The soluble and readily extractable part of the plant extracellular matrix has been termed, the apoplast and contains a wide range of components such as, complex carbohydrates, structural proteins, enzymes and radicals that are known to be responsive to stress and developmental pressures. This thesis describes the development of a technique for the selective enrichment of apoplastic components for a range of subsequent analyses. Using this technique a number of apoplastic proteins were N-terminally sequenced and revealed 2 cell wall related enzymes, an antifungal protein and 3 auxin-binding/germin-like proteins. This technique also provided a novel approach to the further study of auxin-binding proteins via the use of affinity chromatography at their putative site of action, the apoplast. Three potential auxin-binding protiens were identified. Many attempts were made to subject the material extracted from the apoplast to the highly resolving technique of 2-dimensional electrophoresis, and during the process two unusual 2D systems were developed. These systems could be run in a small format that permitted very rapid analysis and/or using an in-gel loading strategy to subject up to 500µg of protein to 2D separation therefore permitting N-terminal sequencing from single 2D gels. Unfortunately 2D separation of apoplastic proteins was never fully achieved within the time frame of this study due to the vast degree of heterogenity present in the sample material. It did however demonstrate the very complex nature of apoplastic components. A series of experiments revealed that the tobacco leaf apoplast contained compartment specific antioxidant enzymes, some of which share physical characteristics with similar enzymes from other species. The activity of these enzymes altered in response to stress and according to the developmental age of the tissue. The reduced activity of these enzymes directly correlated to the degree of oxidative modification of apoplastic proteins illustrating that these enzymes are important in the detoxification of apoplastic radicals. Follow on experiments following the apoplastic generation of the superoxide anion and nitric oxide from impact stressed potato tuber tissue showed that radicals play important roles in the responses of plant tissue to stress, and show the first involvement of nitric oxide in plants in response to abiotic stress.

Despite its numerous side effects, morphine and the opioids derived from this drug, belong among the only effective options for treatment of pain linked to oncological illness. The effect of opioids on the efficiency of cytostatics in vitro has been the subject of many papers but the results are often contradictory, which could be probably caused by the great variability of experimental models and approaches. Some recent studies indicate that the consequences of activation of opioid signaling in astrocytes display certain differences from other cell types. Glioblastoma multiforme, the tumor derived from astrocytes, belong among those with the worst prognosis, mostly for the frequent resistance to cytostatics. In this thesis we focused on the effect of morphine, methadone and DADLE on the efficiency of cytostatics of temozolomide, doxorubicin and vincristine on the cell lines C6 and CCF-STTG1 derived from glioblastomas. Also, we examined the effect of the above mentioned opioids on the level of oxidative cellular stress and using N-acetylcysteine, a ROS scavenger, we verified the role of oxidative stress in cellular systems activated by the effect of the mentioned opioids on the efficiency of cytostatics. We also assessed opioid receptors and the receptor TLR4 in the examined cell lines. The...

O proteassomo é um complexo proteico responsável pela degradação de proteínas poli-ubiquitinadas. É constituído por uma unidade catalítica denominada de proteassomo 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o proteassomo 26S. O 20SPT é capaz de degradar proteínas por um processo independente de ATP e ubiquitina. Este mecanismo é considerado preventivo de agregação proteica, uma vez que o acúmulo de proteínas oxidadas está diretamente associado ao envelhecimento e doenças neurodegenerativas. Foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação nos resíduos de Cys 76 e Cys 221 da subunidade 5. Dados obtidos por microscopia eletrônica de transmissão mostraram que a S-glutatiolação promove a abertura da câmara catalítica e consequentemente o aumento da degradação de proteínas. Recentemente, foram obtidas linhagens com mutações sítio-específicas pela substituição dos dois resíduos de Cys glutatioláveis pela Ser. Ensaios realizados com a linhagem C221 mostraram um aumento da longevidade e resistência ao