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: 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

Orientador: Sisi Marcondes Paschoal / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T22:06:04Z (GMT). No. of bitstreams: 1 Lopes-Pires_MariaElisa_D.pdf: 1241899 bytes, checksum: a13986c42dd2369a280228a76009db4c (MD5) Previous issue date: 2013 / Resumo: A sepse e ainda causa de muitos óbitos em hospitais do mundo todo. A gravidade da sepse esta relacionada ao estado de ativação de plaquetas. Trabalho prévio do nosso grupo mostrou que o tratamento de ratos com lipopolissacarideo (LPS) leva a inibição da agregação plaquetaria e aumento da formação de espécies reativas de oxigênio (EROs) via NADPH oxidase. Entretanto, o efeito inibitório do LPS na agregação não e dependente da liberação de EROs. Portanto, o objetivo do presente trabalho foi investigar as vias de sinalização envolvidas na inibição da agregação e na ativação da NADPH oxidase em plaquetas de ratos tratados com LPS. Para tanto, ratos Wistar machos foram injetados com LPS (1 mg/kg, i.p.) e apos 6h ou 48h o sangue foi coletado. A agregação plaquetaria foi induzida por ADP (10 ?M) na ausência e na presença de diferentes inibidores enzimáticos. A formação de EROs em plaquetas foi determinada por citometria de fluxo utilizando a sonda fluorescente DCFH-DA e a concentração intraplaquetaria de GMPc por imunoensaio. Também foram realizados ensaios de western blotting para a analise da ativação das enzimas c-Src, AKT e NADPH oxidase, bem como para a detecção de proteínas contendo resíduos de nitrotirosina. A analise do western blotting mostrou que a fosforização da c-Src quinase no resíduo Tyr 416, que indica ativação da enzima, foi semelhante em plaquetas de ratos injetados com salina ou LPS em 6h ou 48h. Alem disso, a inibição de Src com PP2 (10 ?M) não modificou a agregação plaquetaria de ratos tratados com LPS. Nos verificamos que a inibição da agregação foi acompanhada por um aumento significativo dos níveis de GMPc, bem como da nitracao de proteínas, em plaquetas de ratos 6h ou 48h apos o tratamento com LPS. A incubação das plaquetas com o sequestrador de peroxinitrito -(-) epigalocatequina gallato (10 ?M) aumentou significativamente a agregação de ratos injetados com LPS em 48h, mas não alterou a agregação em 6h. Entretanto, a inibição da agregação plaquetaria em ratos tratados com LPS em 6h foi revertida pelo inibidor da enzima guanilil ciclase ODQ (25 ?M) ou pelo inibidor de PKG Rp-8-Br (25 ?M). De forma semelhante, o inibidor nao seletivo de PKC GF109203X (10 ?M) reverteu o efeito inibitório do LPS em 6h na agregação e reduziu os niveis de GMPc em plaquetas. Nos mostramos que a fosforização da AKT no resíduo Thr308 foi significativamente maior em plaquetas de ratos tratados com LPS quando comparado com ratos injetados com salina. A incubacao das plaquetas de ratos tratados com LPS com o inibidor de PI3K wortmannin (100 nM) nao modificou a agregação. Entretanto, o inibidor da AKT PPI-1 (20 ?M) aumentou a agregação para níveis semelhantes aos observados nos ratos injetados com salina. A agregação plaquetaria de ratos 48h apos o tratamento com LPS não foi afetada por nenhum dos inibidores enzimáticos utilizados neste trabalho. O aumento da geração de EROs em plaquetas de ratos tratados com LPS em 6h ou 48h foi acompanhado pelo aumento significativo da fosforização do resíduo Ser345 na subunidade p47-phox da NADPH oxidase. A incubação de plaquetas de ratos tratados com LPS com GF109203X inibiu a fosforização da p47-phox bem como reduziu a geração de EROS. A produção aumentada de EROs em plaquetas de ratos tratados com LPS em 6h também foi reduzida em 42% por PP2. A inibição de PI3K ou AKT não modificou a produção de EROS em plaquetas de ratos tratados com LPS. A incubação de plaquetas com ODQ ou com Rp-8-Br (5?M) reduziu significativamente a produção de EROS somente em plaquetas de ratos 48h apos o tratamento com LPS. Portanto, no presente trabalho podemos concluir que a inibição da agregação plaquetaria observada 6h