The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.

Adipose tissue plays a crucial role in nutrient and energy homeostasis. At the time of worldwide pandemy of obesity and consequent metabolic syndrome, a great effort is made to find new treatments with potential to preserve insulin sensitivity, or even counteract development of obesity and type 2 diabetes. There are three principal possibilities how the adipose tissue biology can contribute to this goal: 1) induction of UCP1-dependent energy dissipation in brown adipose tissue; 2) conversion of white adipose depots to brown-like tissue (i.e. "browning"); and 3) stimulation of UCP1-independent thermogenesis in white adipose tissue. This thesis is based on two published works and one article under preparation. Generaly, it is focused on three different approaches targeting the above mentioned processes in adipose tissue of laboratory mouse: 1) diet supplementation with bile acids; 2) combination treatment of ω-3 polyunsaturated fatty acids and calorie restriction; and 3) cold exposure. In the experiments with administration of bile (specifically chenodeoxycholic) acid to mice, we confirm specific induction of UCP1 in both brown and subcutaneous white adipose tissue, as well as reversion of obesity in the response to the treatment. Nevertheless, most of the acute beneficial effects are mediated by...

As exposições precoces às diferentes intervenções, como dietas e estresse, estão associadas a persistentes alterações sobre aspectos neuroquímicos e comportamentais, podendo este ser considerado um gatilho de transtornos psiquiátricos na vida adulta. O presente trabalho objetivou determinar se estas intervenções neonatais poderiam interagir com uma dieta deficiente em ácidos graxos poliinsaturados n-3 (n-3 PUFA), aplicados durante o desenvolvimento, com foco nos níveis centrais (hipocampo) e séricos do fator neurotrófico derivado do encéfalo (BDNF), bem como sobre a atividade enzimática e aspectos morfológicos (e.g. massa, potencial) mitocondriais do hipocampo de ratos machos adultos. Em nossa abordagem experimental animal, as ninhadas foram distribuídas aleatoriamente nos grupos intactos, manipulados neonatalmente [MN, separação mãe-filhote durante 10min/dia, entre o 1° e o 10° dia pós-natal (DPN)] e separado maternalmente [SM, separação mãe-filhote durante 3h/dia, entre o 1° e o 10° DPN]. No DPN 35, os filhotes machos foram agrupados em dieta n-3 PUFA adequada ou deficiente, por 17 semanas de tratamento. O peso e o consumo de ração foram aferidos semanalmente. Após este tratamento, soro e hipocampo foram coletados. Kits comerciais foram utilizados para a medição dos níveis hipocampais e séricos de BDNF (ELISA), bem como a atividade dos complexos da cadeia respiratória mitocondrial (método enzimático, análise espectrofotométrica), massa e potencial mitocondriais (MitoTracker, citometria de fluxo). A expressão gênica de BDNF no hipocampo também foi medido por RT-qPCR. Os testes estatísticos utilizados foram ANOVA de duas vias ou de medidas repetidas. Os níveis de significâncias foram fixados em p<0,05. A dieta deficiente em n-3 PUFA, associada aos estressores neonatais deste estudo (MN, SM) foram capazes de alterar o peso corporal e a ingestão de alimentos de um modo específico, uma vez que os níveis mais elevados destes parâmetros foram encontrados em animais submetidos a SM. Animais submetidos a MN alimentados com uma dieta n-3 PUFA deficiente exibiram uma maior atividade exploratória em resposta a um psicoestimulante (dietilpropiona). Embora os níveis proteicos séricos e hipocampais de BDNF permaneceram inalterados pelos tratamentos aplicados, fomos capazes de demonstrar a redução de sua expressão gênica em animais alimentados com uma dieta n-3 PUFA deficiente. Considerando os padrões mitocondriais e parâmetros de estresse oxidativo, as atividades das enzimas antioxidantes glutationa peroxidase (GPx) e catalase (CAT), bem como a produção de espécies reativas de oxigênio (EROs) se encontraram aumentadas no hipocampo de animais submetidos a uma deficiência crônica em n-3 PUFA. Esta deficiência mostrou interações com o fator estresse neonatal em alguns destes parâmetros (e.g. atividade da GPx, produção de EROs), indicando um possível sinergismo entre estressores neonatais e a deficiência em n-3 PUFA. Os níveis de tióis foram significativamente menores nos grupos estressados (MN, SM), sendo a dieta n-3 PUFA deficiente capaz de aumentar a quantidade de tiol no hipocampo. Por outro lado, animais estressados tratados cronicamente com uma dieta deficiente em n-3 PUFA apresentaram níveis mais elevados de tiol. Contudo, a MN per se foi capaz de diminuir o potencial mitocondrial hipocampal. Adicionalmente, um estudo com humanos teve como objetivo correlacionar os níveis de BDNF periféricos ao consumo de n-3 PUFA em adolescentes. Para este estudo, 137 adolescentes de uma amostra enriquecida para psicopatologias de ansiedade foram submetidos a um questionário de frequência alimentar (QFA), para uma análise quantitativa do consumo de macronutrientes e micronutrientes de n-3 PUFA. As correlações de Spearman foram realizadas para avaliar possíveis associações entre o consumo de n-3 PUFA e os níveis periféricos de BDNF. As amostras de sangue foram coletadas entre 7 a 10h após o período de 10-12h de jejum, sendo o soro armazenado para medir os níveis de BDNF. Todas as análises de BDNF foram realizadas no mesmo dia por ELISA, utilizando anticorpos monoclonais específicos para o BDNF, de acordo com as instruções do fabricante. Os efeitos de possíveis confundidores (e.g. consumo total de gordura, idade, sexo e medida de ansiedade) foram examinados por modelos de regressões lineares. Apesar de algumas limitações apresentadas (e.g. o tamanho reduzido da amostra, alta incidência de adolescentes ansiosos), o que poderia limitar a validade externa deste resultado, fomos capazes de detectar uma correlação entre o consumo de n-3 PUFA e os níveis séricos de BNDF em adolescentes. Como conclusão geral, esta tese demonstra que a manipulação neonatal e separação materna, associada com uma deficiência em n-3 PUFA, são capazes de alterar parâmetros comportamentais e neuroquímicos na idade adulta. Este sinergismo foi capaz de diminuir a expressão gênica de BDNF no hipocampo, embora não apresentando qualquer alteração deste parâmetro perifericamente. A correlação entre os níveis consumidos de n-3 PUFA, em uma população de adolescentes em idade escolar, com os níveis séricos de BDNF também foi encontrada. Ainda, as alterações nas atividades enzimáticas mitocondriais observadas no hipocampo destes animais reforçam a importância da participação desta estrutura, além de sua possível relação com o desenvolvimento de desordens psiquiátricas e de humor. Considerando-se a nossa abordagem metodológica em ratos, a mesma pode ser um protocolo útil para se estudarem as interações entre o ambiente precoce e a nutrição ao curso de vida em diferentes desfechos neuroquímicos. / Early exposure to different interventions, as diets and stress, are associated with persistent alterations in neurochemistry and behavior, and can be considered a trigger of psychiatric disorders in adulthood. This study investigated whether neonatal interventions interact with a diet deficient in n-3 polyunsaturated fatty acids (n-3 PUFA) applied during development, focusing on central (hippocampus) and serum brain-derived neurotrophic factor (BDNF), as well as mitochondrial enzymatic activity and morphology (e.g. mass, potential) in adult male rats. In our animal study, litters were randomized into non-handled (NH), handled [H, mother-offspring separation for 10min/day from 1st-10th postnatal day (PND)] and separated (S, separation for 3h/day from the 1st-10th PND) groups. On PND 35, male pups were randomized into adequate or deficient diet in n-3 PUFA for 17 weeks. The weight and food intake were measured weekly. Serum and hippocampi were collected after this n-3 PUFA treatment. Commercial kits were used for measuring hippocampal and serum BDNF (ELISA), as well as mitochondrial chain respirator complexes (enzymatic method, spectrophotometric analysis), mitochondrial mass and potential (MitoTracker, flow citometry). Hippocampal BDNF gene expression was also measure by RT-qPCR. Statistical testes used were Two-Way or repeated measures ANOVA. Significance levels were set at p<0.05. A n-3 PUFA deficient diet, in association with neonatal stressors used in this study (H, MS) were able to alter body weight and food intake in a specific way, since higher levels of these parameters were found in animals subjected to MS. Animals subjected to H fed an n-3 PUFA deficient diet displayed enhanced exploratory activity in response to a psychostimulant (diethylpropion). Although serum protein levels and hippocampus BDNF remained unchanged by the treatments applied, we were able to demonstrate its gene expression reduction in the hippocampus of animals fed an n-3 PUFA deficient diet. Considering mitochondrial and oxidative stress parameters, the activities of antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) and the production of reactive oxygen species (ROS) were increased in the hippocampus of rats subjected to a deficiency n-3 PUFA. This deficiency displayed interactions with neonatal stress factor in some of these parameters (e.g. GPx activity, ROS), indicating a possible synergism between neonatal stressors and n-3 PUFA deficiency. Thiol levels were significantly decreased by neonatal stressors (H and MS), and the n-3 PUFA deficient diet was able to increase its total amount in hippocampus. On the other hand, chronically stressed animals treated with an n-3 PUFA deficient diet showed higher thiol levels. However, H per se was able to decrease mitochondrial potential in hippocampus. Additionally, a clinical study aimed to correlate peripheral BDNF levels and n-3 PUFA consumption in adolescents. For this study, 137 adolescents from a sample enriched for psychopathology