estresse oxidativo quando comparada com a linhagem selvagem, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Foi verificado também que a população de proteassomo isolada da linhagem C221 apresenta maior proporção da forma aberta da câmara catalítica. Resultados opostos foram observados na linhagem C76S. No entanto, constatamos um aumento de proteínas oxidadas e de agregados proteicos na linhagem C221 em comparação a selvagem. Esses dados não condizem com o aumento do tempo de vida cronológico desta linhagem, porém acreditamos que esses agregados estejam relacionados a um tipo de sequestro de proteínas potencialmente prejudiciais, como será discutido neste trabalho. / The proteasome is a protein complex responsible for the degradation of poly-ubiquitinated proteins. It comprises a catalytic unit called 20S proteasome (20SPT) and regulatory units (19S) coupled at one or both ends to form the 26S proteasome. The 20SPT is able to degrade proteins by an ATP and ubiquitin independent process. This mechanism is considered preventive of protein aggregation, since the accumulation of oxidized proteins is directly related to aging and neurodegenerative diseases. It was observed by our group that the yeast 20SPT (S. cerevisiae) is modified by S-glutathiolation in the residues Cys 76 and Cys 221 of the 5 subunit. Data obtained by transmission electron microscopy showed that the S-glutathiolation promotes the opening of the catalytic chamber and consequently increased degradation of oxidized proteins. Recently, strains were obtained by site-specific mutations by replacing the two glutathiolable Cys residues by Ser. Tests performed with the C221 strain showed an increased longevity and resistance to oxidative stress when compared to the wild type strain, whereas the C76 strain showed a slower growth. It was also found that the isolated population of the 5-C221S proteasome presents the highest frequency of open catalytic chamber conformation. Opposite results were observed in the C76S lineage. However, we noted an increased pool of oxidized proteins and protein aggregates in the C221 strain compared to the wild type. These data do not match with the increase of chronological life span observed in this lineage, but we believe that these aggregates are related to a kind of \"sequestration\" of potentially damaging proteins, as will be discussed in this work.

The objective of this study was to evaluate the effect of thorium (Th) on the metabolism of silver catfish (Rhamdia quelen) through biochemical parameters from the muscle tissue (glycogen, glucose, lactate, protein and ammonia), lipidic peroxidation levels (TBARS), catalase (CAT) and glutathione-S-transferase (GST) in the hepatic and muscular tissues and cytogenetic parameters through the evaluation of nuclear abnormalities in blood cells. Silver catfish juveniles (8.78 ± 0.10cm; 6.41 ± 0.17g) were exposed to different waterborne concentrations of ²³²Th (in μg.L-1): 33.6±8.7; 106.5±37.1; 191.6±19.0 and 758.4±150.4 for 15 days. The levels of muscle glycogen were significantly reduced in fish exposed to 106.5 μg.L-¹ Th, while glucose and protein increased in those exposed to 758.4 μg.L-¹ Th. Lactate levels were higher in fish maintained at 191.6 μg. L-¹ Th and ammonia was higher in those exposed to 33.6, 106.5 and 191.6 μg.L-¹ Th. The lipidic peroxidation levels were diminished in the liver of silver catfish exposed to all tested concentrations of Th. In the muscle lipidic peroxidation was higher in juveniles maintained at 106.5 μg.L-¹ Th and lower in those exposed to 191.6 and 758.4 μg.L-¹ Th. The CAT activity was higher in the hepatic tissue (but not muscle) of fish exposed to all tested concentrations of Th. The GST activity in the liver was lower in fish exposed to 33.6 and 106.5 μg.L-¹ Th, and in the muscular tissue of those maintained at 758.4 μg.L-1 Th. Silver catfish exposed to 106.5 μg.L-¹ presented a significant induction of micronuclei, but no alterations in other erythrocyte abnormalities were observed. These results suggest that exposure to waterborne Th induces changes in the metabolic state, increase of lipidic peroxidation in the liver, some alterations of CAT and GST, and DNA damage. / O objetivo deste estudo foi avaliar o efeito de tório (Th) sobre parâmetros metabólicos (glicogênio, glicose, lactato, proteína e amônia), em tecido muscular de jundiás (Rhandia quelen), níveis de lipoperoxidação (TBARS), catalase (CAT) e glutationa-S-transferase(GST) nos tecidos hepático e muscular e parâmetros citogenéticos através da avaliação de anormalidades nucleares em células sanguíneas. Juvenis de jundiás (8,78±0,10cm; 6,41± 0,17g) foram expostos a diferentes concentrações de ²³²Th 33,6±8,7; 106,5±37,1; 191,6±19,0 e 