Diss. (sammanfattning) Stockholm : Karolinska instututet, 2006. / Härtill 4 uppsatser.

La prévalence de l’obésité est en constante augmentation dans les pays occidentaux, en raison d’une sédentarisation accompagnée d’une alimentation malsaine. L’obésité est souvent associée à une dysfonction endothéliale et à un risque athérogène élevé. Plusieurs observations cliniques ont montré que la modification du mode de vie, incluant la pratique régulière d’une activité physique et l’adoption d’un mode alimentaire sain, représente une stratégie efficace pour combattre l’obésité et ses complications cardiovasculaires. Cependant, de nombreux mécanismes précisant les effets thérapeutiques directs de l’exercice physique sur le risque athérogène lié à l’obésité sont encore largement inconnus. Le but principal de ce travail a donc été d’identifier, en utilisant un modèle de rat rendu obèse par un régime enrichi en graisse, les mécanismes athéro-protecteurs de l’exercice physique seul et/ou associé à une modification du régime alimentaire (du régime riche en graisse au régime standard). Nos résultats montrent que l’exercice physique, indépendamment de la diète utilisée, corrige la dysfonction endothéliale installée au cours de l’obésité. Cet effet bénéfique a été associé à une diminution du stress oxydatif au niveau vasculaire. En effet, nos résultats indiquent que l’exercice diminue l’activité de la NADPH oxydase au niveau aortique. De plus, nous montrons pour la première fois que l’exercice physique seul, indépendamment de la diète utilisée, est capable de moduler la translocation de la sous-unité de la NADPH oxydase p47phox (principal acteur dans l’activation de ce complexe enzymatique) vers la membrane. Nos résultats indiquent également que l’exercice physique, avec ou sans modification du régime, améliore la voie Akt/eNOS phosphorylée, suggérant une augmentation de la production du NO. Ainsi, l’exercice physique, même sans l’associer à un changement du mode alimentaire, peut être considéré comme une stratégie non-pharmacologique efficace pour le traitement du risque athérogène généré par l’obésité / The prevalence of obesity is increasing at an alarming rate in the western countries. It has been attributed to sedentariness and abundance of unhealthy food. Obesity is often associated with endothelial dysfunction and a high atherogenic risk. Several clinical investigations have reported that life style modification included physical exercise and the adoption of healthydiet was an efficient strategy to combat cardiovascular complications linked to obesity. However, numerous mechanisms by which exercise exerts the direct therapeutic effect on atherogenic risk linked to obesity are still unknown. Using the experimental model of high fat diet-induced obesity rat, the general aim of this study, was to identify the possible molecularmechanisms through which exercise with or without diet modification (high fat to standard diet) exerts an antiatherogenic action. Our results show that exercise independently of diet used, corrected the endothelial dysfunction induced by obesity. This benefit effect was associated with the decreased vascular oxidative stress. In effect, our results show that exercise alone was able to decrease NADPH oxidase activity in aortic tissue. Furthermore, we show for the first time that exercise, independently diet used, was able to modulate the translocation of p47phox subunit to membrane (which plays a pivotal role in NADPH oxidase activation). Ours results show also, that exercise with or without diet modification improves the Akt/eNOS phosphorylation pathway, suggesting that exercise increases NO production. In summary, exercise training even without diet modification, may be a non-pharmacological strategy treatment for atherogenic risk linked to obesity