of anxiety were subjected to a food frequency questionnaire (FFQ), in order for measuring the quantitative analysis of n-3 PUFA macronutrients and micronutrients consumption. Spearman correlations were performed to assess the association between n-3 PUFA consumption and serum BDNF levels. Blood samples were collected between 7 and 10h after fasting period of 10-12h, and serum was stored in order to measure BDNF levels. All BDNF measurements were performed in the same day by sandwich-ELISA using monoclonal antibodies specific for BDNF, according to the manufacturer’s instructions. Effects of potential confounders (e.g. total fat consumption, age, gender, anxiety) were examined using linear regression models. Although some limitations were presented (e.g. small sample size with high incidence of eager teenagers), which could limit the external validity of this result, we were able to detect a correlation between the consumption of n-3 PUFA and BDNF serum levels in adolescents. As a general conclusion, this thesis reports that neonatal handling and maternal separation, associated with a nutritional deficiency in n-3 PUFA, were able to change behavioral and neurochemical parameters in adulthood. This synergism was able to decrease BDNF gene expression in the hippocampus, while not presenting any change of this parameter peripherally. A correlation between the consumption levels of n-3 PUFA, in a population of schoolchildren with BDNF serum levels was also found. Still, changes in mitochondrial enzymatic activities observed in the hippocampus of these animals reinforce the importance of this structure participation and its relation to the development of psychiatric and mood disorders. Considering our rat methodological approach, it can be a useful tool for studying the interactions between early life environment and life-course nutrition on different neurochemical outcomes.

As exposições precoces às diferentes intervenções, como dietas e estresse, estão associadas a persistentes alterações sobre aspectos neuroquímicos e comportamentais, podendo este ser considerado um gatilho de transtornos psiquiátricos na vida adulta. O presente trabalho objetivou determinar se estas intervenções neonatais poderiam interagir com uma dieta deficiente em ácidos graxos poliinsaturados n-3 (n-3 PUFA), aplicados durante o desenvolvimento, com foco nos níveis centrais (hipocampo) e séricos do fator neurotrófico derivado do encéfalo (BDNF), bem como sobre a atividade enzimática e aspectos morfológicos (e.g. massa, potencial) mitocondriais do hipocampo de ratos machos adultos. Em nossa abordagem experimental animal, as ninhadas foram distribuídas aleatoriamente nos grupos intactos, manipulados neonatalmente [MN, separação mãe-filhote durante 10min/dia, entre o 1° e o 10° dia pós-natal (DPN)] e separado maternalmente [SM, separação mãe-filhote durante 3h/dia, entre o 1° e o 10° DPN]. No DPN 35, os filhotes machos foram agrupados em dieta n-3 PUFA adequada ou deficiente, por 17 semanas de tratamento. O peso e o consumo de ração foram aferidos semanalmente. Após este tratamento, soro e hipocampo foram coletados. Kits comerciais foram utilizados para a medição dos níveis hipocampais e séricos de BDNF (ELISA), bem como a atividade dos complexos da cadeia respiratória mitocondrial (método enzimático, análise espectrofotométrica), massa e potencial mitocondriais (MitoTracker, citometria de fluxo). A expressão gênica de BDNF no hipocampo também foi medido por RT-qPCR. Os testes estatísticos utilizados foram ANOVA de duas vias ou de medidas repetidas. Os níveis de significâncias foram fixados em p<0,05. A dieta deficiente em n-3 PUFA, associada aos estressores neonatais deste estudo (MN, SM) foram capazes de alterar o peso corporal e a ingestão de alimentos de um modo específico, uma vez que os níveis mais elevados destes parâmetros foram encontrados em animais submetidos a SM. Animais submetidos a MN alimentados com uma dieta n-3 PUFA deficiente exibiram uma maior atividade exploratória em resposta a um psicoestimulante (dietilpropiona). Embora os níveis proteicos séricos e hipocampais de BDNF permaneceram inalterados pelos tratamentos aplicados, fomos capazes de demonstrar a redução de sua expressão gênica em animais alimentados com uma dieta n-3 PUFA deficiente. Considerando os padrões mitocondriais e parâmetros de estresse oxidativo, as atividades das enzimas antioxidantes glutationa peroxidase (GPx) e catalase (CAT), bem como a produção de espécies reativas de oxigênio (EROs) se encontraram aumentadas no hipocampo de animais submetidos a uma deficiência crônica em n-3 PUFA. Esta deficiência mostrou interações com o fator estresse neonatal em alguns destes parâmetros (e.g. atividade da GPx, produção de EROs), indicando um possível sinergismo entre estressores neonatais e a deficiência em n-3 PUFA. Os níveis de tióis foram significativamente menores nos grupos estressados (MN, SM), sendo a dieta n-3 PUFA deficiente capaz de aumentar a quantidade de tiol no hipocampo. Por outro lado, animais estressados tratados cronicamente com uma dieta deficiente em n-3 PUFA apresentaram níveis mais elevados de tiol. Contudo, a MN per se foi capaz de diminuir o potencial mitocondrial hipocampal. Adicionalmente, um estudo com humanos teve como objetivo correlacionar os níveis de BDNF periféricos ao consumo de n-3 PUFA em adolescentes. Para este estudo, 137 adolescentes de uma amostra enriquecida para psicopatologias de ansiedade foram submetidos a um questionário de frequência alimentar (QFA), para uma análise quantitativa do consumo de macronutrientes e micronutrientes de n-3 PUFA. As correlações de Spearman foram realizadas para avaliar possíveis associações entre o consumo de n-3 PUFA e os níveis periféricos de BDNF. As amostras de sangue foram coletadas entre 7 a 10h após o período de 10-12h de jejum, sendo o soro armazenado para medir os níveis de BDNF. Todas as análises de BDNF foram realizadas no mesmo dia por ELISA, utilizando anticorpos monoclonais específicos para o BDNF, de acordo com as instruções do fabricante. Os efeitos de possíveis confundidores (e.g. consumo total de gordura, idade, sexo e medida de ansiedade) foram examinados por modelos de regressões lineares. Apesar de algumas limitações apresentadas (e.g. o tamanho reduzido da amostra, alta incidência de adolescentes ansiosos), o que poderia limitar a validade externa deste resultado, fomos capazes de detectar uma correlação entre o consumo de n-3 PUFA e os níveis séricos de BNDF em adolescentes. Como conclusão geral, esta tese demonstra que a manipulação neonatal e separação materna, associada com uma deficiência em n-3 PUFA, são capazes de alterar parâmetros comportamentais e neuroquímicos na idade adulta. Este sinergismo foi capaz de diminuir a expressão gênica de BDNF no hipocampo, embora não apresentando qualquer alteração deste parâmetro perifericamente. A correlação entre os níveis consumidos de n-3 PUFA, em uma população de adolescentes em idade escolar, com os níveis séricos de BDNF também foi encontrada. Ainda, as alterações nas atividades enzimáticas mitocondriais observadas no hipocampo destes animais reforçam a importância da participação desta estrutura, além de sua possível relação com o desenvolvimento de desordens psiquiátricas e de humor. Considerando-se a nossa abordagem metodológica em ratos, a mesma pode ser um protocolo útil para se estudarem as interações entre o ambiente precoce e a nutrição ao curso de vida em diferentes desfechos neuroquímicos. / Early exposure to different interventions, as diets and stress, are associated with persistent alterations in neurochemistry and behavior, and can be considered a trigger of psychiatric disorders in adulthood. This study investigated whether neonatal interventions interact with a diet deficient in n-3 polyunsaturated fatty acids (n-3 PUFA) applied during development, focusing on central (hippocampus) and serum brain-derived neurotrophic factor (BDNF), as well as mitochondrial enzymatic activity and morphology (e.g. mass, potential) in adult male rats. In our animal study, litters were randomized into non-handled (NH), handled [H, mother-offspring separation for 10min/day from 1st-10th postnatal day (PND)] and separated (S, separation for 3h/day from the 1st-10th PND) groups. On PND 35, male pups were randomized into adequate or deficient diet in n-3 PUFA for 17 weeks. The weight and food intake were measured weekly. Serum and hippocampi were collected after this n-3 PUFA treatment. Commercial kits were used for measuring hippocampal and serum BDNF (ELISA), as well as mitochondrial chain respirator complexes (enzymatic method, spectrophotometric analysis), mitochondrial mass and potential (MitoTracker, flow citometry). Hippocampal BDNF gene expression was also measure by RT-qPCR. Statistical testes used were Two-Way or repeated measures ANOVA. Significance levels were set at p<0.05. A n-3 PUFA deficient diet, in association with neonatal stressors used in this study (H, MS) were able to alter body weight and food intake in a specific way, since higher levels of these parameters were found in animals subjected to MS. Animals subjected to H fed an n-3 PUFA deficient diet displayed enhanced exploratory activity in response to a psychostimulant (diethylpropion). Although serum protein levels and hippocampus BDNF remained unchanged by the treatments applied, we were able to demonstrate its gene expression reduction in the hippocampus of animals fed an n-3 PUFA deficient diet. Considering mitochondrial and oxidative stress parameters, the activities of antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) and the production of reactive oxygen species (ROS) were increased in the hippocampus of rats subjected to a deficiency n-3 PUFA. This deficiency displayed interactions with neonatal stress factor in some of these parameters (e.g. GPx activity, ROS), indicating a possible synergism between neonatal stressors and n-3 PUFA deficiency. Thiol levels were