Hipertensão do avental branco significa uma elevação persistente da pressão arterial no consultório médico ou clínica, com pressão normal em quaisquer outras circunstâncias. Existem diversos questionamentos a respeito da origem, significado clínico, prognóstico e tratamento desta manifestação. Em relação à etiologia, nossa hipótese é que uma alteração endotelial, resultando em deficiência na produção ou utilização de óxido nítrico endógeno, constitua um fator primário para a ocorrência da hipertensão do avental branco. Este estudo, desenvolvido entre moradores do município de Dumont - São Paulo, Brasil, teve como objetivos caracterizar os participantes em relação a fatores demográficos, alterações fisiológicas e metabólicas para posteriormente identificar e comparar os níveis plasmáticos de nitrato - produto da degradação do óxido nítrico ? entre os sujeitos da pesquisa. De uma amostra de 441 voluntários, selecionamos 109 indivíduos, que foram divididos em três grupos: normotensão (no=58), hipertensão essencial (no=33) e hipertensão do avental branco (no=18), após medidas de pressão arterial com aparelho oscilométrico e exame de Monitorização Ambulatorial da Pressão Arterial. Realizamos entrevista, mensuração de dados e coleta de exames laboratoriais para comparação das variáveis encontradas entre os grupos. Para o tratamento estatístico, foram utilizados os testes ANOVA e Tukey. Os resultados foram expressos como médias ± erros padrões das médias. As diferenças foram consideradas estatisticamente significativas para p<0,05. A prevalência de hipertensão do avental branco foi de 34,1%, com predominância do sexo feminino (83,3%), média de idade de 45,28 anos, sendo a maioria natural do Estado de São Paulo (66,7%), de cor branca (88,9%), alfabetizada (33,3%), casada (72,2%), com histórico familiar para doenças cardiovasculares (72,2%). A análise da quantificação de nitrato plasmático apontou diferença significativa entre os grupos hipertensão do avental branco e normotensão em comparação aos hipertensos, com elevação dos níveis de nitrato sérico em portadores de hipertensão essencial. Também encontramos diferença estatisticamente significativa para índice de massa corporal, relação cintura/quadril, glicemia e creatinina plasmáticas, na comparação entre hipertensos do avental branco e normotensos. As distinções observadas entre os grupos e a presença de variações clínicas, demográficas e bioquímicas possibilitam inferir que a hipertensão do avental branco é uma condição que deve ser analisada de maneira distinta em relação a indivíduos normotensos e portadores de hipertensão essencial. / The white coat hypertension is understood as a persistent increase in arterial pressure in the medical office or clinic, while normal blood pressure is observed in any other circumstances. There are several issues regarding the origin, clinical meaning, prognosis and treatment of this condition. Concerning the etiology, our hypothesis is that an endothelial alteration, leading to deficiency either in the production or utilization of endogenous nitric oxide, may constitute a primary factor for the occurrence of white coat hypertension. This study, developed with the population of the city of Dumont São Paulo, Brazil, aims to characterize the participants in relation to demographical factors and metabolic and physiological changes to afterwards identify and compare plasma levels of nitrate product of nitric oxide degradation among the researchs subjects. We selected 109 individuals, from a sample of 441, who were divided in three groups: normotensive (n=58), essential hypertension (n=33) and white coat hypertension (n=18), following arterial pressure measures with oscilometric device and Arterial Pressure Monitoring Exam. Interviews, data measures and laboratory exams were accomplished as to enable the comparison of the variables found between groups. For the statistical treatment, ANOVA and Tukeys test were used. Results were expressed in terms of means ± means standard deviations. The significance level adopted was p<0,05. White coat hypertension prevalence was of 34,1% with predominance of the feminine gender (83,3%), mean age 45,28, most of the participants original from the state of São Paulo (66,7%), white (88,9%), alphabetized (33,3%), married (72,2%) and with family history of cardiovascular diseases (72,2%). Quantification of plasma nitrate showed significant difference between the white coat hypertension group and the normotensive group in comparison to hypertensive patients, with increased levels of serum nitrate in essential hypertension patients. We also found statistically significant difference for corporal mass index, hip/waist ratio, plasma glucose and creatinine, in the comparison between white coat hypertensive and normotensive patients. The distinctions observed between groups and the presence of clinical, demographical and biochemical variations allow us to suggest that the white coat hypertension is a condition which must be analyzed in a distinct way in relation to normotensive and essential hypertension patients.

