Conselho Nacional de Desenvolvimento Científico e Tecnológico / Hypothyroidism is a syndrome resulting from decreased production and secretion of triiothyronine (T3) and thyroxine (T4) hormones, frequently diagnosed in women. Clinical and experimental evidence support the association between reduction in thyroid hormones, neuropsychiatric disorders and oxidative stress. Selenium (Se) is a trace element essential to thyroid functions. The present study investigated the effect of diphenyl diselenide supplementation in a model of methimazole-induced hypothyroidism in female rats. We evaluated behavioral parameters related to memory and spatial learning (Article 1) and depressive-like behavior (Manuscript 1). Biochemical parameters were also investigated in relation to oxidative stress, MAO activity and the expression of antioxidant enzymes genes (Manuscripts 1 and 2). In Article 1, we observed a significant effect of diet supplemented with diphenyl diselenide in improving memory and spatial learning deficits in hypothyroid animals undergoing the task of Morris Water Maze. In Manuscript 1 we observed that methimazole-induced hypothyroidism caused a depressive-like behavior assessed in the forced swimming test and that supplementation with diphenyl diselenide caused a reversal of this effect. Furthermore, animals supplemented with diphenyl diselenide performed better in evaluating the antidepressant-like effect. In both (Article 1 and Manuscript 1), there were no motor and/or anxiety alterations, which could interfere with the results. Biochemical parameters in Manuscript 1 and 2 demonstrated that diphenyl diselenide improved analyzes related to oxidative stress (TBARS, ROS and NP-SH) in brain structures and restored the activity of MAO-B (Manuscript 1). In addition, Manuscript 2 evaluated the effects of hypothyroidism and supplementation with diphenyl diselenide on gene expression of antioxidant enzymes and transcription factor NRF-2. The results showed that hypothyroidism caused a significant increase in the expression of antioxidant enzymes, positively correlated to the transcription factor NRF-2. This effect was observed predominantly in the cerebral cortex and hippocampus, whereas supplementation with diphenyl diselenide was effective in normalizing the levels of antioxidant gene expression. Although hypothyroidism has caused a significant increase in gene expression levels of SOD, CAT and GPx were not observed changes in enzyme activity. Finally, the results of this study indicate through different experimental protocols that methimazole-induced hypothyroidism caused a deficit in spatial learning and memory, and depressive-like behavior. In addition, there were changes related to oxidative stress and the regulation of gene expression related to the antioxidant defense system. Diphenyl diselenide presented significant effects in promoting the reversal of behavioral effects and restore the biochemical parameters, and its role in regulating the expression of genes related to antioxidant defense system, adding a new interpretation to its pharmacological effects and demonstrates its potential to treat the complications caused by hypothyroidism. / O hipotireoidismo é uma síndrome caracterizada pela produção e secreção insuficiente dos hormônios tireóideos, triiodotironina (T3) e tiroxina (T4), frequentemente diagnosticada em mulheres. Evidências clínicas e experimentais têm relatado a associação entre redução dos hormônios tireóideos, doenças neuropsiquiátricas e estresse oxidativo. O selênio (Se) é um elemento traço essencial às funções tireóideas. Este estudo investigou o efeito da suplementação com disseleneto de difenila sobre parâmetros comportamentais e bioquímicos em um modelo de indução ao hipotireoidismo pelo metimazol, em ratas. Foram avaliados parâmetros comportamentais relacionados à memória e à aprendizagem espacial (Artigo 1) e ao comportamento semelhante à depressão (Manuscrito 1). Parâmetros bioquímicos também foram investigados em relação ao estresse oxidativo, à atividade da enzima MAO e à expressão gênica relacionada ao sistema de defesa antioxidante (Manuscritos 1 e 2). No Artigo 1, observamos um efeito significativo da dieta suplementada com disseleneto de difenila, ao reverter o déficit relacionando à memória e à aprendizagem espacial nos animais hipotireóideos submetidos à tarefa do Labirinto Aquático de Morris. No Manuscrito 1 observamos que o hipotireoidismo induzido pelo metimazol causou um comportamento semelhante à depressão avaliada no Teste do Nado Forçado e que a suplementação com disseleneto de difenila provocou a reversão desse efeito. Além disso, os animais suplementados com o disseleneto de difenila apresentaram um desempenho melhor na avaliação do efeito semelhante ao antidepressivo. Em ambos (Artigo 1 e Manuscrito 1), não ocorreram alterações motoras e/ou relacionadas à ansiedade, que pudessem interferir nos resultados. Os parâmetros bioquímicos avaliados nos Manuscritos 1 e 2 mostraram que o disseleneto de difenila melhorou os marcadores de estresse oxidativo (TBARS, ROS e NP-SH) nas estruturas cerebrais e restaurou a atividade da enzima MAO B (Manuscrito 1). Além disso, o Manuscrito 2 avaliou os efeitos do hipotireoidismo e da suplementação com disseleneto de difenila sobre a expressão gênica das enzimas antioxidantes e do fator de transcrição NRF-2. Os resultados mostraram que o hipotireoidismo provocou um aumento significativo na expressão das enzimas antioxidantes, correlacionado positivamente ao fator de transcrição NRF-2. Esse efeito foi predominantemente observado no córtex cerebral e no hipocampo, sendo que a suplementação com disseleneto de difenila foi efetiva em normalizar os níveis de expressão gênica antioxidante. Apesar de o hipotireoidismo ter causado um aumento significativo nos níveis de expressão gênica das enzimas SOD, CAT e GPx não se observaram alterações nas atividades enzimáticas. A partir desse estudo pode-se concluir através dos diferentes protocolos experimentais realizados que o hipotireoidismo induzido pelo metimazol causou déficit de memória e de aprendizagem espacial, bem como o comportamento semelhante à depressão. Além disso, observaram-se as alterações relacionadas ao estresse oxidativo e à regulação da expressão gênica relacionada ao sistema de defesa antioxidante. O disseleneto de difenila apresentou efeitos significativos na reversão dos efeitos comportamentais e na restauração dos parâmetros bioquímicos avaliados, sendo que a regulação da expressão de genes relacionados ao sistema de defesa antioxidante acrescenta uma nova interpretação aos seus efeitos farmacológicos e demonstra o seu potencial para tratar as complicações causadas pelo hipotireoidismo.

Philosophiae Doctor - PhD / This study explores the roles of exogenously applied nitric oxide, exogenously applied caffeic acid and salt stress on the ontioxidant 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 NO induced 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.

Philosophiae Doctor - PhD