemed altered. All of these components are major players involved in the development of human IBD. Moreover, Caco-2/15 cells were treated with purified PON2, which increased their antioxidative defence. All of these results suggest that PONs are implicated in the antioxidative and anti-inflammatory response in intestinal epithelial cells and makes them potentially important players for the aetiology and pathogenesis of IBD.

Background: Oxidative stress is supposed to be implicated in the pathogenesis of several diseases which are connected with increased formation of reactive oxygen and nitrogen species (RONS). Oxidative stress could play an important role in the pathogenesis of inflammation and sepsis, acute and chronic pancreatitis or in the development of cancer. Organisms are protected against RONS from antioxidant system that is composed of antioxidant enzymes and non-enzymatic antioxidants. To the most important antioxidant enzymes belong superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, glutathione reductase and paraoxonase (PON). The aim of this Doctoral Thesis was to investigate the behaviour of three of these antioxidant enzymes - CuZnSOD, CAT and PON1 in different pathophysiological states. Materials and methods: The activities of CuZnSOD, CAT and PON1 were measured in six different pathophysiological states. Forty patients with metabolic syndrome (MetS), 35 women with depressive disorder (DD), 30 septic patients (SP), 50 patients with pancreatic cancer (PC), 50 patients with chronic pancreatitis (CP) and 13 patients with acute pancreatitis (AP) were included in different studies together with sex- and age-matched healthy controls (CON). Patients with AP and SP were observed in the course...

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods. Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.

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