Orientadores: Carlos Arturo Levi D'Ancona, Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T05:49:51Z (GMT). No. of bitstreams: 1 Pereira_MarcyLancia_D.pdf: 636046 bytes, checksum: 9238264528136d3bd8cf921cf3ab4552 (MD5) Previous issue date: 2014 / Resumo: A hiperplasia prostática benigna (HPB) ou aumento benigno da próstata (ABP) leva a disfunção do trato urinário, que pode ser mimetizada outras espécies animais, como roedores, por meio de obstrução parcial da uretra (OPU). O óxido nítrico (NO), sintetizado a partir da L-arginina por meio de três isoenzimas (iNOS, eNOS e nNOS) vem sendo estudado por ser apontado como responsável por alterações morfológicas e funcionais decorrentes do processo obstrutivo uretral. Este trabalho teve como objetivo caracterizar as alterações vesicais crônicas em camundongos com OPU e tratados cronicamente com L-NAME (inibidor competitivo não seletivo da NOS) e aminoguanidina (inibidor competitivo seletivo para iNOS). Os animais foram divididos em 6 grupos experimentais: Sham, Sham + L-NAME, Sham + aminoguanidina, OPU, OPU + L-NAME e OPU + aminoguanidina. A realização da OPU foi feita por meio de laparotomia e ligadura parcial ao nível do colo vesical utilizando-se cateter como guia externo. Após 5 semanas do procedimento cirúrgico, os animais foram avaliados quanto a cistometria, estudos farmacológicos em banho para órgão isolado e peso vesical. Os animais OPU apresentaram disfunção vesical observada por meio de aumento de contrações não miccionais (CNM) e da capacidade vesical, além de menor resposta contrátil muscarínica e elétrica. A inibição das três isoformas de NOS levou a diminuição da capacidade vesical em animais OPU. O tratamento com L-NAME levou a aumento de CNM, prevenção ao ganho de peso vesical e aumento das respostas contráteis a estimulação muscarínica e elétrica em animais OPU. A aminoguanidina diminuiu as CNM, mas não evitou o aumento do peso da bexiga em animais OPU e não aumentou as respostas contráteis vesicais. Tais achados sugerem que as NOS constitutivas (eNOS e nNOS) parecem ter papel mais relevante na fisiopatologia da OPU crônica do que a iNOS / Abstract: Benign prostatic hyperplasia (BPH) or Benign prostatic enlargement (BPE) leads to urinary tract dysfunction, which can be seen in experimental models, like rodents, by causing bladder outlet obstruction (BOO). Nitric oxide (NO), synthetized from L-arginine by three isoforms (iNOS, eNOS and nNOS) has been studied because it can be responsible for the urinary morphofunctional alterations. This study aimed to evaluate chronic bladder function in mice with BOO and treated chronically with L-NAME (non-selective NOS inhibitor) and aminoguanidine (iNOS selective inhibitor). Animals were divided into 6 experimental groups: Sham, Sham + L-NAME, Sham + aminoguanidine, BOO, BOO + L-NAME e BOO + aminoguanidine. BOO induction was made by laparotomy and partial ligature of bladder neck with a catheter as external guide. After 5 weeks of surgical procedure, animals were evaluated and filling cystometry, tissue bath contractile studies and bladder weight. BOO animals showed increase of non voiding contractions (NVC) and bladder capacity, and also less contractile response to Carbachol and Electric Field Stimulation. Inhibition of NOS isoforms diminished bladder capacity in BOO animals. L-NAME caused more NVC, prevented bladder weight gain and leaded to augmented contractile responses at muscarinic and electric stimulation. Aminoguanidine diminished NVC, but did not avoid bladder weight gain in BOO animals and did not cause increase in contractile responses. These results suggest that constitutive NOS (eNOS and nNOS) seem to be more important in chronic BOO pathophysiology than iNOS / Doutorado / Fisiopatologia Cirúrgica / Doutora em Ciências