758,4±150,4 em μg. L-¹ (três repetições por tratamento) por 15 dias. Os níveis de glicogênio muscular diminuíram significativamente a 106,5 μg. L-¹ Th. Glicose e proteína aumentaram na 758,4 μg. L-¹ Th. Os níveis de lactato apresentaram-se elevados em 106,5 μg. L-¹ Th. A amônia aumentou a 33,6; 106,5 e 191,6 μg.L-¹ Th. Os níveis de peroxidação lipídica diminuíram no fígado dos jundiás expostos a todas as concentrações de Th testadas. No músculo esquelético aumentaram a 106,5 μg. L-¹Th e diminuíram a 191,6 e 758,4 μg.L-¹ Th. A atividade da CAT no tecido hepático apresentou aumento em todas as concentrações testadas de Th. Nenhuma alteração foi observada no tecido muscular. A GST diminui no fígado a 33,6 e 106,5 μg.L-¹Th. No tecido muscular diminui a 758,4 μg.L-1 Th. Jundiás expostos a 106,5 μg. L-¹ apresentaram maior indução de micronúcleos. Não foi observadas alterações para outras anormalidades nucleares eritrocíticas. Os resultados obtidos sugerem mudanças nos intermediários metabólitos devido ao estresse provocado pelo Th, aumento da lipoperoxidação no fígado, sendo observado níveis variados no músculo esquelético, alterações das enzimas CAT e GST além de danos no DNA dos peixes expostos ao Th, comparando aos grupos controles.

O proteassomo é um complexo proteico responsável pela degradação de proteínas poli-ubiquitinadas. É constituído por uma unidade catalítica denominada de proteassomo 20S (20SPT) e por unidades regulatórias (19S) acopladas em uma ou ambas as extremidades para formar o proteassomo 26S. O 20SPT é capaz de degradar proteínas por um processo independente de ATP e ubiquitina. Este mecanismo é considerado preventivo de agregação proteica, uma vez que o acúmulo de proteínas oxidadas está diretamente associado ao envelhecimento e doenças neurodegenerativas. Foi observado pelo grupo que o 20SPT da levedura S.cerevisiae sofre S-glutatiolação nos resíduos de Cys 76 e Cys 221 da subunidade 5. Dados obtidos por microscopia eletrônica de transmissão mostraram que a S-glutatiolação promove a abertura da câmara catalítica e consequentemente o aumento da degradação de proteínas. Recentemente, foram obtidas linhagens com mutações sítio-específicas pela substituição dos dois resíduos de Cys glutatioláveis pela Ser. Ensaios realizados com a linhagem C221 mostraram um aumento da longevidade e resistência ao estresse oxidativo quando comparada com a linhagem selvagem, enquanto que a linhagem C76 mostrou uma dificuldade no crescimento. Foi verificado também que a população de proteassomo isolada da linhagem C221 apresenta maior proporção da forma aberta da câmara catalítica. Resultados opostos foram observados na linhagem C76S. No entanto, constatamos um aumento de proteínas oxidadas e de agregados proteicos na linhagem C221 em comparação a selvagem. Esses dados não condizem com o aumento do tempo de vida cronológico desta linhagem, porém acreditamos que esses agregados estejam relacionados a um tipo de sequestro de proteínas potencialmente prejudiciais, como será discutido neste trabalho. / The proteasome is a protein complex responsible for the degradation of poly-ubiquitinated proteins. It comprises a catalytic unit called 20S proteasome (20SPT) and regulatory units (19S) coupled at one or both ends to form the 26S proteasome. The 20SPT is able to degrade proteins by an ATP and ubiquitin independent process. This mechanism is considered preventive of protein aggregation, since the accumulation of oxidized proteins is directly related to aging and neurodegenerative diseases. It was observed by our group that the yeast 20SPT (S. cerevisiae) is modified by S-glutathiolation in the residues Cys 76 and Cys 221 of the 5 subunit. Data obtained by transmission electron microscopy showed that the S-glutathiolation promotes the opening of the catalytic chamber and consequently increased degradation of oxidized proteins. Recently, strains were obtained by site-specific mutations by replacing the two glutathiolable Cys residues by Ser. Tests performed with the C221 strain showed an increased longevity and resistance to oxidative stress when compared to the wild type strain, whereas the C76 strain showed a slower growth. It was also found that the isolated population of the 5-C221S proteasome presents the highest frequency of open catalytic chamber conformation. Opposite results were observed in the C76S lineage. However, we noted an increased pool of oxidized proteins and protein aggregates in the C221 strain compared to the wild type. These data do not match with the increase of chronological life span observed in this lineage, but we believe that these aggregates are related to a kind of \"sequestration\" of potentially damaging proteins, as will be discussed in this work.

