O óxido nítrico (NO) é um versátil agente biológico, atuando em diversas partes do organismo, tais como cérebro, artérias, sistema imunológico, fígado e pulmões. Alterações nos níveis basais de NO são relacionadas a causas de diversas doenças como hipertenção, câncer, doença de Raymond´s e agregação plaquetária. Visando suprir tal deficiência, mostrou-se de grande valia a obtenção de fármacos capazes de liberar quantidades significativas de NO e de maneira controlada. Uma das possibilidades é o uso de compostos de coordenação, com centro metálico rutênio coordenado ao ligante nitrosil, os quais possuem como característica liberação de NO por estímulo externo. Neste trabalho foram estudados as propriedades químicas, fotoquímicas, farmacológicas e tumoricidas do complexo trans-[RuC(MAC)NO]2+, onde MAC é 1,4,8,11-tetraazaciclotetradecano (cyclam) ou 1,4,8,12-tetraazaciclopentadecano ([15]aneN4), na presença do fotossensibilizador [Ru(NH3)5pz]2+. Os estudos fotoquímicos mostraram que o fotossensibilizador utilizado, [Ru(NH3)5pz]2+, possui tempo de vida no estado excitado de 113 ps, e apresenta processos de transferências eletrônicas e de energia fotoinduzida com o oxigênio molecular, gerando espécies reativas de oxigênio (EROS) como oxigênio singlete e ânion superóxido. Na presença do complexo macrocíclico trans-[RuC(MAC)NO]2+ a irradiação luminosa de [Ru(NH3)5pz]2+ na região do visível proporciona trasferência eletrônica fotoinduzida entre o fotosenssibilizador e o complexo nitrosilo com concomitante formação de NO. O rendimento quântico de NO (NO) é dependente do meio e é de cerca de 10-4 Einstein.mol.cm-1 em meio aquoso sob argônio e 10-2 Einstein.mol.cm-1 em meio oxigenado. Os estudos farmacológicos mostraram que a mistura proposta, em tampão fosfato pH 7,4, promoveu 75% de dilatação em musculatura de aorta pré-contraída com KC enquanto que, quando incorporados em sistemas carreadores de fármacos como emulsão óleo-em-água, HEG gel e membrana sol-gel, promoveram 40%, 35% e 35% de dilatação, respectivamente. A via vasodilatadora foi detectada como sendo pela ativação da GMPc (guanosina monofosfato cíclica), através da quantificação da concentração de cálcio citosólico. Os estudos das atividades anti-tumorais com os complexos doadores de NO foram realizados com linhagens celulares do tipo B16F10, Melan A, HL-60 WMs e Jurkat. Estes experimentos mostraram redução da viabilidade celular dos melanomas entre 50% e 75%, não apresentou ação em células normais (MelanA), mostrando-se seletivos ás células neoplasicas; e 95% de redução da viabilidade celular das células leucêmica (HL-60). Os resultados obtidos e descritos neste trabalho permitem vislumbrar que o uso de espécies doadoras de NO e oxigênio singlete possam ser utilizados no futuro em terapia clínica. / Nitric oxide (NO) is a multipurpose biological agent that shows important role in brain, arteries, immunological system, liver and lungs. The NO basal level alterations have correlation with several diseases as hypertension, cancer, Raymonds disease and platelet aggregation. Aiming supply this deficient NO level, it was necessary propose new compounds that can release NO at controlled manner. One of the possibilities is utilize nitrosyl ruthenium complexes that have ability to release NO under external stimulus. In this work we have studied, the chemical, photochemical, pharmacological and anti-tumoral properties of the trans-[RuC(MAC)NO]2+ complex, were MAC is 1,4,8,11-tetraazacyclotetradecane (cyclam) or 1,4,8,12-tetraazacyclopentadecane ([15]aneN4), in the presence of the sensitizer [Ru(NH3)5pz]2+. The photochemical studies of [Ru(NH3)5pz]2+ complex showed excited state lifetime of 113 ps and presented photoinduced electron and energy transfer with oxygen resulting in reactive oxygen species (ROS) generation as singlet oxygen and superoxide anion. In the presence of trans-[RuC(MAC)NO]2+ complex, the visible light irradiation of [Ru(NH3)5pz]2+ causes photoinduced electron transfer between the photosensitizer and the nitrosyl ruthenium complex resulting NO generation. The quantum yield (NO) is milieu dependent and showed 10-4 Einstein.mol.cm-1 in inert atmosphere and 10-2 Einstein.mol.cm-1 in aerated aqueous solution. The pharmacological studies showed that the proposed mixture in phosphate buffer, under visible irradiation, induce 75% of relaxation in aorta pre-contracted with KC while this system incorporated into drug delivery system as water-in-oil emulsion, HEG gel and sol-gel membrane, the nitric oxide generated induced 40%, 35% and 35% of relaxation, respectively. The quantification of cytosolic calcium concentration, using confocal image, showed that vasodilatation pathway observed was via cGMP (cyclic guanosine monophosphate) activation. The anti-tumoral studies using NO donors were carried out for B16F10, Melan A, HL-60, WMs and Jurkat cells. Those experiments showed decrease from 50% to 75% of cell viability of tumor cells and no toxicity in melanoma cells and 95% of reduction of leukemic cells (HL-60). The obtained results described in this work permit to glimpse that the use of NO donor species and singlet oxygen maybe is a useful tool in the future in clinical therapy.

