Foram investigadas as propriedades, a reatividade e a estrutura eletrônica dos complexos trans-[RuII(NH3)4(L)(NO)](X)3 [(1) L = 4-pic, (2) L = P(OEt)3] e trans-[RuIII(NH3)4(4-pic)(L\')](CF3SO3)3 [(3) L\' = NH3, (4) L\' = H2O] através de técnicas espectroscópicas, eletroquímicas e cálculos teóricos. A reatividade dos complexos foi avaliada com glutationa (GSH) e L-cisteína (CisSH). Nitroxil (HNO) e óxido nítrico (NO•) são os produtos da reação entre os complexos 1 e 2 com GSH e CisSH em solução aquosa (pH 4,0 e 7,5). Óxido nítrico, livre (NO•) e coordenado (RuII-NO•), foi identificado através de quimiluminescência (NOA) e EPR, respectivamente. HNO foi sugerida indiretamente por RMN 15N após a identificação da formação de amônia (NH3), hidroxilamina (NH2OH) e sulfinamida (GS(O)NH2). Os complexos 3 e 4 foram isolados e estudados experimentalmente por EPR, UV-vis, Voltametria Cíclica, Cristalografia de Raios X e teoricamente por LFT e DFT. A acidez (pKa) e a constante de substituição do ligante H2O pelo íon Cl- no complexo 3 foram calculados como 3,65 ± 0.05 (25 °C) e 2,5×10-4 M-1s-1 (55 ºC), respectivamente. Os espectros de EPR do complexo 3 apresentaram uma simetria axial (g⊥ > g||) mas com um crescente componente rômbico que demonstrou ser função da basicidade do solvente e da sua interação com o ligante H2O. Não foi observado efeito similar para o complexo 4 ou mesmo para o complexo [RuIII(NH3)5(H2O)](CF3SO3)3. A partir de cálculos de DFT demonstrou-se que a capacidade doadora π do ligante H2O é intensificada quando a interação com moléculas do solvente promove sua rotação no eixo de ligação N(pic)-Ru-O(aq) contendo o ligante receptor π 4-picolina. Uma análise usando LFT demonstrou que de fato um componente rômbico pode ser evidenciado de uma simetria C2v por uma simples torção do ligante H2O no eixo C2. Os complexos 3 e 4 também foram investigados quanto a sua capacidade de oxidar glutationa em solução aquosa nos pH 4,0 e 7,5. A reação teve como produtos os íons trans-[RuII(NH3)4(4-pic)(H2O)]2+(λmax = 394 nm, ε = 5400 M-1cm-1) e [RuII(NH3)5(4-pic)]2+ (λmax = 399 nm, ε = 6400 M-1cm-1) identificados por UV-vis, além de GSSG, originado da reação do radical GSo, e identificado por RMN 1H. A velocidade da formação dos complexos de Ru(II) nestas reações apresentou-se mais lenta na presença de EDTA porém voltando a aumentar com a adição do captador de radical PBN. Respectivamente, essas variações foram atribuídas ao efeito catalítico promovido por íons de metais de transição em solução e à captação do radical GS•, potencialmente um agente oxidante dos complexos de Ru(II). / The proprieties, reactivity and electronic structure of trans-[RuII(NH3)4(L)(NO)](X)3 [(1) L = 4-pic, (2) L = P(OEt)3] e trans-[RuIII(NH3)4(4-pic)(L\')](CF3SO3)3 [(3) L\' = NH3, (4) L\' = H2O] were studied through spectroscopic and electrochemical techniques, and theoretical calculations. The reactivity study of these complexes was performed with the glutathione (GSH) and L-cysteine (CisSH). Nitroxil (HNO) and nitric oxide (NO •) are the products from the reaction of 1 and 2 with GSH and CisSH in aqueous solution (pH 4.0 and 7.5). Free nitric oxide (NO •) and coordinated nitric oxide (RuII-NO •) were identified by chemiluminescence (NOA) and EPR, respectively. HNO was indirectly suggested by 15N NMR data by identifying ammonia (NH3), hydroxylamine (NH2OH) and sulfinamide (RS(O)NH2). The complexes 3 and 4 were isolated and studied experimentally by EPR, UV-vis, cyclic voltammetry, X-Ray Crystallography and theoretically by DFT and LFT. The acidity (pKa), and the constant replacement of the H2O ligand in the complex 3 by the ion Cl- were calculated as 3,65 ± 0.05 (25 °C) and 2.5 × 10-4 M-1s-1 (55 °C), respectively. The EPR spectra of complex 3 showed an axial symmetry (g⊥ > g||) but with a growing rhombic component that proved to be due to the solvent basicity and its interaction with the H2O ligand. No similar effect was observed for the compound 4 or to the [RuIII(NH3)5(H2O)](CF3SO3)3 complex. According to DFT calculations the H2O π-donor ability increased when the interaction with solvent molecules promotes its rotation on the bonding axis N(pic)-Ru-O(aq) containing the pi;-acceptor ligand 4-picoline. A LFT analysis showed that a rhombic component can be evidenced in a C2v symmetry by a simple twist of the H2O ligand in the C2 axis. The complexes 3 and 4 were also investigated for their ability to oxidize glutathione in aqueous solution at pH 4.0 and 7.5. The reaction produced the trans-[RuII(NH3)4(4-pic)(H2O)]2+ (λ max = 394 nm, ε = 5400 M-1cm-1) and [RuII(NH3)5(4-pic)]2+ (λ ;max = 399 nm, ε = 6400 M-1cm-1), besides of GSSG, originated from GSo radical reaction, which was identified by 1H NMR. The rate for Ru(II) formation in these reactions were slower in the presence than in absence of EDTA. This late effect can be reduce by the addition of the radical scavenger PBN. These variations have been interpreted respectively on basis of the transition metal ions catalytic effect and to the scavenger of GS • radical, a potentially oxidizing agent of Ru(II) complexes.

