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Tetraaminas de rutênio (II) como transportadoras de óxido nítrico e benznidazol e sua ação sobre o Trypanosoma cruzi / Ruthenium tetrammines as a carrier of nitric oxide and benznidazole and their action on Trypanosoma cruziSilva, Jean Jerley Nogueira da 28 April 2008 (has links)
Uma nova e promissora alternativa quimioterápica para o tratamento da doença de Chagas usando tetraaminas de rutênio (II) como transportadoras de óxido nítrico (NO) ou Benznidazol (Bz) são apresentados aqui. Os resultados mostraram que os complexos do tipo trans-[Ru(NO)(NH3)4L]n+ e cis-[Ru(NO)(bpy)2L] n+ são mais eficientes para tratar casos agudos da doença (modelo murino) que o Bz livre. Os nitrosilos trans-[Ru(NO)(NH3)4isn](BF4)3, trans-[Ru(NO)(NH3)4imN](BF4)3, cis-[Ru(NO)(bpy) 2imN](PF6)3 e cis-[Ru(NO)(bpy) 2SO3]PF6 são capazes de eliminar in vivo as formas extracelulares e intracelulares do T. cruzi tanto na corrente sanguínea quanto no tecido miocárdio, assegurando a sobrevida de 60-100% dos animais infectados em doses de 900 (400 nmol kg-1) até 3.850 vezes menores (100 nmol kg-1) que a dose considerada ótima para o Bz (385 µmol kg-1 = 100 mg kg-1). Além disso, uma concentração de trans-[Ru(NO)(NH3)4imN](BF4)3 ou cis-[Ru(NO)(bpy) 2imN](PF6)3 dez vezes menor que a recomendada pela Organização Mundial de Saúde (OMS) para tratar sangue infectado com o violeta de genciana (Vg) é capaz de eliminar 100% dos parasitos após 1 hora de incubação a 37 oC. Os experimentos conduzidos in vitro demonstraram que o NO liberado após redução ou reativos intermediários do NO são os responsáveis pelo efeito anti-proliferativo ou tripanocida. O nitrosilo trans-[Ru(NO)(NH3)4isn](BF4)3 (IC50epi = 67 µmol L-1) foi mais ativo que o nitroprussiato de sódio (SNP) (IC50epi = 244 µmol L-1) e o trans-[Ru(NO)(NH3)4imN](BF4)3 e o cis-[Ru(NO)(bpy) 2imN](PF6)3 (IC50tri = 52 e 58 µmol L-1, respectivamente) foram mais eficientes que o Vg (IC50tri = 536 µmol L-1). O transportador do Bz, trans-[Ru(Bz)(NH3)4S(IV)]n+, S(IV) = SO2, HSO3- ou SO32- (RuBz), é mais hidrosolúvel e mais ativo (IC50tri/1h = 79 ± 3 µmol L-1) que o Bz livre (IC50tri/1h = 4,800 ± 70 µmol L-1). Este complexo exibiu baixa citotoxicidade in vitro (IC50esplenócitos > 1 mmol L-1) e in vivo (400 µmol kg-1 < LD50 < 600 µmol kg-1) e a formação do derivado hidroxilamínico é mais favorável no RuBz que no Bz em 9,6 Kcal mol-1. Em diversos modelos murinos de infecção aguda, o RuBz também foi mais ativo que o Bz ainda quando somente uma dose foi administrada. Analogamente, o RuBz a uma dose 1.000 vezes que a dose ótima para o Bz provou ser suficiente para proteger todos os animais infectados, eliminando os ninhos de amastigotas nos seus corações, fígados e músculos esqueléticos. Em experimentos conduzidos com células Vero, os nitrosilos trans-[Ru(NO)(NH3)4imN](BF4)3, cis-[Ru(NO)(bpy)2imN](PF6)3 e o RuBz foram capazes de eliminar as formas intracelulares do T. cruzi e reduzir a porcentagem de células infectadas. Portanto, todos os dados observados sugerem a maior solubilidade em água somada à acessibilidade do potencial de redução do par NO2/NO3.