The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Grace Y. Sun. "December 2009" Includes bibliographical references

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 156 pages. Includes Vita. Includes bibliographical references.

Orientador: Valdecir Farias Ximenes / Coorientador: Luiz Marcos da Fonseca / Banca: Cibele Bonacorsi / Banca: Iracilda Zepponi Carlos / Resumo: Helicobacter pylori é um dos principais causadores de gastrite crônica e úlcera péptica, e embora o mecanismo envolvido na inflamação gástrica por esta bactéria não esteja completamente elucidado, sabe-se do importante papel das espécies reativas de oxigênio (EROs) produzidas por polimorfonucleares neutrófilos, que são atraídos e ativados pelo agente da infecção, sem que, entretanto, consigam debelar a mesma, mas que contribuem fortemente para a lesão tecidual e o processo inflamatório crônico. Diante disso, tem aumentado a busca de novas estratégias de tratamento que possam levar à redução do estresse oxidativo gerado no sítio da infecção, com consequente redução do processo inflamatório. Neste sentido, ácido gálico, juntamente com seus ésteres (galato de metila, propila, hexila e octila), foram utilizados neste estudo, com o objetivo de avaliar suas ações como inibidores da liberação de EROs por neutrófilos ativados, bem como seus efeitos antimicrobianos sobre H. pylori. Para a avaliação da atividade antioxidante dessas substâncias foram realizados ensaios livres de células (efeito supressor sobre o radical DPPH e sobre radicais peroxila), e o efeito anti-EROs foi avaliado utilizando neutrófilos isolados de sangue humano estimulados por H. pylori, Zymosan ou PMA, através de ensaio quimiluminescente dependente de luminol ou lucigenina, ensaio com WST-1, ensaio de inibição da produção de HOCl, e o teste do NBT. Ensaio antimicrobiano foi realizado através da técnica de microd... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Helicobacter pylori is one of major cause of chronic gastritis and peptic ulcer disease, and although the mechanism involved in gastric inflammation by this bacterium is not fully understood, it is know the important role of reactive oxygen species (ROS) produced by polymorphonuclear neutrophils (PMNs), which are attracted and activated by infection agent, without, however, to be able to overcome the same, but which contribute strongly to the tissue damage and chronic inflammation. Therefore, it has increased the search for new strategies of treatment that can lead to the reduction of the oxidative stress generated at the infection site, with consequent reduction of the inflammatory process. In this sense, gallic acid, together with its esters (methyl, propyl, hexyl and octyl gallate), were used in this study, with the aim of evaluating their actions as inhibitors of ROS release by activated neutrophils, as well as their antimicrobial effects on H. pylori. Cell-free assays (suppressor effect on the DPPH radical and peroxyl radicals) were performed to evaluate the antioxidant activity of these substances, and the anti-EROs effect was evaluated using neutrophils isolated from human blood, stimulated by H. pylori, Zymosan or PMA, through luminol-dependent or lucigenin-dependent chemiluminescent assay, WST-1 assay, inhibition of HOCl production assay, and the NBT assay. Antimicrobial assay was performed by broth microdilution technique. The presence of the carbonic side chain led... (Complete abstract click electronic access below) / Mestre

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

