Cigarette smoking is a complex mixture of many constituents identified, among them reactive substances (reactive oxygen and nitrogen species), which are capable of initiating or promoting oxidative damage. Lungs and brain are affected by cigarette smoke exposure. Exposure to cigarette smoke is related to development of diseases in children and adults. However, children are more susceptible than adults to damage caused by cigarette smoke. Thus, the use of antioxidants is a good alternative to protect tissues of oxidative damage caused by cigarette smoke exposure. Diphenyl diselenide [(PhSe)2] is an organoselenium compound that presents pharmacological effects, among them the antioxidant effect. Nevertheless, the mechanism involved in antioxidant effect of (PhSe)2 was not been elucidated. Therefore, this study was performed to study the effects of cigarette smoke passive exposure in lungs and brain of rat pups in two experimental protocols of oxidative stress. Moreover, the antioxidant effect of (PhSe)2 in these experimental protocols was studied. Besides, the mechanisms involved in the antioxidant effect of (PhSe)2 were investigated. Rat pups that were exposed to two experimental protocols were used. In a first experimental protocol (P1), the effect of exposure to one, two and three cigarettes during the first, second and third weeks of live, respectively, was studied. In a second experimental protocol (P2), the effect of exposure to four, five and six cigarettes during the first, second and third weeks of life, respectively, was carried out. The duration of each exposure was 15 min. Animals were exposed to cigarette smoke during 20 days (3 weeks). Immediately before each exposure, animals that were treated with (PhSe)2 received daily an oral dose of 0.5 mg/kg. At the end of the experimental exposure period (3 weeks), rat pups were euthanized, and lungs and brain were removed for analyses of lipid peroxidation, enzymatic antioxidant defenses (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferase (GST) activities) and non-enzymatic defenses (non-protein thiols (NPSH) and ascorbic acid levels). Rat pups were daily weighed, before each exposure. The weight of animals was not changed after cigarette smoke exposure neither to P1 nor to P2. In both experimental protocols, animals that were exposed to cigarette smoke showed an increase in lipid peroxidation in lungs. In brain, an increase in thiobarbituric acid reactive species (TBARS) was observed only in P2. Levels of non-enzymatic antioxidant defenses decreased in lungs of animals exposed to P1 and P2. In brain, exposure to P1 increased ascorbic acid levels and exposure to P2 reduced NPSH and ascorbic acid levels. Enzymatic antioxidant defenses changed in P2 in lungs of rat pups. In brain, the activity of CAT was reduced after exposure to P1, while SOD and CAT activities were decreased after exposure to P2. Treatment with (PhSe)2 restored the oxidative damage caused by cigarette smoke exposure in lungs and brain of rat pups. Moreover, NPSH levels, ascorbic acid content and GST activity showed an increase per se in lungs of animals treated with (PhSe)2. The mechanisms involved in antioxidant effect of (PhSe)2 (1 - 50 μM) were studied. To this end, dehydroascorbate (DHA) reductase and GST activities were determined. (PhSe)2 at concentration of 5 μM demonstrated DHA reductase and GST-like activities. Furthermore, the scavenger effect of 2,2 -diphenyl-1-picrylhydrazyl (DPPH ) radical and 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS +) radicals, and the protection of Fe2+ autooxidation were studied. However, the compound had no scavenger effect or protected Fe2+ autooxidation, discarding that these mechanisms are involved in the antioxidant effect of (PhSe)2. Synthesis of glutathione (GSH) was also studied as a possible mechanism involved in antioxidant effect of (PhSe)2. For this, buthionine sulfoximine (BSO), a substance that inhibits γ-glutamilcystheine synthase activity, an enzyme involved in the synthesis of GSH was used. BSO blocked the protective effect of (PhSe)2 in reducing NPSH levels caused by cigarette smoke passive exposure in lungs and liver of rat pups. In this study it was concluded that (PhSe)2 had antioxidant effect by different mechanisms. The action of (PhSe)2 to increase NPSH and ascorbic acid levels, and GST activity is one of mechanisms of the antioxidant effect of this compound. (PhSe)2 presented DHA reductase and GST-like activities, demonstrating a new mechanism of antioxidant effect of the compound. Furthermore, the synthesis of GSH is involved in the antioxidant role of (PhSe)2, since blocking the synthesis of GSH, presented the antioxidant effect of this compound. / A fumaça do cigarro é uma mistura complexa de diversos constituintes identificados, entre eles, substâncias reativas (espécies reativas de oxigênio e nitrogênio), as quais podem estar relacionadas com o desenvolvimento de várias doenças em adultos e crianças. Os pulmões e cérebro estão entre os órgãos mais afetados pela exposição à fumaça do cigarro. Entretanto, as crianças são mais suscetíveis aos danos causados pela exposição passiva à fumaça do cigarro do que os adultos. Para proteger os tecidos do dano oxidativo causado por esta exposição são utilizados antioxidantes. O disseleneto de difenila [(PhSe)2] é um composto orgânico de selênio que apresenta diversos efeitos farmacológicos descritos, entre eles, o antioxidante. Entretanto, o mecanismo pelo qual este composto exerce seus efeitos antioxidantes ainda não foi elucidado. Portanto, o presente trabalho visa estudar os efeitos da exposição passiva à fumaça do cigarro nos pulmões e no cérebro de ratos jovens, em dois protocolos experimentais de estresse oxidativo, e verificar o papel protetor do (PhSe)2 nestes protocolos. Além disso, investigaram-se os mecanismos envolvidos no efeito antioxidante desse composto. Para isso, foram utilizados ratos jovens que foram submetidos a dois protocolos experimentais de estresse oxidativo. Em um primeiro protocolo experimental (P1) verificou-se o efeito da exposição passiva à fumaça de um, dois e três cigarros nas primeira, segunda e terceira semanas de vida, respectivamente. Em um segundo protocolo experimental (P2) foi verificado o efeito da exposição passiva à fumaça de quatro, cinco e seis cigarros nas primeira, segunda e terceira semanas de vida, respectivamente. Todos os animais em ambos os protocolos experimentais foram expostos à fumaça do cigarro diariamente, 15 minutos cada exposição, por um período de 20 dias. O animais que foram tratados com o (PhSe)2, receberam diariamente pela via oral a dose de 0,5 mg/kg, imediatamente antes de cada exposição. No 21° dia, os animais foram eutanasiados e os pulmões e cérebro foram retirados para a análise da peroxidação lipídica (níveis de espécies reativas ao ácido tiobarbitúrico TBARS), das defesas antioxidantes enzimáticas (atividade das enzimas superóxido dismutase (SOD), catalase (CAT), glutationa redutase (GR), glutationa peroxidase (GPx) e glutationa S-transferase (GST)), das defesas antioxidantes não-enzimáticas (níveis de tióis não-protéicos (SHNP) e ácido ascórbico) e da atividade da δ-aminolevulinato desidratase (δ-ALA-D). Os animais foram pesados diariamente, antes de cada exposição. O peso dos animais não alterou após a exposição à fumaça do cigarro em nenhum grupo experimental, em nenhum dos protocolos. Nos P1 e P2, os animais que foram expostos à fumaça do cigarro apresentaram um aumento da peroxidação lipídica no pulmão. Entretanto no cérebro, o aumento no TBARS foi verificado apenas no P2. Em relação às defesas antioxidantes não-enzimáticas, foi observado uma redução nos níveis dos parâmetros estudados nos animais expostos ao P1 e P2. Enquanto no cérebro, a exposição ao P1 aumentou os níveis de ácido ascórbico e a exposição ao P2 reduziu os níveis de SHNP e de ácido ascórbico. Quanto às defesas antioxidantes enzimáticas, elas alteraram no pulmão apenas no P2. No cérebro, a atividade da CAT reduziu após a exposição