Orientador: José Barreto Campello Carvalheira, Marcelo Gonzarolli de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T01:22:50Z (GMT). No. of bitstreams: 1 Dias_MariliaMeira_D.pdf: 3051857 bytes, checksum: 7b4e0fbf888dc0d19226feb71ecec132 (MD5) Previous issue date: 2012 / Resumo: A S-nitrosação é uma modificação pós-traducional dinâmica e reversível de proteínas que controla importantes funções celulares através da alteração de resíduos tióis de cisteínas pelo óxido nítrico (NO). Assim como a fosforilação, a S-nitrosação é capaz de alterar a conformação, a atividade e a função de proteínas e contribui em grande parte para a influência ubíqua do NO nas vias de transdução de sinal celulares. Inúmeras evidências apontam para um papel crucial da S-nitrosação não apenas na fisiologia dos organismos, mas também em inúmeras doenças humanas, entre elas o câncer. Entretanto, trata-se de um mecanismo ainda pouco explorado de modulação de proteínas envolvidas com a progressão tumoral, como as presentes na via do mTOR. Assim, o objetivo do trabalho é analisar os mecanismos pelos quais o NO afeta a função do mTOR. A análise da S-nitrosação do mTOR foi realizada através do Biotin Switch Method e demonstra que o doador exógeno de NO, GSNO, aumenta os níveis de mTOR S-nitrosada. Além disso, a GSNO reduz a fosforilação das proteínas mTOR, p70S6K e 4EBP-1 de forma tempo- e dose-dependente. A fosforilação da proteína p70S6K, reduzida pela exposição ao NO, é restaurada pela adição do agente redutor e denitrosante DTT. Entretanto, o pré-tratamento das linhagens celulares PC-3, DU145 e MCF-7 com o inibidor da guanilil ciclase, ODQ, ou com o sequestrador de NO livre, PTIO, não reverte o efeito da GSNO na fosforilação das proteínas mTOR, p70S6K e 4EBP-1, corroborando a hipótese de que o mTORC1 é modulado por S-nitrosação. A viabilidade das linhagens tumorais humanas PC3, DU145, MDA-MB-468, MCF-7, HT-29, CACO-2 e A549 também diminui após exposição aos doadores de NO, sendo observada redução da proliferação e aumento da morte celular. As linhagens PC-3, MCF-7 e MDA-MB-468, que apresentam mutações ativadoras na cascada de sinalização PI3K-mTOR, são mais sensíveis à presença do NO. Assim, a S-nitrosação do mTORC1 é uma importante modificação pós-traducional capaz de modular a atividade quinase da proteína mTOR e reduzir a proliferação de células tumorais humanas, apresentando potenciais implicações terapêuticas / Abstract: S-Nitrosation is a dynamic and reversible post-translational modification of proteins that controls important cellular functions through the modification of cysteine thiol side chains by nitric oxide (NO). mTOR signaling pathway deregulation is involved in various cancer types and contributes to cancer cell proliferation as well as growth factor independence. The aim of this work was to analyze the mechanisms by which S-nitrosogluthatione (GSNO) affects mTOR function. S-nitrosation of mTOR was assessed by the Biotin Switch Method. GSNO was shown to S-nitrosate mTOR with a consequent time- and dose-dependent decrease in the phosphorylation of mTOR and S6K proteins, which were reversed by the addition of the denitrosating agent DTT. Pre-treatments of cells with the inhibitor of soluble guanylyl cyclase, ODQ, or with the NO scavenger, PTIO, had no effect on the GSNO-mediated decrease in phosphorylation of mTOR and its substrates p70S6K and 4EBP-1. Results also demonstrated that cancer cell line viability decreased after exposure to the NO donors GSNO and S-nitroso-N-acetyl-cysteine (SNAC), altering proliferation and increasing cell death. PC-3, MCF-7 and MDA-MB-468 cell lines, which have mutations in the PI3K-mTOR pathway, showed a greater response to GSNO. Therefore, S-nitrosation is a novel post-translational modification capable of modulating mTOR activity, with possible therapeutic implications / Doutorado / Fisiopatologia Médica / Doutor em Ciências