apos a injeção de LPS e mediada pela via NO/GMPc/PKG e também e modulada pela PKC e AKT, enquanto que, o efeito inibitório do LPS em 48h e essencialmente dependente da formação de peroxinitrito. A produção aumentada de EROs em plaquetas de ratos tratados com LPS envolve a fosforização da subunidade p47-phox da NADPH oxidase pela PKC. Alem da PKC, são importantes no aumento da liberação de EROs em plaquetas a Src em 6h e a via GMPc/PKG em 48h apos a injeção de LPS / Abstract: Sepsis is still a cause of high mortality in hospitals all over the world and its severity is directly related to platelet activity. A previous work of our group showed that the treatment of rats with lipopolysaccharide (LPS) inhibited platelet aggregation and also increased reactive oxygen species (ROS) production which was mediated especially by NADPH oxidase. However, the inhibitory effect of LPS on platelet aggregation is independent of ROS formation. Therefore, in the present work we investigated the signaling pathways involved in the aggregation inhibition as well as in the increased ROS formation in platelets of LPS-treated rats. Male Wistar rats were injected with LPS (1 mg/kg, i.p.) and blood was collected after 6h or 48h. Platelet aggregation was induced by ADP (10 ?M) in the absence or in the presence of different enzymatic inhibitors. ROS formation in platelets was determined through flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFHDA) and cGMP intraplatelet levels by enzyme immunoassay kit. Western blotting assays were carried out to analyze AKT and NADPH oxidase activation and the presence of nitrated proteins in platelets. In the present work, we observed that the inhibition of aggregation was accompanied by a significant increase of cGMP levels as well as protein nitration in platelets of LPS-treated rats. Incubation of platelets with the peroxynitrite scavenger -(-) epigallocatechingallate (10 ?M) significantly increased aggregation of LPS-treated rats at 48h, but did not modify it at 6h. However, the inhibitory effect of LPS at 6h on platelet aggregation was reversed by the guanylyl cyclase (sGC) inhibitor ODQ (25 ?M) or by the PKG inhibitor Rp-8-Br (25 ?M). Likewise, the PKC inhibitor GF109203X (10 ?M) reversed the inhibition of aggregation and the increased cGMP levels in platelets of LPS-treated rats at 6h. We demonstrated that AKT phosphorylation at Thr308 was significantly higher in platelets of LPS-injected rats than in the saline group. The AKT inhibitor PPI-1 (20 ?M) increased platelet aggregation of rats 6h after LPS-injection to the levels comparable to the saline group, despite of the PI3K inhibitor wortmannin (100 nM) has had no effect. Platelet aggregation of rats 48h after LPS injection was not affected by any enzymatic inhibitors used in this work. Increased ROS formation in platelets of LPS injected rats at 6h or 48h was followed by a marked increase of the NADPH oxidase subunit p47-phox phosphorylation at Ser345. Incubation of platelets of LPS-injected rats with GF109203X inhibited the p47-phox phosphorylation as well as ROS generation. The increased ROS production in platelets of rats 6h after LPS-injection was reduced 42% by PP2. Inhibition of both PI3K and AKT did not change ROS production in platelets of LPS-injected rats. Incubation of either ODQ or Rp-8-Br (5 ?M) reduced significantly the ROS production just in platelets of rats 48h after LPS-injection. Therefore, our results show that the inhibition of ADP-induced platelet aggregation of rats 6h after LPS injection is mediated by NO/cGMP/PKG-dependent mechanisms, and PKC and AKT probably act upstream upregulating this pathway. On the other hand, the inhibitory effect of LPS at 48h on platelet aggregation is essentially dependent on ONOO- production. In addition, our results show that the augmented ROS production in platelets of LPS-treated rats is mediated by PKCdependent phosphorylation of p47-phox. Besides PKC, the increased ROS formation in platelets is also modulated by Src at 6h after LPS injection, while NO/cGMP/PKG pathway takes part of this effect at 48h / Doutorado / Farmacologia / Doutora em Farmacologia