Made available in DSpace on 2016-06-07T17:12:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-06-02. Added 1 bitstream(s) on 2016-06-07T17:16:46Z : No. of bitstreams: 1 000866188.pdf: 703355 bytes, checksum: 0b29948d0caf17093c2ef18fe5f398a8 (MD5) / A insuficiência cardíaca caracteriza-se pela diminuição da capacidade física com exacerbação precoce de fadiga e dispneia. Embora alterações intrínsecas da musculatura esquelética têm sido responsabilizadas por esses sintomas, os mecanismos envolvidos nas anormalidades musculares ainda não estão completamente esclarecidos. O estresse oxidativo miocárdico e sistêmico está aumentado na insuficiência cardíaca. Entretanto, há pouca informação sobre seu papel nas alterações da musculatura esquelética e sobre as fontes de geração de espécies reativas de oxigênio (EROs). Apesar do complexo NADPH oxidase constituir importante fonte geradora de EROs, poucos estudos avaliaram seu comportamento em músculos esqueléticos durante a insuficiência cardíaca. No miocárdio, a apocinina pode inibir o complexo NADPH oxidase e reduzir o estresse oxidativo durante agressão cardíaca. Objetivo: Avaliar os efeitos do tratamento com apocinina sobre alterações cardíacas e anormalidades fenotípicas e do estresse oxidativo do músculo sóleo de ratos com insuficiência cardíaca induzida por estenose aórtica supravalvar. Métodos: Vinte semanas após a indução da estenose aórtica, ratos Wistar foram alocados nos grupos estenose aórtica (EAo), EAo tratado com apocinina (EAo-apo) e controle (Sham). A apocinina foi administrada por oito semanas na água de beber (16 mg/kg/dia). Ecocardiograma transtorácico foi realizado antes e após o tratamento com apocinina. A atividade das enzimas antioxidantes superóxido dismutase, glutationa peroxidase e catalase e a concentração de hidroperóxido de lipídeo foram avaliadas por espectrofotometria no músculo sóleo. A concentração sérica de malonaldeído, a atividade muscular da NADPH oxidase e a geração total de EROs no músculo sóleo foram analisadas por HPLC. No sóleo, a composição das cadeias pesadas de miosina foi avaliada por eletroforese e a área seccional das fibras foi... / Heart failure is characterized by a decreased functional capacity with early fatigue and dyspnea. Although intrinsic skeletal muscle changes may be at least partially responsible for these symptoms, mechanisms involved in muscle abnormalities are not completely clear. Myocardial and systemic oxidative stress is increased during heart failure. However, there is scarce information on the sources of oxygen reactive species (ROS) and on the role of oxidative stress in inducing skeletal muscle changes. NADPH oxidase complex is an important source of ROS; only a few studies have evaluated its activity in skeletal muscles during heart failure. In myocardial, apocynin can inhibit NADPH oxidase activity and reduce oxidative stress during cardiac injury. Purpose: In this study we evaluated the effects of the antioxidant apocynin on cardiac alterations and soleus muscle oxidative stress and phenotypic characteristics in rats with supravalvar aortic stenosis-induced heart failure. Methods: Twenty weeks after inducing aortic stenosis, Wistar rats were assigned into three groups: aortic stenosis (AS), AS treated with apocynin (AS-apo, 16 mg/kg/day dissolved in drinking water for eight weeks), and control (Sham). Transthoracic echocardiogram was performed before and after apocynin treatment. Activity of the antioxidant enzymes superoxide dismutase, gluthatione peroxidase, and catalase, and lipid hydroperoxide concentration were assessed by spectrophotometry in the soleus muscle. Serum concentration of malondialdehyde and soleus muscle NADPH oxidase activity and total ROS generation were analyzed by HPLC. Soleus myosin heavy chain composition was evaluated by electrophoresis and fiber cross-sectional areas were measured in hematoxylin and eosin-stained histological sections. Statistical analysis: one-way analysis of variance complemented by Bonferroni or Mann- Whitney test. Results: Before treatment, AS and AS-apo groups presented concentric hypertrophy ...