Pacientes em hemodiálise (HD) estão sob estresse oxidativo, o que leva à formação de espécies reativas de oxigênio (EROs). Toxinas urêmicas, a própria hemodiálise e o comprometimento do sistema antioxidante induzem a formação de EROs. Dentre os antioxidantes está o Selênio (Se), mineral que a literatura já relata deficiência em pacientes em HD. Considerando a importância da atividade antioxidante nestes pacientes com doença renal crônica (DRC) em HD, o objetivo deste estudo foi avaliar o efeito da suplementação com castanha-do-brasil nos níveis de Se e na atividade da glutationa peroxidase (GSH-Px) em pacientes nestas condições. Foram estudados 81 pacientes em HD (52,0 ± 15,2 anos, tempo média de HD de 82,3 ± 91,4 meses e IMC de 24,4 ± 4,4 kg/(m)2) das clínicas RenalCor e RenalVida do Rio de Janeiro. Os pacientes receberam uma castanha (≈ 5g), cerca 290µg Se, por dia, durante três meses. A concentração de Se foi determinada por espectrometria de absorção atômica com geração de hidretos (HITACHI®, Z-500). A atividade da enzima GSH-Px foi medida usando kit comercial RANDOX®. Os níveis de Se no plasma (18,8 ± 17,4 µg/L) e no eritrócito (72,4 ± 37,4 µg/L) antes da suplementação estavam abaixo da faixa da normalidade (60-120 µg/L no plasma e de 90-190 µg/L no eritrócito) e após a suplementação os níveis plasmáticos e eritrocitários aumentaram para 104,0 ± 65,0 µg/L e 244,1 ± 119,5 µg/L (p< 0,0001), respectivamente. A atividade de GSH-Px também aumentou após a suplementação passando de 46,4 ± 14,9 U/gHb para 55,9 ± 23,6 U/gHb (p< 0,0001). Antes da suplementação 11% dos pacientes apresentaram atividade da GSH-Px abaixo da normalidade (27,5 - 73,6 U/gHb) e, após a suplementação todos pacientes apresentaram atividade da GSH-Px normal. Os resultados mostraram que os pacientes estudados apresentaram Se plasmático e eritrocitário abaixo dos valores normais e o consumo de apenas uma castanha-do-brasil por dia (5g) foi eficaz no aumento da concentração de Se e na atividade da GSH-Px nos pacientes em HD; além disto, ela pode contribuir para a melhora da condição de estresse oxidativo desses pacientes. / Hemodialysis (HD) patients may undergo oxidative damage which causes reactive oxygen species (ROS) formation. Uremic toxins, HD treatment and the decrease in antioxidant system are known to result in the ROS generation. Selenium (Se) has a role as an antioxidant and studies report Se deficiency in these patients. Considering the importance of the antioxidant activity in patients with chronic kidney disease (CKD) in HD, the aim of this work was evaluate the effect of Brazilian nuts (main food source) supplementation on levels of Se and glutathione peroxidase (GSH-Px) activity in HD patients. A total of 81 HD patients (52.0 ± 15.2 years old, average time on dialysis was 82.3 ± 91.4 months and BMI of 24.9 ± 4.4 kg/m2) from RenalCor and RenalVida Clinics in Rio de Janeiro, Brazil were studied. The patients received one nut (≈ 5g), average of 290 µg Se, each day during three months. The Se concentration was determined by atomic absorption spectrophotometry with hydride generation (HITACHI®, Z-500). The levels of GSH-Px were measured by using RANDOX® commercial kits. The Se levels in plasma (18.8 ± 17.4 µg/L) and erythrocyte (72.4 ± 37.9 µg/L) before supplementation were below the normal range (60-120 µg/L for plasma and 90-190 µg/L for erythrocyte) and after nut supplementation, the plasma levels increased to 104.0 ± 65.0 µg/L and erythrocytes to 244.1 ± 119.5 µg/L (p<0.0001). The activity of GSH-Px also increased after supplementation from 46.6 ± 14.9 U/ gHb to 55.9 ± 23.6 U/gHb (p<0.0001). Before supplementation, 11% of patients had activity of GSH-Px below the normal range (27.5 - 73.6 U/gHb) and, after supplementation, all patients showed activity of GSH-Px within the normal range. The data revealed that the investigated patients had erythrocyte and plasma Se levels below the normal values and the consumption of just one Brazil nut each day (5g) was effective to increase on Se concentration and GSH-Px activity of the patients in HD, thus contributing to improve the antioxidant condition of these patients.