Neste trabalho propusemos a utilização do fotossensibilizador ZnPC associado ao complexo nitrosilo de rutênio Ru-tpy por meio de lipossomas de longo tempo de circulação, com o objetivo de analisar o efeito sinérgico das espécies reativas de oxigênio (EROs) e espécies reativas de nitrogênio (ERONs) geradas respectivamente pelos mesmos, atuando sobre uma linhagem celular neoplásica. Foram estudadas as propriedades fotofísicas e fotoquímicas do sistema misto ZnPC/Ru-tpy empregando-se técnicas de espectroscopia no estado estacionário e resolvido no tempo. Com isto foi possível determinar importantes parâmetros que elucidaram seu perfil fotodinâmico, confirmando sua viabilidade para aplicação em estudos in vitro e in vivo. Estes estudos também indicaram uma possível interação entre a ZnPC e o complexo Ru-tpy através do mecanismo de transferência de elétron do fotossensibilizador para o complexo de rutênio, desta forma liberando o NO. Realizamos os estudos em meio biológico utilizando a linhagem neoplásica B16-F10, avaliando a toxicidade dos sistemas lipossomais na ausência e na presença de luz. Os nossos resultados demonstraram que o sistema misto ZnPC/Ru-tpy em meio lipossomal apresenta propriedades fotofísicas e fotobiológicas úteis, gerando as espécies reativas (EROs e ERONs) atuando sinergicamente pela TFD. / We proposed in this work the use of photosensitizer Zinc phthalocyanine (ZnPC) associated with the nitrosyl ruthenium complex (Ru-tpy) in long circulation liposomes (stealth liposome), with the aim of analyzing the synergistic effects of the reactive of oxygen species (EROs) and reactive of nitrogen species (ERONs) generated, acting on the neoplastic cell line. The photophysical and photochemical properties of the mix system ZnPC/Ru-tpy were studied being used spectroscopic techniques in the stationary state and resolved in the time. With these studies were possible to determine important parameters that elucidated its photodynamic profile, confirming the viability for application in studies in vitro and in vivo. These studies also indicated the possible interaction between ZnPC and the complex Ru-tpy through the electron transfer process from the photosensitizer to the nitrosyl ruthenium complex which leads to release nitric oxide (NO). We accomplished the biological studies using the neoplastic cell line B16-F10, evaluating the toxicity of the liposomes in the absence and presence of light. Our results demonstrated that the system ZnPC/Ru-tpy in the stealth liposome presents useful photophysical and photobiological properties, generating the species reactives (EROs and ERONs) to work synergically for the Photodynamic Therapy (PDT).