significantly decreased by neonatal stressors (H and MS), and the n-3 PUFA deficient diet was able to increase its total amount in hippocampus. On the other hand, chronically stressed animals treated with an n-3 PUFA deficient diet showed higher thiol levels. However, H per se was able to decrease mitochondrial potential in hippocampus. Additionally, a clinical study aimed to correlate peripheral BDNF levels and n-3 PUFA consumption in adolescents. For this study, 137 adolescents from a sample enriched for psychopathology of anxiety were subjected to a food frequency questionnaire (FFQ), in order for measuring the quantitative analysis of n-3 PUFA macronutrients and micronutrients consumption. Spearman correlations were performed to assess the association between n-3 PUFA consumption and serum BDNF levels. Blood samples were collected between 7 and 10h after fasting period of 10-12h, and serum was stored in order to measure BDNF levels. All BDNF measurements were performed in the same day by sandwich-ELISA using monoclonal antibodies specific for BDNF, according to the manufacturer’s instructions. Effects of potential confounders (e.g. total fat consumption, age, gender, anxiety) were examined using linear regression models. Although some limitations were presented (e.g. small sample size with high incidence of eager teenagers), which could limit the external validity of this result, we were able to detect a correlation between the consumption of n-3 PUFA and BDNF serum levels in adolescents. As a general conclusion, this thesis reports that neonatal handling and maternal separation, associated with a nutritional deficiency in n-3 PUFA, were able to change behavioral and neurochemical parameters in adulthood. This synergism was able to decrease BDNF gene expression in the hippocampus, while not presenting any change of this parameter peripherally. A correlation between the consumption levels of n-3 PUFA, in a population of schoolchildren with BDNF serum levels was also found. Still, changes in mitochondrial enzymatic activities observed in the hippocampus of these animals reinforce the importance of this structure participation and its relation to the development of psychiatric and mood disorders. Considering our rat methodological approach, it can be a useful tool for studying the interactions between early life environment and life-course nutrition on different neurochemical outcomes.

As exposições precoces às diferentes intervenções, como dietas e estresse, estão associadas a persistentes alterações sobre aspectos neuroquímicos e comportamentais, podendo este ser considerado um gatilho de transtornos psiquiátricos na vida adulta. O presente trabalho objetivou determinar se estas intervenções neonatais poderiam interagir com uma dieta deficiente em ácidos graxos poliinsaturados n-3 (n-3 PUFA), aplicados durante o desenvolvimento, com foco nos níveis centrais (hipocampo) e séricos do fator neurotrófico derivado do encéfalo (BDNF), bem como sobre a atividade enzimática e aspectos morfológicos (e.g. massa, potencial) mitocondriais do hipocampo de ratos machos adultos. Em nossa abordagem experimental animal, as ninhadas foram distribuídas aleatoriamente nos grupos intactos, manipulados neonatalmente [MN, separação mãe-filhote durante 10min/dia, entre o 1° e o 10° dia pós-natal (DPN)] e separado maternalmente [SM, separação mãe-filhote durante 3h/dia, entre o 1° e o 10° DPN]. No DPN 35, os filhotes machos foram agrupados em dieta n-3 PUFA adequada ou deficiente, por 17 semanas de tratamento. O peso e o consumo de ração foram aferidos semanalmente. Após este tratamento, soro e hipocampo foram coletados. Kits comerciais foram utilizados para a medição dos níveis hipocampais e séricos de BDNF (ELISA), bem como a atividade dos complexos da cadeia respiratória mitocondrial (método enzimático, análise espectrofotométrica), massa e potencial mitocondriais (MitoTracker, citometria de fluxo). A expressão gênica de BDNF no hipocampo também foi medido por RT-qPCR. Os testes estatísticos utilizados foram ANOVA de duas vias ou de medidas repetidas. Os níveis de significâncias foram fixados em p<0,05. A dieta deficiente em n-3 PUFA, associada aos estressores neonatais deste estudo (MN, SM) foram capazes de alterar o peso corporal e a ingestão de alimentos de um modo específico, uma vez que os níveis mais elevados destes parâmetros foram encontrados em animais submetidos a SM. Animais submetidos a MN alimentados com uma dieta n-3 PUFA deficiente exibiram uma maior atividade exploratória em resposta a um psicoestimulante (dietilpropiona). Embora os níveis proteicos séricos e hipocampais de BDNF permaneceram inalterados pelos tratamentos aplicados, fomos capazes de demonstrar a redução de sua expressão gênica em animais alimentados com uma dieta n-3 PUFA deficiente. Considerando os padrões mitocondriais e parâmetros de estresse oxidativo, as atividades das enzimas antioxidantes glutationa peroxidase (GPx) e catalase (CAT), bem como a produção de