Segundo a Organização Mundial de Saúde (OMS), mais de um bilhão de pessoas estão infectadas com uma ou mais doenças tropicais endêmicas encontradas especialmente entre as populações pobres da África, Ásia e América Latina. Nos últimos 25 anos, apenas 1% de todos os medicamentos desenvolvidos no mundo foram destinados ao tratamento de doenças tropicais, como a doença de Chagas. A doença de Chagas é causada por um protozoário intracelular, o Trypanosoma cruzi, e atualmente apenas dois compostos tem sido empregados para tratamento etiológico da doença de Chagas: nifurtimox e benznidazol. Entretanto, ambos os compostos apresentam considerável toxicidade sistêmica. Nesse contexto, o objetivo do presente trabalho foi desenvolver complexos híbridos de rutênio, com potencial atividade tripanocida, que possam atuar com maior eficácia em sítios biológicos específicos do Trypanosoma cruzi e com reduzida toxicidade sistêmica. Para tal efeito, moléculas com conhecida atividade microbicida como derivados aminoglicosídeos, óxido nítrico e benznidazol foram coordenados a complexos de rutênio originando as espécies Ru(desoxiestreptamina), Ru(neamina), cis-[Ru(bpy)2(Bz)(NO)](PF6)3 e cis-[Ru(NO2)(bpy)2(Bz)]PF6. Os complexos foram caracterizados por espectroscopia de absorção no UV-vis e FTIR, espectrometria de massa, análise elementar, voltametria cíclica e voltametria de pulso diferencial. Nossos estudos sugerem que os complexos Ru(desoxiestreptamina), Ru(neamina), cis-[Ru(bpy)2(Bz)(NO)](PF6)3 e cis-[Ru(NO2)(bpy)2(Bz)]PF6 apresentam propriedades químicas que suportam o sucesso da coordenação dos ligantes ao íon metálico rutênio(II) ou (III). Dentre todos os compostos estudados, cis-[Ru(NO2)(bpy)2(Bz)]PF6, apresentou maior potência tripanocida desprovida de citotoxicidade, a julgar pelos estudos in vitro. IC50 foi ao redor de 0,24 ?mol.L-1, o que é 47 vezes menor do que a droga comumente usada em tratamento de Doença de Chagas. Isto determinou avaliação in vivo, no que foi observado aumento da sobrevida dos animais infectados com tripomastigotas, para 60 dias. Os estudos desenvolvidos com complexos de rutênio no presente trabalho reafirmam o sucesso na obtenção de tais complexos inicialmente propostos, implementando importantes informações e perspectivas no que diz respeito a complexos nitrosilos de rutênio e seus potenciais terapêuticos na doença de Chagas. / Ru(bpy)2(Bz)(NO)](PF6)3 and cis-[Ru(NO2)(bpy)2(Bz)]PF6 complexes. The compounds were characterized by UV-vis absorption spectroscopy, FTIR, mass spectrometry, elemental analysis, and cyclic voltammetry and differential pulse voltammogram. The chemical characterization presented in this work gave evidencie that support the coordination of biological ligand in ruthenium ion such as in Ru(desoxiestreptamina), Ru(neamina), cis-[Ru(bpy)2(Bz)(NO)](PF6)3 and cis-[Ru(NO2)(bpy)2(Bz)]PF6 complexes. Subsequently, in vitro and in vivo studies have been conducted evaluating the cytotoxicity and trypanocidal activity of the ligands and ruthenium complexes. In vitro analysis suggested us that ruthenium complexes are greatly effective against T. cruzi. Among all complexes synthesized cis-[Ru(NO2)(bpy)2(Bz)]PF6 showed higher in vitro trypanocidal activity, which has determined the in vivo assays with this compound. IC50 was around 0,24 ?mol.L-1, which is 47 times less than usual drugs used in Chagas diseases treatment. This was performed using animals infected with T. cruzi in trypomastigote form, animal survivals until 60 days. The studies developed for ruthenium complexes reaffirm the success in obtaining of the complexes originally proposed, implementing important information and perspectives regarding the nitrosyl ruthenium complex and its therapeutic potential in Chagas disease.

Paracoccidioidomicose é adquirida pela via respiratória e o óxido nitrico (NO) está envolvido na eliminação de patógenos. Nós propusemos investigar o papel do NO na doença em animais NO-/- sintase deficientes (iNOS-/-) e seu grupo WT. Na 2ª semana de infecção, a ausência de NO resultou em doença menos grave com aumento dos níveis de TNF-a acompanhado com o intenso afluxo de linfócitos T e macrófagos para os pulmões. Na 10ª semana, os animais iNOS-/- desenvolveram alta carga fúngica nos pulmões com menor afluxo de celulas T ativadas e macrófagos e presença de células T reguladoras CD4+CD25+FoxP3+ nos pulmões. Somente os animais iNOS-/- desenvolveram lesões pulmonares organizadas em granulomas, embora não foi detectado diferença na mortalidade para ambos os grupos. As diferenças morfológicas nas lesões foram abolidas pela depleção de TNF-a que induziu nos animais iNOS-/- uma mortalidade precoce e intenso afluxo inflamatório nos pulmões. Além disso, a depleção de linfócitos TCD8+ resultou em doença mais grave e menor recrutamento celular pulmonar nos animais iNOS-/-. / Paracoccidioidomycosis is acquired by the respiratory route and nitric oxide (NO) is involved in the killing of pathogens, we aimed to investigate the role of NO in the course of the disease using NO- synthase deficient (iNOS-/-) and WT mice. At week 2 postinfection, NO absence resulted in less severe infection associated with increased TNF-a levels besides a massive influx of activated T cells and macrophages to the lungs. By week 10, iNOS-/- mice developed increased fungal burdens allied with less pronounced influx of activated T cells and macrophages and increased presence of regulatory CD4+CD25+FoxP3+ T cells to the lungs. Only iNOS-/- mice developed organized pulmonary granulomas, although no differences in the mortality rates were detected. The differences in the morphology of lesions were partially abrogated by TNF-a depletion which, induced a precocious mortality of iNOS-/- and massive influx of inflammatory pulmonary cells. Indeed, the CD8+T cells depletion developed a more severe infection with less recruitment of pulmonary cells in iNOS-/- mice.