Le cancer colorectal est un cancer majeur en termes de fréquence et de mortalité. Il s’agit de la deuxième cause de décès par cancer, avec 17 500 décès en France en 2011. Le traitement des stades avancés repose sur différentes molécules anti-cancéreuses telles que l’oxaliplatine. Cependant le développement de résistances entraine des échecs thérapeutiques expliquant le faible taux de survie observé. Il est donc crucial d’identifier les mécanismes de résistance et ses acteurs et de découvrir de nouvelles pistes de traitements.Dans un premier temps, nous nous sommes donc intéressés aux rôles joués par les calpaïnes et NOX1 dans le développement de la résistance à l’oxaliplatine en étudiant des cellules tumorales colorectales résistantes à cette drogue. Ce qui nous a permis d'identifier une voie de signalisation impliquée dans la résistance à cette chimiothérapie.Dans un second temps nous nous sommes intéressés à étudier la réversion de cette résistance à l’oxaliplatine. En criblant différentes chimiothérapies ce qui a permis de mettre en évidence une inversion du statut résistant/sensible dans nos cellules sélectionnées.La première partie de nos données met en évidence de nouvelles régulations de Nox1 qui diffèrent en fonction de la sensibilité des cellules à l’oxaliplatine. Nos résultats montrent aussi que p38 MAPK pourrait être une cible thérapeutique de choix. Dans la deuxième partie nous avons identifié un nouveau traitement permettant l'induction de l'apoptose dans nos cellules résistantes. Ainsi la gemcitabine pourrait être une solution pour traiter les patients ne répondant pas ou plus aux protocoles basés sur l’oxaliplatine / Colorectal cancer is a major cancer in terms of frequency and mortality. This is the second leading cause of cancer death, with 17,500 deaths in France in 2011. The treatment of advanced stages is based on different chemotherapeuties including oxaliplatin. However, the development of resistance leads to therapeutic failures explaining the low survival rate. It is therefore crucial to identify the mechanisms of resistance and the actors involved and to discover new therapeutic approaches. We first investigated the role played by calpains and NOX1 in the development of resistance to oxaliplatin, studying oxaliplatin-resistant colorectal tumor cells. This allowed us to identify a signaling pathway involved in resistance to this chemotherapy.Secondly, we have studied the reversion of this resistance to oxaliplatin. A screening of different chemotherapies revealed a reversal of the resistant / sensitive status in our selected cells In the first part, our data highlight novel Nox1 regulations which differ according to the sensitivity of the cells to oxaliplatin. Our results also show that p38 MAPK could be a therapeutic target for treating colorectal cancers resistant to oxaliplatin. In the second part, we have identified a new treatment to induce apoptosis in our resistant cells. Indeed, gemcitabine may be a solution to treat patients who do not respond to oxaliplatin-based protocols.

Les cellules dendritiques jouent un rôle prépondérant dans le développement des réponses immunitaires ; les nombreuses activités fonctionnelles de ces cellules dont leur importante capacité de phagocytose leur confèrent le pouvoir d'activer les différents mécanismes de défense de l'organisme qu'ils fassent parties de l'immunité innée ou adaptative. Or de nombreux travaux ont montré que lors du développement des réponses immunitaires, les mécanismes oxydatifs ont un rôle essentiel et sont donc étroitement contrôlés. Dans le cas des cellules dendritiques, la production de radicaux oxygénés est en très grande partie relié au fonctionnement d'une NADPH Oxydase (NOX2) que notre laboratoire a été le premier à identifier dans ces cellules, il y a quelques années. Ce complexe enzymatique à une position centrale dans la production en premier des radicaux superoxydes puis des différentes formes réactives de l'oxygène (FRO) qui en découlent. Au cours de ma thèse, j'ai tout d'abord mis en place un protocole permettant l'étude de la production des FRO dans les cellules dendritiques. Les différentes méthodes employées m'ont permis de démontrer que d'autres protéines du système oxydant tels NOX1 et SOD3, étaient également produites par les cellules dendritiques. Nos résultats montrent que ces protéines, à l'instar de ce qui avait été montré pour NOX2, jouent un rôle dans la formation et le contrôle des FRO. J'ai également étudié la localisation intracellulaire de ces protéines, ce qui m'a permis de mieux comprendre les rôles respectifs de ces dernières dans le contrôle de la production et de l'activité des FRO dans les cellules dendritiques. Il avait été préalablement montré que la dégradation des pathogènes dans les cellules dendritiques, après qu'ils aient été phagocytés, était en partie liée à l'activité NOX2. Le travail réalisé dans le cadre de cette thèse nous permet d'envisager un impact des FRO résultant de l'activité de NOX1 sur le