espécies reativas de oxigênio (EROs) se encontraram aumentadas no hipocampo de animais submetidos a uma deficiência crônica em n-3 PUFA. Esta deficiência mostrou interações com o fator estresse neonatal em alguns destes parâmetros (e.g. atividade da GPx, produção de EROs), indicando um possível sinergismo entre estressores neonatais e a deficiência em n-3 PUFA. Os níveis de tióis foram significativamente menores nos grupos estressados (MN, SM), sendo a dieta n-3 PUFA deficiente capaz de aumentar a quantidade de tiol no hipocampo. Por outro lado, animais estressados tratados cronicamente com uma dieta deficiente em n-3 PUFA apresentaram níveis mais elevados de tiol. Contudo, a MN per se foi capaz de diminuir o potencial mitocondrial hipocampal. Adicionalmente, um estudo com humanos teve como objetivo correlacionar os níveis de BDNF periféricos ao consumo de n-3 PUFA em adolescentes. Para este estudo, 137 adolescentes de uma amostra enriquecida para psicopatologias de ansiedade foram submetidos a um questionário de frequência alimentar (QFA), para uma análise quantitativa do consumo de macronutrientes e micronutrientes de n-3 PUFA. As correlações de Spearman foram realizadas para avaliar possíveis associações entre o consumo de n-3 PUFA e os níveis periféricos de BDNF. As amostras de sangue foram coletadas entre 7 a 10h após o período de 10-12h de jejum, sendo o soro armazenado para medir os níveis de BDNF. Todas as análises de BDNF foram realizadas no mesmo dia por ELISA, utilizando anticorpos monoclonais específicos para o BDNF, de acordo com as instruções do fabricante. Os efeitos de possíveis confundidores (e.g. consumo total de gordura, idade, sexo e medida de ansiedade) foram examinados por modelos de regressões lineares. Apesar de algumas limitações apresentadas (e.g. o tamanho reduzido da amostra, alta incidência de adolescentes ansiosos), o que poderia limitar a validade externa deste resultado, fomos capazes de detectar uma correlação entre o consumo de n-3 PUFA e os níveis séricos de BNDF em adolescentes. Como conclusão geral, esta tese demonstra que a manipulação neonatal e separação materna, associada com uma deficiência em n-3 PUFA, são capazes de alterar parâmetros comportamentais e neuroquímicos na idade adulta. Este sinergismo foi capaz de diminuir a expressão gênica de BDNF no hipocampo, embora não apresentando qualquer alteração deste parâmetro perifericamente. A correlação entre os níveis consumidos de n-3 PUFA, em uma população de adolescentes em idade escolar, com os níveis séricos de BDNF também foi encontrada. Ainda, as alterações nas atividades enzimáticas mitocondriais observadas no hipocampo destes animais reforçam a importância da participação desta estrutura, além de sua possível relação com o desenvolvimento de desordens psiquiátricas e de humor. Considerando-se a nossa abordagem metodológica em ratos, a mesma pode ser um protocolo útil para se estudarem as interações entre o ambiente precoce e a nutrição ao curso de vida em diferentes desfechos neuroquímicos. / Early exposure to different interventions, as diets and stress, are associated with persistent alterations in neurochemistry and behavior, and can be considered a trigger of psychiatric disorders in adulthood. This study investigated whether neonatal interventions interact with a diet deficient in n-3 polyunsaturated fatty acids (n-3 PUFA) applied during development, focusing on central (hippocampus) and serum brain-derived neurotrophic factor (BDNF), as well as mitochondrial enzymatic activity and morphology (e.g. mass, potential) in adult male rats. In our animal study, litters were randomized into non-handled (NH), handled [H, mother-offspring separation for 10min/day from 1st-10th postnatal day (PND)] and separated (S, separation for 3h/day from the 1st-10th PND) groups. On PND 35, male pups were randomized into adequate or deficient diet in n-3 PUFA for 17 weeks. The weight and food intake were measured weekly. Serum and hippocampi were collected after this n-3 PUFA treatment. Commercial kits were used for measuring hippocampal and serum BDNF (ELISA), as well as mitochondrial chain respirator complexes (enzymatic method, spectrophotometric analysis), mitochondrial mass and potential (MitoTracker, flow citometry). Hippocampal BDNF gene expression was also measure by RT-qPCR. Statistical testes used were Two-Way or repeated measures ANOVA. Significance levels were set at p<0.05. A n-3 PUFA deficient diet, in association with neonatal stressors used in this study (H, MS) were able to alter body weight and food intake in a specific way, since higher levels of these parameters were found in animals subjected to MS. Animals subjected to H fed an n-3 PUFA deficient diet displayed enhanced exploratory activity in response to a psychostimulant (diethylpropion). Although serum protein levels and hippocampus BDNF remained unchanged by the treatments applied, we were able to demonstrate its gene expression reduction in the hippocampus of animals fed