ao P1, e a exposição ao P2 reduziu a atividade da CAT e da SOD. A atividade da δ-ALA-D foi alterada no P1 no pulmão e no P2 no cérebro. O tratamento com (PhSe)2 restaurou os danos causados pela exposição passiva à fumaça do cigarro nos pulmões e cérebro dos ratos jovens. Além disso, os níveis de SHNP, o conteúdo de ácido ascórbico e a atividade da GST apresentaram um aumento per se nos pulmões dos animais tratados com (PhSe)2. Para estudar o mecanismo pelo qual o (PhSe)2 apresentou efeito antioxidante, verificou-se o efeito mimético in vitro deste composto orgânico de selênio (1 - 50μM) na atividade das enzimas dehidroascobato (DHA) redutase e GST. O (PhSe)2 a partir da concentração de 5μM apresentou efeito mimético da atividade destas enzimas. Além disso, estudou-se o efeito do (PhSe)2 (10 - 50μM) como scavenger dos radicais 1,1-difenil-2-picril-hidrazil (DPPH ) e 2,2 -azino-bis(3-etilbenztiazolina-6-ácido sulfônico) (ABTS +), e na proteção da auto-oxidação do Fe2+. Entretanto, o composto não apresentou efeito scavenger, nem protegeu da auto-oxidação do Fe2+, descartando que esses mecanismos estariam envolvidos na ação antioxidante do composto. O envolvimento na síntese da glutationa (GSH) também foi estudado na tentativa de explicar os mecanismos pelos quais o (PhSe)2 apresenta efeito antioxidante. Para isso, foi utilizado a butionina sulfoximina (BSO), uma substância que inibe a γ-glutamilcisteína sintetase, uma enzima envolvida na síntese da GSH. Verificou-se que o BSO bloqueou o efeito protetor do (PhSe)2 na redução dos níveis de SHNP provocada pela exposição passiva à fumaça do cigarro no pulmão e fígado de ratos jovens. Com o presente trabalho conclui-se que o (PhSe)2 apresenta efeito antioxidante por diferentes mecanismos. A ação do (PhSe)2 em aumentar os níveis de SHNP, ácido ascórbico e a atividade da GST é um dos mecanismos do efeito antioxidante deste composto. O (PhSe)2 apresentou atividade DHA redutase e GST-like, demonstrando um novo mecanismo do efeito antioxidante do composto. Além disso, a síntese da GSH está envolvida no efeito antioxidante do (PhSe)2, uma vez que, bloqueando a síntese da GSH, ocorre um bloqueio no efeito antioxidante do composto.

La broncho-pneumopathie chronique obstructive (BPCO) est une maladie inflammatoire chronique des voies aériennes dont le facteur de risque principal est l’exposition chronique à la fumée de cigarette. L’histoire de la maladie est fréquemment associée à une colonisation bactérienne des voies aériennes et ponctuée d’épisodes aigus d’exacerbation de la maladie associés à une morbi-mortalité importante. Ces exacerbations sont principalement d’origine infectieuses et plus particulièrement, associées à une bactérie dans 50 % des cas, majoritairement Haemophilus influenzae, Streptococcus pneumoniae et Moraxella catarrhalis. Le contrôle de ces infections bactériennes implique en particulier une réponse immunitaire de type Th17 efficace. Cette immunité Th17 médiée principalement par les cytokines IL-17A et IL-22 est impliquée dans la physiopathologie de la BPCO mais n’a été que peu étudiée dans le contexte des exacerbations. Notre hypothèse est que la réponse Th17 aux pathogènes est altérée dans la BPCO, mécanisme qui serait impliqué dans la susceptibilité aux infections respiratoires observée chez les patients.Différentes approches ont été utilisées pour tester cette hypothèse. Tout d’abord, une approche ex vivo, à partir de cellules mononucléées circulantes (PBMC) de patients atteints de BPCO comparées à celles issues de sujets sains non fumeurs et de sujets fumeurs sans obstruction bronchique, montrant un défaut de production par les PBMC des cytokines IL-17A et IL-22 mais également des cytokines IL-6 et IL-23 produites par les cellules présentatrices d’antigènes (CPA) et impliquées dans l’activation de cette immunité Th17, en réponse à une activation par S. pneumoniae. Ensuite, une approche in vitro, avec un modèle de cellules dendritiques dérivées de monocytes (MDDC) exposées à la fumée de cigarette. Ces MDDC présentaient un défaut de maturation, de production de cytokines pro-Th17 et de leur capacité à activer une réponse Th17 lymphocytaire en réponse à S. pneumoniae. Enfin, une approche in vivo, utilisant un modèle murin de souris exposées de façon chronique à la fumée de cigarette confirmant ces résultats avec un défaut de réponse IL-17A et IL-22 mais également de production des cytokines pro-Th17 IL-1β et IL-23 par les CPA en réponse à S. pneumoniae. Dans ce modèle, l’apport d’IL-22 permettait d’améliorer la clairance bactérienne et de réduire les lésions pulmonaires, suggérant des possibilités thérapeutiques pour améliorer la prise en charge de ces exacerbations infectieuses.Ces trois approches permettent d’apporter des arguments forts en faveur d’un défaut de réponse Th17 au cours des exacerbations bactériennes de la BPCO, hypothèse confortée par d’autres travaux de notre équipe montrant dans le modèle murin d’exposition chronique à la fumée de cigarette la présence d’un défaut de production d’IL-22 dans la réponse à Haemophilus influenzae. Ces travaux qui doivent maintenant être d’une part, confirmés par une étude clinique incluant des patients atteints de BPCO en exacerbation, et d’autre part, complétés pour préciser l’impact sur les cellules lymphoïdes innées productrices de ces cytokines et sur la réponse de l’épithélium bronchique à l’infection, ouvrent la voies à des perspectives thérapeutiques dans la prise en charge de ces exacerbations bactériennes. / Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the airways mainly due to chronic exposure to cigarette smoke. Evolution of the disease is often associated with bacterial colonization of the airways and punctuated by acute exacerbation of the disease with a frequent related morbi-mortality. These exacerbations are mainly due to infection and almost 50 % are associated with bacteria, often Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Th17 immune response is particularly involved in control of bacterial infection and is principally mediated by IL-17A and IL-22 cytokines. This Th17 inflammation is involved in COPD physiopathology but there is paucity of data focusing on this immune response during COPD exacerbations. Our hypothesis is that Th17 immune response to pathogens is defective in COPD leading to airways infection susceptibility.We have tested our hypothesis by different approaches. First, the ex vivo responses to Streptococcus pneumoniae of peripheral blood mononuclear cells (PBMC) from COPD patients, healthy non smokers and healthy smokers were compared showing decreased production of IL-17A and IL-22 but also of pro-Th17 cytokines IL-6 and IL-23 which are produced by antigen presenting cells (APC). Second, we used an in vitro model of monocyte-derived dendritic cells (MDDC) exposed to cigarette smoke showing a defective MDDC maturation, pro-Th17 cytokines production and ability to promote T-cells Th17 response, in response to S. pneumoniae. Finally, an in vivo murine model of mice chronically exposed to cigarette smoke showing defective production of IL-17A and IL-22 but also of pro-Th17 cytokines IL-1β and IL-23 produced by APC, in response to S. pneumoniae. In this model, supplementation with IL-22 restored bacterial clearance and limited lung alterations suggesting therapeutic opportunities to improve infectious COPD exacerbation management.Altogether, these results strengthen our hypothesis of a defective Th17 immune response during bacterial COPD exacerbations. They are comforted by other studies in our team showing a defective IL-22 production in response to Haemophilus influenzae in our in vivo model of mice chronically exposed to cigarette smoke. Now we have to confirm these results in a clinical trial including COPD patients in exacerbation and to further explore the impact on innate lymphoid cells, which produced these cytokines, and on the innate immune response of epithelial cells to infection, in order to develop new infectious COPD exacerbation therapeutics.