contrôle de l'apoptose des cellules dendritiques ou sur certaines voies de signalisation au niveau de noyau. De plus, la dismutation du superoxyde en peroxyde (H2O2) pourrait ne pas être une réaction spontanée comme plusieurs auteurs l'ont proposé, mais pourrait être lié à la présence de SOD3 qui interviendrait dans les compartiments d'endocytose. / Dendritic cells play an important role in the development of immune responses; the numerous functional activities of DC, among them their important phagocytic potential, give to these cells the capacity to activate both innate and acquire immune responses. Many authors have shown that during the development of these processes, oxidative mechanisms have a very important role and consequently must be strictly controlled. In dendritic cells, oxygen radicals production is mainly related to an NADPH Oxidase activity (NOX2), that our laboratory was the first to identify in these cells few years ago. This enzymatic complex has an important function in the production of firstly oxygen superoxide radicals and secondly, various reactive oxygen species (ROS) that are produced like hydrogen peroxide. During my PhD, I have developed various protocols, which enabled us to analyse ROS production by dendritic cells. The different results allowed me to demonstrate that other proteins belonging to the family of the Red/Ox controlling elements are produced by dendritic cells: NOX1 and SOD3. My results showed that these proteins play an important role in ROS formation and control, in complement to what was previously demonstrated for NOX2. I have studied also their intracellular localisation, which permitted a better understanding of their respective role in dendritic cells. It was already shown that, after phagocytosis, pathogen degradation in dendritic cells was partially related to NOX2 activity. The work performed in this thesis let us consider a potential impact of NOX1 derived ROS on the control of the apoptosis of dendritic cells or on signalling pathways at nucleus level. Moreover, superoxyde anion dismutation in hydrogen peroxide (H2O2) could be due to the presence of SOD3 that may be found in endocytic compartments rather than the result of spontaneous reactions most often proposed by authors.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The nitric oxide (NO) constitutes one of the most important mediators of the processes intra and extracellular. Amongst its biological functions, there is a role of neuromodulador. In Podarcis hispanica, diverse research had described the citoarchitecture of the cerebral cortex of this animal, however, there isn't data published about neurons that producers neuronal nitric oxide synthetase (nNOS). The aim of this work was to identify the positive NADPHd/nNOS neurons in the cerebral cortex of P. hispanica. For such, it was used the histochemistry technique NADPHd and imunochemistry to nNOS and soluble Guanylyl Cyclase (sGC α1). The results showed that the cerebral cortex of P. hispanica has a population of 1897 diaphorase positive neurons, being, 1090 (57,5%) in young animals, 528 (27,8%) in adults and 279 (14,7%) in age. Among the telencephalics levels, pre-comisural, comisural and post-comisural showed 423 (22,3%); 459 (24,2%) and 1015 (53,5%) neurons, respectively. In relation the cortical areas, the medial cortex (CM), dorsomedial (CDM), dorsal (CD) and lateral (CL) showed 86 (4,5%), 530 (27,9%), 1243 (65,5%) and 38 (2%) neurons, respectively. Inner and outer plexiforme layers showed, respectively, 1264 (66,6%), 533 (28,1%) and 100 (5.3%) neurons. 1737 (91,5%) NADPHd positive neurons had been found in the regions Timm-positive and 160 (8,5%) in the Timm-negative. 15 types of morphological NOS positive neurons were identified: piramidal, bouquet double, deep stellate, sarmentous, polymorphous, ectopic vertical multipolar, coral, granular unipolar, large radial, horizontal bipolar, horizontal unipolar, smooth vertical, spheroidal and fusiforme. In conclusion, the cerebral cortex of Podarcis hispanica showed neurons with NADPHd activity, as well as immunoreactive to the anti-nNOS and sGC α1 antibodies; NADPHd positive neurons are of three types: Type I, II and III; There is a significant difference in the number of NADPHd positive neurons among animals of different ages and among the cortical areas and layers and were found mainly in the Timm-positive regions. / O óxido