Le symporteur Na+/I- (NIS) médie le captage de l'iode dans la glande thyroïde et cette propriété est exploitée depuis de nombreuses années en thérapeutique pour traiter les cancers différenciés de la thyroïde à l’iode radioactif 131 (Radiothérapie métabolique ou RAI). Cependant, 5 à 10% des patients deviennent réfractaires à la RAI, ce qui indique un mauvais pronostic. La réduction de l'expression NIS et son internalisation sont caractéristiques de ce processus. La mutation activatrice BRAFV600E est la plus fréquemment identifiée au sein des cancers différenciés de la thyroïde de type papillaires (CPT), qui est le type le plus répandu. Dans les thyrocytes de souris, BRAF muté induit la sécrétion de TGF qui active ensuite la voie Smad ce qui entraîne la répression du NIS. La NADPH oxydase 4 (NOX4), enzyme génératrice d’espèces réactives de l'oxygène (ROS), est un médiateur clé de la signalisation du TGFß dans de nombreux types cellulaires et sa surexpression a été détectée dans le cancer de la thyroïde. L'objectif de ce travail est d'évaluer si NOX4 est un médiateur de la répression NIS induite par BRAFV600E dans des lignées cellulaires thyroïdiennes. En utilisant une lignée cellulaire de thyroïde normal de rat (PC-BRAF), nous avons démontré que le TGF-β ou l'expression BRAFV600E promeut la diminution de l'ARNm NIS ainsi que celle du captage de l'iodure en revanche ils augmentent l’expression de l'ARNm NOX4. Le silençage de Nox4 par siRNA ou un traitement des cellules par SIS3, un inhibiteur de pSmad3, inhibe la répression du NIS médiée par BRAFV600E, indiquant l'implication de la voie Smad3 et de Nox4 dans cette répression. Dans la lignée tumorale BCPAP derivée d’un CPT humain et porteuse de la mutation BRAFV600E, nous avons également observé une augmentation de l'expression de l'ARNm NIS lorsque BRAFV600E ou NOX4 sont inhibés. Dans les cellules BCPAP, un traitement par H2O2 augmente l'expression de la protéine ADN méthyltransférase 1 (DNMT1) dans la fraction cellulaire enrichie en protéines liées à la chromatine. Un traitement par des antioxydants ou le silençage de NOX4 réduise ce recrutement. Le TGF augmente le niveaux de protéines DNMT1 dans la fraction cellulaire enrichie en chromatine, lequel qui est renversé par un traitement par un inhibiteur de NADPH oxydase : le Diphenyleneiodonium (DPI). La méthylation de l'ADN induite par le TGF et l'hypoacétylation de l'histone H3K9/K14, qui sont des marques de répression de la transcription, sont détectées au niveau du promoteur NIS. L’ensemble de ces données suggère que Nox4 est un acteur clé de la signalisation TGF-BRAFV600 et joue un rôle répressif sur l'expression du NIS, probablement par des mécanismes épigénétiques. Cette étude apporte des données fonctionnelles pour le développement de nouveaux outils thérapeutiques. / The co-transporter Na+/ I- (NIS) mediates iodide uptake in thyroid gland which is a key step in hormonal biosynthesis. Iodide accumulation by thyrocytes is the basis of radioiodine therapy (RAI) which is the standard post-surgery therapeutic approach to efficiently eliminate remaining cancer lesions and metastasis of differentiated thyroid cancer (DTC). However, 5-10% of DTC patients become RAI-refractory which is indicative of poor prognosis. Reduced NIS expression and NIS internalization are in this process. BRAFV600E mutation is the most common genetic event in papillary thyroid cancers (PTCs), the most prevalent type of DTC. In rat thyrocytes, BRAFV600E induces secretion of TGFβ that activates Smad pathway resulting in NIS downregulation and overexpression of TGFβ is associated with NIS repression in patients. NADPH oxidase NOX4, a professional reactive oxygen species (ROS) generating enzyme, is a key mediator of TGFβ signaling in many cell types and has been previously demonstrated to be overexpressed thyroid cancers. To better understand the molecular mechanisms involved in PTC loss of iodine avidity, the aim of