Araucaria angustifolia é uma conífera nativa do Brasil de importância econômica, que, após intenso desmatamento, possui sua distribuição limitada a aproximadamente 3% da área original. Atualmente está incluída na lista de espécies ameaçadas de extinção, na categoria de perigo crítico de acordo com a IUCN (International Union for Conservation of Nature). A embriogênese somática é um processo em que, através da técnica de cultivo in vitro, células isoladas ou um pequeno grupo de células somáticas dão origem a embriões. Este é um sistema com potencial de aplicação em conservação, reflorestamento, propagação em larga escala, e melhoramento vegetal em espécies arbóreas recalcitrantes e em risco de extinção. Um dos principais fatores limitantes, nos sistemas de embriogênese somática em arbóreas, é a baixa freqüência de maturação e conversão de embriões somáticos em plântulas. Durante a fase de maturação, os embriões somáticos acumulam substâncias de reserva, reduzem a atividade metabólica e adquirem tolerância à desidratação. A suplementação do meio de cultura com determinados reguladores de crescimento e agentes osmóticos, os quais permitem a progressão do desenvolvimento normal dos embriões somáticos, promove um estresse hídrico. Esta situação mimetiza aquela que ocorre durante a embriogênese zigótica quando do desenvolvimento da semente. O óxido nítrico (NO) e espécies reativas de oxigênio (ROS) são moléculas que atuam nas respostas das plantas aos vários estresses ambientais e estão envolvidas em processos de desenvolvimento como embriogênese e germinação de sementes, gravitropismo, formação de raízes. Este trabalho teve como objetivo avaliar o efeito de promotores de maturação, ABA e os agentes osmóticos, PEG e maltose, no conteúdo de NO e ROS em culturas embriogênicas de A. angustifolia. Foi observado que os agentes promotores da maturação reduzem a síntese endógena de NO e ROS. Essa redução é dose dependente. Foram pesquisadas duas linhagens de células com diferentes capacidades de maturação. Para as culturas embriogênicas com competência de maturação esses promotores induzem a maturação e nas culturas que não maturam, promovem a morte celular programada. / Araucaria angustifolia is a native conifer of economic importance in Brazil. Due its massive deforestation, this species is now limited to only 3% of the original area. A. angustifolia is currently on the list of endangered species in the category of critically endangered according to IUCN (International Union for Conservation of Nature). Somatic embryogenesis is a process that, through the technique of in vitro culture, isolated cells or a small group of somatic cells gives rise to embryos. This is a system with potential application in conservation, reforestation, large-scale propagation and breeding of tree species . A limiting factor in the systems of somatic embryogenesis in trees is the low frequency of embryo maturation and conversion into plantlets. During the maturation, somatic embryos accumulate storage substances, reduce metabolic activity and acquire tolerance to dehydration. The water stress is made by supplementation of culture medium with growth regulators and osmotic agents, which allow the normal development of somatic embryos. This situation mimics that occurs during development of the seed. Nitric oxide (NO) and reactive oxygen species (ROS) are molecules that act in plant responses to various environmental stresses and are involved in developmental processes such as embryogenesis and seed germination, gravitropism, root formation. This study aimed to evaluate the effect of promoters of maturation, ABA and osmotic agents, PEG and maltose in the generation of NO and ROS in embryogenic cultures of A. angustifolia. It was observed that the promoters of maturation reduces the endogenous synthesis of NO and ROS. We investigated two cell lines with different maturation capacities. For responsive cell line these promoters induce maturation and for blocked cell line, promote programmed cell death.

O óxido nítrico (NO) é um mensageiro biológico que tem importância vital em muitos processos fisiológicos e apresenta uma multiplicidade de funções de regulação no corpo humano, tais como neurotrasmissão, vasodilatação, participa nas respostas imunes e em vários processos associados com o desenvolvimento dos tumores. Vários estudos fornecem evidências sobre as propriedades tumoricidas do óxido nítrico e doadores de NO que podem ser usados para o tratamento de tumores malignos e são objetos de interesse na atualidade. Baseado nisto o objetivo deste trabalho foi de sintetizar e descrever aspectos estruturais dos compostos [Ru(bpy)2(dcbpy)] (dcbpy = ácido 4,4\'-dicarboxi-2,2\'-bipiridina, bpy = 2,2\'-bipyridina) [Ru(bpy)(dcbpy)2], cis-[RuCl2(dcbpy)2], cis-[Ru(dcbpy)2NO(L)]n+ (L = cloreto), [Ru(TERPY)(dcbpy)Cl] (TERPY = 2,2\': 6\'\', 2\'\' - terpiridina) and [Ru(TERPY)(dcbpy)NO], além de ensaios de citotoxicidade in vitro para alguns dos complexos sintetizados. Ensaios de citotoxicidade com solução aquosa de complexos rutênio-nitrosilo em linhagem de células metastáticas B16-F10 mostrou resultados relativamente baixos. Medidas de viabilidade celular mostrou que estes decrescem cerca de 10 % em relação ao controle. Este resultado foi interpretado como sendo devido a baixa interação entre o complexo e a célula. Bioconjugação de rutênio-nitrosilo com anticorpo policlonal IgG foi obtido por interação convalente e mostrou maior especificidade com um alvo na célula. Cromatografia de exclusão foi utilizada para separar o