nítrico (NO) constitui um dos mais importantes mediadores dos processos intra e extracelulares. Dentre suas funções biológicas, destaca-se seu papel de neuromodulador. Em Podarcis hispanica, diversos trabalhos descreveram a citoarquitetura do córtex cerebral desse animal, no entanto, nenhuma informação referente aos neurônios produtores da isoforma óxido nítrico neuronal (nNOS) nessa região telencefálica foi publicada até o presente momento. Neste trabalho, objetivou-se identificar os neurônios NADPHd/nNOS positivos no córtex cerebral do referido animal. Para tal, utilizou-se a técnica histoquímica NADPHd e imunohistoquímica frente a nNOS e Guanilil Ciclase Solúvel (sGC α1). Os resultados demonstraram que o córtex cerebral de P. hispanica apresenta uma população de 1897 neurônios diaforase positivos, sendo, 1090 (57,5%) em animais jovens, 528 (27,8%) em adultos e 279 (14,7%) em senis. Entre os níveis telencefálicos, o pré-comissural, o comissural e o pós-comissural apresentaram 423 (22,3%); 459 (24,2%) e 1015 (53,5%) neurônios, respectivamente. Em relação as áreas corticais, o córtex medial (CM), dorsomedial (CDM), dorsal (CD) e lateral (CL) apresentaram 86 (4,5%), 530 (27,9%), 1243 (65,5%) e 38 (2%) neurônios, respectivamente. Nas camadas plexiformes interna e externa e camada celular foi verificado, respectivamente, 1264 (66,6%), 533 (28,1%) e 100 (5,3%) neurônios. 1737 (91,5%) neurônios NADPHd positivos foram encontrados nas regiões Timm-positivas e 160 (8,5%) nas Timm-negativas. 15 tipos morfológicos de neurônios NOS positivos foram identificados, são eles: piramidal, buquê duplo, estrelado, sarmentoso, polimorfo, ectópico vertical, multipolar, coral, granular unipolar, radial grande, horizontal bipolar, horizontal unipolar, vertical liso, esferoidal e fusiforme. Então, concluiu-se que: o córtex cerebral de Podarcis hispanica apresenta neurônios com atividade NADPHd, como também imunorreativos aos anticorpos anti-nNOS e sGC α1; os neurônios NADPHd positivos são de três tipos: Tipo I, II e III; há uma diferença significativa na expressão do número de neurônios NADPHd positivos entre animais de diferentes faixas etárias e entre as áreas e camadas corticais; córtex cerebral de P. hispanica apresenta uma variedade de 15 morfologias de neurônios NADPHd positivos; os neurônios NADPHd positivos foram encontrados principalmente nas zonas Timm-positivas.

Avaliamos importância da NADPH oxidase 2 (NOX2) na produção de espécies reativas de oxigênio (EROs) em ilhotas de ratas alimentadas ou em jejum, incubadas na presença de 2,8 mM ou 16,7 mM de glicose, associada ou não a leucina, com ou sem inibição da NOX2. As ilhotas dos animais alimentados ou em jejum apresentaram reduzida secreção de insulina e altas concentrações de EROs na presença de 2,8 mM de glicose. Esses parâmetros foram invertidos pela adição de inibidores da NOX2. A leucina, que é metabolizada no Ciclo dos Ácidos Tricarboxílicos, também aumentou a secreção de insulina por aumento de ATP, e diminuiu as EROs, devido ao aumento de NADPH, um substrato do sistema antioxidante. Desse modo, quando as ilhotas são submetidas ao jejum, a diminuição da atividade secretória é fundamental para impedir que quantidades maiores de hormônio sejam secretadas, podendo levar a uma hipoglicemia. Porém, na presença de alta concentração de glicose, a ativação das defesas antioxidantes da célula b atenua o excesso de EROS, liberando a secreção de insulina. / We sought to evaluate the importance of NADPH oxidase 2 (NOX2) in the production of reactive oxygen species (ROS) in islets from rats fed or fasted, incubated in the presence of glucose 2.8 mM or 16.7 mM, with or without leucine or inhibition of NOX2. Islets of fed or fasted animals showed reduced insulin secretion and high concentration of ROS in the presence of 2.8 mM glucose. These parameters were reversed by addition of inhibitors NOX2. Leucine that is metabolized in the cycle of Tricarboxylic Acids (TCA) also increased insulin secretion, by increasing ATP, and ROS decreased due to the increase of NADPH, a substrate of the antioxidant system. Thus, when the islets are subjected to fasting, decreased secretory activity is essential to prevent amounts of the hormone be secreted and may lead to hypoglycaemia. However, in the presence of high glucose levels, activation of antioxidant defenses of b cell attenuates the excess of ROS, releasing insulin secretion.