O Óxido Nítrico (NO) faz parte de uma família de radicais livres que está envolvida em várias funções do organismo como controle cardiovascular, homeostase, formação óssea, neurotransmissão e funções imunológicas. Tem sido descrito que o NO atuaria na resposta de defesa do hospedeiro frente à infecção dos tecidos orais. Por outro lado, tem sido descrito também que quantidades excessivas de NO podem contribuir para a destruição tecidual na periodontite. Além destes aspectos muito importantes na boca, o NO é uma das moléculas de maior importância para a saúde humana, pois a sua liberação pelo endotélio dos vasos é um componente fundamental para o controle da pressão arterial. Já foi demonstrado que bactérias comensais da boca seriam responsáveis pela produção de nitritos que, no estômago, seriam convertidos a NO e este absorvido. Como as quantidades de nitrito na saliva são cerca de mil vezes maiores do que aquelas detectadas no sangue total, acredita-se que o nitrito da saliva possa ter repercussão no NO do sangue total e possivelmente possa também ter algum efeito na regulação de eventos sistêmicos, como a própria pressão arterial. O número de trabalhos sobre NO na saliva encontrados na literatura científica de língua inglesa não soma 2 dezenas, e pela importância do assunto, faz-se necessário conhecer em detalhes as concentrações de nitrito nos diferentes tipos de saliva, no sangue total, eritrócitos e plasma em pacientes com doença periodontal e controles, antes e depois do tratamento. As hipóteses deste estudo são: 1- Que as concentrações de nitrito sejam menores na saliva total de indivíduos com doença periodontal e que estas aumentem após o tratamento periodontal; 2- Que haveria o mesmo perfil de variação nas concentrações de nitrito no sangue total, eritróticos e plasma vista na saliva total. No presente estudo avaliaram-se as concentrações de NO na saliva total, saliva sumandibular/sublingual, parotídea, no sangue total, eritrócitos e plasma de pacientes controle e pacientes com doença periodontal crônica, com coletas antes e 3 meses após o tratamento periodontal não cirúrgico. A seguir testou-se se eram significativas as diferenças entre os valores obtidos antes e após o tratamento e foram comparados os perfis dos resultados de nitrito na saliva e sangue total. Foram realizadas as dosagens de NO pelo método de análise de quimiluminescência. Os resultados obtidos mostraram que em todos os parâmetros clínicos houve diferença estatisticamente significante antes e após o tratamento periodontal (DPA e DPD), nos grupos controle antes e depois do tratamento (CA e CD) e nos controles antes e doentes antes do tratamento (CA e DPA).Sangramento a sondagem: (DPA versus DPD) p<0,0001, (CA versus CD) p=0,0005 , (CA versus DPA) p<0,0001. Nivel clinico de inserção: (DPA versus DPD) p<0,0001, (CA versus CD) p=0,01 , (CA versus DPA) p<0,0002. Profundidade de sondagem: (DPA versus DPD) p<0,0001, (CA versus DPA) p<0,0001. Sítios maiores que 4 mm: (DPA versus DPD) p<0,0001, (CA versus DPA) p<0,0001. Tal fato confirma um tratamento periodontal realizado com sucesso. Na saliva total, houve significante diminuição das concentrações de NO entre os controles antes e os doentes antes do tratamento (CA versus PDA), com p= 0,04. No sangue total houve diminuição das concentrações de NO antes e após o tratamento periodontal (DPA versus DPD), p=0,026. Já nos eritrócitos antes e após o tratamento (DPA versus DPD) houve significante diminuição, p=0,02. As concentrações de nitrito na saliva total tiveram medianas igual a 0,69 (Q1: 0,46/ Q3: 1,44) no grupo CA, mediana de 0,55 (Q1: 0,39/ Q3: 1,55) no grupo CD, mediana de 0,44 (Q1: 0,26/ Q3: 0,81) no grupo DPA e mediana de 0,38 (Q1: 0,22/ Q3: 0,69) no grupo DPD. As concentrações de nitrito na saliva submandibular/sublingual tiveram medianas igual a 1,86 (Q1: 0,61/ Q3: 4,59) no grupo CA, mediana de 0,64 (Q1: 1,82/ Q3: 4,37) no grupo CD, mediana de 2,37 (Q1: 0,47/ Q3: 4,79) no grupo DPA e mediana de 2,69 (Q1: 1,69/ Q3: 3,43) no grupo DPD. As concentrações de nitrito na saliva parotídea apresentaram médias de 0,58, e desvio padrão de 0,32 no grupo CA, média de 0,69 e desvio padrão de 0,33 no grupo CD, média de 0,69 e desvio padrão de 0,36 no grupo DPA e média de 0,85 e desvio padrão de 0,58 no grupo DPD. As concentrações de nitrito no sangue total tiveram medianas igual a 1,41 (Q1: 1,1/ Q3: 2,21) no grupo CA, mediana de 0,99 (Q1: 0,58/ Q3: 2,11) no grupo CD, mediana de 1,61 (Q1: 1,12/ Q3: 2,3) no grupo DPA e mediana de 1,45 (Q1: 0,61/ Q3: 0,87) no grupo DPD. As concentrações de nitrito nos plasma tiveram medianas igual a 0,51 (Q1: 0,36/ Q3: 1,09) no grupo CA, mediana de 0,63 (Q1: 0,56/ Q3: 1,82) no grupo CD, mediana de 0,60 (Q1: 0,37/ Q3: 0,97) no grupo DPA e mediana de 0,5 (Q1: 0,39/ Q3: 0,83) no grupo DPD. As concentrações de nitrito no eritrócitos apresentaram médias de 0,45 e desvio padrão de 0,41 no grupo CA, média de 0,46 e desvio padrão de 0,47 no grupo CD, média de 