Orientadores: Gilberto de Nucci, Fabiola Taufic Monica Iglesias / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T09:35:48Z (GMT). No. of bitstreams: 1 RojasMoscoso_JulioAlejandro_D.pdf: 775214 bytes, checksum: 63d1a32bc16687d42f750db1ef690fb0 (MD5) Previous issue date: 2012 / Resumo: A enzima alvo do NO, a guanilato ciclase solúvel (GCs) é responsável em converter o trifosfato de guanosina (GTP) em monofosfato de guanosina cíclico (GMPc). É sabido que a via do NO-GCs-GMPc está alterada em diversas patologias, como no diabetes mellitus, na hipertensão arterial e pulmonar, na disfunção erétil assim como nas alterações do baixo trato urinário. O estresse oxidativo presente nestas patologias pode ser um dos responsáveis por influenciar o estado redox da GCs promovendo a oxidação da mesma e, portanto, criando um estado de refratariedade aos tratamentos usuais. O uso de nitratos orgânicos não é eficiente no tratamento destas patologias já que o uso contínuo leva à tolerância. Sendo assim, compostos que atuem na via NO-GCs-GMPc seja ativando ou estimulando a enzima GCs constituem importantes alvos no tratamento das disfunções causadas por anormalidades da via do NO. Baseado no exposto acima o objetivo do presente trabalho foi avaliar o estado redox da enzima GCs de ratos hipertensos renovasculares (2K1C) e espontaneamente hipertensos (SHR) e seus respectivos grupos controles através de curvas concentração resposta à fenilefrina (PE), acetilcolina (ACh), nitroprussiato de sódio (SNP), ao estimulador (BAY 41-2272) e ativador (BAY 60-2770) da GCs em artéria mesentérica superior isolada. Em alguns experimentos os inibidores da GCs (quinoxalin-1-one 1H[1,2,4] oxidiazolo [4,3-a]) (ODQ) ou da sintase de óxido nítrico (NOS), N (G)-nitro-Larginine methyl ester (L-NAME) foram incubados previamente ao relaxamento induzido pelo BAY 41-2272 ou BAY 60-2770. Os parâmetros de potência (pEC50) e resposta máxima (Emax) foram determinados. A pressão sistólica (PS) e o peso foram determinados semanalmente. A PS dos animais SHR mostrou elevada (188,23 ± 3,46 mm/Hg) na 8ª semana em relação ao respectivo grupo controle (118,83 ± 2,67 mm/Hg), sendo este aumento ainda maior na 16ª (203,75 ± 3,61 mm/Hg), enquanto que o peso corporal dos animais SHR apresentou-se menor na 8ª semana de vida. Nos ratos 2K1C os animais apresentaram aumento na PS de, aproximadamente, 25 e 40 % na 1ª e 8ª semana pós-clipagem, respectivamente. Os resultados funcionais mostraram que a contração a fenilefrina foi significativamente maior nos animais SHR em comparação ao controle e este efeito não foi observado nos animais 2K1C. Em animais SHR e 2K1C o relaxamento a ACh foi maior em comparação ao grupo controle, com aumento da resposta máxima e deslocamento da potência (aproximadamente 3 vezes), respectivamente. Em relação ao SNP, tanto nos animais SHR como nos 2K1C houve deslocamento da pEC50 de, aproximadamente, 67 e 5 vezes para a direita, respectivamente em relação aos respectivos grupos controles, sem alteração da Emax. O relaxamento ao estimulador da GCs, BAY 41-2272 encontrou-se deslocado para a direita nos animais SHR (2,2 vezes), sem nenhuma alteração nos ratos 2K1C. A adição de L-NAME e ODQ tanto nos animais hipertensos como normotensos diminui a potência do BAY 41-2272, sugerindo que o relaxamento desta substância é dependente do acúmulo de GMPc e atua sinergicamente com NO. Interessantemente, o ativador BAY 60-2770 induziu relaxamento nos animais SHR que foi, aproximadamente, 47 vezes mais potente em relação ao controle. Nenhuma diferença foi observada nos animais 2K1C. Diferentemente do BAY 41-2272, a presença de LNAME ou ODQ potencializou o