an n-3 PUFA deficient diet. Considering mitochondrial and oxidative stress parameters, the activities of antioxidant enzymes glutathione peroxidase (GPx) and catalase (CAT) and the production of reactive oxygen species (ROS) were increased in the hippocampus of rats subjected to a deficiency n-3 PUFA. This deficiency displayed interactions with neonatal stress factor in some of these parameters (e.g. GPx activity, ROS), indicating a possible synergism between neonatal stressors and n-3 PUFA deficiency. Thiol levels were significantly decreased by neonatal stressors (H and MS), and the n-3 PUFA deficient diet was able to increase its total amount in hippocampus. On the other hand, chronically stressed animals treated with an n-3 PUFA deficient diet showed higher thiol levels. However, H per se was able to decrease mitochondrial potential in hippocampus. Additionally, a clinical study aimed to correlate peripheral BDNF levels and n-3 PUFA consumption in adolescents. For this study, 137 adolescents from a sample enriched for psychopathology of anxiety were subjected to a food frequency questionnaire (FFQ), in order for measuring the quantitative analysis of n-3 PUFA macronutrients and micronutrients consumption. Spearman correlations were performed to assess the association between n-3 PUFA consumption and serum BDNF levels. Blood samples were collected between 7 and 10h after fasting period of 10-12h, and serum was stored in order to measure BDNF levels. All BDNF measurements were performed in the same day by sandwich-ELISA using monoclonal antibodies specific for BDNF, according to the manufacturer’s instructions. Effects of potential confounders (e.g. total fat consumption, age, gender, anxiety) were examined using linear regression models. Although some limitations were presented (e.g. small sample size with high incidence of eager teenagers), which could limit the external validity of this result, we were able to detect a correlation between the consumption of n-3 PUFA and BDNF serum levels in adolescents. As a general conclusion, this thesis reports that neonatal handling and maternal separation, associated with a nutritional deficiency in n-3 PUFA, were able to change behavioral and neurochemical parameters in adulthood. This synergism was able to decrease BDNF gene expression in the hippocampus, while not presenting any change of this parameter peripherally. A correlation between the consumption levels of n-3 PUFA, in a population of schoolchildren with BDNF serum levels was also found. Still, changes in mitochondrial enzymatic activities observed in the hippocampus of these animals reinforce the importance of this structure participation and its relation to the development of psychiatric and mood disorders. Considering our rat methodological approach, it can be a useful tool for studying the interactions between early life environment and life-course nutrition on different neurochemical outcomes.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.

Impaired endothelial vasomotor function is a hallmark of many chronic disease states, including essential hypertension and type 2 diabetes mellitus. Loss of the homeostatic role of the endothelium in large conduit arteries can contribute to the pathogenesis of cardiovascular conditions in these vessels (e.g. stroke, atherosclerosis). A fundamental understanding of mechanisms controlling endothelial function in hypertension and type 2 diabetes mellitus is required for appropriate clinical strategies targeting the cardiovascular conditions associated with these diseases. The vast majority of basic science studies examining endothelial function in animal models of hypertension and type 2 diabetes have been conducted in males. Studying endothelial function in females is imperative for determining potential sex-specific mechanisms of dysfunction and thus appropriate therapeutic strategies. Thus the global purpose of this thesis is to identify and characterize the pathways controlling impaired vasomotor function in female animal models of two chronic disease states: hypertension and type 2 diabetes mellitus. Chapters 2 and 3 of this thesis examine sex differences in endothelium-dependent vasorelaxation (EDR) and vasocontraction (EDC) of aortic segments isolated from male and female spontaneously hypertensive rats (SHR), a model of essential hypertension, as the animals age between 16 and 30 wk old. All endothelial vasomotor data presented in the Abstract are peak responses to 10⁻⁵ M acetylcholine. Endothelial vasomotor impairment is represented by lower EDR or by higher EDC. These present data confirmed well-established findings from the literature that 16 wk old male SHR exhibit endothelial vasomotor impairments (EDR: 77±4 %; EDC: 76±7 %) compared to normotensive Wistar-Kyoto (WKY; EDR: 89±6 %; EDC: 59±8 %; p<0.05) controls, and that this impairment worsens with ageing in 30 wk male SHR (EDR: 63±2 %; EDC: 91±3 %; p<0.05). The observation that EDR was reduced in 30 wk female