Orientador: Marina Politi Okoshi / Coorientador: Paula Felippe Martins / Banca: Bertha Furlan Polegato / Banca:Silméia Garcia Zanati Bazan / Banca: Denise de Castro Fernandes / Banca: Fábio Rodrigues Ferreira Silva / Resumo: A insuficiência cardíaca caracteriza-se pela diminuição da capacidade física com exacerbação precoce de fadiga e dispneia. Embora alterações intrínsecas da musculatura esquelética têm sido responsabilizadas por esses sintomas, os mecanismos envolvidos nas anormalidades musculares ainda não estão completamente esclarecidos. O estresse oxidativo miocárdico e sistêmico está aumentado na insuficiência cardíaca. Entretanto, há pouca informação sobre seu papel nas alterações da musculatura esquelética e sobre as fontes de geração de espécies reativas de oxigênio (EROs). Apesar do complexo NADPH oxidase constituir importante fonte geradora de EROs, poucos estudos avaliaram seu comportamento em músculos esqueléticos durante a insuficiência cardíaca. No miocárdio, a apocinina pode inibir o complexo NADPH oxidase e reduzir o estresse oxidativo durante agressão cardíaca. Objetivo: Avaliar os efeitos do tratamento com apocinina sobre alterações cardíacas e anormalidades fenotípicas e do estresse oxidativo do músculo sóleo de ratos com insuficiência cardíaca induzida por estenose aórtica supravalvar. Métodos: Vinte semanas após a indução da estenose aórtica, ratos Wistar foram alocados nos grupos estenose aórtica (EAo), EAo tratado com apocinina (EAo-apo) e controle (Sham). A apocinina foi administrada por oito semanas na água de beber (16 mg/kg/dia). Ecocardiograma transtorácico foi realizado antes e após o tratamento com apocinina. A atividade das enzimas antioxidantes superóxido dismutase, glutationa peroxidase e catalase e a concentração de hidroperóxido de lipídeo foram avaliadas por espectrofotometria no músculo sóleo. A concentração sérica de malonaldeído, a atividade muscular da NADPH oxidase e a geração total de EROs no músculo sóleo foram analisadas por HPLC. No sóleo, a composição das cadeias pesadas de miosina foi avaliada por eletroforese e a área seccional das fibras foi... / Abstract: Heart failure is characterized by a decreased functional capacity with early fatigue and dyspnea. Although intrinsic skeletal muscle changes may be at least partially responsible for these symptoms, mechanisms involved in muscle abnormalities are not completely clear. Myocardial and systemic oxidative stress is increased during heart failure. However, there is scarce information on the sources of oxygen reactive species (ROS) and on the role of oxidative stress in inducing skeletal muscle changes. NADPH oxidase complex is an important source of ROS; only a few studies have evaluated its activity in skeletal muscles during heart failure. In myocardial, apocynin can inhibit NADPH oxidase activity and reduce oxidative stress during cardiac injury. Purpose: In this study we evaluated the effects of the antioxidant apocynin on cardiac alterations and soleus muscle oxidative stress and phenotypic characteristics in rats with supravalvar aortic stenosis-induced heart failure. Methods: Twenty weeks after inducing aortic stenosis, Wistar rats were assigned into three groups: aortic stenosis (AS), AS treated with apocynin (AS-apo, 16 mg/kg/day dissolved in drinking water for eight weeks), and control (Sham). Transthoracic echocardiogram was performed before and after apocynin treatment. Activity of the antioxidant enzymes superoxide dismutase, gluthatione peroxidase, and catalase, and lipid hydroperoxide concentration were assessed by spectrophotometry in the soleus muscle. Serum concentration of malondialdehyde and soleus muscle NADPH oxidase activity and total ROS generation were analyzed by HPLC. Soleus myosin heavy chain composition was evaluated by electrophoresis and fiber cross-sectional areas were measured in hematoxylin and eosin-stained histological sections. Statistical analysis: one-way analysis of variance complemented by Bonferroni or Mann- Whitney test. Results: Before treatment, AS and AS-apo groups presented concentric hypertrophy ... / Doutor