- no RuBz são os responsáveis pela maiores atividades anti-proliferativa e tripanocida com respeito ao Bz ou Vg. Adicionalmente, cis-[Ru(NO)(bpy)2imN](PF6)3, cis-[Ru(NO)(bpy) 21-miN](PF6)3 e cis-[Ru(NO)(bpy) 2SO3]PF6 provaram ser capazes de inibir a atividade catalítica da gGAPDH de 89-97% a uma concentração de 260 µmol L-1, enquanto os complexos da série trans-[Ru(NO)(NH3)4L]n+, os precursores cis-[Ru(bpy) 2L(NO2)]n+, cis-[Ru(H2O)(bpy) 2L]n+ bem como os complexos RuBz, cis-[Ru(NO)(NH3)4 (NO2)]Cl2 não exibiram atividade inibitória significativa. / A novel and promising chemotherapeutic alternatives for the treatment of Chagas\' disease by use of ruthenium tetraammnine complexes as a carrier of nitric oxide (NO) or Benznidazole (Bz) are presented herein. The results showed that the trans- [Ru(NO)(NH3)4L]n+ and cis-[Ru(NO)(bpy) 2L] n+ compounds are more efficient in the treatment of acute cases of the disease (mouse model) than free Bz. The trans- [Ru(NO)(NH3)4isn](BF4)3, trans-[Ru(NO)(NH3)4imN](BF4)3, cis- [Ru(NO)(bpy) 2imN](PF6)3, and cis-[Ru(NO)(bpy) 2SO3]PF6 are able to eliminate, in vivo, extracelullar as well as intracellular forms of T. cruzi in the bloodstream and myocardium tissue and to assure the survival of 60-100% of infected mice at doses of 900 (400 nmol kg-1) up to 3,850 fold smaller (100 nmol kg-1) than that considered the optimal dose for Bz (385 µmol kg-1 = 100 mg kg-1). Furthermore, a dose of trans-[Ru(NO)(NH3)4imN](BF4)3 or cis-[Ru(NO)(bpy) 2imN](PF6)3 ten-fold smaller than that recommended by World Health Organization (WHO) for gentian violet, Vg, a phenyl methane currently recommended by WHO in the treatment of blood banks in endemic and non-endemic areas to prevent the transmission of Chagas\' disease by blood transfusion, is able to lyses 100% of the parasites after 1 hour of incubation. The tests conducted in vitro demonstrated that the NO liberated upon reduction of these nitrosyls is responsible for the observed antiproliferative and trypanocidal activities. The trans-[Ru(NO)(NH3)4isn](BF4)3 (IC50epi = 67 µmol L-1) was found to be more efficient than the classic NO-donor, sodium nitroprusside (SNP) (IC50epi = 244 µmol L-1) and trans-[Ru(NO)(NH3)4imN](BF4)3 and cis-[Ru(NO)(bpy) 2imN](PF6)3 (IC50try = 52 and 58 µmol L-1, respectively) were more efficient than the gentian violet (IC50try = 536 µmol L-1). The Bz carrier, trans-[Ru(Bz)(NH3)4S(IV)] n+, S(IV) = SO2, HSO3 - or SO3 -2 (RuBz), is more hydrosoluble and more active (IC50try/1h = 79 ±?3 µmol L-1) than free Bz (IC50try/1h = 4,800 ±?70 µmol L-1). This complex exhibits low acute citotoxicity in vitro (IC50trysplenocytes > 1 mmol L-1) and in vivo (400 µmol kg-1 < LD50try < 600 µmol kg-1) and the formation of hydroxylamine is more favorable in RuBz than in Bz by 9.6 Kcal mol-1. In murine acute models of Chagas\' disease, RuBz also was more active than Bz even when only one dose was administrated. Moreover, RuBz at a thousand-fold smaller concentration than the considered optimal dose for Bz proved to be sufficient to protect all infected mice, eliminating the amastigotes nests in their hearts, livers, and skeletal muscles as observed in H?E micrographics. Experiments conduced with infected Vero cells, the trans- [Ru(NO)(NH3)4imN](BF4)3, cis-[Ru(NO)(bpy) 2imN](PF6)3, and RuBz are able to eliminate the intracellular forms of T. cruzi and to reduce the percentage of infected cells. In all, the observed data strongly suggest that the higher solubility in water and accessibility for reduction of NO2/NO2 .- couple in RuBz are responsible for higher antriproliferative and trypanocidal activities regarding to Bz or Vg. Additionally, the cis- [Ru(NO)(bpy) 2imN](PF6)3, cis-[Ru(NO)(bpy) 21-miN](PF6)3, and cis- [Ru(NO)(bpy) 2SO3]PF6 proved to be able to inhibit the catalytic activity of gGAPDH of 89-97% at a concentration of 260 µmol L-1, whereas their precursors cis- [Ru(bpy) 2L(NO2)]n+ and cis-[Ru(H2O)(bpy) 2L]n+ and the trans-[Ru(NO)(NH3)4L] 3+ and cis-[Ru(NO)(NH3)4 (NO2)]Cl2 nitrosyls and RuBz complex do not exhibited significative inhibition.
