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Made available in DSpace on 2018-11-26T17:41:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-11-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) National Institute of Health Ru(terpy)(bdq)NO](3+) (TERPY) is a nitric oxide (NO) donor that promotes relaxation of the mesenteric artery and aorta in rats. We sought to investigate whether it acts as both an NO donor and endothelial NO synthase (eNOS) activator, as shown previously for nitroglycerin. Human umbilical vein endothelial cells (HUVECs) and human embryonic kidney 293 cells transfected with empty vector (HEK) or eNOS cDNA (HEK-eNOS) were treated with TERPY (1 mu M) for different lengths of time. eNOS expression, dimerization, and Ser(1177) phosphorylation, caveolin-1 (Cav-1) oligomerization, Cav-1 Tyr(14) phosphorylation were evaluated by Western blotting. Studies also assessed the production of reactive oxygen/nitrogen species (ROS/RNS) in HUVECs and HEK-eNOS cells. In HEK cells devoid of eNOS, TERPY released NO without additional stimulus indicating that is an NO donor. Moreover, in HEK-eNOS cells, TERPY-induced NO production that was blocked by L-NAME. In addition, TERPY increased ROS and ONOO- production which were blocked by more than 80% by BH4 (essential eNOS co-factor) and eNOS siRNA. These results suggest that TERPY-induced ROS and ONOO- production were originated from eNOS. HUVECs stimulated with TERPY showed increased eNOS Ser(1177) and Cav-1 Tyr(14) phosphorylation, and decreased eNOS dimerization, Cav-1 oligomerization, and Cav-1/eNOS interaction after 20 min. It suggests that TERPY induces eNOS hyperactivation and uncoupling by disrupting Cav-1/eNOS interaction and depleting BH4. Endothelium-dependent vasodilation in response to NO donor TERPY is associated with eNOS activation and uncoupling, and thereby appears to be mediated, at least in part, via eNOS-dependent ROS/RNS production. Sao Paulo State Univ, Sch Dent, Dept Basic Sci, Programa Multicentr Posgrad Ciencias Fisol, Aracatuba, Brazil Univ Sao Paulo, Sch Pharmaceut Sci, Dept Phys & Chem, Ribeirao Preto, Brazil Univ Illinois, Dept Med, Chicago, IL USA Univ Illinois, Dept Anesthesiol, Chicago, IL USA Univ Illinois, Dept Pharmacol, Chicago, IL USA Sao Paulo State Univ, Sch Dent, Dept Basic Sci, Programa Multicentr Posgrad Ciencias Fisol, Aracatuba, Brazil CNPq: 400164/2014-0 CNPq: 232217/2014-9 National Institute of Health: HL60678 National Institute of Health: HL125356

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Toxoplasma gondii é um protozoário parasito intracelular obrigatório que infecta uma ampla variedade de vertebrados de sangue quente, incluindo seres humanos. Muitos estudos demonstram que a ciclooxygenase-2 (COX-2) é um potente modulador da resposta imune em vários tipos de infecção. No entanto, o papel da ciclooxygenase ainda não é claro durante uma infecção causada por T. gondii. Neste sentido, o objetivo deste estudo foi verificar o papel funcional da COX-2 em roedores Calomys callosus infectados por T. gondii, um excelente modelo experimental in vivo tradicionalmente utilizado para estudar a toxoplasmose. Para este fim, fêmeas de C. callosus foram infectadas com cinquenta cistos de T. gondii (cepa ME49), tratadas com inibidores da ciclooxygenase Meloxicam (inibidor de COX-1 e COX-2) e Celecoxibe (inibidor seletivo de COX-2), e avaliadas a cada 48 horas para a verificação da alteração de peso corporal e morbidade durante 40 dias consecutivos. Como controle, os animais foram infectados e não tratados com inibidores da ciclooxygenase. Depois de 40 dias de infecção, os cérebros foram coletados e processados para imuno-histoquímica ou PCR em tempo real para detecção de T. gondii, enquanto o soro dos animais foi avaliado para detecção de citocinas. Além disso, macrófagos peritoneais de C. callosusnão infectados ou infectados com a cepa RH de T. gondii foram tratados com Meloxicam ou Celecoxibe a fim de avaliar a proliferação intracelular do parasito por ensaio de beta-galactosidase, produção de citocinas por ELISA e produção de nitrito por meio do método de Griess, após 24 h de infecção. Os resultados mostraram que a morbidade dos animais foi alterada após a infecção por T. gondii, independentementedo tratamento. Em relação à alteração de peso corporal, animais tratados com Celecoxibe apresentaram uma perda de peso menor quando comparados com seu respectivo controle. A imuno-histoquímica e PCR em tempo real realizados no tecido cerebral mostraram uma redução significativa de cistos teciduais em animais tratados com ambos os inibidores, quando comparados com animais infectados e não tratados. Além disso, observou-se que ambos os tratamentos com os inibidores foram capazes de controlar a proliferação intracelular de T. gondii em macrófagos