0,57 e desvio padrão de 0,44 no grupo DPA e média de 0,29 e desvio padrão de 0,22 no grupo DPD. As concentrações de nitrito foram da ordem de micromolar na saliva e de nanomolar no sangue total, guardando uma relação de cerca de 1000 vezes entre estes 2 fluidos. As concentrações de nitrito obtidas foram decrescentes na seguinte ordem: saliva total, saliva sumandibular/sublingual, saliva parotídea, eritrócitos sangue total e plasma. Na saliva, os pacientes do grupo DPA e DPD apresentaram concentrações menores de nitrito em relação ao CA e CD (p<0.05), e o tratamento periodontal não teve efeito sobre as concentrações de nitrito. Também houve diminuição estatisticamente significante nas concentrações de nitrito no sangue total e eritrócitos após o tratamento periodontal (DPA versus DPD). Os resultados sugerem que não há relação entre as concentrações salivares e sanguíneas de nitrito. As concentrações salivares diminuídos depois do tratamento em pacientes com doença periodontal podem talvez estar associados com aumento dos níveis de arginase. Além disso, aumento nas concentrações sanguíneas de nitrito parecem estar associados aos aumentos de mediadores inflamatórios como IL-6, proteina C reativa, entre outros. As concentrações de nitrito no sangue total sugerem que a diminuição após o tratamento possa ser resultado da diminuição na atividade inflamatória nos pacientes com doença periodontal, já caracterizada anteriormente neste grupo de pacientes. / The Nitric Oxide (NO) is part of a family of free radicals that are involved in various bodily functions such as cardiovascular control, homeostasis, bone formation, neurotransmission and immune functions. It has been reported that NO act in response to host defense against infection of the oral tissues. Moreover, it has also been described that excessive amounts of NO can contribute to the periodontal tissue destruction. In addition to these very important aspects in the mouth, NO is a molecule of major importance to human health, since its release by the endothelium of blood vessels is a critical component to blood pressure control. It has been demonstrated that commensal bacteria of the mouth would be responsible for production of nitrites, in the stomach, be converted to NO and this be absorbed. The amount of nitrite in the saliva is about one thousand times greater than those detected in whole blood, it is believed that the nitrite saliva may have the effect of whole blood and NO can also possibly have an effect on regulation of systemic events as the proper blood pressure. The number of papers on NO in the saliva found in the literature of the English language does not add 2 tens, and the importance of the subject, it is necessary to know in detail the concentrations of nitrite in different types of saliva, whole blood, erythrocytes and plasma in patients with periodontal disease and controls before and after treatment. The hypotheses of this study are: 1 - That the nitrite concentrations are lower in whole saliva of individuals with periodontal disease and that they increase after periodontal treatment, 2 - What there would be the same profile of variation in the concentrations of nitrite in blood, erythrocytes and plasma seen in the whole saliva. In the present study were evaluated the concentrations of NO in whole saliva, salivasumandibular / sublingual, parotid, whole blood, erythrocytes and plasma of control patients and patients with chronic periodontits, with collections before and three months after non-surgical periodontal treatment. The following were tested if significant differences between the values obtained before and after treatment were compared and the profiles of the the results of nitrite in the saliva and blood. Were performed measurements of NO by Chemiluminescence analysis method. The results showed that in all clinical parameters were statistically significant differences before and after periodontal treatment (DPA and DPD) in the control groups before and after treatment (CA and CD) and controls before and patients before treatment (CA and DPA). Bleeding on Probe (DPA versus DPD) p<0,0001, (CA versus CD) p=0,0005, (CA versus DPA) p<0,0001. Clinical attachment level: (DPA versus DPD) p<0,0001, (CA versus CD) p=0,01 , (CA versus DPA) p<0,0002. Probing depth: (DPA versus DPD) p<0,0001, (CA versus DPA) p<0,0001. Sites larger than 4 mm: (DPA versus DPD) p<0,0001, (CA versus DPA) p<0,0001. This fact confirms a periodontal treatment performed successfully. In whole saliva, a significant decrease in NO concentration between controls and patients before treatment (CA versus PDA), with p = 0.04. Whole blood decreased NO concentration before and after periodontal treatment (DPA versus DPD), p = 0.026. Already in erythrocytes before and after treatment (DPA versus DPD) there was a significant decrease, p = 0.02. The concentrations of nitrite in whole saliva had median equal to 0.69 (Q1: 0.46 / Q3:1.44) in group CA, a median of 0.55 (Q1: 0.39 / Q3: 1.55) in CD group, median 0.44 (Q1: 0.26 / Q3: 0.81) in the DPA and a median of 0.38 (Q1: 0.22 / Q3: 0.69) in the DPD. The concentrations of nitrite in saliva submandibular / sublingual medians were equal to 1.86 (Q1: 0.61 / Q3: 4.59) in group CA, a median of 0.64 (Q1: 1.82 / Q3: 4.37 ) in the CD and a median of 2.37 (Q1: 0.47 / Q3: 4.79) in the DPA and a median of 2.69 (Q1: 1.69 / Q3: 3.43) in the DPD. The nitrite concentrations in parotid saliva showed mean of 0.58 and standard desviation of 0.32 in the CA group, mean 0.69 and standard deviation of 0.33 in CD group, mean 0.69 and standard deviation of DPA and 0.36 in the group average of 0.85 and standard deviation of 0.58 in the DPD group. The concentrations of nitrite in whole blood were median equal to 1.41 (Q1: 1.1 / Q3: 2.21) in group CA, a median of 0.99 (Q1: 0.58 / Q3: 2.11) in CD group, median 1.61 (Q1: 1.12 / Q3: 2.3) in the DPA and a median of 1.45 (Q1: 0.61 / Q3: 0.87) in the DPD. The plasma concentrations of nitrite in the medians were equal to 0.51 (Q1: 0.36 / Q3: 1.09) in group CA, a median of 0.63 (Q1: 0.56 / Q3: 1.82) in group CD and a median of 0.60 (Q1: 0.37 / Q3: 0.97) in the DPA and a median of 0.5 (Q1: 0.39 / Q3: 0.83) in the DPD. The concentrations of nitrite in erythrocytes showed averages of 0.45 and standard deviation of 0.41 in the CA group, mean 0.46 and standard deviation of 0.47 in the CD group, mean 0.57 and standard deviation of 0, 44 DPA and the group average of 0.29 and standard deviation of 0.22 in DPD group. The nitrite concentrations were in the order of micromolar and nanomolar saliva in whole blood, keeping a ratio of about 1000-fold between the two fluids. The concentrations of nitrite were obtained in the following descending order: whole saliva, saliva submandibular / sublingual, parotid saliva, whole blood, erythrocytes and plasma. In saliva, the patients in the PDA and DPD showed lower concentrations of nitrite in relation to CA and CD (p <0.05), and periodontal treatment had no effect on concentrations of nitrite. There was also a statistically significant decrease in nitrite concentrations in whole blood and erythrocytes after periodontal treatment (DPA versus DPD). The results suggest that there is no relationship between the salivary and blood concentrations of nitrite. Salivary levels decreased after treatment in patients with periodontal disease may perhaps be associated with increased levels of arginase. Furthermore, increased blood concentration of nitrite may be associated with increases in inflammatory mediators such as IL-6, C-reactive protein, among others. The nitrite concentrations in whole blood suggest that reduced after treatment may be a consequence of diminished inflammatory activity in patients with periodontal disease, characterized already earlier in this group of patients.

How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.

Background: The incidence of coronary heart diseases, including atherosclerosis, increases with ageing. The factors which influence arterial function, and which may be changed with ageing, are multiple but effects of perivascular adipose tissue (PVAT) on large arteries have not previously been considered. A key role for nitric oxide (NO) in mediating the anti-contractile capacity of PVAT has been suggested. Caveolin-1 (Cav-1) modulates the production of NO in vivo by tonic inhibition of eNOS. The influence of aortic PVAT and the contribution of NO to vascular reactivity in ageing C57BL/6 mice, atherosclerotic ApoE knockout mice (ApoE-/-), Cav-1 knockout mice (Cav-1-/-) and atheroprotected ApoECav-1 double knockout mice (ApoE-/-Cav-1-/-) is unknown. Hypothesis: The influence of PVAT on vascular function is modulated by ageing and the development of atherosclerosis via NO bioavailability. Methods: Male mice were used in this study. C57BL/6 mice were obtained at 4 weeks of age and maintained on a normal rodent diet (ND) for 8, 16 or 26 weeks. ApoE-/- and Cav-1-/- mice were bred from in-house colonies and ApoE-/-Cav-1-/- mice were generated by interbreeding ApoE-/- and Cav-1-/- mice. Upon weaning, ApoE-/-, Cav-1-/- and ApoE-/-Cav-1-/- mice were maintained on either a ND or Western-type diet (WD) for 8, 16 or 26 weeks. Vascular reactivity studies on isolated aortic ring preparations were performed in the presence or absence of PVAT. The contribution of NO to the vascular reactivity of aortic PVAT was determined using pharmacological inhibition of NO synthase. Aortic PVAT was assessed for evidence of morphological and/or compositional changes associated with ageing or a WD. Results: NO mediated an anti-contractile effect of aortic PVAT in C57BL/6 mice fed a ND up to 16 weeks. The anti-contractile capacity of aortic PVAT was lost after 26 weeks on a ND and preceded endothelial dysfunction. Loss of the PVAT anti-contractile effect was accompanied by alterations in PVAT morphology and composition. Aortic PVAT from ND-fed ApoE-/- mice was