Os genes relógio são responsáveis pelo ritmo circadiano e homeostase de diversos sistemas biológicos, incluindo o pâncreas endócrino. Nas células β são de grande importância para a regulação do metabolismo e da secreção de insulina (SI), e sua ausência pode levar ao desenvolvimento do diabetes. A NADPH oxidase (NOX) é um complexo enzimático responsável pela produção do ânion superóxido através da redução do oxigênio molecular. Em ilhotas pancreáticas, a NOX participa da regulação do metabolismo da glicose e da SI, através da modulação do estado redox intracelular. O objetivo do nosso estudo foi verificar se a desregulação dos genes relógio mediada pela ausência de Bmal1 seria capaz de modular a NOX e o estado redox nas células β pancreáticas, influenciando assim a SI. Observamos que a ausência de Bmal1 alterou a atividade e expressão da NOX, desregulando o estado redox intracelular. Essas alterações levaram à redução da viabilidade celular e mudanças na resposta à estimulação com glicose, resultando em uma deficiência na principal função da célula β a SI. / Clock genes are responsible for homeostasis and circadian rhythm in various biological systems, including endocrine pancreas. In β -cells, they are important for the regulation of metabolism and insulin secretion (IS), and its absence can lead to development of diabetes. NADPH oxidase (NOX) is an enzymatic complex responsible for production of superoxide anion by reducing molecular oxygen. In pancreatic islets, NOX regulates glucose metabolism and IS through modulation of the intracellular redox state. The aim of our study was to investigate whether dysregulation of clock genes mediated by Bmal1 suppression would be able to modulate NOX activity and redox state in pancreatic β cells, thus influencing the SI. In this work, the lack of Bmal1 altered the activity and expression of NOX, deregulating the intracellular redox state. These changes led to reduced cell viability and changes in cell response after stimulation with glucose, resulting in a deficiency in β cell main function, GSIS.

Linhagens secretoras de insulina, como a BRIN-BD11, são utilizadas para o estudo do mecanismo de secreção de insulina estimulado pela glicose (GSIS). Essas células expressam o GPR40, que quando ativado por ácidos graxos (AG), potencializam a GSIS. Considerando que as espécies reativas de oxigênio (EROs) podem estar relacionadas com a secreção de insulina mediada por AG, este estudo tem como foco avaliar se há envolvimento do GPR40 na GSIS e na produção de EROs, via NADPH oxidase (NADPHox), em células BRIN-DB11. A ativação do GPR40 foi feita com agonista GW9508 (GW). A secreção de insulina (SI), o conteúdo de superóxido (CS) e ativação da NADPHox foram analisados em resposta a glicose na presença ou ausência de GW (20mM). O envolvimento da NADPHox no CS induzido pelo GW foi analisado após inibição da NADPHox. O aumento da concentração de glicose paralela com aumento da SI, causa redução no CS. A presença do GW potencializa a SI em altas concentrações de glicose, aumenta o CS, e aumenta a translocação da subunidade p47phox do plasma para a membrana. Com a inibição da NADPHox o efeito do GW não ocorre. Os dados confirmam um efeito positivo do GPR40 na secreção de insulina estimulada pela glicose. O GW ativa a NADPHox resultando em um aumento do CS nas células BRIN-BD11. / BRIN-BD11, an Insulin secreting cell line, are used to study the mechanisms of glucose-stimulated insulin secretion (GSIS). These cells express GPR40, which is activated by fatty acids (FA) and potentiates GSIS. Considering that reactive oxygen species (ROS) is a signal that modulates insulin secretion (IS) induced by FA, this study aimed to investigate the involvement of GPR40 in ROS production, via NADPH oxidase (NADPHox), and in GSIS. To activate the GPR40 its agonist, GW9508 (GW), was used. IS and superoxide production (SP) were analyzed in response to increasing glucose concentrations (IGC), with or without GW (20mM). The activation of NADPHox was analyzed. The involvement of NADPHox on SP induced by GW was also evaluated using a small interference RNA or using the NADPHox inhibitor, VAS2870 (12mM). The presence of GW potentiated the IS at high glucose concentrations, together with an increased effect on SC. This increase was paralleled by an increase in the translocation of p47phox to the plasma membrane. Moreover, the inhibition of NADPHox abolished the GW9508 effect. These results show a positive effect of the GPR40 activation in GSIS. Additionally, GW9508 activates NADPHox resulting in an increase in the SC in BRIN-BD11 cells.