this work is to evaluate whether NOX4 is a key player of BRAFV600E-mediated NIS repression in thyroid cell lines. Using a normal rat thyroid cell line (PC-BRAF) we demonstrated that TGF-β administration or expression of BRAFV600E resulted in reduced NIS mRNA, reduced iodine uptake and increased NOX4 mRNA expression. NOX4 silencing or treatment with SIS3 an inhibitor of Smad pathway partially inhibited NIS repression indicating the implication of both Smad pathway and NOX4. To confirm this results we used a human thyroid cancer cell lines that harbors BRAFV600E mutation (BCPAP and 8505c) and observed an increase in NIS expression followed by BRAFV600E or NOX4 downregulation. Exogenous H2O2 induced DNA methyl-transferase 1 (DNMT1) enrichment in tight-chromatin protein fraction which was decreased by antioxidants or NOX4 silencing in BCPAP cells. TGF increased DNMT1 protein levels in chromatin-enriched cell fraction which was reversed by NADPH oxidase inhibitor, Diphenyleneiodonium (DPI). TGF-mediated DNA methylation and histone H3K9/K14 hypoacetylation were detected in NIS promoter, which is a repressive transcriptional mark. The data obtained suggest that NOX4 is a mediator of BRAFV600-TGF signaling and has a repressive role over NIS expression probably through epigenetic mechanisms. These results should lead to a better understanding of NIS expression regulation in thyroid cancer, bringing functional data for the development of new therapeutic tools.

Several epidemiological studies found that hypertensive patients have an increased risk to develop kidney cancer. Hyperaldosteronism frequently results in arterial hypertension and contributes to the development and progression of kidney injury, with reactive oxygen species (ROS) playing an important role. ROS are thought to be associated with many pathological conditions such as cancer and other disorders, like cardiovascular complications , which often go along with hypertension. The aim of the present work was to investigate whether the effects of elevated aldosterone concentrations might be involved in the increased cancer incidence of hypertensive individuals. First, the potential capacity of aldosterone to induce oxidative stress and DNA damage was investigated in vitro and in vivo. In LLC-PK1 porcine kidney cells and MDCK canine kidney cells the significant formation of ROS, and especially of superoxide (O2˙ˉ) was assessed. With two genotoxicity tests, the comet assay and the micronucleus frequency test, the DNA damaging potential of aldosterone was quantified. In both genotoxicity tests a dose-dependent increase in aldosterone-induced structural DNA damage was observed. Oxidative stress and DNA damage were prevented by antioxidants, suggesting ROS as a major cause of DNA damage. Furthermore, the oxidatively modified DNA lesion 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG), was found to be significantly elevated. In kidneys of rats with desoxycorticosterone acetate (DOCA)/salt-induced hypertension, which is a model of severe mineralocorticoid-dependent hypertension, elevated levels of ROS and superoxide were found, compared to kidneys of sham rats. Also DNA strand breaks, measured with the comet assay and double strand breaks, visualized with antibodies against the double strand break-marker gamma-H2AX were significantly elevated in kidneys of DOCA/salt-treated rats. In addition, significantly increased amounts of 8-oxodG were detected. Proliferation of kidney cells was found to be increased, which theoretically enables the DNA damage to manifest itself as mutations, since the cells divide. Second, the effects of aldosterone on the activation of transcription factors and signaling pathways were investigated. A significant activation of the potentially protective transcription factor Nrf2 was observed in LLC-PK1 cells. This activation was triggered by an increase of ROS or reactive nitrogen species (RNS). In response to oxidative stress, glutathione synthesis and detoxifying enzymes, such as the subunits of the