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Evidências têm mostrado que as espécies reativas de oxigênio (ROS) geradas pela NAD(P)H oxidase são importantes moduladores de diversas funções celulares como migração, crescimento, proliferação e sobrevivência. Estudos recentes demonstraram o envolvimento da atividade da NAD(P)H oxidase no crescimento e sobrevivência de células de melanoma. Neste trabalho, investigamos o efeito da inibição da NAD(P)H oxidase por difenileneiodônio (DPI) sobre o crescimento das células de melanoma humano MV3 e observamos que este composto reduziu o crescimento destas células em aproximadamente 50%. A inibição da NAD(P)H oxidase induziu mudanças no formato celular, com arredondamento, diminuição do espraiamento e descolamento celular. Esta redução foi acompanhada por um rearranjo do citoesqueleto de actina, diminuição da fosforilação no resíduo Tyr397 da quinase de adesão focal (FAK) e redução na associação de FAK com actina e com a tirosina quinase c-Src. Isto indica que a inibição da geração de ROS está modulando negativamente vias de sinalização ativadas por integrinas, o que freqüentemente conduz a um tipo particular de morte celular conhecida por anoikis. Comprovando a ocorrência deste fenômeno, observamos que a inibição da atividade da NAD(P)H oxidase aumentou a apoptose das células de melanoma e induziu a ativação da caspase-3. Nossos resultados mostram ainda que a inibição da viabilidade celular por DPI foi revertida com o pré-tratamento das células MV3 com um inibidor de tirosina fosfatases (ortovanadato de sódio). Em resumo, este estudo mostra que a geração de ROS por NAD(P)H oxidase está envolvida nos mecanismos de sobrevivência em células de melanoma, uma vez que afetam as vias de sinalização dependentes de FAK-Src, através da inibição da atividade de proteína tirosina fosfatases. / NAD(P)H oxidase-derived reactive oxygen species (ROS) have emerged as critical mediators of several cell functions as diverse as migration, growth, proliferation and survival. Recent evidence has show that NAD(P)H oxidase activity is essential to melanoma proliferation and survival. We reported that NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) inhibited melanoma growth. NAD(P)H oxidase inhibition induced changes in cell shape with cell spreading decrease, rounding up and detachment. These phenomena were accompanied by rearrangement of actin network and a decrease in both focal adhesion (FAK) phosphorylation in Tyr397 residue and in FAK association to actin and c-Src, indicating that inhibition of ROS generation would down- modulate integrin-mediated signaling, what often results in a particular type of apoptotic cell death, known as anoikis. We observed that NAD(P)H oxidase inhibitor induced apoptosis in melanoma cells with activation of caspase-3. We results show that the effects promoted by NAD(P)H oxidase inhibition on melanoma growth were completely abolished by the pre-treatment of MV3 cells with the protein tyrosine phosphatases inhibitor sodium orthovanadate. In conclusion, our results strongly suggest that ROS generated by NAD(P)H oxidase complex transmit cell survival signals in melanoma cells through the FAK-Src pathway, probably inhibiting protein tyrosine phosphatases.

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

Le polynucléaire neutrophile (PMN), cellule clé de l’immunité innée, est la première cellule à être recrutée sur le site inflammatoire. Après avoir détruit l’agent pathogène, il entre en apoptose puis est éliminé par les macrophages pour éviter le déversement de son contenu lytique, dangereux pour l’environnement. La régulation de la balance survie/apoptose du neutrophile est donc une étape cruciale de la résolution de l’inflammation. Notre laboratoire a mis en évidence la présence du Proliferating Cell Nuclear Antigen (PCNA) dans le neutrophile mature. PCNA est exprimé dans le noyau des cellules proliférantes, où il est impliqué dans la réplication/réparation de l’ADN et dans le contrôle du cycle cellulaire. PCNA est une protéine trimérique conservée au cours de l’évolution dépourvue d’activité enzymatique. En effet, PCNA constitue une “plateforme” qui interagit avec différents partenaires protéiques et orchestre leurs fonctions. De plus, pour assurer sa fonction, PCNA doit être obligatoirement sous forme trimérique. Dans le neutrophile mature, il a été démontré que PCNA avait une localisation exclusivement cytosolique et qu’il contrôlait spécifiquement la survie du neutrophile. La translocation de PCNA du noyau au cytosol a lieu pendant la différenciation granulocytaire. Elle est dépendante d'une séquence d'export nucléaire (NES) accessible et fonctionnelle que lorsque PCNA est monomérique. Le but de ma thèse a été d’étudier la plateforme de PCNA dans le cytosol du neutrophile afin d'identifier les protéines associées à PCNA afin de