Orientador: Antonio Condino Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T09:19:02Z (GMT). No. of bitstreams: 1 Prando_CarolinaCardosodeMello_D.pdf: 7021288 bytes, checksum: e93bf24a96be00102eb5a19f5d7c8880 (MD5) Previous issue date: 2008 / Resumo: O sistema NADPH oxidase fagocítico humano possui um papel importante na defesa contra microorganismos intracelulares, incluindo micobactérias. Mutações nas subunidades deste sistema resultam na Doença Granulomatosa Crônica (DGC). O gene CYBB, localizado no cromossomo X, codifica a subunidade gp91phox, e mutações neste gene são responsáveis por cerca de 60% dos casos de DGC. Cerca de 40 anos depois da identificação de DGC, foi identificado o primeiro dos 13 defeitos genéticos associados à Susceptibilidade Mendeliana à Micobacteriose, participantes do eixo IL12/23-IFN-?. Baseado no fato de que ambas as doenças predispõem a infecções por micobactérias e que o IFN-? is é um importante ativador do gene CYBB os autores se propuseram a estudar as características clinicas de pacientes latino-americanos com DGC e o sistema NADPH oxidase e expressão do gene CYBB em pacientes com defeitos no eixo IL-12/23-IFN-?. Em relação às características clínicas: história familiar de infecções graves e/ou de repetição, bem como reação adversa à vacina BCG, linfadenopatia, abscessos de pele e profundos estavam associados à DGC, em comparação com não-DGC avaliados pelo laboratório. Defeitos nos receptores IFNGR1 e IFNGR2 e cadeia B1 do receptor de IL-12 podem apresentar expressão do gene CYBB e atividade do sistema NADPH oxidas e diminuída ou abolida, chegando a níveis comparáveis a um paciente com DGC. O IFN- ? e seus receptores são essenciais para o desenvolvimento e ativação do sistema NADPH oxidase, e pacientes com comprometimento da função deste sistema devem também ser avaliados para defeitos do eixo IL12/23-IFN-? afetando secundariamente o sistema NADPH oxidase. / Abstract: The NADPH phagocytic oxidase system plays a crucial role in host defense against intracellular microorganisms, including mycobacteria. Mutations affecting subunits of this system result in Chronic Granulomatous Disease (CGD). The CYEE gene, located in the X chromosome, encodes gp91 phox, and mutations on this gene account for more than 60% of CGD cases. Almost 40 years after, the first of 13 different genetic disorders associated with Mendelian Susceptibility to Mycobacterial Diseases (MSMD), was identified. The genes responsible for MSMD are part of the IL12/23-IFN-? axis. Based on the fact that both the diseases predispose to mycobacterial infections and that IFN-? is an important activator of CYBB gene, the authors aimed to describe clinical aspects of Latin American CGD patients and investigate ifthe NADPH oxidase system function and gp91phox expression would be affected in patients with defects in the IL-12/23-IFN-? axis. Regarding clinical features familial history of recurrent and sever infections, as well as adverse reactions to BCG vaccine, lymphadenopathy, skin and profound abscess were associated to DGC when compared to clinical features of non-CGD evaluated in the laboratory. Defects of IFNGR1 and IFNGR2 and IL12RB1 may present diminish.ed or abolished gene expression of CYBB and activity of NADPH oxidase system ate levels of a CGD patient. Based on that, we can conclude that IFN- ? and its receptor are essential for development and activation 9f NADPH oxidase system. In addition, patients who present an impaired superoxide release and/or failure on expressing gp91phox should also be evaluated for IL12/23-IFN-? axis affecting secondarily the NADPH oxidase system. / Doutorado / Doutor em Farmacologia