La bronchopneumopathie chronique obstructive (BPCO) est définie par une obstruction bronchique progressive et irréversible associant deux lésions anatomiques différentes : le remodelage des petites voies aériennes et l'emphysème. Le remodelage des petites voies aériennes est localisé au niveau des bronchioles ayant un diamètre interne de moins de 2 mm et est principalement caractérisé par une fibrose péribronchiolaire. Les mécanismes conduisant au remodelage bronchiolaire, incluent essentiellement l’inflammation chronique et les facteurs de croissance, mais ne sont pas encore tous connus. La télomérase, qui a été récemment associée à la fibrose pulmonaire interstitielle et à l’emphysème, pourrait avoir des rôles dans la pathogenèse du remodelage bronchiolaire liée à la BPCO. La télomérase est en effet une enzyme responsable de l’allongement télomères et présente des fonctions extra-télomériques supplémentaires impliquées dans de nombreux processus cellulaires tels la prolifération cellulaire, l’apoptose et le stress oxydant. Le TA-65, une petite molécule utilisée dans la pharmacopée traditionnelle chinoise, est connu comme un activateur de télomérase. Notre étude avait pour but d’étudier les effets d’une modulation pharmacologique positive de l'activité télomérase, en utilisant le TA-65, sur le développement du remodelage bronchiolaire après exposition à la fumée de cigarette chez la souris. Les souris ont été quotidiennement exposées à la fumée de cigarette pendant 4 semaines, avec ou sans administration concomitante de TA-65 (25 mg / kg / jour, par gavage) ou d’imetelstat (un inhibiteur de la télomérase, à raison de 45 mg / kg / jour en administration intrapéritonéale), débutant 7 jours avant le début des expositions à la fumée de cigarette. Les souris témoins ont été exposées à l’air filtré dans les mêmes conditions. Le remodelage bronchiolaire a été caractérisé par analyses morphométriques et immunohistochimie. La production de mucus, la différenciation des myofibroblastes, le dépôt de collagènes ainsi que du TGF-β1 (Tranforming Growth Factor bêta 1) dans la paroi des bronchioles ont ainsi été examinés. La mesure de l'activité de la télomérase mesurée par la méthode TRAP (Telomere Repeat Amplification Protocol) et la différenciation des myofibroblastes ont été réalisées sur des fibroblastes pulmonaires de souris obtenus par culture primaire puis exposés au TGF-β1 avec ou sans prétraitement avec des modulateurs de la télomérase. La fumée de cigarette a induit un remodelage bronchiolaire, caractérisé par une fibrose péribronchiolaire chez la souris après 4 semaines d’exposition. La fibrose péribronchiolaire a été révélée par une augmentation du dépôt de collagènes (collagène total, collagènes I et III) et de l'expression d’α-SMA dans la paroi des bronchioles, en l'absence de surproduction de mucus. Le TA-65, qui induit une augmentation de l'activité de la télomérase dans les fibroblastes pulmonaires et de l'expression protéique de hTERT dans la paroi des bronchioles, a protégé les bronchioles de cette fibrose péribronchiolaire. D'autre part, le TA-65 inhibe l’augmentation de l'expression du TGF-β1 en réponse à l'exposition à la fumée de cigarette dans les bronchioles. Enfin, le TA-65 a protégé les fibroblastes pulmonaires murins contre la différenciation myofibroblastique induite par le TGF-β. Nos résultats suggèrent fortement que la télomérase est un acteur essentiel du remodelage bronchiolaire et fournissent ainsi de nouvelles perspectives dans la compréhension de la pathogenèse et le traitement de la BPCO. / Small airway remodeling (SAR) is an important cause of airflow obstruction in smokers with chronic obstructive pulmonary disease (COPD). Small airways are the bronchioles having an internal diameter inferior to 2 mm. SAR results in an increased thickness of small airway wall, with a combination of peribronchiolar fibrosis with increased deposition of fibrous tissue and accumulation of mesenchymal and epithelial cells. SAR pathogenesis is still unclear and recent data suggest that alterations in telomerase activity could represent a possible underlying mechanism. Our study was therefore dedicated to identify a potential protective role of TA-65, a pharmacological telomerase activator in a cigarette smoke (CS) model of SAR in mice. C57BL/6J mice were daily exposed to air or CS during 4 weeks with or without a concomitant administration of TA-65, starting 7 days before CS exposure. Morphological analyses were performed, and mucus production, myofibroblast differentiation, collagen deposition, as well as transforming growth factor-β1 (TGF-β1) expression in the small airway wall were examined. Finally, the measurement of telomerase activity measured by the TRAP method (Telomere Repeat Amplification Protocol) and the effect of TA-65 treatment on TGF-β-induced fibroblast-to-myofibroblast differentiation were evaluated in primary cultures of pulmonary fibroblast and then exposed to TGF-β1 with or without pretreatment with modulators of telomerase. Exposure to CS during 4 weeks induced SAR in mice, characterized by a small airway wall thickening and a peribronchiolar fibrosis (increased deposition of collagen (total collagen, collagen I and III), expression of α-SMA in small airway wall), in the absence of mucus overproduction. Treatment of mice with TA-65 protected them from CS-induced SAR. This effect was associated with the prevention of CS-induced TGF-β1 expression in vivo, and a blockade of TGF-β-induced myofibroblast differentiation in vitro. CS-induced SAR was prevented by a pharmacological activator of telomerase. Our findings strongly suggest that telomerase is a critical player of SAR and therefore provide new insights in the understanding and treatment of COPD pathogenesis.

Background: The appetite-stimulating hormone ghrelin is a fundamental regulator of human energy metabolism. A series of studies support the notion that long-term appetite and weight regulation may be already programmed in early life and it could be demonstrated that the intrauterine environment affects the ghrelin system of the offspring. Animal studies have also shown that intrauterine programming of orexigenic systems persists even until adolescence/adulthood. Methods: We hypothesized that plasma ghrelin concentrations in adulthood may be associated with the intrauterine exposure to cigarette smoke. We examined this hypothesis in a sample of 19-year-olds followed up since birth in the framework of the Mannheim Study of Children at Risk, an ongoing epidemiological cohort study of the long-term outcome of early risk factors. Results: As a main finding, we found that ghrelin plasma concentrations in young adults who had been exposed to cigarette smoke in utero were significantly higher than in those without prenatal smoke exposure. Moreover, individuals with intrauterine nicotine exposure showed a significantly higher prevalence of own smoking habits and lower educational status compared to those in the group without exposure. Conclusion: Smoking during pregnancy may be considered as an adverse intrauterine influence that may alter the endocrine-metabolic status of the offspring even until early adulthood.