bioconjugado --IgG, que foi caracterizado por teste Western Blotting. Após a bioconjugação, o --IgG foi submetido a estudos de citotoxicidade com células metastáticas e a viablidade celular avaliada com ensaios MTT. Os resultados exibiram um incrível aumento na citotoxicidade de células B16-F10. Viabilidade celular foi determinada e valores de morte celular de cerca de 90 % obtida para uma das frações de --IgG. Nossos resultados demonstraram que o bioconjugado --IgG pode aumentar a resposta citotóxica e quiçá ser útil em terapia clínica. / Nitric oxide (NO) is a biological messenger that has vital importance in many physiological processes and shows a multitude of regulatory roles in the human body, such as neurotrasmission, vasodilatation, immune responses and also participates in various processes associated with cancer development. Several studies provide evidences of the tumoricidal properties of NO donors that can be used for the treatment of malignant tumors and nowadays are objects of interest. Based on this the aim of this work was synthesize compounds that in a controlled manner can deliver NO in a biological process. The synthesis, structural aspects, and in vitro cytotoxic properties of [Ru(bpy)2(dcbpy)] (dcbpy = 2,2\'-bipyridine-4,4\'-dicarboxylic acid), 2\'-bipyridine; bpy = 2,2\'-bipyridine) [Ru(bpy)(dcbpy)2], cis-[RuCl2(dcbpy)2], cis-[Ru(dcbpy)2NO(L)]n+ (L = chloride), [Ru(TERPY)(dcbpy)Cl] (TERPY = 2,2\': 6\'\', 2\'\' - terpiridine) and [Ru(TERPY)(dcbpy)NO] are described. Citotoxicity assays with aqueous nitrosyl ruthenium complex in metastatic B16-F10 cells displayed very little effect. Cell viability measurement showed decrease around 10 % in comparison to the control. It was associated due to the low interaction between nitrosyl ruthenium specie and the cell. Bioconjugation of nitrosyl ruthenium specie with polyclonal antibody IgG was achieved by covalent interaction and showed more specific interaction between bioconjugated and target cell. Exclusion chromatography was used to isolate --IgG conjugated, which was characterized by Western Blotting test. Following bioconjugation, the --IgG was submitted to cytotoxic studies with metastatic cells and the viability evaluated by MTT assay. The results displayed incredible increase of citotoxicity for B16F10 cells. Cell viability was achieved to decrease until to 90 % in comparison to the control when one fraction of --IgG was used. Taken together, the present findings demonstrate that the --IgG complex may elicit citotoxicity responses that may find useful applications in clinical therapy.

A doença periodontal é a doença crônica mais prevalente nas doenças orais. Dentre os mediadores desse processo contam-se a Resolvina E1 (RvE1), um mediador pró-resolução da inflamação capaz de diminuir a perda óssea secundária á doença periodontal em coelhos, e o oxido nítrico (NO), o qual pode ser produzido em grandes quantidades pela ação de citocinas e estimular a diferenciação e atividade osteoclástica. O objetivo deste estudo foi investigar os efeitos da RvE1 em osteoclastos (OCs) em cultura e os mecanismos envolvidos, bem como o papel do NO na progressão da periodontite experimental em ratos e as alterações sistémicas resultantes de danos oxidativos. A diferenciação de OCs foi induzida em células de medula óssea de camundongos C57BL/6 em cultura (7 dias) e tratadas com diferentes doses de RvE1. NFkB e Akt fosforilada foram analisadas por Western blotting e a expressão gênica de NO sintase (NOS) induzível (iNOS) por \"real-time\" PCR. A participação dos receptores ChemR23 e BLT-1 na resposta à RvE1 foi estudada em membranas isoladas de OCs empregando radioligantes. A perda óssea alveolar e danos em órgãos periféricos foram analisados em ratos com periodontite induzida por ligadura (P) e sob tratamento de longo prazo com o inibidor não-seletivo de NOS, L-NAME. Os animais receberam L-NAME durante as 2 semanas prévias à indução da periodontite e até o momento do sacrifício (3, 7 ou 14 após a ligadura). A perda óssea alveolar foi avaliada radiograficamente, e análises do conteúdo de proteínas contendo nitrotirosina (NT), espécies reativas do ácido tiobarbitúrico (TBARs) e atividade da mieloperoxidase (MPO) foram realisadas em amostras de coração, baço, rim, fígado e pulmão. RvE1 (3 ng/mL) através da ativação do receptor para BLT-1 (mas não ChemR23) inibiu a diferenciação e atividade de OCs (p<0,05) após 5 ou 7 dias de cultura, assim como a fosforilação dos dois sítios da Akt e a traslocação do NF-<font face=\"symbol\">kB para o núcleo, um evento chave tanto na diferenciação de OCs (p<0,05) como na diminuição da expressão de iNOS. In vivo, ratos P (dia 7) mostraram um aumento na expressão de NT cardíaca e MPO renal em comparação ao grupo Sham (S; p<0,05). L-NAME resultou em aumento de NT hepática no grupo P no dia 3 (p<0,05), mas diminuição da NT cardíaca no dia 7 (p<0,01). Em comparação ao grupo P, ratos P+LN mostraram um aumento significativo na MPO hepática, cardíaca e renal no dia 3 (p<0,05), mas diminuição de MPO (dia 7) e TBARs esplênico (dia 3, p<0,05). Em resumo, mostramos que a RvE1 ligando-se ao receptor para BLT-1 inibe a diferenciação e atividade de OCs interferindo com a sinalização de Akt e NF-<font face=\"symbol\">kB, e consequentemente inibindo a expressão da iNOS, e que o NO tem um papel central na periodontite, não só relacionado a consequências locais na perda óssea alveolar, como também em órgãos periféricos distantes. / The periodontal disease is the most prevalent chronic disease in oral diseases. Among