Pacientes em hemodiálise (HD) estão sob estresse oxidativo, o que leva à formação de espécies reativas de oxigênio (EROs). Toxinas urêmicas, a própria hemodiálise e o comprometimento do sistema antioxidante induzem a formação de EROs. Dentre os antioxidantes está o Selênio (Se), mineral que a literatura já relata deficiência em pacientes em HD. Considerando a importância da atividade antioxidante nestes pacientes com doença renal crônica (DRC) em HD, o objetivo deste estudo foi avaliar o efeito da suplementação com castanha-do-brasil nos níveis de Se e na atividade da glutationa peroxidase (GSH-Px) em pacientes nestas condições. Foram estudados 81 pacientes em HD (52,0 ± 15,2 anos, tempo média de HD de 82,3 ± 91,4 meses e IMC de 24,4 ± 4,4 kg/(m)2) das clínicas RenalCor e RenalVida do Rio de Janeiro. Os pacientes receberam uma castanha (≈ 5g), cerca 290µg Se, por dia, durante três meses. A concentração de Se foi determinada por espectrometria de absorção atômica com geração de hidretos (HITACHI®, Z-500). A atividade da enzima GSH-Px foi medida usando kit comercial RANDOX®. Os níveis de Se no plasma (18,8 ± 17,4 µg/L) e no eritrócito (72,4 ± 37,4 µg/L) antes da suplementação estavam abaixo da faixa da normalidade (60-120 µg/L no plasma e de 90-190 µg/L no eritrócito) e após a suplementação os níveis plasmáticos e eritrocitários aumentaram para 104,0 ± 65,0 µg/L e 244,1 ± 119,5 µg/L (p< 0,0001), respectivamente. A atividade de GSH-Px também aumentou após a suplementação passando de 46,4 ± 14,9 U/gHb para 55,9 ± 23,6 U/gHb (p< 0,0001). Antes da suplementação 11% dos pacientes apresentaram atividade da GSH-Px abaixo da normalidade (27,5 - 73,6 U/gHb) e, após a suplementação todos pacientes apresentaram atividade da GSH-Px normal. Os resultados mostraram que os pacientes estudados apresentaram Se plasmático e eritrocitário abaixo dos valores normais e o consumo de apenas uma castanha-do-brasil por dia (5g) foi eficaz no aumento da concentração de Se e na atividade da GSH-Px nos pacientes em HD; além disto, ela pode contribuir para a melhora da condição de estresse oxidativo desses pacientes. / Hemodialysis (HD) patients may undergo oxidative damage which causes reactive oxygen species (ROS) formation. Uremic toxins, HD treatment and the decrease in antioxidant system are known to result in the ROS generation. Selenium (Se) has a role as an antioxidant and studies report Se deficiency in these patients. Considering the importance of the antioxidant activity in patients with chronic kidney disease (CKD) in HD, the aim of this work was evaluate the effect of Brazilian nuts (main food source) supplementation on levels of Se and glutathione peroxidase (GSH-Px) activity in HD patients. A total of 81 HD patients (52.0 ± 15.2 years old, average time on dialysis was 82.3 ± 91.4 months and BMI of 24.9 ± 4.4 kg/m2) from RenalCor and RenalVida Clinics in Rio de Janeiro, Brazil were studied. The patients received one nut (≈ 5g), average of 290 µg Se, each day during three months. The Se concentration was determined by atomic absorption spectrophotometry with hydride generation (HITACHI®, Z-500). The levels of GSH-Px were measured by using RANDOX® commercial kits. The Se levels in plasma (18.8 ± 17.4 µg/L) and erythrocyte (72.4 ± 37.9 µg/L) before supplementation were below the normal range (60-120 µg/L for plasma and 90-190 µg/L for erythrocyte) and after nut supplementation, the plasma levels increased to 104.0 ± 65.0 µg/L and erythrocytes to 244.1 ± 119.5 µg/L (p<0.0001). The activity of GSH-Px also increased after supplementation from 46.6 ± 14.9 U/ gHb to 55.9 ± 23.6 U/gHb (p<0.0001). Before supplementation, 11% of patients had activity of GSH-Px below the normal range (27.5 - 73.6 U/gHb) and, after supplementation, all patients showed activity of GSH-Px within the normal range. The data revealed that the investigated patients had erythrocyte and plasma Se levels below the normal values and the consumption of just one Brazil nut each day (5g) was effective to increase on Se concentration and GSH-Px activity of the patients in HD, thus contributing to improve the antioxidant condition of these patients.