La fumée de cigarette est un aérosol complexe constitué d’une phase gazeuse et d’une phase particulaire. Elle est issue de la combustion et de la pyrolyse des différents ingrédients de la cigarette lors de son fumage. Sa toxicité est intimement liée à sa composition chimique et à son comportement dans l'organisme. L’objectif de ce travail de thèse a été d’approfondir notre connaissance de la composition chimique de la matière particulaire des fumées de cigarette inhalées par le fumeur actif (MSS). La démarche entreprise a été celle d’une analyse non-ciblée assurant la mise en évidence du plus grand nombre possible de constituants de la fumée de cigarette. Un préalable à cette étude a été la mise en place d’une méthode répétable de fumage des cigarettes et de collecte de la phase gazeuse et particulaire. Cette méthode a pu être validée par le suivi de composés traceurs : les BTEX (benzène, toluène, éthyl-benzène, ortho- méta- et para-xylène) pour la phase gazeuse et la nicotine pour la phase particulaire. L’analyse non-ciblée de la matière particulaire a ensuite pu être menée directement par désorption/ionisation laser (LDI) et par électro-nébulisation (ESI). De manière systématique, un spectromètre de masse à résonance cyclotronique des ions et à transformée de Fourier (FT–ICR MS) a permis des mesures de masse à très haute résolution et très haute précision. La complémentarité des analyses LDI et ESI pour l'analyse de la fumée de cigarette a pu être établie. Les analyses ESI assurent la détection des composés les plus polaires comme ceux possédant un groupement pyridine ou pyrrolidinyl. Celles menées par LDI apparaissent plus sensibles aux espèces hétéro-aromatiques poly-condensées. L’ensemble des résultats obtenus montre l’influence de la morphologie de la cigarette, de son origine et de la présence potentielle d’agents de saveur (menthol ou clou de girofle) sur la composition chimique de la matière particulaire des MSS / The cigarette smokes are complex mixtures, composed of a gaseous phase and a particulate phase. They are generated by the combustion and the pyrolysis of the cigarette ingredients (tobacco, additive and paper). The toxic effects of cigarette smoke are directly correlated with its chemical composition as well as the morphology of the aerosol itself. The aim of this thesis is to increase our knowledge of the chemical composition of the particulate matter of the cigarette smoke inhaled by an active smoker (MSS). The used approach is a non-targeted analysis ensuring the detection of as many as possible of the cigarette smoke constituents. A prior to this study was the establishment of a repeatable method of smoking cigarettes and collecting the gas phase and the particulate phase. This method has been validated by the monitoring of tracer compounds: BTEX (benzene, toluene, ethylbenzene, ortho- meta- and para-xylene) for the gas phase and the nicotine for the particulate phase. Non-targeted analysis of the particulate matter is carried out without pretreatment by laser desorption/ionization (LDI) or by electrospray ionization (ESI) after extraction. A Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) was systematically used. The very high resolution and the very high accuracy measurement achieved by this instrument are required for the examination of such extremely complex samples. The complementarity of LDI and ESI ionization for the analysis of the particle cigarette smoke compounds was established. The ESI analyses evidenced polar compounds and components with a pyridine group and LDI ensured the detection of poly-condensed heteroaromatic species. All the results obtained in this study showed the influence of the morphology of the cigarette, of its origin and the presence of flavoring agents (cloves) on the chemical composition of the particulate matter of the MSS

Unexpected findings that contradict well-established relationships between exposures and outcomes are often referred to as “paradoxes” in the epidemiologic literature. For example, the “birth weight paradox” refers to the observed protective association between smoking during pregnancy and infant mortality among low birth weight infants. A recent body of literature suggests that this and several other well-known epidemiologic paradoxes can be attributed to collider bias. Collider bias results from conditioning on a variable that is caused by the exposure or shares common cause with the exposure and is caused by the outcome or shares common causes with the outcome. Several recent epidemiology textbooks and methodological studies further suggest that collider bias is the graphical representation of selection bias, suggesting that these two biases are synonymous. This structural approach to bias is conceptually very useful for defining, describing, and identifying selection bias, but it introduces paradoxes of its own due to contradictory conclusions in the selection and collider bias methodologic literatures about their likely impact on study results in terms of magnitude, direction, and strata affected. Resolving these discrepancies is essential for our theoretical understanding of the relationship between selection and collider bias and has important practical implications for how we teach epidemiology, design studies, and evaluate and quantify the potential effects of bias on our results. For example, while patterns of collider bias coincide qualitatively with the birth weight paradox, the magnitude of collider bias would have to be substantial to reverse the sign of the association, contrary to prevailing beliefs that collider bias only minimally affects our results. To date, the plausibility of collider bias as an explanation for the birth weight paradox has not been empirically evaluated using data in which the paradox is observed.Taken together, these inconsistencies and contradictions suggest that our understanding of selection bias and collider bias remains incomplete. The overarching goal of this dissertation was to advance the theoretical and quantitative understanding of the impact of collider bias on study results to clarify the relationship between selection and collider bias. I began by systematically reviewing the methodologic literature on selection and collider bias. I found that selection bias and collider bias are increasingly treated as synonyms, but that conclusions about the magnitude and direction of selection and collider bias, the stratum affected, and the conditions under which the effects of each type of bias were evaluated are highly inconsistent. This suggested that divergent findings about the impact of selection and collider bias might be resolved by considering the impact of collider bias under a broader set of circumstances. I used microsimulations grounded in the sufficient component cause model to examine collider bias not under the null; interrogate why multiplicative interaction appeared central to the impact of collider bias; and clarify which stratum or strata are affected by collider bias. I identified clear patterns for the magnitude, direction, and strata affected by collider bias and successfully reconciled discrepancies with the selection bias literature. This work also enabled me to interrogate both the causal mechanisms and mathematical principles that underlie collider bias, which revealed how collider bias leads to non-exchangeability and when stratifying on a collider results in bias. Finally, I applied this deeper understanding of the mechanisms underlying collider bias to empirically evaluate the plausibility of collider bias as an explanation for the birth weight paradox. Using microsimulations parameterized with 2015 National Center for Health Statistics Cohort Linked Birth-Infant Mortality, I identified scenarios that successfully reproduced the paradox and all observed relationships between smoking during pregnancy, infant mortality, and low birth weight. These findings strengthen the evidence for the role of collider bias in producing the paradox and shed light on the potential magnitude of unmeasured confounding and direct effects of smoking and low birth weight on infant mortality that may be required for the observed magnitude of the paradox to arise. This work clarifies that almost all selection bias is collider bias; that the effects of collider bias vary in magnitude and direction; that selecting on a collider always leads to bias, but this bias may not occur in the stratum that coincides with our analytical sample; and that collider bias may resolve the birth weight paradox, but is unlikely to explain all epidemiologic paradoxes.