peritoneais. No soro de C.callosus, os dados do ELISA mostraram que animais tratados com Meloxicam apresentaram uma tendência para maior produção de MIF quando comparados ao controle. Já no sobrenadante de macrófagos peritoneais, houve um aumento na produção de TNF-α e uma redução de IL-6, e o ensaio de Griess demonstrou que ambos os tratamentos induziram uma alta produção denitrito. Todos estes resultados indicam que as ciclooxygenases são capazes de aumentar a susceptibilidade a infecção por T. gondii, uma vez que a inibição destes mediadores induz o controle significativo da infecção. Em conclusão, os nossos resultados mostram que a COX-1 e COX-2 são importantes para aumentar o processo infeccioso por T. gondii no cérebro e macrófagos peritoneais de C.callosus. Toxoplasma gondii is an obligate intracellular protozoan parasite that infects a wide range of warm-blooded vertebrates, including humans. Many studies show that the ciclooxygenase-2 (COX-2) is a potent modulator of immune response in multiple types of infection. However, the role of cyclooxygenase during an infection by T. gondii is still not clear. Therefore, the aim of this study was to investigate the functional role of COX-2 in Calomys callosus rodents infected with T. gondii, an excellent in vivo experimental model traditionally used for studying toxoplasmosis. For this purpose, C. callosus females were infected with fifty cysts of T. gondii (ME49 strain), treated with ciclooxygenase inhibitors as Meloxicam, COX-1 and COX-2 inhibitor, and Celecoxib, COX-2 selective inhibitor, and evaluated every 48 hours to check the body weight change and morbidity for 40 consecutive days. As a control, the animals were infected and not treated with ciclooxygenase inhibitors. After 40 days of infection was performed collection and processing of the brains for immunohistochemistry or real-time PCR for the detection of T. gondii, while evaluating the serum of animals for the detection of cytokines by ELISA. Furthermore, the peritoneal macrophages of C. callosus, uninfected or infected with T. gondii RH strain, were treated with Meloxicam or Celecoxib in order to evaluate the parasite intracellular proliferation by beta-galactosidase assay, and the supernatant was collected for cytokine detection by ELISA and production of nitrite by Griess method, after 24h of infection. The results showed that the morbidity of the animals changed after infection by T. gondii, regardless of both treatments. Regarding the change in body weight, animals treated with Celecoxib had a smaller loss of weight when compared to their respective control. Immunohistochemistry and real-time PCR performed on brain tissue showed a significant reduction of tissue cysts in animals treated with both inhibitors when compared to infected and untreated animals. Besides, it was observed that both treatments with the inhibitors were able to control the T. gondii intracellular proliferation in peritoneal macrophages. On the serum of C. callosus, the ELISA data shows that animals treated with Meloxicam showed a tendency to increase the MIF production when compared to the PBS control. In the supernatant of peritoneal macrophages, there was an increase in TNF-α production and IL-6 reduction, and Griess assay showed that both treatments induced a high production of nitrite. All these results indicate that cyclooxygenases are capable of favoring infection by T. gondii, since inhibition of these mediators induced significant control of infection. In conclusion, our results show that COX-1 and COX-2 are important to facilitate the T. gondii infection in the brain and peritoneal macrophages of C. callosus. Dissertação (Mestrado)

Immune complex (IC) deposition in tissues triggers the release of harmful oxidant and lytic compounds by neutrophils. We examined how ten 3-phenylcoumarin derivatives affect the reactive oxygen species (ROS) production by IC-stimulated human neutrophils. Most of the 3-phenylcoumarins inhibited the luminol-enhanced chemiluminescence (CL-lum) more strongly than they inhibited the lucigenin-enhanced chemiluminescence (CL-luc), without clear signs of toxicity. The most effective CL-lum inhibitors, 6,7-dihydroxy-3-[3′,4′-methylenedioxyphenyl]-coumarin (5) and 6,7-dihydroxy-3-[3′,4′-dihydroxyphenyl]-coumarin (19), also inhibited myeloperoxidase activity more potently and had higher hypochlorous acid scavenging ability, but did not affect the NADPH-oxidase activity. The type, number, and position of the substituent influenced the pharmacological effects of 3-phenylcoumarins; however, the structural requirements for CL-lum and CL-luc inhibition were a little different. Compounds 5 and 19 are promising prototypes of therapeutic molecules to modulate ROS production by neutrophils in IC-mediated inflammatory diseases.