comprendre sa fonction dans les neutrophiles. Nous avons montré la présence de la forme monomérique et de la forme trimérique de PCNA dans le cytosol du neutrophile mature. Nous avons démontré une activité anti-apoptotique de la forme monomérique dans des cellules PLB985 différenciées en neutrophiles. De plus, nous avons identifié des peptides exposés sur la surface monomérique de PCNA qui sont utilisé comme des compétiteurs pour déplacer les interactions entre PCNA et ses partenaires dans le cytosol des neutrophiles. Ces peptides modulent la survie des neutrophiles. Grâce à des analyses de Spectrométrie de Masse, nous avons identifié des nouveaux partenaires de PCNA dans le cytosol du neutrophile impliqués dans plusieurs voies métaboliques. Cela suggère que PCNA régule la survie du neutrophile en interagissant avec différents protéines cytosoliques. Parmi les partenaires identifiés, nous avons trouvé les sous-unités cytosoliques de la NADPH oxydase, enzyme responsable de la production de formes réactives de l’oxygène, à la base de l’activité microbicide du neutrophile. Nous avons montré en particulier l’interaction entre p47phox et PCNA. Nous avons enfin étudié l’implication fonctionnelle de l’interaction de PCNA avec la NADPH oxydase dans des cellules PLB985 et également dans des neutrophiles humains. L’ensemble des résultats suggère que PCNA cytoplasmique maintient le neutrophile dans un état de repos, et aide l’assemblage de la NADPH oxydase lors de son activation. Le réseau protéique associé à PCNA régule l’activité et la survie du neutrophile en modulant différentes voies de signalisation. / Polymorphonuclear neutrophils (PMN), key cells of innate immunity are the first cell recruited to the inflammatory site. After destroying the pathogen, neutrophils undergo apoptosis and are cleared by macrophages to prevent the spillage of their lytic content that is dangerous for the environment. The regulation of the survival/apoptosis balance of neutrophil is a crucial step in the inflammation resolution. Our laboratory has shown the presence of Proliferating Cell Nuclear Antigen (PCNA) in mature neutrophils. PCNA is expressed in the nucleus of proliferating cells, where it is involved in DNA replication/repair and in cell cycle control. PCNA is a trimeric protein conserved during evolution and deprived of enzymatic activity. Indeed, PCNA is a “platform” that interacts with different partner proteins and orchestrates their functions. Furthermore, PCNA must be in trimeric form to play its role. In mature neutrophils, PCNA has an exclusively cytosolic localization where it specifically controls their survival. The PCNA translocation from nucleus to the cytosol happened during the granulocytic differentiation. This nuclear-to-cytosol relocalisation is dependent on a nuclear export sequence (NES), which is accessible and functional when PCNA is monomeric. The aim of my thesis was to study the PCNA platform in the neutrophil cytosol to identify the proteins associated with PCNA in order to understand its function in neutrophils. We have shown the expression of monomeric and trimeric forms of PCNA in the cytosol of mature neutrophils. We have demonstrated the anti-apoptotic activity of the monomeric form in PLB985 cells differentiated in neutrophils. Moreover, we have identified the surface-exposed peptides from the monomeric PCNA which are used as competitors of interactions between PCNA and its partner in the cytosol of neutrophils. These peptides modulate neutrophils survival. Thanks to the analysis of Mass Spectrometry, we have identified new partners of PCNA in the neutrophil cytosol involved in several metabolic pathways. This suggests that PCNA regulates neutrophil survival by interacting with different cytosolic proteins. Among the identified partners, we have found the cytosolic subunits of the NADPH oxidase, the enzyme responsible of the reactive oxygen species production, at the base of the neutrophil microbicidal activity. We have shown especially the interaction between p47phox and PCNA. Finally, we have investigated the functional implication of the interaction of PCNA with the NADPH oxidase in PLB985 cells and also in human neutrophils. Taken altogether, results suggest that the cytosolic PCNA maintains the resting state of neutrophils, and it helps the assembly of the NADPH oxidase when activated. The protein network associated with PCNA regulates the activity and the survival of neutrophil by modulating several pathways.