L’athérosclérose est la principale cause d’infarctus du myocarde, de mort subite d’origine cardiaque, d’accidents vasculaires cérébraux et d’ischémie des membres inférieurs. Celle-ci cause près de la moitié des décès dans les pays industrialisés. Lorsque les obstructions artérielles athérosclérotiques sont tellement importantes que les techniques de revascularisation directe ne peuvent être effectuées avec succès, la sévérité de l’ischémie tissulaire résiduelle dépendra de l’habilité de l’organisme à développer spontanément de nouveaux vaisseaux sanguins (néovascularisation). La néovascularisation postnatale est le résultat de deux phénomènes : la formation de nouveaux vaisseaux à partir de la vasculature existante (angiogenèse) et la formation de vaisseaux à partir de cellules souches progénitrices (vasculogenèse). Notre laboratoire a démontré que plusieurs facteurs de risque associés aux maladies cardiovasculaires (tabagisme, vieillissement, hypercholestérolémie) diminuaient également la réponse angiogénique suite à une ischémie. Cependant, les mécanismes précis impliqués dans cette physiopathologie sont encore inconnus. Un point commun à tous ces facteurs de risque cardiovasculaire est l’augmentation du stress oxydant. Ainsi, le présent ouvrage visait à élucider l’influence de différents facteurs de risque cardiovasculaire et du stress oxydant sur la néovascularisation. Nos résultats démontrent que l’exposition à la fumée de cigarette et le vieillissement sont associés à une diminution de la néovascularisation en réponse à l’ischémie, et que ceci est au moins en partie causé par une augmentation du stress oxydant. De plus, nous démontrons que les acides gras dérivés de la diète peuvent affecter la réponse à l’ischémie tissulaire. La première étude du projet de recherche visait à évaluer l’impact de l’exposition à la fumée de cigarette sur la néovascularisation post-ischémique, et l’effet d’une thérapie antioxydante. L’exposition à la fumée de cigarette a été associée à une réduction significative de la récupération du flot sanguin et de la densité des vaisseaux dans les muscles ischémiques. Cependant, une récupération complète de la néovascularisation a été démontrée chez les souris exposées à la fumée de cigarette et traitées au probucol ou aux vitamines antioxydantes. Nous avons démontré qu’une thérapie antioxydante administrée aux souris exposées à la fumée de cigarette était associée à une réduction significative des niveaux de stress oxydant dans le plasma et dans les muscles ischémiques. De plus, les cellules endothéliales progénitrices (EPCs) exposées à de l’extrait de fumée de cigarette in vitro présentent une diminution significative de leur activité angiogénique (migration, adhésion et incorporation dans les tissus ischémiques) qui a été complètement récupérée par le probucol et les vitamines antioxydantes. La deuxième étude avait pour but d’investiguer le rôle potentiel de la NADPH oxydase (Nox2) pour la modulation de la néovascularisation post-ischémique dans le contexte du vieillissement. Nous avons trouvé que l’expression de la Nox2 est augmentée par le vieillissement dans les muscles ischémiques des souris contrôles. Ceci est associé à une réduction significative de la récupération du flot sanguin après l’ischémie chez les vieilles souris contrôles comparées aux jeunes. Nous avons aussi démontré que la densité des capillaires et des artérioles est significativement réduite dans les muscles ischémiques des animaux vieillissants alors que les niveaux de stress oxydant sont augmentés. La déficience en Nox2 réduit les niveaux de stress oxydant dans les tissus ischémiques et améliore la récupération du flot sanguin et la densité vasculaire chez les animaux vieillissants. Nous avons aussi démontré que l’activité fonctionnelle des EPCs (migration et adhésion à des cellules endothéliales matures) est significativement diminuée chez les souris vieillissantes comparée aux jeunes. Cependant, la déficience en Nox2 est associée à une récupération de l’activité fonctionnelle des EPCs chez les animaux vieillissants. Nous avons également démontré une augmentation pathologique du stress oxydant dans les EPCs isolées d’animaux vieillissants. Cette augmentation de stress oxydant dans les EPCs n’est pas présente chez les animaux déficients en Nox2. La troisième étude du projet de recherche a investigué l’effet des acides gras dérivés de la diète sur la néovascularisation postnatale. Pour ce faire, les souris ont reçu une diète comprenant 20% d’huile de maïs (riche en oméga-6) ou 20% d’huile de poisson (riche en oméga-3). Nos résultats démontrent qu’une diète riche en oméga-3 améliore la néovascularisation post-ischémique au niveau macro-vasculaire, micro-vasculaire et clinique comparée à une diète riche en oméga-6. Cette augmentation de la néovascularisation postnatale est associée à une réduction du ratio cholestérol total/cholestérol HDL dans le sérum et à une amélioration de la voie VEGF/NO dans les tissus ischémiques. De plus, une diète riche en acides gras oméga-3 est associée à une augmentation du nombre d’EPCs au niveau central (moelle osseuse) et périphérique (rate). Nous démontrons aussi que l’activité fonctionnelle des EPCs (migration et incorporation dans des tubules de cellules endothéliales matures) est améliorée et que le niveau de stress oxydant dans les EPCs est réduit par la diète riche en oméga-3. En conclusion, nos études ont permis de déterminer l’impact de différents facteurs de risque cardiovasculaire (tabagisme et vieillissement) et des acides gras dérivés de la diète (oméga-3) sur la néovascularisation post-ischémique. Nous avons aussi identifié plusieurs mécanismes qui sont impliqués dans cette physiopathologie. Globalement, nos études devraient contribuer à mieux comprendre l’effet du tabagisme, du vieillissement, des oméga-3, et du stress oxydant sur l’évolution des maladies vasculaires ischémiques. / Atherosclerosis is the main cause of myocardial infarction, sudden cardiac death, stroke and lower limb ischemia. It is responsible for nearly half of all deaths in industrialized countries. When atherosclerotic arterial obstructions are so important that direct revascularization techniques cannot be successfully performed, the severity of residual tissue ischemia depends on the ability of the organism to spontaneously develop new blood vessels (neovascularization). Postnatal neovascularization is the result of two phenomena: the formation of new bloods vessels from the existing vasculature (angiogenesis) and vessel formation from progenitor cells (vasculogenesis). Our laboratory has demonstrated that several cardiovascular risk factors (smoking, aging, and hypercholesterolemia) also impair the angiogenic response after ischemia. However, the precise mechanisms involved in that pathophysiology are still unknown. A common feature of all the cardiovascular risk factors is increased oxidative stress. Therefore, the purpose of the present work was to elucidate the influence of cardiovascular risk factors and oxidative stress on neovascularization. Our results demonstrate that exposure to cigarette smoke and aging are associated with impaired neovascularization in response to ischemia, and that this is at least in part due to increased oxidative stress. In addition, we demonstrate that fatty acids derived from the diet can modulate the response to tissue ischemia. The first study of the research project evaluated the effect of cigarette smoke exposure on neovascularization in response to ischemia, and the effect of an antioxidant therapy. Exposure to cigarette smoke was associated with a significant reduction in the recovery of blood flow perfusion and vessel density in ischemic muscles. However, a complete recovery of neovascularization was demonstrated in mice exposed to cigarette smoke that were treated with probucol or antioxidant vitamins. We found that antioxidant therapy in mice exposed to cigarette smoke was associated with a significant reduction of oxidative stress levels in the plasma and in ischemic muscles. In addition, endothelial progenitor cells (EPCs) exposed to cigarette smoke extracts in vitro showed a significant decrease in their angiogenic activities (migration, adhesion and homing into ischemic tissues) that was completely rescued by probucol and antioxidants vitamins. The goal of the second study was to investigate the potential role of NADPH oxidase (Nox2) in the modulation of ischemia-induced neovascularization in the context of aging. We found that the expression of Nox2 is increased by aging in ischemic muscles of control mice. This is associated with a significant reduction of blood flow recovery after ischemia in older compared to young control mice. We also demonstrated that the density of capillaries and arterioles is significantly reduced in ischemic muscles of older animals, whereas oxidative stress levels are increased. Nox-2 deficiency reduces oxidative stress levels in ischemic tissues and improves blood flow recovery and vascular densities in older animals. We also demonstrated that the functional activities of EPCs (migration and adhesion to mature endothelial cells) were significantly reduced in older compared to young mice. However, Nox2 deficiency is associated with preserved EPCs functional activities in older animals. We also demonstrated an age-dependent pathological increase of oxidative stress in EPCs that is not found in Nox2-deficient animals. The third study of the research project investigated the effect of fatty acids derived from the diet on postnatal neovascularization. To this end, mice received a diet containing either 20% corn oil (rich in omega-6) or 20% fish oil (rich in omega-3). Our results demonstrate that an omega-3 rich diet increases neovascularization in response to ischemia at the macrovascular, microvascular and clinical level compared to an omega-6 rich diet. This increased postnatal neovascularization is associated with decreased total cholesterol/HDL cholesterol ratio in the serum and improved VEGF/NO pathway in ischemic tissues. In addition, the omega-3 rich diet is associated with a significant increase of central (bone marrow) and peripheral (spleen) EPCs. We also show that the functional activities of EPCs (migration and incorporation into tubules) are improved and oxidative stress level in EPCs is reduced by the omega-3 rich diet. In conclusion, our studies have clarified the impact of cardiovascular risk factors (smoking and aging) and fatty acids derived from the diet (omega-3) on ischemia-induced neovascularization. We have also identified several mechanisms involved in that physiopathology. Globally, our studies should contribute to a better understanding of the effects of cigarette smoking, aging and omega-3 on the evolution of ischemic vascular diseases.