dysfunctional and did not exert an anti-contractile effect. Furthermore, a WD did not alter the influence of PVAT on vascular reactivity in ApoE-/- mice and PVAT morphology and composition was unchanged. NOS inhibition did not alter the contractile responses. The aortic PVAT of ND-fed Cav-1-/- mice did not exert an anti-contractile effect and PVAT composition was unchanged with increasing age. However, after 26 weeks on a WD, aortic PVAT from Cav-1-/- mice potentiated contractions to phenylephrine and white adipocyte hypertrophy was observed. NOS inhibition revealed a pro-contractile effect of aortic PVAT from Cav-1-/- mice. Loss of Cav-1-/- conferred significant protection against the development of atherosclerosis in WD-fed ApoE-/-Cav-1-/- mice despite a proatherogenic lipid profile. Aortic PVAT from ND-fed ApoE-/-Cav-1-/- mice did not exhibit an anti-contractile capacity and PVAT morphology was unchanged with ageing. Additionally, a WD did not influence the effect of PVAT on vascular reactivity in ApoE-/-Cav-1-/- mice although white adipocyte hypertrophy was observed after 26 weeks of high fat feeding. NOS inhibition revealed a pro-contractile effect of aortic PVAT in 8-week ND-fed ApoE-/-Cav-1-/- mice. Conclusions: This work has produced novel insights into the influence of aortic PVAT and NO on vascular reactivity and the morphology of aortic PVAT in ageing C57BL/6 mice, atherosclerotic ApoE-/- mice, Cav-1-/- mice and athero-protected ApoE-/-Cav-1-/- double knockout mice. Ageing to pre-middle age in C57BL/6 mice results in a loss of the anti-contractile effect of PVAT prior to endothelial dysfunction. This is associated with altered NO bioavailability and changes to the morphology and composition of PVAT. This may reveal potential therapeutic targets to restore the anti-contractile capacity of PVAT if comparable age-related PVAT dysfunction is observed in humans. Aortic PVAT of ApoE-/- mice does not exert an anti-contractile effect which may be attributed to decreased basal eNOS activity. A WD does not alter the vascular reactivity of PVAT. In addition, aortic PVAT from Cav-1-/- mice does not exhibit an anti-contractile capacity yet it exerts a pro-contractile effect after 26 weeks on a WD. The aortic PVAT of ApoE-/-Cav-1-/- mice does not modulate vascular reactivity and this is unaltered with feeding of a WD although white adipocyte hypertrophy was observed within the PVAT. The critical role of Cav-1 in the initiation and progression of atherosclerosis is reinforced by the atheroprotected phenotype of the ApoE-/-Cav-1-/- mice even though a severely proatherogenic lipid profile is observed in both the ND and WD-fed mice. Therapeutically targeting LDL transcytosis into the arterial wall could potentially prevent or halt the development of atherosclerosis. Aortic PVAT of ND-fed Cav-1-/- and ApoE-/-Cav-1-/- mice may not be dysfunctional but unable to modulate vascular reactivity due to attenuated vasoconstrictor responses of PVAT-denuded aortic rings as a result of excess NO, although this requires further investigation.

Lu Lifang. / "November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. (192-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Raymond Hiu Yeung, Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 51-69). / Acknowledgment --- p.iii / Abstract --- p.iv / List of Abbreviations --- p.vii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1. --- Parkinson's Disease --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Clinical symptoms --- p.2 / Chapter 1.1.3 --- Neuropathology --- p.3 / Chapter 1.2 --- Proposed mechanisms of Neuronal Cell Death in PD / Chapter 1.2.1 --- Oxidative Stress --- p.4 / Chapter 1.2.2 --- Mitochondrial Dysfunction --- p.5 / Chapter 1.2.3 --- Excitotoxicity --- p.6 / Chapter 1.2.4 --- Genetic Factor --- p.8 / Chapter 1.2.5 --- Aging --- p.9 / Chapter 1.3 --- "1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a PD model" / Chapter 1.3.1 --- The discovery of MPTP --- p.10 / Chapter 1.3.2 --- The mechanism of MPTP toxicity --- p.10 / Chapter 1.4 --- Reactive Oxygen Species (ROS) and Antioxidants in CNS / Chapter 1.4.1 --- Superoxide and Superoxide Dismutases --- p.13 / Chapter 1.4.2 --- "Hydrogen Peroxide, Catalase and Glutathione System" --- p.14 / Chapter 1.4.3 --- Hydroxyl Radicals --- p.15 / Chapter 1.4.4 --- Nitric Oxide (NO) --- p.16 / Chapter 1.5 --- Astrocytes / Chapter 1.5.1 --- Characteristics of astrocytes --- p.20 / Chapter 1.5.2 --- The role of astrocytes in PD --- p.21 / Chapter 1.6 --- The aim of this project --- p.24 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Astrocyte cultures --- p.27 / Chapter 2.2 --- MPTP treatment --- p.28 / Chapter 2.3 --- Lactate Dehydrogenase Assay --- p.29 / Chapter 2.4 --- Determination of nitrite and nitrate levels in cultured astrocytes --- p.30 / Chapter 2.5 --- Assay for Cyclic GMP production --- p.32 / Chapter 2.6 --- Inhibition of NO by L-NAME and Dexamethasone --- p.33 / Chapter 2.7 --- NFkB immunostaining --- p.33 / Chapter 2.8 --- Superoxide Dismutase Assay --- p.34 / Chapter 2.9 --- Statistics --- p.36 / Chapter CHAPTER 3: --- RESULTS / Chapter 3.1 --- Lactate dehydrogenase (LDH) activities after MPTP treatment --- p.37 / Chapter 3.2 --- The effects of MPTP on nitrite levels --- p.37 / Chapter 3.2.1 --- Mesencephalic astrocytes --- p.37 / Chapter 3.2.2 --- Striatal astrocytes --- p.38 / Chapter 3.2.3 --- Cortical astrocytes --- p.38 / Chapter 3.3 --- The effects of L-NAME on nitrite levels after MPTP treatment --- p.38 / Chapter 3.4 --- The effects of dexamethasone on nitrite levels after MPTP treatment --- p.39 / Chapter 3.5 --- Change in intracellular cyclic GMP in astrocytes after MPTP treatment --- p.40 / Chapter 3.6 --- The effects of MPTP on NFkB distribution in astrocytes --- p.40 / Chapter 3.7 --- The effects of MPTP on SOD activity in astrocytes --- p.41 / Chapter 3.7.1 --- Mesencephalic astrocytes --- p.41 / Chapter 3.7.2 --- Striatal astrocytes --- p.41 / Chapter 3.7.3 --- Cortical astrocytes --- p.42 / Chapter CHAPTER 4: --- DISCUSSION AND CONCLUSION --- p.43 / REFERENCES --- p.51

Chan Hoi Yun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGMENTS --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.v / ABSTRACT IN CHINESE --- p.viii / CONTENTS --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Steroid Hormones --- p.1 / Chapter 1.1.1. --- Synthesis of estrogens and progesterone --- p.1 / Chapter 1.2. --- Cellular Mechanisms of Female Steroid Hormones --- p.5 / Chapter 1.2.1. --- Genomic actions of female steroid hormones --- p.5 / Chapter 1.2.2. --- Non-genomic actions of female steroid hormones --- p.7 / Chapter 1.2.3. --- Estrogen antagonists --- p.7 / Chapter 1.2.3.1. --- Classification of estrogen antagonists --- p.7 / Chapter 1.2.3.2. --- Mechanisms of estrogen antagonists --- p.9 / Chapter 1.3. --- Chronic (genomic) Effects of 17β-Estradiol and Progesterone --- p.10 / Chapter 1.3.1. --- Effects of lipid metabolism --- p.10 / Chapter 1.3.2. --- Effects on cell proliferation --- p.11 / Chapter 1.3.3. --- Effects on endothelial cells --- p.12 / Chapter 1.4. --- Acute Effects of 17β-Estradiol and Progesterone --- p.13 / Chapter 1.4.1. --- Role of endothelium in 17β-estradiol or progesterone Relaxation --- p.13 / Chapter 1.4.2. --- Involvement of plasma membrane estrogen receptors --- p.14 / Chapter 1.4.3. --- Role of Ca2+ and K+ channel in estrogen relaxation --- p.14 / Chapter 1.4.4. --- Interaction with vasoconstrictors --- p.15 / Chapter 1.4.5. --- Interaction with endothelium-dependent dilators --- p.16 / Chapter 1.4.6. --- Interaction with adrenergic response --- p.17 / Chapter 1.5. --- Clinical Studies --- p.19 / Chapter 1.6. --- Therapeutic Values of Estrogen and Progesterone --- p.20 / Chapter 1.7. --- Objectives of the Present Study --- p.22 / Chapter Chapter 2 --- Method and Materials / Chapter 2.1. --- Tissue Preparation --- p.25 / Chapter 2.1.1. --- "Preparation of the rat aorta, mesenteric artery and carotid Artery" --- p.25 / Chapter 2.1.2. --- Removal of the functional endothelium --- p.26 / Chapter 2.2. --- Organ Bath Set-up --- p.26 / Chapter 2.3. --- Force Measurement --- p.28 / Chapter 2.3.1. --- Vascular action of 17β-estradiol and progesterone --- p.29 / Chapter 2.3.1.1. --- Role of endothelium/nitric oxide in 17β-estradiol- or progesterone-induced relaxation --- p.29 / Chapter 2.3.1.2. --- Role of inducible nitric oxide in progesterone-induced relaxation --- p.30 / Chapter 2.3.1.3. --- Effect of estrogen receptor inhibitor on 17β-estradiol- induced relaxation --- p.30 / Chapter 2.3.1.4. --- Interaction between progesterone and 17β-estradiol --- p.31 / Chapter 2.3.1.5. --- Effect of 17β-estradiol on protein kinase C-mediated contraction --- p.31 / Chapter 2.3.1.6. --- Synergistic interaction between β-adrenoceptor agonists and 17β-estradiol --- p.32 / Chapter 2.4. --- Porcine Coronary Artery Experiments --- p.33 / Chapter 2.4.1. --- Vessel preparation --- p.33 / Chapter 2.4.2. --- Force measurement --- p.33 / Chapter 2.4.3. --- Experimental protocol --- p.34 / Chapter 2.4.3.1. --- Effect of physiological level of 17β-estradiol on β- adrenoceptor agonist-induced relaxation --- p.34 / Chapter 2.4.3.2. --- Effect of physiological level of 17β-estradiol on phosphodiesterase inhibitor-induced relaxation --- p.34 / Chapter 2.5. --- Ovariectomy --- p.35 / Chapter 2.5.1. --- Method of ovariectomy --- p.35 / Chapter 2.5.2. --- Preparation of blood vessels --- p.36 / Chapter 2.5.3. --- Experimental protocols --- p.38 / Chapter 2.5.3.1. --- Effect of ovariectomy on contractility of rat carotid arteries --- p.38 / Chapter 2.5.3.2. --- Effect of ovariectomy on relaxation of rat carotid arteries --- p.38 / Chapter 2.6. --- Chemicals and Solutions --- p.39 / Chapter 2.7. --- Statistical Analysis --- p.42 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Role of Endothelium/Nitric Oxide in 17β-Estradiol- and Progesterone-induced