ABSTRACT EFFECT OF SALT STRESS ON ANTIOXIDANT DEFENSE SYSTEMS OF SENSITIVE AND RESISTANT CULTIVARS OF LENTIL Cicerali, Iin Nur M.Sc., Department of Biotechnology Supervisor: Prof. Dr. Meral Y&uuml / cel Co-supervisor: Asst. Prof. Dr. F&uuml / sun (nci) Eyidoan June 2004, 90 pages In this study, two lentil cultivars (Lens culinaris, Medik.) (ILL5582-salt tolerant and ILL590) were characterized and compared due to their NaCl susceptibility and antioxidant mechanism was examined under laboratory conditions. Physiological parameters such as wet-dry weight, root-shoot lengths, cell membrane stability, lipid peroxidation in terms of malondialdehyde (MDA), H2O2, proline contents were determined. The activities of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2) were examined and analyzed in 14 days old plant seedlings after 9 days of normal growth and 5 days of 100mM and 200mM NaCl stress conditions. Shoot-root length and wet-dry weight percent decrease were more in ILL590. Especially shoot tissues were affected more from the stress conditions when compared to root tissues. ii According to malondialdehyde (MDA) content and membrane stability results, lipid peoxidation was higher in ILL590 and significant increases were observed in shoot tissues. Proline concentration showed a remarkable increase in salt concentration dependent manner. Higher concentrations of proline in ILL5582 might be the reason of higher salt tolerance when compared to ILL590. Among the antioxidant enzymes SOD was the one which showed highest activity increase. At organ level roots showed highest activity when compared to leaves. In the organelle higher activity percent contribution was achieved by cytosolic Cu/ZnSOD isozyme. Higher percent increase of this isozyme was observed in ILL5582. This might be one of the tolerance mechanisms that get activated against NaCl stress. APX activity showed similar alterations in both cultivars. In leaf tissues significant increase was observed but in root tissues ascorbate peroxidase activity did not change significantly. Glutathione Reductase activity increase was significant in both cultivars leaf tissues but although ILL5582 showed a stress concentration dependent increase, ILL590 did not. The activity of CAT enzyme in leaf and root tissues of both cultivars did not significantly change under increasing salt stress conditions. The results suggested that the leaves were more susceptible to salt stress. Also when two cultivars were compared ILL5582 was found to be more tolerant against salt stress than ILL590 under laboratory conditions and SOD enzyme seemed to be the most active component of the salt tolerant mechanism.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Seizures can occur at any age, affecting at least 1-2% of the world population, they are far more common in children than adults. Prolonged seizures in the early developmental period can cause brain damage and lead to serious consequences later in life. In the present study the potential neurotoxicity of diphenyl diselenide, as measured by the manifestation of seizures in rat pups (postnatal day, PND, 12-14) was evaluated. The results suggest that the latency for the appearance of tonic-clonic seizures, characterized by rearing and falling of rat pups body, was dependent of the dose tested. Diphenyl diselenide at high doses induced seizure episodes in rat pups. The highest dose of diphenyl diselenide (500mg/kg) increased the levels of lipid peroxidation and catalase activity as well as decreased d-ALA-D (d-aminolevulinate dehydratase) and Na+, K+-ATPase activity in brain of rat pups. Our results indicate the possible involvement of free radical oxygen injury in diphenyl diselenide-induced seizures. The data obtained with the dose of 150 mg/kg in the brain of rats that exhibiting seizures are: an increase in lipid peroxidation levels; the lack of effect on catalase activity; an inhibition of d-ALA-D activity, supporting that the enzyme activity is more sensitive than other parameters analyzed as an indicator of oxidative stress. The lowest dose of diphenyl diselenide