the mediators of this process, is the Resolvin E1 (RvE1), a novel mediator pro-resolving of inflammation that is capable to decrease alveolar bone loss secondary to periodontal disease in rabbits; and the nitric oxide (NO), that can be produced in large amount, induced by cytokines and it can stimulate the osteoclast differentiation and activity. The aim of this study is to investigate the effects of RvE1 on osteoclasts (OCs) culture and the pathway involved, also the role of NO in the progression of experimental periodontitis in rats and systemic alterations due to oxidative damage. The OCs differentiation was induced in bone marrow cell culture from C57BL/6 mice (7 days) and treated with various doses of RvE1. NF<font face=\"symbol\">kB and Akt phosphorylation were analyzed with Western blotting and the genic expression of NO synthase (NOS) inducible (iNOS) with \"Real Time\" PCR. The role of receptors ChemR23 and BLT-1 was accessed in OCs isolated membranes performing radioligants. The alveolar bone loss and peripheral organ damage was assessed in rats with ligature-induced periodontitis (P) under a long-term treatment of a NOS inhibitor, L-NAME. The animals received L-NAME from two weeks prior to periodontitis induction and until their sacrifice (3, 7 and 14 days after ligature). The alveolar bone loss was evaluated radiographically, and the protein nitrotyrosine (NT) content, reactive species of thiobarbituric acid (TBARs) and myeloperoxidase activity (MPO) were analyzed in samples of heart, spleen, kidney, lungs and kidneys. RvE1 (3 ng/mL) trough BLT-1 receptor activation (but not ChemR23) inhibits the OCs differentiation and activity (p<0.05) after 5 or 7 days of the culture, as well as the Akt phosphorylation and NF-<font face=\"symbol\">kB translocation to the nucleus, a key event both in OCs differentiation (p<0.05) and iNOS expression decreases. In vivo, P rats (day 7) show an increase of heart NT and renal MPO, but lower lung MPO activity in comparison to the Sham group (S; p<0.05). L-NAME leads to an increase the liver NT expression in P rats on day 3 (p<0.05), but decreases the cardiac NT on day 7 (p<0.01). In comparison with the P group, P+LN rats showed significantly increased liver, heart and kidney MPO content on day 3 (p<0.05), but lower lung MPO (day 7) and spleen TBARs (day 3) content (p<0.05). In summary we have shown that RvE1 binding on BLT-1 receptor inhibits OCs differentiation and activity by interfering with Akt and NF-<font face=\"symbol\">kB signaling and consequently iNOS inhibition, and NO has a central role on periodontitis, not only related to the local consequences on alveolar bone resorption, but also on distant peripheral organs.

Recently, it has emerged that the NO metabolites, nitrite and nitrate can be chemically reduced in vivo to biologically active nitric oxide (NO). This generation of NO is dependent on reduction of nitrate to nitrite by facultative anaerobes on the dorsal surface of the tongue, entry of the nitrite into the enterosalivary circuit, transit to the stomach, and absorption through the gut wall into the circulation. Conversion of nitrite to NO is then facilitated by vascular nitrite reductase enzymes. This nitrate-nitrite-NO pathway has been shown to exert a number of beneficial effects in healthy volunteers e.g. lowering of blood pressure, however whether this pathway is affected by cardiovascular disease (CVD) is currently unknown. Ozone chemiluminescence was used to determine and compare nitrite and nitrate levels in 2 models of CVD. To study atherosclerosis wild type (WT) and apolipoprotein E knock out (ApoE KO) mice were used and for hypertension wistar kyoto (WKY) rats as controls vs. spontaneously hypertensive rats (SHR). Assessment of nitrite reductase activity was conducted in the compartment which showed the most consistent differences in distribution, the red blood cell (RBC) and in homogenates of liver tissue. The impact of dietary nitrite and nitrate on distribution of the 2 anions throughout the cardiovascular system was assessed to determine the utility of this approach in restoring levels of these anions in CVD. Finally, using flow cytometry I investigated whether dietary nitrate supplementation could be used to influence inflammatory responses as a mechanism to improve CVD. Compared to WT mice, nitrate levels were reduced in ApoE KO mice in the plasma and across most of the tissues. In contrast in SHRs, reduction of the anions was only apparent in RBCs with no differences compared to WKY in all other tested tissues. Furthermore I have demonstrated that the most efficient way to restore nitrate levels back up to baseline is through a dietary nitrate strategy and that a dose of 15mM nitrate in the drinking water is sufficient to achieve this. In addition I have shown that nitrite reductase activity is enhanced in CVD particularly at the level of the RBC in both atherosclerosis and hypertension and that this enhanced activity is due, in part, to upregulation of xanthine oxidoreductase (XOR). Finally I have shown that dietary nitrate is an effective way to modulate an acute inflammatory response. This modulation is mediated through interfering with the ability of the neutrophil to firmly adhere to the vascular endothelium. These changes were shown to be dose-dependent and concomitant with dose-dependent increases in plasma nitrite and plasma nitrate. These data suggest that utilization of the nitrate-nitrite-NO pathway with dietary nitrate may represent an effective approach for the treatment of CVD.