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Ancoramento de nitrosilo complexo de rutênio em dendrímeros PAMAM e estudo de suas propriedades químicas e biológicas / Anchoring ruthenium nitrosyl complex on PAMAM dendrimer and chemical and biological propertiesRoveda Júnior, Antonio Carlos 14 July 2011 (has links)
O ancoramento do complexo trans-[RuIII(NH3)4(SO4)ina]Cl em dendrímeros PAMAM de geração 0 e 2 (G0 e G2) foi realizada por meio de uma ligação peptídica, e esses produtos foram submetidos à reação com NO(g) gerando os respectivos nitrosilo complexos G0/RuNO e G2/RuNO. A caracterização desses compostos por infravermelho, UV-vis, voltametria cíclica, RMN de 1H e 13C, e análise elementar indica que os nitrosilo complexos foram imobilizados na superfície dos PAMAM G0 e G2. Os espectros de infravermelho para G0/RuNO e G2/RuNO apresentaram apenas um estiramento νNO+, respectivamente em 1933 e 1937 cm-1, e para o produto RuNO (não ligado ao dendrímero) em 1933 cm-1. O espectro eletrônico para esses três compostos apresentou bandas nas regiões de 230, 270 e 330 nm, e por meio de voltametria cíclica observou-se o processo eletroquímico relativo a NO+/NO0 com ENO+/NO0 vs ECS igual a -0,173 V para G0/RuNO, -0,178 V G2/RuNO e -0,175 V para RuNO. O espectro de 1H RMN do complexo RuNO apresentou dois dubletos com deslocamentos químicos centrados em 8,73 e 8,35 ppm, referentes aos hidrogênios aromáticos respectivamente nas posições orto e meta do ligante ina coordenado ao metal. Para G0/RuNO e G2/RuNO esses sinais foram observados em 8,73 e 8,36 ppm, e os sinais referentes aos dendrímeros nesses produtos foram verificados entre 2,7 e 4,0 ppm. O espectro de RMN 13C para o complexo RuNO apresentou quatro sinais, e para G0/RuNO e G2/RuNO, respectivamente, dez e doze sinais, conforme esperado para esses compostos. Apesar dos resultados supracitados indicarem que o ancoramento ocorreu de forma satisfatória, os dados de análise elementar apresentaram desvios significativos entre o valor teórico e o experimental, principalmente para G2/RuNO. Em adição, foram realizados ensaios em células do baço de camundongos para verificar a toxicidade dos nitrosilo complexos às células saudáveis, e os resultados indicaram baixa citotoxicidade (<15%) para RuNO, G0/RuNO e G2/RuNO. Também foram realizados experimentos sobre a atividade in vitro desses compostos contra os parasitos Trypanosoma cruzi e Leishmania major. Os melhores resultados, ainda que preliminares, foram obtidos com a maior concentração, 200µM (em relação à Ru), em que observou-se atividade tripanocida (média) em torno de 88% para G2/RuNO, 82% para G0/RuNO e 72% para RuNO, enquanto que para o Bz (referência) esse valor foi de 96%. Já a atividade leishmanicida (concentração de 200µM) desses compostos ficou entre 60 a 70% (65% para G2RuNO, 69% para G0/RuNO e 60% para RuNO). / The anchoring of the complex trans-[RuIII(NH3)4(SO4)ina]Cl on PAMAM dendrimers of generation 0 and 2 (G0 and G2) was performed by a peptide bond, and the products were submitted to reaction with NO (g) generating the related nitrosyl complexes G0/RuNO and G2/RuNO. The characterization of these compounds by IR, UV-vis, cyclic voltammetry, 1H and 13C NMR, and elemental analysis indicated that the nitrosyl complexes were immobilized on the surface of PAMAM G0 and G2. Infrared spectra for G0/RuNO and G2/RuNO showed only one νNO+ band in 1933 and 1937 cm-1 respectively, and for RuNO (complex not bounded to the dendrimer) at 1933 cm-1. Electronic spectra for these three compounds showed bands in the regions of 230, 270 and 330 nm, and by cyclic voltammetry it was possible to observe the electrochemical process relative to NO+/NO0 with ENO+/NO0 equal to -0.173 V vs SCE for G0/RuNO , -0.178 V for G2/RuNO and -0.175 V for RuNO. The 1H NMR spectra for RuNO complex showed two doublets with chemical shifts centered at 8.73 and 8.35 ppm, respectively referring to the aromatic hydrogens in the ortho and meta positions of the ina ligand coordinated to the metal. The same signals obtained for G0/RuNO and G2/RuNO were