relaxamento ao BAY 60-2770, mostrando que a oxidação da GCs ou até mesmo a ausência de NO favorecem este efeito. Em conclusão, nossos dados mostram que nas mesentéricas dos animais SHR a enzima GCs pode estar oxidada (Fe3+), uma vez que observamos diminuição e aumento da potência do BAY 41-2272 e BAY 60-2770, respectivamente. Por sua vez, nos vasos de animais 2K1C nenhuma alteração foi observada nas respostas a estas substâncias, porém, o relaxamento ao SNP encontrou-se diminuído, sugerindo diminuição da biodisponibilidade do NO. Assim, os estimuladores e ativadores da enzima GCs constituem importante ferramenta farmacológica para avaliar o estado redox da GCs / Abstract: The target enzyme NO, the soluble guanylate cyclase (sGC) is responsible to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). It is known that, via the NO-cGMP GCs is altered in various pathologies, such as diabetes mellitus, arterial hypertension and pulmonary in erectile dysfunction as well as changes in the lower urinary tract. The oxidative stress can be present in these conditions a significant role in influencing the redox state of sGC promoting oxidation thereof and, thus creating a state of refractory to usual treatments. The use of organic nitrates is not effective in treatment of pathologies since the continuous use leads to tolerance. Thus, compounds that act via the NO-sGC-cGMP is activating or stimulating the enzyme sGC are important targets in the treatment of disorders caused by abnormalities of the NO pathway. Based on the above the aim of this study was to evaluate the redox state of the enzyme sGC renovascular hypertensive rats (2K1C) and spontaneously hypertensive rats (SHR) and their respective controls by concentration-response curves to phenylephrine (PE), acetylcholine (ACh), sodium nitroprusside (SNP), the stimulator (BAY 41-2272) and activator (BAY 60-2770) of the sGC in superior mesenteric artery isolated. In some experiments inhibitors sGC (quinoxalin-1-one 1 H [1,2,4] oxidiazolo [4,3-a]) (ODQ) or nitric oxide synthase (NOS), N (G)-nitro-L- arginine methyl ester (LNAME), were incubated prior to the relaxation induced by BAY 41-2272 or BAY 60- 2770. The power parameters (pEC50) and maximal response (Emax) was determined. Systolic blood pressure (SBP) and weight were determined weekly. The PBS of the SHR showed higher (188.23 ± 3.46 mm/Hg) at week 8 compared to respective control group (118.83 ± 2.67 mm/Hg), and this increase was even higher at 16 (203.75 ± 3.61 mm/Hg), whereas the body weight of SHR was lower at 8 weeks of age. In rats, 2K1C animals showed an increase in PBS of approximately 25 and 40% in the 1st and 8th week after clipping, respectively. Functional results showed that the contraction to phenylephrine was significantly higher in SHR compared to control and this effect was not observed in 2K1C animals. In SHR and 2K1C relaxation to ACh was greater in the control group, with increased maximum response and power shift (approximately 3- fold), respectively. Concerning the SNP, both in SHR and in 2K1C displacement pEC50 was approximately 67 and 5 times to the right, respectively in relation to the respective control group, without changing the Emax. The relaxation of the sGC stimulator, BAY 41- 2272 was found displaced to the right in SHR (2.2 times), with no change in 2K1C rats. The addition of L-NAME and ODQ both in normotensive and hypertensive rats reduces the power of BAY 41-2272, suggesting that the relaxation of this substance is dependent on the accumulation of cGMP and acts synergistically with NO. Interestingly, the activator BAY 60-2770 induced relaxation in SHR was approximately 47 times more potent than the control. No difference was observed in 2K1C animals. Unlike BAY 41- 2272, the presence of L-NAME or ODQ potentiated relaxation to BAY 60-2770, showing that the oxidation of sGC or even the absence of NO contribute to this effect. In conclusion, our data show that in the mesenteric SHR sGC the enzyme can be oxidized (Fe3+), since we observed a decrease and increase of power of BAY 41-2272 and BAY 60-2770, respectively. In turn, in 2K1C animals vessels no change was observed in response to these substances, however, found to relax the SNP is decreased, suggesting decreased bioavailability of the NO. Thus, stimulators and activators of the enzyme sGC is an important pharmacological tool to assess the redox state of the GCs / Doutorado / Farmacologia / Doutor em Farmacologia

Orientador: José Butori Lopes de Faria / Textos em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T19:18:35Z (GMT). No. of bitstreams: 1 Faria_AlineMacedo_D.pdf: 2909540 bytes, checksum: fff73b7c4aa0615644542da6920d5af7 (MD5) Previous issue date: 2012 / Resumo: No diabetes mellitus (DM), o estresse oxidativo e a redução do óxido nítrico (NO) contribuem para a patogênese da nefropatia diabética. O desacoplamento da NO sintase endotelial (eNOS) faz com que essa sintase produza superóxido ao invés de NO. O objetivo do presente estudo foi investigar o potencial do chá verde (CV) na melhora do desacoplamento da eNOS no DM. Em ratos com DM induzido por estreptozotocina, a biodisponibilidade NO estava reduzida pelo desacoplamento da eNOS, caracterizada pela redução nos níveis de BH4 e pela redução da estrutura conformacional ativa da eNOS, avaliada pela expressão da razão dímero/monômero. O tratamento com chá verde foi capaz de reverter estas abnormalidades. Além disso, células mesangiais humanas imortalizadas (ihMCs) cultivadas sob condições de alta glicose (30mM) exibiram um aumento na produção de espécies reativas de oxigênio (ERO) e uma redução na biodisponibilidade de NO, que foram revertidos pelo CV. A produção de BH4 e a atividade da guanosina trifosfato ciclohidrolase I (GTPCH I), enzima importante na formação do BH4, diminuíram em ihMCs expostas a alta glicose e foram normalizados pelo CV. Administração exógena de BH4 nas ihMCs reverteu o aumento das ERO e declínio da produção de NO induzido pela alta glicose. Contudo, a co-administração de CV e BH4 não resultou em uma redução adicional na produção de ERO, sugerindo que a redução na produção de ERO pelo CV é um efeito secundário ao desacoplamento da eNOS. Em resumo, CV reverte a redução dos níveis de BH4, induzida pelo DM, melhorando o desacoplamento da eNOS, levando ao aumento da biodisponibilidade de NO e redução do estresse oxidativo, duas anormalidades que são envolvidas na patogêneses da nefropatia diabética / Abstract: The aim of the present study was to investigate the potential of green tea (GT) to improve uncoupling endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes mellitus (DM), nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in BH4 levels and an increase in the eNOS dimer/monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH4 and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and were normalized by GT. Exogenous administration of BH4 in ihMCs reversed the HG-induced rise in ROS and decline in NO production. However, co-administration of GT with BH4 did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes-induced reduction of BH4 levels, ameliorating uncoupling eNOS, thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15728号 / 工博第3342号 / 新制||工||1505(附属図書館) / 28273 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 濵地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Philosophiae Doctor - PhD / This study explores the roles of exogenously applied nitric oxide, exogenously applied caffeic acid and salt stress on the antioxidant system in cereal (exemplified by maize) and