glutathione-cysteine-ligase or heme oxygenase 1 were rapidly induced after 4 h. Nevertheless, after 24 h a decrease of glutathione levels was observed. Since ROS levels were still high after 24 h, but Nrf2 activation decreased, this adaptive survival response seems to be transient and quickly saturated and overwhelmed by ROS/RNS. Furthermore, Nrf2 activation was not sufficient to protect cells against oxidative DNA damage, because the amounts of double strand breaks and 8-oxodG lesions steadily rose up to 48 h of aldosterone treatment. The second transcription factor that was time- and dose-dependently activated by aldosterone in LLC-PK1 and MDCK cells was NF-kappaB. Furthermore, a significant cytosolic and nuclear activation of ERK was detected. Aldosterone induced the phosphorylation of the transcription factors CREB, STAT1 and STAT3 through ERK. Third, the underlying mechanisms of oxidant production, DNA damage and activation of transcription factors and signaling pathways were studied. Aldosterone exclusively acted via the MR, which was proven by the MR antagonists eplerenone, spironolactone and BR-4628, whereas the glucocorticoid receptor (GR) antagonist mifepristone did not show any effect. Furthermore, aldosterone needed cytosolic calcium to exert its negative effects. Calcium from intracellular stores and the influx of calcium across the plasma membrane was involved in aldosterone signaling. The calcium signal activated on the one hand, the prooxidant enzyme complex NAD(P)H oxidase through PKC, which subsequently caused the generation of O2˙ˉ. On the other hand, nitric oxide synthase (NOS) was activated, which in turn produced NO. NO and O2˙ˉ can react to the highly reactive species ONOO- that can damage the DNA more severely than the less reactive O2˙ˉ. In the short term, the activation of transcription factors and signaling pathways could be a protective response against aldosterone-induced oxidative stress and DNA damage. However, a long-term NF-B and ERK/CREB/STAT activation by persistently high aldosterone levels could unfold the prosurvival activity of NF-kappaB and ERK/CREB/STAT in aldosterone-exposed cells. DNA damage caused by increased ROS might become persistent and could be inherited to daughter cells, probably initiating carcinogenesis. If these events also occur in patients with hyperaldosteronism, these results suggest that aldosterone could be involved in the increased cancer incidence of hypertensive individuals. / Mehrere epidemiologische Studien haben ein erhöhtes Nierenkrebsrisko bei Patienten mit Bluthochdruck aufgedeckt. Hyperaldosteronismus führt oft zu arteriellem Bluthochdruck und trägt zur Entwicklung und zum Fortschreiten von Nierenschäden bei, wobei reaktive Sauerstoffspezies (ROS) eine wichtige Rolle spielen. Immer häufiger werden ROS mit Krankheitsbildern wie Krebs und kardiovaskulären Erkrankungen, die mit Bluthochdruck einhergehen, in Verbindung gebracht. Das Ziel dieser Arbeit war es, zu untersuchen, ob erhöhte Aldosteronkonzentrationen an dem gesteigerten Krebsrisiko von hypertensiven Patienten beteiligt sein könnten. Zunächst wurde die potentielle Kapazität von Aldosteron, oxidativen Stress und DNA-Schaden in vitro und in vivo induzieren zu können, untersucht. In der Schweine-Nierenzelllinie LLC-PK1 und der Hunde-Nierenzelllinie MDCK wurde die Entstehung von ROS und speziell die Bildung von Superoxid (O2˙ˉ) nachgewiesen. Das gentoxische Potential von Aldosteron wurde mit zwei Genotoxizitätstests, dem Comet Assay und dem Mikrokernfrequenztest bestimmt. In beiden Genotoxizitätstests konnte ein dosis-abhängiger Anstieg des strukturellen DNA-Schadens beobachtet werden. Antioxidantien konnten den oxidativen Stress und die DNA-Schäden verringern, was annehmen lässt, dass ROS die Hauptursache für die Entstehung der DNA-Schäden sind. Darüberhinaus wurden signifikant erhöhte Mengen der oxidativ modifizierten DNA Läsion 8-Oxo-7,8-dihydro-2´-deoxyguanosin (8-oxodG) gefunden. In Nieren von Ratten mit Desoxycorticosteron-Acetat (DOCA) und Salz-induziertem Bluthochdruck, ein