Orientador: Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T08:48:26Z (GMT). No. of bitstreams: 1 Silva_FabioHenriqueda_D.pdf: 1675795 bytes, checksum: d2c520651641831b55ef2e2707792e5f (MD5) Previous issue date: 2014 / Resumo: A prevalência da disfunção erétil (DE) aumenta progressivamente com o envelhecimento, sendo 29% em homens de meia-idade (40-49 anos). O aumento do estresse oxidativo tem sido apontado como uma das principais causas das complicações vasculares decorrentes do envelhecimento, podendo contribuir para a DE na meia-idade. Uma das principais consequências do aumento do estresse oxidativo é a inativação do óxido nítrico (NO) pelas altas concentrações de ânion superóxido (O2-). O NO pode reagir com o O2-, produzindo peroxinitrito (ONOO-), reduzindo assim a biodisponibilidade de NO e o relaxamento dos corpos cavernosos. Assim, propusemos a hipótese que o aumento da produção de espécies reativas de oxigênio (ERO) nos corpos cavernosos dos ratos de meia-idade (CCRM) contribui para a DE nesta faixa etária. Para tanto, utilizamos ratos jovens (3,5 meses) e de meia-idade (10 meses). Na primeira etapa, estudamos o papel do estresse oxidativo sobre os mecanismos relaxantes de CCRM. A pressão intracavernosa (in vivo) foi significativamente menor nos ratos de meia-idade. Os relaxamentos produzidos pela acetilcolina (ACh; 10 nM ¿ 10 mM), nitroprussiato de sódio (SNP; 10 nM ¿ 10 mM), sildenafil (1 nM ¿ 10 µM), BAY 41-2272 (1 nM ¿ 10 µM) e estimulação elétrica (EFS; 8 ¿ 32 Hz) foram significativamente menores nos CCRM, sendo estes prevenidos pela pré-incubação com apocinina (100 µM) ou superóxido dismutase (SOD; 75 U/ml). O tratamento crônico com apocinina (85 mg/rato/dia, por 4 semanas) também preveniu a redução dos relaxamentos induzidos pela ACh, SNP e EFS em CCRM, sem modificar as respostas nos ratos jovens. O relaxamento induzido pelo 8-Br-GMPc (10 nM ¿ 300 µM) permaneceu inalterado nos CCRM. Os níveis de GMPc basais e estimulados pelo SNP ou BAY 41-2272 foram significantemente menores nos CCRM em comparação com o grupo jovem, sendo restaurados pela apocinina ou SOD. A expressão protéica da nNOS, fosfo-eNOS (Ser-1177) e GCs (subunidades ?1 e ?1) foi significativamente menor nos CCRM, mas nenhuma mudança foi observada para a expressão protéica da eNOS total e fosfo-eNOS (Thr-495). A expressão do RNAm da gp91phox foi maior nos CCRM. A expressão do RNAm da SOD-1 foi similar entre o grupo jovem e de meia-idade. Os níveis basais de ERO foram 64% maiores nos CCRM em comparação com o grupo jovem. Na segunda etapa do nosso estudo, estudamos o papel do estresse oxidativo sobre os mecanismos contráteis dos CCRM. As respostas contráteis induzidas pela EFS (4 ¿ 32 Hz) foram significativamente maiores nos CCRM, as quais foram acompanhadas por um aumento de 2 vezes na expressão protéica da tirosina hidroxilase. As respostas contráteis induzidas pela fenilefrina também foram maiores nos CCRM. O tratamento crônico com apocinina (85 mg/rato/dia, por 4 semanas) restaurou o aumento da contração induzida pela fenilefrina e EFS, assim como a expressão protéica para tirosina hidroxilase e GCs (subunidade ?1 e ?1) nos CCRM. Em suma, nossos resultados mostram que a DE em ratos de meia-idade está associada à redução da biodisponibilidade do NO devido o aumento da expressão do RNAm da gp91phox e redução da expressão protéica da nNOS e fosfo-eNOS (Ser-1177). A DE na meia-idade também está ligada a aumento da expressão da tirosina hidroxilase e da resposta contrátil. A redução da expressão da GCs (subunidades ?1 e ?1) também parece participar da DE em ratos de meia-idade. O tratamento crônico com apocinina reverteu os efeitos deletérios das ERO em ratos meia-idade, melhorando assim a função erétil. Portanto, a diminuição do estresse