O crescimento de uma camada neoíntima é o marcador central do processo aterosclerótico, bem como do remodelamento vascular associado à reestenose após intervenções vasculares terapêuticas, tais como angioplastia ou colocação de stents. A célula neointimal é essencialmente uma célula com fenótipo muscular liso indiferenciado, cuja origem pode ser múltipla e com característica ação secretora de matriz extracelular. Dentre os fatores que coordenam a (des)diferenciação, proliferação e migração destas células, há evidências de que processos redox tenham papel preponderante, porém os mecanismos de tais processos não estão claros. A compreensão mais profunda de tais mecanismos redox tem sido dificultada pela falta de modelos de células neointimais cultivadas. Os objetivos específicos deste trabalho são: 1) Desenvolver um modelo de cultura de células neointimais de artéria ilíaca de coelho obtidas após lesão por catéter-balão. 2) Avaliar em tais células índices do estado redox e potenciais fontes enzimáticas de ERO, com ênfase no complexo NAD(P)H oxidase. 3)Avaliar marcadores de estresse do retículo endoplasmático e sua correlação com os índices do estado redox. 4) Estudar o efeito de estímulos proapoptóticos como deprivação de soro, estressores do RE e particularmente administração exógena de NO na viabilidade e estado redox de células neointimais. Os resultados obtidos demonstram que: é possível obter células neointimais em cultura de modo reproduzível e estável. Células provenientes da artéria lesada mantém um estado persistente de aumento do estresse oxidativo. O estresse oxidativo nessas células decorre de produção aumentada de radical superóxido e ativação do complexo da NADPH oxidase vascular. Diferentemente do observado em vasos lesados, os marcadores do estresse do reticulo endoplasmático não se apresentam alterados se comparados a células musculares lisas normais. Células neointimais têm resposta aumentada a agonistas da NADPH oxidase e estressores celulares. A exposição a óxido nitrico promove aumento da produção de superóxido, particularmente acentuado em células neointimais. As curvas de viabilidade celular indicam uma sensibilidade aumentada a estressores do RE, doadores de NO e particularmente a oxidantes exógenos. Em conjunto, estes resultados permitem concluir que o fenótipo neointimal é um fenótipo de estresse oxidativo acentuado e persistente, mesmo em condições de cultura celular, e que este estresse oxidativo decorre pelo menos em parte da ativação do complexo NADPH oxidase no contexto de uma adaptação à resposta celular integrada ao estresse. Estes dados indicam novas perspectivas no entendimento dos mecanismos envolvidos na fisiopatologia redox da neoíntima, podendo suscitar o desenho de intervenções terapêuticas racionais. / Formation of a neointimal layer is the hallmark of most vascular diseases, such as atherosclerosis and restenosis after angioplasty. Neointimal cells display an undifferentiated noncontractile smooth muscle phenotype with marked extracellular matrix secretion. Their origin can be multiple. Among factors that govern the (de)differentiation, proliferation and migration of neointimal cells, there is evidence for a key role of redox processes, but the underlying mechanisms are unclear. Advancing the knowledge about such redox mechanisms has been difficulted by the absence of a reproducible method of neointimal cell culture. The objectives of this work are: 1) To develop a model of neointimal cell culture, in which cells are harvested from rabbit iliac arteries 14 days after overdistention balloon injury. 2) To assess the redox status in such neointimal cells and the possible enzymatic source of reactive oxygen, with emphasis in the NAD(P)H oxidase complex. 3) To investigate the expression of endoplasmic reticulum stress markers and their corralation with redox status. 4) To investigate the effects of proapoptotic stimuli such as serum deprivation, endoplasmic reticulum stressors and particularly the exogenous administration of nitric oxide in viability and redox status of neointimal cells. Our results show that it is possible to harvest and cultivate neointimal cells after balloon injury. The neointimal cells in culture, even after several passages, exhibit increased indexes of oxidative stress. Oxidative stress in such cells is associated with increased activation of the vascular NAD(P)H oxidase complex. Contrarily to what was observed in healing arteries harvested from in vivo rabbits, markers of ER stress did not show any change when compared with primary smooth muscle cells kept in similar conditions. Oxidative stress response was increased after NADPH oxidase agonists; in particular, exposure to exogenous nitric oxide markedly increased superoxide radical production in neointimal cells. Cell viability curves showed increased sensitivity to ER stressors, NO donors and, particularly, exogenous oxidants. Therefore, the neointimal phenotype is a phenotype of intrinsic sustained oxidative stress even after several passages in culture. Such oxidative stress is due at least in part to activation of the NAD(P)H oxidase complex in the context of adaptation to an integrated stress response. This data provide new perspectives to understand redox mechanisms associated with neointimal pathophysiology and can lead to development of rational therapeutic interventions.