in brain emphasizes the relationship between the appearance of seizures and the latency for the onset of the first episode. Taken together, this paper could add to our understanding of diphenyl diselenide neurotoxic effect demonstrated by the appearance of seizures which are, at least in part, related to the oxidative stress. / As convulsões podem ocorrer em todas as idades, afetando cerca de 1-2% da população mundial, sendo mais comum em crianças do que em adultos. Convulsões prolongadas no período de desenvolvimento podem levar a sérias conseqüências na idade adulta. Neste estudo o potencial neurotóxico do disseleneto de difenila foi avaliado pela manifestação de convulsões em ratos jovens (12-14 dias de vida). Os resultados sugerem que a latência para o aparecimento de convulsões tônicoclônicas, é dependente da dose testada. O disseleneto de difenila em altas doses induziu episódios convulsivos em ratos jovens. A maior dose de disseleneto de difenila (500mg/kg) aumentou os níveis de peroxidação lipídica, atividade da catalase bem como diminuiu as atividades da d-ALA-D (d-aminolevulinatodesidratase) e Na+, K+-ATPase no cérebro dos ratos jovens. Nossos resultados indicam o possível envolvimento dos danos causados pelos radicais livres nas convulsões induzidas pelo disseleneto de difenila. Os dados obtidos na dose de 150 mg/kg no cérebro dos ratos que exibiram convulsão são: um aumento nos níveis de peroxidação lipídica, atividade da catalase e inibição da atividade da d-ALA-D, suportando que a atividade desta enzima é mais sensível que os outros parâmetros analisados como um indicador do estresse oxidativo. A menor dose de disseleneto de difenila no cérebro enfatiza a relação entre o aparecimento das convulsões e a latência para o primeiro episódio convulsivo. Contudo, este trabalho pode demonstrar os efeitos neurotóxicos do disseleneto de difenila, em ratos jovens, caracterizados pelo aparecimento de convulsões, que são em parte, relacionadas ao estresse oxidativo.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-15T14:31:09Z No. of bitstreams: 1 TeseMGP.pdf: 6082207 bytes, checksum: 573b75094c040d929b1f8f03f927018e (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:25:25Z (GMT) No. of bitstreams: 1 TeseMGP.pdf: 6082207 bytes, checksum: 573b75094c040d929b1f8f03f927018e (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:25:35Z (GMT) No. of bitstreams: 1 TeseMGP.pdf: 6082207 bytes, checksum: 573b75094c040d929b1f8f03f927018e (MD5) / Made available in DSpace on 2016-09-16T19:25:46Z (GMT). No. of bitstreams: 1 TeseMGP.pdf: 6082207 bytes, checksum: 573b75094c040d929b1f8f03f927018e (MD5) Previous issue date: 2015-05-29 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Eutrophication of the aquatic environment favors cyanobacteria blooms and affects the biota due to organoleptic water changing and the release of toxins. The objective of this study was to evaluate the effect of microcystin contained in the crude extract of the cyanobacteria Radiocystis fernandoi (R28 strain) in the fish Hoplias malabaricus assessing the degree of structural and functional impairment of the liver, the accumulation potential in muscle and risk to human health. Traíras were injected intraperitoneally with 100 μg MC-LReq kg-1 following two experimental protocols: (i) acute exposure with the application of a single dose of the crude extract and evaluation after 12 and 96 hours and (II) chronic exposure in which a dose was injected every 72 hours during 30 days. Genotoxic, biochemical, physiological and morphological biomarkers were used to evaluate the action of toxins, structural and ultrastructural damage in the liver, the detoxification mechanisms and changes in energy metabolism. The R28 strain produced mainly the MC-RR and MC-YR showing hepatotoxic potential. In the liver there was accumulation of MC-YR after acute and chronic exposure and MC-RR and MC-RR only after chronic exposure. There was not MC accumulation in muscle. The accumulation of the variants (MC-RR and MC-YR) depends on the concentration and the body's defense system for depuration