observed in 8.73 and 8.36 ppm, and signals related to dendrimers between 2.7 and 4.0 ppm. The 13C NMR spectrum for RuNO exhibited four signals, and for G0/RuNO and G2/RuNO, respectively, ten and twelve signals, as expected for these compounds. Despite the results above, which indicate that anchoring occurred satisfactorily, the elemental analysis showed significant deviations between the theoretical and experimental values, especially for G2/RuNO. In adition, in vitro assays were performed on mice spleen cells to determine the toxicity of the nitrosyl complex to healthy cells, and the results showed low cytotoxicity (<15%) for RuNO, G0/RuNO and G2/RuNO. In vitro experiments were also carried out to determine the activity of these compounds against the parasite Trypanosoma cruzi and Leishmania major. The best results (preliminary) were obtained with the highest concentration 200µM (relative to Ru), which was observed trypanocidal activity (average) around 88% for G2/RuNO, 82% for G0/RuNO and 72% for RuNO, while for Bz (reference) it was around 96%. The leishmanicidal activity (concentration of 200µM) of these compounds was in the range of 60 to 70% (65% for G2RuNO, 69% for G0/RuNO and 60% for RuNO).
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A reatividade e as propriedades dos ligantes NO e H2O em tetraaminas de rutênio. Reações com glutationa e cisteina / The reactivity and properties of the NO and H2O ligands in ruthenium tetraammines. The reaction with glutathione and cysteineSouza, Maykon Lima 09 December 2014 (has links)
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.
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Estudo da associação de complexos nitrosilos de rutênio liberadores de NO com o agente fotossensibilizador Zinco ftalocianina ZnPC em sistemas de liberação utilizados na terapia fotodinâmica / Study of NO donors, nitrosyl ruthenium complex, associate with the photosensitizer ZnPc in drug delivery systems applied in photodynamic therapyMaranho, Daniela Silva 26 August 2008 (has links)
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).
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Efeito dos agentes de maturação, ABA, PEG e maltose, na produção de óxido nítrico e de espécies reativas de oxigênio em culturas embriogênicas de Araucaria angustifolia / Effect of maturation promoters, ABA, PEG and maltose in the production of nitric oxide and reactive species in Araucaria angustifolia embryogenic culturesAndrade, Julia Bolanho da Rosa 16 August 2010 (has links)
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.
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Interação anticorpo policlonal-complexo de rutênio como sistemas de liberação de óxido nítrico. Medida da especificidade e avaliação citotóxica / Interaction polyclonal antibody-ruthenium complex such as nitric oxide release systems. Specificity and cytotoxicity measurementRamos, Loyanne Carla Barbosa 27 June 2012 (has links)
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.
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Investigation of the distribution of nitrite and nitrate and nitrite reductase activity in models of cardiovascular diseaseGhosh, Suborno Mukut January 2014 (has links)
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.
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Functional studies on a novel cytochrome c from Rhodobacter sphaeroidesLi, Bor-Ran January 2009 (has links)
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.
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Redox signalling and innate immunity : a role for protein S-nitrosylation in the immune response of Drosophila melanogasterHomem, Rafael Augusto January 2016 (has links)
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.
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Molecular pharmacology of altered cardiopulmonary function in inflammationEl-Awady, Mohammed January 2008 (has links)
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.
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