SHR (EDR: 76±4 %) compared to 16 wk counterparts (EDR: 101±2 %; p<0.05), however, was novel and interesting, as there were previously no reports of vasomotor responses in female SHR older than 19 wk. Moreover, the blunted EDR response of 30 wk female SHR approached the level of impairment exhibited by 30 wk male SHR (but was still slightly greater in females; p<0.05). The limited sex difference of the EDR within 30 wk SHR (males –13 % vs. females; p<0.05) contrasted that of 16 wk SHR (males –24 % vs. females; p<0.05), when the robust and unimpaired relaxation displayed by females was much greater than the significantly blunted response of males. Interestingly, endothelium-dependent contractions in quiescent rings were moderate and similar between 16 wk (EDC: 50±4 %) and 30 wk female SHR (EDC: 59±7 %; p=N/S) as compared to the greater contractions of males that were exacerbated with ageing (see above; p<0.05 both sex and ageing comparison). A major role has been established for the cyclooxygenase (COX)-1-thromboxane A₂/prostaglandin (TP) receptor pathway in the impaired endothelial vasomotor function of male SHR. Indeed, a similar mechanism appears to be responsible for the dysfunction observed in 30 wk female SHR in this thesis since robust endothelial function was restored in these animals with both antagonism of TP receptor (EDR: 111±2 %; EDC: 7±2 %; p<0.05) and preferential inhibition of COX-1 (EDR: 112±3 %; EDC: –5±3 %; p<0.05). In contrast, preferential inhibition of COX-2 only partially tempered endothelial impairments of 30 wk female SHR (EDR: 99±5 %; EDC: 27±3 %; p<0.05), suggesting that, similar to ageing male SHR, this isoform makes at most a secondary contribution to the dysfunction in 30 wk female SHR. Collectively, these data indicate that ageing female SHR exhibit a mechanism of endothelial impairment that is similar to that of male SHR and that is largely COX-1- and TP receptor-dependent. Chapter 4 examines the ability of chronic dietary administration of the n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA, 22:6 n-3), to ameliorate endothelial vasomotor function in adult male SHR with established hypertension. The impaired endothelial function of aortic segments isolated from adult male SHR (EDR: 48±6 %) was not improved following 10–12 wk of DHA feeding (EDR: 45±5 %; p=N/S). This finding was unexpected since it has been shown in the literature that feeding other n-3 PUFAs improves vasomotor responses in younger SHR, in which hypertension and its associated consequences are still developing. This is the first report of the effects of n-3 PUFA on endothelial vasomotor responses in adult SHR with established hypertension. These data suggest that dietary DHA do not improve vasomotor function in adult SHR. Chapter 5 examines α₁ adrenergic contraction and EDR of aortic segments isolated from 14 wk old female Zucker diabetic fatty rats (ZDF), a genetic model of high fat diet-induced obesity and type 2 diabetes, and lean non-diabetic female Zucker Lean rats. Additionally, some ZDF received an 8 wk administration of anti-diabetic metformin drug therapy, aerobic exercise training, or a combination of the two. Maximal α₁ adrenergic contractions were over 2-fold higher in high fat-fed ZDF (1.69±0.16 g) compared to Lean (0.71±0.13 g; p<0.05). This elevation in ZDF was abolished by exercise training alone (1.02±0.17 g; p<0.05) but was not altered by metformin (1.56±0.19 g; p=N/S). In contrast to the severely impaired endothelial vasomotor function reported in male ZDF in the literature, robust EDR was observed in female ZDF (72±7 %) that was similar to Lean (75±6 %; p=N/S) and that was unaltered by exercise training (76±5 %; p=N/S) or metformin (76±6 %; p=N/S). These results indicate that enhanced α₁ adrenergic contraction is a mechanism of altered vasomotor function in female type 2 diabetic ZDF rats and that it could possibly be addressed by a chronic exercise training intervention. The main novelty of the thesis is the extension of the current understanding of endothelial vasomotor function to hypertensive and type 2 diabetic females. The knowledge gained from examining mechanisms involved in endothelial impairments in ageing hypertensive females and from testing the therapeutic potential of currently used anti-diabetic interventions in the type 2 diabetic female vasculature has interesting potential application. This basic scientific information could help direct clinical therapeutic strategies to target population-specific mechanisms of dysfunction. Understanding female sex-specific endothelial behaviour in patient populations is important for describing cardiovascular complications, defining mechanisms, and applying appropriate therapeutic targets. Findings from this thesis indicate a sex-dependence of the total divergence of endothelial function (e.g. female type 2 diabetic rats vs. male counterparts in the literature) and of the interaction of disease variables (e.g. age) and endothelial vasomotor responses.