oxidativo no tecido erétil por terapias antioxidantes pode ser uma boa estratégia farmacológica para o tratamento da DE em estágios iniciais de impotência sexual masculina / Abstract: The prevalence of erectile dysfunction increases progressively with age, being 29% in middle-aged men (40- to 49-year-old men). The increased oxidative stress has been implicated as a major cause of ED-associated vascular complications as and may contribute to ED in middle age. One major consequence of the increased oxidative stress is the inactivation of nitric oxide (NO) by high concentrations of superoxide anion (O2-). The NO can react with O2-, producing peroxynitrite (ONOO-), thus reducing the NO bioavailability and the corpus cavernosum relaxation. Thus, we hypothesized that increases in reactive-oxygen species (ROS) production in corpus cavernosum of middle-aged rats (CCMR) contributes to ED in this age group. Therefore, we used young (3.5-month) and middle-aged (10-month) rats. We first studied the role of stress oxidative on the relaxant mechanisms of CCMR. The ICP was significantly reduced in middle-aged rats. The relaxations induced by acetylcholine (ACh; 10 nM ¿ 10 mM), sodium nitroprusside (SNP; 10 nM ¿ 10 mM), sildenafil (1 nM ¿ 10 µM), BAY 41-2272 (1 nM ¿ 10 µM) and electrical field-stimulation (EFS; 8 ¿ 32 Hz) were significantly decreased in CCMR, all of which prevented by pre-incubation with apocynin (100 µM) or superoxide dismutase (SOD; 75 U/ml). The treatment chronic with apocynin (85 mg/rat/day, 4 weeks) also prevented the reduction of relaxations induced by ACh, SNP and EFS in CCMR, without changing the response in the young rats. Relaxation induced by 8-Br-GMPc (10 nM ¿ 300 µM) remained unchanged. Basal levels of cGMP and stimulated with SNP or BAY 41-2272 were significantly lower in CCMR in comparison with young group, which were restored by apocynin or SOD. Protein expression of nNOS, phosphorylated eNOS (p-eNOS) (Ser-1177) and sGC (?1 and ?1 subunits) was reduced in CCMR, but no changes were observed in the protein expression for total eNOS and p-eNOS (Thr-495). The mRNA expression for the gp91phox was higher in CCMR. The mRNA expression for the SOD-1 was similar between young and middle-aged groups. Basal levels of ROS were 64% higher in the middle-aged in comparison with young group. In a second part, we studied the role of stress oxidative on the contractile mechanisms of CCMR. Contractile responses elicited by EFS (4-32 Hz) were significantly greater in CCMR, which were accompanied by a 2.0-fold higher protein expression of tyrosine hydroxylase. The contractile responses induced by phenylephrine were also greater in middle-aged group. The treatment chronic with apocynin (85 mg/rat/day, 4 weeks) restored the increase of contraction induced by phenylephrine and EFS, as well as the protein expression of tyrosine hydroxylase and sGC (?1 and ?1 subunits) in CCMR. In conclusion, our data shows that ED seen in middle-aged rats is associated with decreased NO bioavailability due to upregulation of mRNA expression for gp91phox and downregulation of nNOS e p-eNOS (Ser-1177). The ED in middle aged is also associated with increased protein expression of tyrosine hydroxylase and contractile responses. Downregulation of GCs (?1 and ?1 subunits) in cavernosal also appears to participate to ED in middle-aged rats. The treatment chronic with apocynin reverted the deleterious effects of ROS in middle-aged rats, thus ameliorating the erectile function. Therefore, decrease of oxidative stress in erectile tissue by antioxidant therapies may be a good pharmacological approach to treat ED at early stages of the male impotence / Doutorado / Farmacologia / Doutor em Farmacologia