legume (using soybean as an example) plants together with their influence on membrane integrity and cell death.This study investigates changes in H2O2 content, root lipid peroxidation, root cell death and antioxidant enzymatic activity in maize roots in response to exogenously applied nitric oxide (NO) and salt stress. This part of the study is based on the partially understood interaction between NO and reactive oxygen species (ROS) such as H2O2 and the role of antioxidant enzymes in plant salt stress responses. The results show that application of salt (NaCl) results in elevated levels of H2O2 and an increase in lipid peroxidation, consequently leading to increased cell death. The study also shows that by regulating the production and detoxification of ROS through modulation of antioxidant enzymatic activities, NO plays a pivotal role in maize responses to salt stress. The study argues for NO as a regulator of redox homeostasis that prevents excessive ROS accumulation during exposure of maize to salinity stress that would otherwise be deleterious to maize. This study extends the role of exogenously applied NO to improve salt stress tolerance in cereals crops (maize) further to its role in enhancing salt stress tolerance in legumes. The effect of long-term exposure of soybean to NO and salt stress on root nodule antioxidant activity was investigated to demonstrate the role of NO in salt stress tolerance. The results show that ROS scavenging antioxidative enzymes like SOD, GPX and GR are differentially regulated in response to exogenous application of NO and salt stress. It remains to be determined if the NOinduced changes in antioxidant enzyme activity under salt stress are sufficient to efficiently reduce ROS accumulation in soybean root nodules to levels close to those of unstressed soybean root nodules. Furthermore, this study investigates the effect of long-term exposure of soybean to exogenous caffeic acid (CA) and salt stress, on the basis of the established role of CA as an antioxidant and the involvement of antioxidant enzymes in plant salt stress responses. The effect of CA on soybean nodule number, biomass (determined on the basis of nodule dry weight, root dry weight and shoot dry weight), nodule NO content, and nodule cyclic guanosine monophosphate (cGMP) content in response to salt stress was investigated. Additionally, CA-induced changes in nodule ROS content, cell viability, lipid peroxidation and antioxidant enzyme activity as well as some genes that encode antioxidant enzymes were investigated in the presence or absence of salt stress. The study shows that long-term exposure of soybean to salt stress results in reduced biomass associated with accumulation of ROS, elevated levels of lipid peroxidation and elevated levels of cell death. However, exogenously applied CA reversed the negative effects of salt stress on soybean biomass, lipid peroxidation and cell death. CA reduced the salt stress-induced accumulation of ROS by mediating changes in root nodule antioxidant enzyme activity and gene expression. These CA-responsive antioxidant enzymes were found to be superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR), which contributed to the scavenging of ROS in soybean nodules under salt stress. The work reported in Chapter 2 has been published in a peer-reviewed journal [Keyster M, Klein A, Ludidi N (2012) Caspase-like enzymatic activity and the ascorbate-glutathione cycle participate in salt stress tolerance of maize conferred by exogenously applied nitric oxide. Plant Signaling and Behavior 7: 349-360]. My contribution to the published paper was all the work that is presented in Chapter 2,whereas the rest of the work in the paper (which is not included in Chapter 2) was contributed by Dr Marshall Keyster.