L’athérosclérose est la principale cause d’infarctus du myocarde, de mort subite d’origine cardiaque, d’accidents vasculaires cérébraux et d’ischémie des membres inférieurs. Celle-ci cause près de la moitié des décès dans les pays industrialisés. Lorsque les obstructions artérielles athérosclérotiques sont tellement importantes que les techniques de revascularisation directe ne peuvent être effectuées avec succès, la sévérité de l’ischémie tissulaire résiduelle dépendra de l’habilité de l’organisme à développer spontanément de nouveaux vaisseaux sanguins (néovascularisation). La néovascularisation postnatale est le résultat de deux phénomènes : la formation de nouveaux vaisseaux à partir de la vasculature existante (angiogenèse) et la formation de vaisseaux à partir de cellules souches progénitrices (vasculogenèse). Notre laboratoire a démontré que plusieurs facteurs de risque associés aux maladies cardiovasculaires (tabagisme, vieillissement, hypercholestérolémie) diminuaient également la réponse angiogénique suite à une ischémie. Cependant, les mécanismes précis impliqués dans cette physiopathologie sont encore inconnus. Un point commun à tous ces facteurs de risque cardiovasculaire est l’augmentation du stress oxydant. Ainsi, le présent ouvrage visait à élucider l’influence de différents facteurs de risque cardiovasculaire et du stress oxydant sur la néovascularisation. Nos résultats démontrent que l’exposition à la fumée de cigarette et le vieillissement sont associés à une diminution de la néovascularisation en réponse à l’ischémie, et que ceci est au moins en partie causé par une augmentation du stress oxydant. De plus, nous démontrons que les acides gras dérivés de la diète peuvent affecter la réponse à l’ischémie tissulaire. La première étude du projet de recherche visait à évaluer l’impact de l’exposition à la fumée de cigarette sur la néovascularisation post-ischémique, et l’effet d’une thérapie antioxydante. L’exposition à la fumée de cigarette a été associée à une réduction significative de la récupération du flot sanguin et de la densité des vaisseaux dans les muscles ischémiques. Cependant, une récupération complète de la néovascularisation a été démontrée chez les souris exposées à la fumée de cigarette et traitées au probucol ou aux vitamines antioxydantes. Nous avons démontré qu’une thérapie antioxydante administrée aux souris exposées à la fumée de cigarette était associée à une réduction significative des niveaux de stress oxydant dans le plasma et dans les muscles ischémiques. De plus, les cellules endothéliales progénitrices (EPCs) exposées à de l’extrait de fumée de cigarette in vitro présentent une diminution significative de leur activité angiogénique (migration, adhésion et incorporation dans les tissus ischémiques) qui a été complètement récupérée par le probucol et les vitamines antioxydantes. La deuxième étude avait pour but d’investiguer le rôle potentiel de la NADPH oxydase (Nox2) pour la modulation de la néovascularisation post-ischémique dans le contexte du vieillissement. Nous avons trouvé que l’expression de la Nox2 est augmentée par le vieillissement dans les muscles ischémiques des souris contrôles. Ceci est associé à une réduction significative de la récupération du flot sanguin après l’ischémie chez les vieilles souris contrôles comparées aux jeunes. Nous avons aussi démontré que la densité des capillaires et des artérioles est significativement réduite dans les muscles ischémiques des animaux vieillissants alors que les niveaux de stress oxydant sont augmentés. La déficience en Nox2 réduit les niveaux de stress oxydant dans les tissus ischémiques et améliore la récupération du flot sanguin et la densité vasculaire chez les animaux vieillissants. Nous avons aussi démontré que l’activité fonctionnelle des EPCs (migration et adhésion à des cellules endothéliales matures) est significativement diminuée chez les souris vieillissantes comparée aux jeunes. Cependant, la déficience en Nox2 est associée à une récupération de l’activité fonctionnelle des EPCs chez les animaux vieillissants. Nous avons également démontré une augmentation pathologique du stress oxydant dans les EPCs isolées d’animaux vieillissants. Cette augmentation de stress oxydant dans les EPCs n’est pas présente chez les animaux déficients en Nox2. La troisième étude du projet de recherche a investigué l’effet des acides gras dérivés de la diète sur la néovascularisation postnatale. Pour ce faire, les souris ont reçu une diète comprenant 20% d’huile de maïs (riche en oméga-6) ou 20% d’huile de poisson (riche en oméga-3). Nos résultats démontrent qu’une diète riche en oméga-3 améliore la néovascularisation post-ischémique au niveau macro-vasculaire, micro-vasculaire et clinique comparée à une diète riche en oméga-6. Cette augmentation de la néovascularisation postnatale est associée à une réduction du ratio cholestérol total/cholestérol HDL dans le sérum et à une amélioration de la voie VEGF/NO dans les tissus ischémiques. De plus, une diète riche en acides gras oméga-3 est associée à une augmentation du nombre d’EPCs au niveau central (moelle osseuse) et périphérique (rate). Nous démontrons aussi que l’activité fonctionnelle des EPCs (migration et incorporation dans des tubules de cellules endothéliales matures) est améliorée et que le niveau de stress oxydant dans les EPCs est réduit par la diète riche en oméga-3. En conclusion, nos études ont permis de déterminer l’impact de différents facteurs de risque cardiovasculaire (tabagisme et vieillissement) et des acides gras dérivés de la diète (oméga-3) sur la néovascularisation post-ischémique. Nous avons aussi identifié plusieurs mécanismes qui sont impliqués dans cette physiopathologie. Globalement, nos études devraient contribuer à mieux comprendre l’effet du tabagisme, du vieillissement, des oméga-3, et du stress oxydant sur l’évolution des maladies vasculaires ischémiques. / Atherosclerosis is the main cause of myocardial infarction, sudden cardiac death, stroke and lower limb ischemia. It is responsible for nearly half of all deaths in industrialized countries. When atherosclerotic arterial obstructions are so important that direct revascularization techniques cannot be successfully performed, the severity of residual tissue ischemia depends on the ability of the organism to spontaneously develop new blood vessels (neovascularization). Postnatal neovascularization is the result of two phenomena: the formation of new bloods vessels from the existing vasculature (angiogenesis) and vessel formation from progenitor cells (vasculogenesis). Our laboratory has demonstrated that several cardiovascular risk factors (smoking, aging, and hypercholesterolemia) also impair the angiogenic response after ischemia. However, the precise mechanisms involved in that pathophysiology are still unknown. A common feature of all the cardiovascular risk factors is increased oxidative stress. Therefore, the purpose of the present work was to elucidate the influence of cardiovascular risk factors and oxidative stress on neovascularization. Our results demonstrate that exposure to cigarette smoke and aging are associated with impaired neovascularization in response to ischemia, and that this is at least in part due to increased oxidative stress. In addition, we demonstrate that fatty acids derived from the diet can modulate the response to tissue ischemia. The first study of the research project evaluated the effect of cigarette smoke exposure on neovascularization in