Modell für massiven Mineralocorticoid-induzierten Bluthochdruck, wurde ebenfalls eine erhöhte Bildung von ROS und O2˙ˉ in Nieren von DOCA/Salz-Ratten im Vergleich zu Sham-Ratten beobachtet. Auch im Comet Assay erfasste DNA-Strangbrüche und Doppelstrangbrüche, die mit Hilfe von Antikörpern gegen den Doppelstrangbruchmarker gamma-H2AX sichtbar gemacht wurden, waren in den Nieren der DOCA/Salz-behandelten Ratten signifikant erhöht. Weiterhin wurden erhöhte 8-oxodG-Spiegel in DOCA/Salz-Ratten beobachtet. Auch eine erhöhte Proliferationsrate in DOCA/Salz-behandelten Ratten konnte festgestellt werden, was theoretisch dazu führen könnte, dass sich die DNA-Schäden als Mutationen manifestieren, da sich die Zellen teilen. Im zweiten Teil der Arbeit wurde der Einfluss von Aldosteron auf die Aktivierung von Transkriptionsfaktoren und Signalwegen untersucht. Zunächst konnte die Aktivierung des potentiell schützenden Transkriptionsfaktors Nrf2 in LLC-PK1 Zellen mittels electrophoretic mobility shift assay (EMSA) beobachtet werden. Diese Aktivierung wurde durch den Anstieg an ROS und reaktiven Stickstoffspezies (RNS) ausgelöst. Als Antwort auf den oxidativen Stress, wurde die Glutathion-Synthese und detoxifizierende Enzyme, wie die Untereinheiten der Glutathion-Cystein-Ligase oder Hämoxygenase 1, nach 4 Stunden rasch hochreguliert. Nichtsdestotrotz konnte nach 24 Stunden eine Abnahme des Glutathionspiegels festgestellt werden. Da die Konzentration an ROS nach 24 Stunden immer noch signifikant erhöht war, die Aktivierung von Nrf2 allerdings stark zurückgegangen ist, scheint diese adaptive Überlebensstrategie nur kurzfristig, und somit schnell durch ROS/RNS gesättigt zu sein. Weiterhin war die Aktivierung von Nrf2 nicht ausreichend, um die Zellen vor dem durch Aldosteron-induzierten DNA-Schaden zu schützen, da Doppelstrangbrüche, sowie 8-oxodG-Läsionen bei bis zu 48-stündiger Inkubation mit Aldosteron stetig anstiegen. Der zweite Transkriptionsfaktor, der zeit- und dosisabhängig durch Aldosteron aktiviert wurde, war NF-kappaB. Ausserdem wurde die cytosolische und nukleäre Aktivierung von ERK nachgewiesen. Aldosteron induzierte weiterhin die Phosphorylierung der Transkriptionsfaktoren CREB, STAT1 und STAT3 durch ERK. Im dritten Teil dieser Arbeit wurden die zugrundeliegenden Mechanismen der Entstehung von ROS/RNS, des DNA-Schadens und der Aktivierung von Transkriptionsfaktoren untersucht. Aldosteron wirkte ausschließlich über den MR, bewiesen durch Einsatz der MR-Antagonisten Eplerenon, Spironolakton und BR-4628. Der Glucocorticoid-Rezeptor-Antagonist Mifepriston zeigte dagegen keinen Effekt. Weiterhin benötigte Aldosteron cytosolisches Calcium, um seine negativen Effekte auszuüben. Es waren intrazelluäres Calcium, sowie ein Calciuminflux über die Plasmamembran am Aldosteronsignal beteiligt. Einerseits wurde der prooxidative Enzymkomplex NAD(P)H-Oxidase von Calcium durch die Proteinkinase C (PKC) aktiviert, was wiederum zur Bildung von O2˙ˉ führte. Andererseits kam es durch erhöhtes cytosolisches Calcium zur Aktivierung der NO-Synthase (NOS), welche daraufhin Stickoxid (NO) produzierte. NO und O2˙ˉ können zu dem hochreaktiven Peroxynitrit (ONOO-) reagieren, welches die DNA mehr schädigen kann als das etwas weniger reaktive O2˙ˉ. Kurzfristig könnte die Aktivierung der Transkriptionsfaktoren und Signalwege eine schützende Wirkung gegen den durch Aldosteron-induzierten oxidativen Stress und DNA-Schaden in den Zellen haben. Allerdings kann eine länger anhaltende Aktivierung von NF-kappaB und ERK/CREB/STAT durch permanent hohe Aldosteronspiegel zur Induktion einer Überlebensstrategie durch NF-kappaB und ERK/CREB/STAT in Aldosteron-exponierten Zellen führen. Der DNA-Schaden, der durch erhöhte ROS-Spiegel entsteht, könnte persistent und somit an Tochterzellen weitervererbt werden, was eventuell zur Entstehung von Krebs beitragen könnte. Falls diese Effekte auch in Patienten mit Hyperaldosteronismus gefunden werden können, dann könnte Aldosteron an der erhöhten Krebsinzidenz bei Bluthochdruck beteiligt sein.