>Magister Scientiae - MSc / Nitric oxide (NO) has been shown to orchestrate multiple defense responses to both abiotic and biotic stress. Importantly, elevation of nitric oxide content in plants by using nitric oxide generating compounds has been shown to enhance plant tolerance to abiotic stresses such as salt and drought via up-regulation of genes involved in the regulation of plant responses to abiotic stress. In this study, the effect(s) of nitric oxide (generated from 10 μM of the nitric oxide donor DET/NO) on the expression of a novel soybean cystatin gene (Glyma20g08800), lipid peroxidation, caspase-like activity and cell death in salt (150 mM)-stressed soybean leaves, roots and nodules were investigated. Salt treatment resulted in elevated lipid peroxidation, caspase-like activity and increased cell death in organs studied while the observed detrimental effects of salt stress were reversed by NO treatment. Salt stress suppressed the expression of Glyma20g08800 while the levels of expression of Glyma20g08800 returned towards those of unstressed plants when the salt-stressed plants were supplemented with nitric oxide (DETA/NO). Furthermore, promoter sequences of GmCYS1p626 and three of its homologues (Glyma20g08800, Glyma14g04250 and Glyma18g12240) were analyzed for putative abiotic stress and/NO cisregulatory elements based on co-expression analyses using bioinformatics. Several abiotic stress induced transcription factors (TFs) were identified and were hypothesized to be co-acting either directly or indirectly through additional factors in the regulation of soybean cystatin expression in response to NO and abiotic stress. Taken together, these results highlight the possibility of using NO to drive high levels of expression of cystatins during salt stress and lead to accumulation of the cystatin to levels that are sufficient to inhibit salt stress-induced caspase-like activity, which will inhibit salt stress-induced cell death and thus enhance the tolerance of the plant to salt stress and possibly tolerance to drought stress as well.

>Magister Scientiae - MSc / Nitric oxide (NO) is a signaling molecule involved in mediating plant responses to various biotic and abiotic stresses. Major abiotic stresses (drought, salinity, cold) induce common cellular responses, causing osmotic stress in plants. This results in oxidative stress due to increased production of reactive oxygen species (ROS). The increased ROS levels simultaneously induce the antioxidative system (including antioxidant enzymes such as superoxide dismutase) that regulates ROS toxicity and enhance stress tolerance in plants. It is suggested that the scavenging of ROS by antioxidant enzymes can be controlled by NO. The aim of this study was to evaluate the role of exogenously applied NO on soybean (Glycine max L. Merr.) during osmotic stress, with the purpose of determining the effects of NO on the superoxide dismutase (SOD) activity in response to osmotic stress. This study also aimed at identifying and characterising SOD isoforms induced in soybean in response to osmotic stress and exogenous NO. To achieve these aims, soybean plants were treated with sorbitol (to induce osmotic stress), an NO donor [2,2'-(hydroxynitrosohydrazono)bis-ethanimine, DETA/NO] and its respective control (Diethylenetriamine, DETA). The results showed that exogenous NO alleviated osmotic stress-induced damage by reducing the superoxide radical content, lipid peroxidation levels and also maintaining cell viability in soybean leaves, nodules and roots. Only two SOD isoforms i.e. manganese SOD (MnSOD) and copper/zinc SOD (CuZnSOD) were identified and characterised in soybean leaves and roots, iron SOD (FeSOD) was not induced. The isoforms identified exhibited low SOD activity in response to osmotic stress, with the exception of a few isoforms that had increased activity. The SOD activity was regulated by exogenously applied NO. The enzymatic activity of SOD isoforms was up-regulated by exogenous NO, except for a few SOD isoforms that were not responsive to NO. The results also showed that the increased SOD activity was associated with reduced lipid peroxidation levels. The results obtained from this study suggest that exogenous NO improves osmotic stress tolerance in soybean by regulating and increasing the SOD activity of only specific isoforms. The increased SOD activity maintains the redox homeostasis balance by detoxifying and controlling the superoxide radical levels, subsequently reducing lipid peroxidation and maintaining cell viability.

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