SHP (Sphaeroides Heme Protein) is a monoheme cytochrome c of unknown function. In general, ligands cannot bind to ferric SHP, but some diatomic molecules, such as O2 or NO, can bind to ferrous SHP. The gene encoding SHP and genes encoding a diheme cytochrome c (DHC) and a b-type cytochrome (Cyt-b) are found in the same chromosome region in different species. In the case of Shewanella oneidensis MR-1, mRNA levels for SHP, DHC, and Cyt-b are up-regulated by nearly 10-fold when grown under anaerobic conditions using nitrate as the electron acceptor. Thus it is possible that the physiological role of SHP may be in nitrate metabolism. However, nitrate is too big to be a candidate substrate for SHP, and some nitrification steps need more than one electron transfer (SHP is a monoheme cytochrome). Therefore, we will focus on the nitrite reductase, nitric oxide reductase and nitric oxide dioxygenase activities of SHP. In this thesis it is shown that SHP can catalyse the reaction between oxygen and nitric oxide to give a nitrate ion as the final product. Thus a possible aerobic function for SHP as a nitric oxide dioxygenase is proposed. Aerobically, SHP is proposed to be a nitric oxide dioxygenase which utilizes the same mechanism as other NO dioxygenases, flavohemoglobin (HMP) and neuroglobin (Ngb). This mechanism is proposed to proceed via an oxy-ferrous complex (SHP2+-O2) which reacts with nitric oxide. A mechanism for the catalytic reaction with ferrous-NO complex is described. SHP2+-NO can be quickly converted back to ferrous SHP by reacting with superoxide liberated by SHP2+-O2 or from another source. In addition it is also found that Shewanella MR-1 wild type reveals a higher NO tolerance than the SHP knockout strain in aerobic conditions. The catalytic mechanism of NO dioxygenase is oxygen-dependent, but the SHP mRNA up-regulation in Shewanella oneidensis MR-1 grown with nitrate under anaerobic conditions indicates that SHP may also perform some anaerobic function and may possibly be involved in nitrate metabolism. This work found that SHP reveals anaerobic nitrite reductase activity. However, the catalytic efficiency of SHP is considerably lower than other nitrite reductases. This infers that although SHP can reduce nitrite in vitro, it is unlikely to function as a nitrite reductase in vivo. Ferrous SHP binds NO with a Kd of less than 1 μM, and does not auto-oxidise. Therefore, under anaerobic conditions SHP2+-NO must be processed by some other mechanism. In addition, biochemical results reveal that the SHP/DHC complex has NO reductase activity under anaerobic conditions. Unfortunately, this function was not proved in vivo. SHP was initially isolated from Rhodobacter sphaeroides and its structure was reported in 2000. Based upon this structure, SHP is clearly a class I cytochrome c with one axial histidine ligand to the heme iron. Unusually, however, it has an asparagine residue as the other axial heme ligand, and as such is unique among cytochromes c. For this reason it may be assumed that the asparagine plays a special role. This study reveals several potential reasons why SHP utilises asparagine as a heme ligand. Firstly, in the ferric form, asparagine 88 binds to the heme iron to prevent small molecules binding. Secondly, in the ferrous form it moves to allow oxygen to bind and form the oxy-ferrous complex, using hydrogen bonding for stability. Thirdly, using asparagine as a heme ligand creates a suitable redox potential for reduction by DHC, thus allowing NO dioxygenation.