A espécie Garcinia brasiliensis, conhecida como "bacupari", é nativa da Amazônia e cultivada em todo o território brasileiro. Na medicina popular, suas folhas são utilizadas para tratar tumores, inflamações das vias urinárias e artrite, bem como para aliviar dores. Este trabalho foi conduzido para avaliar a atividade analgésica e antiinflamatória do extrato etanólico foliar (GbEE) e da 7-epiclusianona, uma benzofenona natural poliprenilada isolada de suas folhas, em modelos animais in vivo. A ação deste benzofenona também foi investigada em um ensaio antiinflamatório ex vivo, enfocando o “burst” respiratório de neutrófilos e as vias bioquímicas envolvidas. Foi constatada atividade atividade antiinflamatória tanto para GbEE v.o. ( 30, 100 3 300 mg /kg) como para 7-epiclusianona v.o.(5, 10 e 15mg/Kg) por reduzir o edema de pata induzido por carragenina e inibir o recrutamento de leucócitos na cavidade peritoneal. Além disso, através do modelo de indução de inflamação crônica foi observado uma redução estatisticamente significativa de formação do tecido granulomatoso. Por outro lado, efeito antinociceptivo periférico foi atribuído ao GbEE sugerindo ser mais eficaz contra dores inflamatórias, enquanto que para a benzofenona foi constatada tanto atividade antinociceptiva periférica como central, através de diferentes mecanismos. O composto 7-epiclusianona (10 a 100µg) também foi capaz de reduzir e até mesmo suprimir a liberação de ânion superóxido por fagócitos inflamatórios através de um mecanismo controlado pela fosforilação da proteína tirosina e pela estimulação direta da proteína quinase C, sugerindo novas abordagens terapêuticas para o tratamento de processos inflamatórios e desenvolvimento de novos fármacos. Os resultados mostraram atividades antiinflamatória e analgésica das folhas de G. brasiliensis e da benzofenona isolada, confirmando o uso tradicional dessa espécie para tratamento de dores e inflamações. / The species Garcinia brasiliensis, known as “bacupari”, is native to the Amazon and cultivated throughout Brazil. Their leaves are used in folk medicine to treat tumors, inflammation of the urinary tract and arthritis as well as to relief pain. This study was conducted to evaluate the anti-inflammatory and analgesic effects of the ethanolic extract of leaves (GbEE) and the 7-epiclusianone, a natural polyisoprenilated benzophenone isolated from G. brasiliensis, in several animal models. The action of the benzophenone was also investigated ex vivo throughout an anti-inflammatory assay, focusing neutrophil respiratory burst and the biochemical pathway. Antiinflammatory activity was found for both GbEE (30, 100 and 300 mg /kg) p.o. and for 7-epiclusianone (5, 10 and 15mg/kg) p.o. to reduce the paw edema induced by carrageenan and to inhibit leukocyte recruitment into the peritoneal cavity. In addition, through the induction model of chronic inflammation, was observed a statistically significant reduction of granulomatous tissue formation. Moreover, peripheral antinociceptive effect was attributed to GbEE suggesting that this extract could be more effective against inflammatory pain, whereas for benzophenone was verified both peripheral and central antinociceptive activity through different mechanisms.The compound 7-epiclusianone (10 to 100 μg) was also able to reduce and even suppress the release of superoxide anion by inflammatory phagocytes through a mechanism controlled by protein tyrosine phosphorylation and by direct stimulation of protein kinase C, suggesting new therapeutic approaches for the treatment of inflammatory processes and development of new drugs. So, it was demonstrated the anti-inflammatory and antinociceptive activities of the leaves of G. brasiliensis and the benzophenone isolated which supports previous claims of the traditional use of this species against inflammation and pain. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Neutrophils, essential innate immune cells, recognize danger signals through receptors on their surface. Upon receptor ligation, neutrophils may undergo priming, a process involving limited reactive oxygen species (ROS) generation and partial degranulation. Priming facilitates neutrophil migration and prepares the cell for an enhanced response to a secondary stimulus, including a spike in ROS generation by NADPH oxidase 2 (NOX2). It is well established that NOX2-derived oxidants are involved in pathogen killing and that off-target effects can cause host tissue damage; however, several lines of recent evidence also support an anti-inflammatory function for NOX2 oxidants. First, patients with chronic granulomatous disease exhibit sterile inflammatory phenomena. Second, neutrophils lacking NOX2 function (genetically or pharmacologically) have an inflammatory phenotype under resting conditions. Finally, NOX2-deficient mice exhibit enhanced localized inflammation in several disease models. The goals of this thesis were to investigate an anti-inflammatory function for NOX2 during systemic inflammation and to further elucidate mechanisms of neutrophil priming, with particular focus on priming through Toll-like receptor 2 (TLR2). Using a murine model of sterile systemic inflammatory response syndrome (SIRS), we observed that NOX2-deficient mice had dramatically increased mortality compared to WT mice. While both genotypes developed SIRS, characterized by hypothermia, hypotension, and leukopenia, the WT mice recovered within 48 h whereas the NOX2-deficient mice did not. Moreover, NOX2 function limited the extent of pulmonary pathology as significant lung injury was noted in the NOX2-deficient mice compared to the WT mice. Plasma analysis revealed that several inflammatory cytokines were persistently elevated in the NOX2-deficient mice, likely contributing to the ongoing inflammatory response. One of the complications seen in human SIRS patients is the development of multiple organ dysfunction syndrome (MODS). Thus, we next investigated the role of NOX2 in the progression from SIRS to MODS. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histology showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation. As we utilized a TLR2/6 agonist (zymosan) to induce SIRS in our in vivo model, we wanted to investigate neutrophil priming through TLR2 in an in vitro model. Notably, we determined that a TLR2/6 agonist, FSL-1, primed neutrophils from all donors to a similar extent, evidenced by direct and primed ROS generation, MAPK signaling, limited degranulation, and cytokine secretion. Surprisingly, Pam3CSK4, a TLR2/1 agonist, primed neutrophils from a subset of donors to a much greater extent than neutrophils from other donors. We demonstrated that the different neutrophil priming responses were the consequence of a common TLR1 polymorphism. In sum, the data presented here significantly advance our understanding of the roles of NOX2 and TLR2 signaling in host inflammation and neutrophil priming. This research could advance the development of therapies that target pathogenic neutrophil subsets in inflammatory conditions without compromising innate immune function

Les formes réactives de l'oxygène (FRO) ont été analysées dans différents compartiments cellulaires en utilisant des méthodes spectroscopiques (UV/VIS, fluorescence, infrarouge, résonance paramagnétique électronique). L'identité et les mécanismes catalytiques des enzymes qui produisent les FRO dans les membranes plasmiques (MP) et les mitochondries ont été étudiés, ainsi que le rôle protectif de l'oxydase terminale plastidiale (PTOX) des chloroplastes. Cd2+ s'est révélé être un inhibiteur de la NADPH oxydase des MP. In vivo Cd2+ inhibait la production extracellulaire de O2•- mais stimulait l'accumulation de H2O2. Dans des mitochondries isolées, Cd2+ a augmenté la production de FRO. Antimycin A a entraîné une élévation du H2O2 extracellulaire, confirmant que la mitochondrie est le site principal de production de l'H2O2 extracellulaire induite par Cd2+ in vivo. Une quinone réductase (QR) génératrice de FRO a été isolée des MP. La déprotonation pH-dépendante du quinole a produit des formes intermédiaires instables qui génèrent des FRO par réaction avec O2. Des espèces quinoniques ont été détectées dans la MP et pourraient servir de substrat aux QR in vivo. La protection de la chaine photosynthétique de transfert d'électron par la plastoquinol:O2 oxydoréductase a été étudiée chez des plantes PTOX+ surexprimant PTOX. En raison de leur réponse altérée en conditions de faible et forte intensité lumineuse, il a été proposé que pour fonctionner comme enzyme protectrice, PTOX est couplée à une SOD. Chez les lignées PTOX+, le niveau de SOD chloroplastique n'était pas plus élevé, limitant probablement leur capacité à détoxifier les taux élevés de O2•- généré.