response to ischemia, and the effect of an antioxidant therapy. Exposure to cigarette smoke was associated with a significant reduction in the recovery of blood flow perfusion and vessel density in ischemic muscles. However, a complete recovery of neovascularization was demonstrated in mice exposed to cigarette smoke that were treated with probucol or antioxidant vitamins. We found that antioxidant therapy in mice exposed to cigarette smoke was associated with a significant reduction of oxidative stress levels in the plasma and in ischemic muscles. In addition, endothelial progenitor cells (EPCs) exposed to cigarette smoke extracts in vitro showed a significant decrease in their angiogenic activities (migration, adhesion and homing into ischemic tissues) that was completely rescued by probucol and antioxidants vitamins. The goal of the second study was to investigate the potential role of NADPH oxidase (Nox2) in the modulation of ischemia-induced neovascularization in the context of aging. We found that the expression of Nox2 is increased by aging in ischemic muscles of control mice. This is associated with a significant reduction of blood flow recovery after ischemia in older compared to young control mice. We also demonstrated that the density of capillaries and arterioles is significantly reduced in ischemic muscles of older animals, whereas oxidative stress levels are increased. Nox-2 deficiency reduces oxidative stress levels in ischemic tissues and improves blood flow recovery and vascular densities in older animals. We also demonstrated that the functional activities of EPCs (migration and adhesion to mature endothelial cells) were significantly reduced in older compared to young mice. However, Nox2 deficiency is associated with preserved EPCs functional activities in older animals. We also demonstrated an age-dependent pathological increase of oxidative stress in EPCs that is not found in Nox2-deficient animals. The third study of the research project investigated the effect of fatty acids derived from the diet on postnatal neovascularization. To this end, mice received a diet containing either 20% corn oil (rich in omega-6) or 20% fish oil (rich in omega-3). Our results demonstrate that an omega-3 rich diet increases neovascularization in response to ischemia at the macrovascular, microvascular and clinical level compared to an omega-6 rich diet. This increased postnatal neovascularization is associated with decreased total cholesterol/HDL cholesterol ratio in the serum and improved VEGF/NO pathway in ischemic tissues. In addition, the omega-3 rich diet is associated with a significant increase of central (bone marrow) and peripheral (spleen) EPCs. We also show that the functional activities of EPCs (migration and incorporation into tubules) are improved and oxidative stress level in EPCs is reduced by the omega-3 rich diet. In conclusion, our studies have clarified the impact of cardiovascular risk factors (smoking and aging) and fatty acids derived from the diet (omega-3) on ischemia-induced neovascularization. We have also identified several mechanisms involved in that physiopathology. Globally, our studies should contribute to a better understanding of the effects of cigarette smoking, aging and omega-3 on the evolution of ischemic vascular diseases.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cigarette smoke exposure is a major risk factor to the development of cardiovascular diseases, neurocognitive and neurobiological deficits. Nowadays, phytotherapy is widely employed in the treatment of many illnesses. Curcumin, a polyphenol obtained from the rhizomes of Curcuma longa and commonly used in the oriental culinary and traditional medicine, has several pharmacological properties such as antioxidant, antiaggregant and neuroprotective. Despite its wide-ranging spectrum of pharmacological properties, curcumin possess potential to prevent the noxious effects caused by cigarette smoke exposure. In this context, the purpose of this study was to evaluate the effect of curcumin on memory and parameters involved in the homeostasis of central nervous system (CNS) in rats passively exposed to cigarette smoke. The experiments were performed in two different stages, being the first divided in two sets. In the first set, animals were randomly assigned into four groups: vehicle; curcumin 12.5 mg/kg; curcumin 25 mg/kg; and curcumin 50 mg/kg. In the second set, animals were randomly assigned into five groups: vehicle, cigarette smoke; curcumin 12.5 mg/kg along with cigarette smoke; curcumin 25 mg/kg along with cigarette smoke; and curcumin 50 mg/kg along with cigarette smoke. In the second experimental stage, animals were randomly divided into ten groups: vehicle; curcumin 12.5 mg/kg; curcumin 25 mg/kg; curcumin 50 mg/kg; nanoencapsulated curcumin 4 mg/kg; cigarette smoke; curcumin 12.5 mg/kg along with cigarette smoke; curcumin 25 mg/kg along with cigarette smoke; curcumin 50 mg/kg along with cigarette smoke; and nanoencapsulated curcumin 4 mg/kg along with cigarette smoke. The treatment with curcumin and cigarette smoke was carried out once a day, 5 days each week, during 30 days. Curcumin was administered orally and, approximately 10 minutes later, the smoking groups were exposed to the sidestream smoke of four commercial cigarettes (nicotine 0.9 mg, tar 10 mg each) inside a whole-body smoke exposure chamber. After thirty days, the animals were euthanized, the blood collected and the brain dissected in cerebral cortex, hippocampus, hypothalamus, striatum and cerebellum. The group of rats exposed to cigarette smoke showed an increase in the activity of the enzymes E-NTPDase (ATP as substrate) and E-5 -NT, and a reduction in the activity of the enzyme E-NTPDase (ADP as substrate) in platelets; an increase in the activities of the enzymes E-NTPDase, E-5 -NT and AChE in synaptosomes from the cerebral cortex; an increase in the activity of AChE in cerebellum, cerebral cortex, hippocampus, striatum, hypothalamus and peripheral blood; a decrease in the activities of the enzymes Na+,K+-ATPase and Ca2+-ATPase and a redox imbalance. Furthermore, in the same group of animals, it was observed a cognitive impairment evaluated through the inhibitory avoidance test and the object recognition test. We conclude that the use of both formulations of curcumin, free and nanostructured, prevents the effects observed in the purinergic and cholinergic system, in the enzymes involved in the ion homeostasis and in the oxidative stress parameters. Finally, the results obtained in this study indicate that curcumin administration as lipid-core nanocapsules may be an alternative to increase its efficacy, probably by the increase of its bioavailability when administered orally. / A exposição à fumaça do cigarro é um fator de risco para o desenvolvimento de doenças cardiovasculares, déficits neurocognitivos e neurobiológicos. Atualmente, o emprego de fitoterápicos é uma das alternativas para o tratamento de diversas doenças. A curcumina, um polifenol obtido a partir de rizomas de Curcuma longa e amplamente utilizado na culinária e na medicina tradicional oriental, possui diversas propriedades farmacológicas como antioxidante, anti-agregante e neuroprotetora. Em virtude de seu amplo espectro de propriedades farmacológicas a curcumina possui potencial para a prevenção dos efeitos causados pela exposição à fumaça do cigarro. Neste contexto, o objetivo deste estudo foi avaliar o efeito da curcumina sobre a memória e parâmetros