Over the past three decades, nitric oxide (NO) has been recognised as one of the most versatile and important players in many aspects of physiology, including immune responses. More recently, S-nitrosylation, the incorporation of a NO moiety into a protein thiol group, has emerged as a major post-translational modification (PTM) during pathophysiological responses in plants and animals. The main goal of this work was to investigate the role of S-nitrosylation in physiology and innate immunity of animals using the genetic reference system, Drosophila melanogaster. The S-nitrosylated derivative of glutathione (GSH), S-nitrosoglutathione (GSNO), is the main non-protein S-nitrosothiol (SNO) in the cell and extracellular fluids. GSNO can trans-S-nitrosylate other thiols and is considered a reservoir of NO bioactivity. The levels of GSNO and total S-nitrosylation have been shown to be controlled by S-nitrosoglutathione reductase (GSNOR) in yeast, plants and mammals. By employing an overlapping deletion technique to knock-out gsnor, a role for S-nitrosylation in the immune response of D. melanogaster is proposed. Compared to wild type flies, gsnor overlapping deletion flies presented lower expression of antimicrobial peptides in response to infections, and succumbed more rapidly to both Gram-positive bacterial and fungal pathogens. As the Toll pathway mediates responses against these pathogens, key components of this network were tested for their propensity to being S-nitrosylated. Two CLIP-domain serine proteases of the Toll signalling pathway, Persephone (PSH) and Spätzle-Processing Enzyme (SPE), were shown to be S-nitrosylated both in vitro and in vivo and this process seemed to control the quaternary structure of these proteins and interfere with the immune response of D. melanogaster. At least for PSH, S-nitrosylation at C254 has an immune significance as the expression of non-Snitrosylable PSHC254S in gsnor knock-out flies partially recovered the resistance of these animals to infections with the entomopathogenic fungus Beauveria bassiana. These findings might represent a novel mechanism by which NO and S-nitrosylation regulate immunity. Further results presented in this thesis reveal an interplay between reactive oxygen species (ROS) and reactive nitrogen species (RNS) in D. melanogaster physiology and immunity. Similarly to what has been reported in Arabidopsis thaliana, gsnor knock-out flies presented higher tolerance to the herbicide paraquat, an inducer of superoxide (O2 -) production. Moreover, additional mutations in Catalase (Cat), a hydrogen peroxide (H2O2) scavenger enzyme, partially restored the immunodeficiency phenotypes of gsnor knock-out flies. These findings suggest an inter-relation between the levels of ROS and RNS during stress responses of plants and animals. In addition, CRISPR/Cas9 technology was employed to generate gsnor knock-outs in the genome of D. melanogaster. These flies were shown to have no GSNOR activity, presented lower tolerance to pharmacological-induced nitrosative stress and succumbed faster to infections with B. bassiana compared to wild type flies. These results support the role played by GSNOR in regulating NO homeostasis and immunity in D. melanogaster.

Inflammation has incompletely characterized effects on cardiopulmonary vascular reactivity. Sepsis is a major inflammatory disease characterized by two main vasomotor complications, generalized vasodilation with hyporesponsiveness to vasoconstrictors and pulmonary hypertension. The main aim of this study is to examine the molecular mechanisms involved in cardiopulmonary vascular reactivity changes induced by the powerful inflammatory stimulus lipopolysaccharide (LPS). Pulmonary and aortic rings from male Wistar rats (250-300g) were isolated and incubated for 20 h in culture medium (DMEM+10% FBS) with or without LPS (E. coli O55:B5, 10 μg.ml⁻¹). The effect of organ culture and LPS type, concentration and incubation time in addition to tissue contraction to endothelin-1 (ET-1), phenylephrine, 80 mM KCl, and U46619; and relaxation responses to ACh, sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY 41-2272, T-0156, nifedipine, SKF-96365, Ro-31-8425, and Y-27632 were measured by standard organ bath techniques. Nitric oxide (NO) production was measured by the Griess method and SNP-induced cGMP production was measured by ELISA. mRNAs expression levels of eNOS, iNOS, ET-1, ETA and ETB were measured by qRT-PCR and the expression levels of PKC, sGCα₁, sGCβ₁ and PDE5 and phosphorylation of MLC₂₀, ROKα, CPI-17 and MYPT1 were measured by immunoblotting. The effect of endothelium removal, indomethacin, trolox, external Ca²⁺ removal, 1400W, ODQ, glibenclamide, iberiotoxin and cycloheximide in addition to changes in intracellular Ca²⁺ ([Ca²⁺]i) in aortic vascular smooth muscle cells (VSMCs) induced by ET-1 were also measured. LPS selectively induced vascular hyporeactivity to different vasoconstrictors in rat aorta but not in the pulmonary artery, which is not due to organ culturing and is not affected by changing the LPS type, but is enhanced by increasing LPS concentration or the incubation time. This aortic hypocontractility to ET-1 is largely mediated by NO-independent activation of sGC and depends on external Ca²⁺ influx through non-VOCCs, but not on ET-1 receptor expression or Ca²⁺ sensitization. In addition, this aortic hyporeactivity to ET-1 is dependent on protein synthesis. The pulmonary artery is not affected because LPS induces a desensitization of the sGC/cGMP dependent pathway by decreasing protein expression levels of sGCβ₁, and hence sGC activity, and increasing PDE5 activity. Neither the endothelium, cyclooxygenase, reactive oxygen species nor K⁺ channels are involved in these LPS effects. Therefore, it is likely that both Ca²⁺ homeostasis and the sGC/cGMP pathway play important roles in vasomotor complications in sepsis. sGC and/or PDE5-selective inhibitors, together with manipulating VSMC [Ca²⁺]i, could be important in controlling systemic and pulmonary vasomotor complications in sepsis.