and biomagnification. Acute and chronic exposure caused inhibition of the serine protein phosphatase / threonine type of PP2A activity, increased alanine aminotransferase and aspartate activity and direct bilirubin in plasma indicating liver damage. Macroscopically occurred changes in the color, texture and liver mass; microscopically the morphology of hepatocytes and intracellular organelles were altered and there were dilated sinusoids and hyperemia. After 30 days, the frequency of changes increase, and fibrous tissue disrupted the architecture of hepatic tissue. Changes in intermediary metabolism as glucose, liver glycogen, pyruvate and lactate showed increased energy demand and physiological stress. Moreover, there was activation of phase I of the biotransformation system and alterations in cellular antioxidant systems. Morphological signs of recovery in the liver were observed after 96 hours (single dose) to MC, but not after 30 days and no signs of cell regeneration. DNA damage were observed as well. The biochemical changes and liver structure showed functional impairment of the liver. In conclusion, the microcystin present in the crude extract of cyanobacteria R. fernandoi (R28 strain) combined with other substances in the extract may compromise the health and survival of the animal. / A eutrofização do ambiente aquático favorece as florações de cianobactérias e afeta a biota devido à alteração organoléptica da água e a liberação de toxinas. O objetivo deste estudo foi avaliar o efeito de microcistinas contidas no extrato bruto da cianobactéria Radiocystis fernandoi (cepa R28) no peixe Hoplias malabaricus quanto ao grau de comprometimento estrutural e funcional do fígado, o potencial de acumulação no músculo e possível risco para a saúde humana. Traíras foram injetadas intraperitonealmente com 100 μg MC-LReq kg-1 seguindo 2 protocolos experimentais: (i) exposição aguda com a aplicação de uma única dose do extrato bruto e avaliação após 12 e 96h e (II) exposição crônica com dose injetada a cada 72h durante 30 dias. Biomarcadores genotóxicos, bioquímicos, fisiológicos e morfológicos foram utilizados para avaliar a ação das toxinas, danos estruturais e ultraestruturais, mecanismos de desintoxicação no fígado e alterações no metabolismo energético. A cepa R28 produziu majoritariamente as variantes MCRR e MC-YR evidenciando potencial hepatotóxico. A MC-YR acumulou no fígado após exposição aguda e crônica e a MC-RR apenas após exposição crônica. Não ocorreu acúmulo de MC no músculo. O potencial de acumulação das variantes (MC-RR e MC-YR) depende da sua concentração, sistema de defesa do organismo para sua depuração e da biomagnificação. A exposição aguda e crônica causou inibição da atividade da proteína fosfatase de serina/treonina do tipo PP2A, aumento da atividade da alanina e aspartato aminotransferases e bilirrubina direta no plasma evidenciando danos hepáticos. Macroscopicamente ocorreu alteração da massa, cor e textura do fígado; microscopicamente a morfologia dos hepatócitos e as organelas intracelulares foram alteradas e ocorreu dilatação dos sinusóides e hiperemia. Após 30 dias, a frequência de alterações aumentou e o fígado apresentou tecido fibroso descaracterizando a arquitetura do parênquima hepático. As alterações no metabolismo intermediário como glicose, glicogênio hepático, piruvato e lactato em fígado, músculo e plasma evidenciaram aumento da demanda energética e estresse fisiológico. Além disso, ocorreu ativação do sistema de biotransformação de fase I e alterações no sistema antioxidante celular. Sinais de recuperação morfofuncional do fígado foram observados após 96 horas de exposição (dose única) a MC, mas não após 30 dias e não houve sinais de regeneração celular. Danos no DNA também foram observados em células hepáticas. As alterações bioquímicas e estrutura do fígado evidenciaram comprometimento funcional do órgão. Em conclusão, as microcistinas presentes no extrato bruto da cianobactéria R. fernandoi (cepa R28) associadas a outras substâncias presentes no extrato podem comprometer a saúde e sobrevicência do animal.