envolvidos na homeostase do sistema nervoso central (SNC) em ratos expostos de forma passiva à fumaça do cigarro. Os experimentos foram realizados em duas etapas, sendo a primeira delas dividida em duas fases. Na primeira fase, os animais foram divididos aleatoreamente em quatro grupos, denominados: veículo; curcumina 12,5 mg/kg; curcumina 25 mg/kg; e curcumina 50 mg/kg. Na segunda fase, os animais foram divididos aleatoreamente em cinco grupos, denominados: veículo; cigarro; cigarro + curcumina 12,5 mg/kg; cigarro + curcumina 25 mg/kg; e cigarro + curcumina 50 mg/kg. Na segunda etapa experimental, os animais foram divididos aleatoreamente em dez grupos, denominados: veículo; curcumina 12,5 mg/kg; curcumina 25 mg/kg; curcumina 50 mg/kg; curcumina nanoencapsulada 4 mg/kg; cigarro; cigarro + curcumina 12,5 mg/kg; cigarro + curcumina 25 mg/kg; cigarro + curcumina 50 mg/kg; cigarro + curcumina nanoencapsulada 4 mg/kg. O tratamento com a curcumina e com a fumaça do cigarro foi realizada uma vez por dia, cinco dias por semana, durante trinta dias. A curcumina foi administrada de forma oral e, após aproximadamente dez minutos, os grupos fumantes eram expostos à fumaça de quatro cigarros comerciais (0,9 mg de nicotina, 10 mg de alcatrão cada) dentro de uma câmara de exposição. Após trinta dias, os animais foram eutanasiados, o sangue coletado e o encéfalo dissecado em córtex cerebral, hipocampo, hipotálamo, estriado e cerebelo. O grupo de ratos expostos à fumaça do cigarro apresentou um aumento na atividade das enzimas E-NTPDase (ATP como substrato) e E-5 -NT, e uma redução na atividade da enzima E-NTPDase (ADP como substrato) em plaquetas; um aumento nas atividades das enzimas E-NTPDase, E-5 -NT e AChE em sinaptossomas de córtex cerebral; um aumento na atividade da enzima AChE em cerebelo, córtex cerebral, hipocampo, estriado, hipotálamo e sangue periférico; uma redução nas atividades das enzimas Na+,K+-ATPase e Ca2+-ATPase e um desequilíbrio no balanço redox. Além disso, neste mesmo grupo de animais observou-se um déficit cognitivo avaliado através dos testes da esquiva inibitória e do reconhecimento de objetos. Concluimos que o uso de ambas as formulações de curcumina livre e nanoestruturada previne os efeitos observados nas atividades das enzimas do sistema purinérgico, colinérgico, nas enzimas envolvidas na formação do gradiente iônico e nos parâmetros de estresse oxidativo. Por fim, os resultados obtidos neste estudo indicam que a administração da curcumina através de nanocápsulas de núcleo lipídico possa ser uma alternativa para o aumento de sua eficácia, provavelmente pelo aumento da biodisponibilidade da curcumina administrada de forma oral.

L’intoxication tabagique, source d’oxydants, est un facteur de risque important de développement de l’emphysème pulmonaire et du pneumothorax spontané primitif. Les macrophages alvéolaires contribuent pour une large part à l’inflammation pulmonaire au cours de ces pathologies en produisant des métalloprotéases et des espèces réactives de l’oxygène à l’origine du déséquilibre des balances protéase/anti-protéase et oxydant/antioxydant. L'hème oxygénase-1 (HO-1), exprimée principalement par les macrophages, est une enzyme clé des défenses anti-oxydantes pulmonaires. Nous avons initialement étudié l’expression et la localisation cellulaire de l’HO-1 et de ses régulateurs potentiels (Nrf2, Keap1, Bach1 et HIF-1a) dans les macrophages alvéolaires au cours de l’emphysème pulmonaire post-tabagique et du pneumothorax spontané primitif. Les voies de régulation de l’expression de ces protéines ont été analysées in vitro sur des macrophages dérivés de la lignée THP-1 exposés ou non au condensat de fumée de cigarette et à l’hypoxieréoxygénation visant à mimer une partie des effets de l’atélectasie-réexpansion observée lors de la prise en charge thérapeutique des pneumothorax récidivants. Les travaux présentés dans cette thèse nous ont permis de mettre en évidence une altération de l’expression de la voie Nrf2/Keap1-Bach1 associée à une diminution de l’expression des enzymes anti-oxydantes, dont l’HO-1, dans les macrophages alvéolaires au cours de l’emphysème pulmonaire sévère post-tabagique, malgré un stress oxydant important. In vitro, ces altérations pourraient être liées à une activation spécifique des MAPKinases ERK1/2 et JNK par le condensat de fumée de cigarette. Nous avons également montré que la stimulation du système de l’HO-1 était probablement orchestrée par la voie du facteur HIF-1a, et non par celle de Nrf2, dans les macrophages alvéolaires au cours du pneumothorax spontané primitif récidivant du sujet fumeur. Ces résultats pourraient contribuer à une meilleure connaissance de la physiopathologie de l’emphysème pulmonaire et permettre d’envisager de nouvelles approches thérapeutiques basées sur la préservation et/ou la restauration de l’équilibre Nrf2/Keap1-Bach1. Nos travaux suggèrent également que la physiopathologie du pneumothorax spontané primitif est différente chez les patients fumeurs et non fumeurs. Le pneumothorax du sujet fumeur est associé à un stress oxydant pulmonaire et à une induction de l’HO-1 probablement orchestrée par HIF-1a. Ces résultats, confirmés in vitro, mettent en évidence une interaction potentielle entre le stress oxydant et l’hypoxie-réoxygénation / Chronic cigarette smoking, a source of oxidants, is an important risk factor for lung emphysema and primary spontaneous pneumothorax development. Alveolar macrophages are mainly involved in lung inflammation observed in these pathologies through the production of metalloproteases and reactive oxygen species resulting to protease/anti-protease and oxidant/anti-oxidant imbalances. Heme oxygenase-1 (HO-1), mainly expressed in macrophages, is a key enzyme in pulmonary anti-oxidant defences. Therefore, the first aim of our studies was to investigate the expression and cellular localisation of HO-1 and its potential regulators (Nrf2, Keap1, Bach1 and HIF-1a) in alveolar macrophages from smoking related lung emphysema and primary spontaneous pneumothorax. Regulation pathways involved in expression of these proteins were assessed in vitro in macrophage cell line THP-1 exposed or not to cigarette smoke condensate and with or without hypoxia-reoxygenation mimicking parts of events induced by atelectasia-reexpansion during recurrent pneumothorax constitution and treatment. In these studies, we showed an altered expression of Nrf2/Keap1- Bach1 pathway associated with a reduced expression of anti-oxidants enzymes, like HO-1, in alveolar macrophages from smoking related lung emphysema patients, despite an important oxidative stress. These alterations might be related to cigarette smoke condensate activated ERK1/2 and JNK MAPKinases as observed in THP-1 cells. Furthermore, we showed that HO- 1 system induction was mediated by HIF-1a instead of Nrf2 pathway in alveolar macrophages from smoking related recurrent primary spontaneous pneumothorax. These findings may contribute to a better knowledge of the pathophysiology of lung emphysema and could provide new therapeutic approaches based on preservation and/or restoration of Nrf2/Keap1-Bach1 equilibrium. Our results also suggest that the pathophysiology of primary spontaneous pneumothorax could be different in smokers and non smokers. Spontaneous pneumothorax in smokers is associated with lung oxidative stress and the orchestrated induction of HO-1 probably via HIF-1a. These results provide a new link between oxidative stress and hypoxia/reoxygenation