Fundamento: O óxido nítrico inalatório (NOi) tem sido utilizado para controle e tratamento da hipertensão pulmonar (HP) durante ou imediatamente após cirurgias cardiotorácicas. Entretanto, seus efeitos comparados a outras medicações vasodilatadoras ainda são controversos. Objetivo: Revisar sistematicamente os efeitos do NOi comparado com outras medicações vasodilatadoras administrado durante ou imediatamente após cirurgias cardiotorácicas, sobre variáveis hemodinâmicas e oxigenação, em pacientes com HP. Métodos: Foi realizada uma busca sistemática nas bases de dados Cochrane CENTRAL, MEDLINE, Lilacs, PEDro e Embase, além de busca manual em referências de estudos publicados até maio de 2015. Foram incluídos ensaios clínicos randomizados (ECRs) que compararam NOi vs. outras medicações vasodilatadoras (inalatórias ou intravenosas) e que analisaram índice de oxigenação (relação PaO2/FiO2) e variáveis hemodinâmicas como pressão média da artéria pulmonar (PMAP), resistência vascular pulmonar (RVP), fração de ejeção do ventrículo direito (FEVD), saturação venosa mista de oxigênio (SvO2), índice cardíaco (IC), pressão venosa central (PVC), frequência cárdica (FC), pressão arterial média sistêmica (PAM), e resistência vascular sistêmica (RVS). Resultados: Foram identificados 2.561 artigos, totalizando seis ECRs incluídos. O uso do NOi comparado com outras medicações vasodilatadoras não promoveu alterações na relação PaO2/FiO2, PMAP, RVP, IC, FEVD, SvO2, PVC e RVS, entretanto houve redução na FC (-5,47 bpm; IC95%: -10,87 a -0,06) comparado com medicações inalatórias e aumento na PAM (9,30 mmHg; IC95%: 3,94 a 14,65; I2: 86%) comparado com medicações vasodilatadoras intravenosas. Conclusões: O uso do NOi não promoveu alterações na relação PaO2/FiO2 e nas variáveis hemodinâmicas comparado com outras medicações vasodilatadoras, exceto na FC onde houve redução e na PAM onde houve aumento, durante ou imediatamente após cirurgias cardiotorácicas em pacientes adultos com HP. / Introduction: The inhaled nitric oxide (INO) has been used for control and treatment of pulmonary hypertension (PH) during or immediately after cardiothoracic surgery. Although, the effects when compared to other vasodilator medications are still controversial. Objectives: The purpose of this study was to systematically review the effects of INO compared with other vasodilatory drugs administered during or immediately after cardiothoracic surgeries on hemodynamics and oxygenation variables in patients with PH. Methods: A systematic research was conducted in the databases Cochrane CENTRAL, MEDLINE, Lilacs, PEDro e Embase, in addition a manual search at references of published studies until May 2015. Randomised trials (RCTs) were included, comparing INO vs. other vasodilator medications (inhaled or intravenous), that analyzed the oxygenation index (ratio PaO2/FiO2) and hemodynamic variables: mean pulmonary artery pressure (MPAP), pulmonary vascular resistance (PVR), right ventricular ejection fraction (RVEF), mixed venous oxygen saturation (SvO2) , cardiac index (CI), central venous pressure (CVP), cardia rate (HR), mean systemic arterial pressure (MAP) and systemic vascular resistance (SVR). Results: We identified 2561 articles, being only six ECRs included. The use of NOi compared to other vasodilator medications did not change the ratio PaO2/FiO2, MPAP, PVR, CI, RVEF, SvO2, CVP e SVR, however there was a reduction in HR (-5,47 bpm; IC95%: -10,87 a -0,06) compared to inhaled medications and increased MAP (9,30 mmHg; IC95%: 3,94 a 14,65; I2: 86%) compared with intravenous vasodilator medications. Conclusion: The use of INO did not change ratio PaO2/FiO2 and hemodynamic variables, compared with other vasodilator medications, except HR where has been found a reduction, and MAP where has been found an increasing, during or immediately after thoracic cardiovascular surgery in adult patients with HP.