Relaxations --- p.43 / Chapter 3.1.1. --- Relaxant response of 17β-estradiol --- p.43 / Chapter 3.1.2. --- Effects of inhibitors of nitric oxide activity on 17β- estradiol-induced relaxation --- p.46 / Chapter 3.1.3. --- Relaxant response of progesterone --- p.46 / Chapter 3.1.4. --- Effects of inhibitors of nitric oxide activity on progesterone-induced relaxation --- p.50 / Chapter 3.2. --- Effect of Estrogen Receptor Inhibitor on 17β-Estradiol- induced Relaxation --- p.56 / Chapter 3.3. --- Interaction between Progesterone and 17β-Estradiol --- p.56 / Chapter 3.4. --- Effect of Female Sex Steroid Hormones on Protein Kinase C-mediated Contraction --- p.59 / Chapter 3.4.1. --- Effect of 17β-estradiol on phorbol ester-induced contraction --- p.59 / Chapter 3.4.2. --- Effect of progesterone on phorbol ester-induced contraction --- p.59 / Chapter 3.5. --- Effects of β-adrenoceptor Agonists on 17β-Estradiol- induced Relaxations --- p.62 / Chapter 3.5.1. --- Effect of isoproterenol on 17β-estradiol-induced relaxation --- p.62 / Chapter 3.5.2. --- Role of endothelium/nitric oxide on the isoproterenol potentiation of 17β-estradiol-induced relaxation --- p.63 / Chapter 3.5.3. --- Role of cyclic AMP on isoproterenol-enhancement of 17β- estradiol-induced relaxation --- p.69 / Chapter 3.5.4. --- Effects of β-adrenoceptor antagonists --- p.69 / Chapter 3.6. --- Effects of Physiological Concentration of 17β-EstradioI onβ-adrenoceptor Agonists-induced Relaxationsin Porcine Coronary Artery --- p.77 / Chapter 3.6.1. --- Effect of 17β-estradiol on isoproterenol-induced relaxations --- p.77 / Chapter 3.6.2. --- Effect of 17β-estradiol on fenoterol-induced relaxations --- p.11 / Chapter 3.6.3. --- Effect of 17β-estradiol on dobutamine-induced relaxations --- p.81 / Chapter 3.6.4. --- Effect of 17β-estradiol on IBMX-induced relaxation --- p.86 / Chapter 3.7. --- Effect of Ovariectomy on the Vascualr Reactivity --- p.88 / Chapter 3.7.1. --- Effect of ovariectomy on the contractile activity of rat carotid artery --- p.88 / Chapter 3.7.1.1. --- Effect of ovariectomy on phenylephrine-induced contraction --- p.88 / Chapter 3.7.1.2. --- Effect of ovariectomy on U46619-induced contraction --- p.96 / Chapter 3.7.1.3. --- Effect of ovariectomy on high K+- induced contraction --- p.102 / Chapter 3.7.1.4. --- Effect of ovariectomy on acetylcholine-induced relaxation --- p.106 / Chapter Chapter 4 --- Discussions / Chapter 4.1. --- Role of Endothelium/Nitric oxide in 17β-Estradiol- and Progesterone-induced Relaxations --- p.110 / Chapter 4.2. --- Effect of Estrogen Receptor Inhibitor on 17β-Estradiol- induced Relaxation --- p.113 / Chapter 4.3. --- Interaction between Progesterone and 17β-Estradiol --- p.114 / Chapter 4.4. --- Effects of Female Sex Steroid Hormones on Protein Kinase C-mediated Contraction --- p.115 / Chapter 4.5. --- Effects of β-Adrenoceptor Agonists on 17β-Estradiol- induced Relaxations --- p.116 / Chapter 4.6. --- Effects of 17β-Estradiol on β-Adrenoceptor Agonists- induced Relaxations in Porcine Coronary Artery --- p.121 / Chapter 4.7. --- Effect of Ovariectomy on the Vascular Reactivity --- p.125 / Chapter 4.8. --- Conclusions --- p.129 / References --- p.131 / Publications --- p.145

Chong Lung-wun. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 49-58). / Abstracts in English and Chinese. / Acknowledgement --- p.1 / Contents --- p.2 / Abstract --- p.4 / Chapter I. --- Introduction --- p.6 / Chapter 1. --- Nitrate therapy for angina patients --- p.7 / Chapter 1.1 --- The mode of action of nitrates --- p.8 / Chapter 1.2 --- Hypotheses for the nitrate tolerance --- p.9 / Chapter 1.2.1 --- The Sulfhydryl depletion hypothesis --- p.9 / Chapter 1.2.2 --- Neurohormonal hypothesis --- p.10 / Chapter 1.2.3 --- Plasma volumc expansion hypothesis --- p.10 / Chapter 1.2.4 --- Free-radical hypothesis --- p.11 / Chapter 2. --- Testosterone therapy for angina patients --- p.12 / Chapter 3. --- Nitric Oxide and Steroidogenesis --- p.13 / Chapter II. --- Materials and Methods --- p.14 / Chapter 1. --- Subjects --- p.14 / Chapter 2. --- Method for measuring nitrite and nitrate in plasma --- p.16 / Chapter 3. --- Method for the determination of total and free testosterone in blood --- p.24 / Chapter 3.1 --- Reagent preparation --- p.24 / Chapter 3.2 --- Assay procedure for total testosterone --- p.28 / Chapter 3.3 --- Assay procedure for free testosterone --- p.29 / Chapter 3.4 --- Determination of working antiserum for free testosterone assay --- p.30 / Chapter 4. --- Method for the determination of serum Cortisol --- p.31 / Chapter III. --- Result --- p.33 / Chapter 1. --- Summary of nitric oxide assay performance --- p.33 / Chapter 2. --- Summary of total testosterone assay performance --- p.34 / Chapter 3. --- Summary of free testosterone assay performance --- p.34 / Chapter 4. --- Data analysis --- p.35 / Chapter IV. --- Discussion --- p.43 / Chapter V. --- Conclusion --- p.47 / Chapter VI. --- References --- p.49