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Thesis (PhD(Agric) (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: Salinity is one of the major limiting abiotic stresses on legume plant yield, leading to early senescence of root nodules. This occurs because of accumulation of reactive oxygen species (ROS) in plant cells under salinity stress. Concurrent with the increase in cellular reactive oxygen species levels is the increase in cellular antioxidants and corresponding antioxidant enzymes. This feature is observed mostly in the shoots and roots of more tolerant genotypes compared to the susceptible genotypes. It is accepted that the mechanism of plant tolerance to stress is dependent upon the response of the antioxidant systems. Most studies carried out on shoot tissues suggest that scavenging of ROS by the plant antioxidant system is modulated by nitric oxide (NO). However, the pathways by which NO mediates such antioxidant responses are not fully understood. For legumes, salinity stress has adverse effects on yield and this is in part due to inhibition of nitrogen fixation in the root nodules of the legumes, which causes severe nitrogen starvation in nitrogen-deficient soils. Nodules are specialized organs comprising of both the rhizobia and the plant tissue, hence the physiological aspects may vary from the findings from the leaves. It was therefore deemed necessary to establish the role of NO on the nodule antioxidant system in the absence and presence of salinity stress. For the purposes of this study, the effect of both exogenously applied NO and endogenous NO on superoxide dismutase, glutathione peroxidase and glutathione content was determined. The studies involved the use of nitric oxide donors like sodium nitroprusside (SNP) and diethylenetriamine/nitric oxide adduct (DETA/NO), their respective fixed controls potassium ferricyanide and diethylenetriamine (DETA), plus a nitric oxide synthase inhibitor (to inhibit nitric oxide production by the enzyme nitric oxide synthase) on nodulated roots. The data obtained in this work points out specifically at roles played by nitric oxide in regulating superoxide dismutases, glutathione peroxidase and glutathione during salinity stress and proposes a link between nitric oxide-mediated changes in these antioxidant systems and salinity stress tolerance. Both the exogenously applied and endogenous nitric oxide increases the enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR). However, there is both time dependency and nitric oxide concentration dependency on the enzyme activities. The total SOD enzyme activity increases upon nitric oxide exposure and with time of exposure. The individual SOD isoforms identified and studied in the root nodules all contribute to this increase in SOD activity upon nitric oxide treatment except for MnSOD I. This increase in activity is regulated at transcriptional level as the RT-PCR results targeting the individual isoforms reveals an increase in transcript levels after 6 hours of nitric oxide treatment. However, the CuZn SOD I isoform transcripts are reduced upon nitric oxide treatment. A similar response was also observed in GPX enzyme activity in which nitric oxide increased the GPX activity above all the controls. The GR enzyme activity exhibits an opposite response because the activity decreases with time of exposure to NO and concentration of NO. In order to determine the effect of NO under saline conditions, an experiment was set up that involved incubation of nodulated roots in solutions containing 150 mM NaCl. The stressed nodules exhibited generally higher levels of enzyme activities than the non-stressed nodules. Furthermore, exposure to nitric oxide donor in combination with NaCl induced even higher activities of SOD and GPX than NaCl or nitric oxide donor alone. There were also higher levels of reduced glutathione and total glutathione recorded under stress compared to optimal conditions. Nitric oxide increased the concentration of these forms of glutathione, suggesting an improved redox status based on the GSH/GSSG ratios under salinity stress in the presence of nitric oxide. Attenuation of nitric oxide synthesis with L-Nω-Nitroarginine methyl ester (L-NAME) reverses all the recorded effects of nitric oxide on antioxidant enzymes and glutathione pool. This was observed in salinity stressed nodules and non-stressed nodules. This work further establishes that NO plays a pivotal role in modulating the enzymatic activities through a pathway that is mediated by guanosine 3,5-cyclic monophosphate (cGMP). The experiment involving the inhibition of soluble guanylyl cyclase (sCG) (an enzyme that catalyzes the biosynthesis of cGMP), cell-permeable cGMP anaologue and L-NAME revealed that GPx activity is modulated through a cGMP-dependent pathway and NO is positioned up-stream of cGMP in the pathway leading to improved GPX activity. Cyclic GMP also modulates the GPX activity in a concentration dependent manner. NO improves the redox status of the cell under both saline conditions and non-saline conditions and this effect is modulated through a cGMP-dependent pathway. It is thus rational to conclude that; in the root nodules of legumes, like in other plant tissues, the increased accumulation of antioxidants and the increased activity of their corresponding enzymes, as modulated through the cGMP-dependent pathway by nitric oxide, confer root nodule tolerance to salinity. This concept directly points out at an attractive strategy for developing legumes that are genetically improved for enhanced root nodule tolerance to salinity; via differential regulation of antioxidants and antioxidant enzyme genes in the root nodules under abiotic stress. Towards attaining the goal for such genetic improvement, experiments involving construction of an abiotic stress-responsive and nodule-specific chimeric promoter were carried out. By fusing the 5-untranslated (5-UTR) region of the LEA gene that contains an abiotic stress-responsive cis-acting element (from theGmPM9 promoter) to the nodulin N23 promoter bearing the highly functional cluster of motifs for nodule specificity, the candidate nodule specific promoter that is abiotic stress responsive (ASREF/NSP) was constructed. The construct harbouring this ASREF/NSP chimeric promoter was fused to the -glucuronidase (GUS) reporter gene so as to study the functionality of the promoter in Medigaco truncatula plants. The construct was delivered into the Medicago plants through Agrobacterium rhyzogenes mediated transformation to produce composite Medicago plants. The transgenic roots have been cultured for futher manipulation and to confirm the functionality of the promoter. Furthermore several strategies can be deployed via the use of this chimeric promoter so as to enhance the nodular antioxidant system. This would involve either gene regulator-chimeric promoter fusion or the use of a single gene approach. As part of this work, the MtNOA gene homologous to AtNOAs, has been cloned from Medicago trancatula and put as ASREF/NSP fusion in a binary vector pBINPLUS and delivered into Medicago trancatula for nodule-specific and abiotic stress-induced nitric oxide synthesis. Since there is no plant NOS identified to date, the possibility of the use of a regulatory gene in this aspect is still limited. There are other options involving the use of the chimeric promoter with the individual genes encoding the antioxidant enzyme genes such as genes encoding SOD, GPX and the glutathione synthatase to enhance the plant antioxidant system during abiotic stress. / AFRIKAANSE OPSOMMMING: Geen opsomming was ingedien met die tesis

Growth performance of Bur oak (Q. macrocarpa Michx.) and Red oak (Q. rubra L.) under salinity conditions was assessed by growing seedlings in the presence of increasing levels of NaCl. Salinity reduced root growth in both species, although its repressive effect was more pronounced in Red oak. Exposure to 75 mM NaCl for three weeks almost arrested root growth in Red oak, while it reduced it only by 40 % in Bur oak. Red oak roots showed extensive necrosis and limited branching. Salinity also induced leaf injury, which at a NaCl level of 25 mM was less severe in Bur oak possibly due the higher expression of dehydroascorbate reductase (DHAR) and ascorbate peroxidase (APX), enzymes participating in the detoxification of reactive oxygen species (ROS). Salinity also altered nutrient uptake and accumulation in root and leaf tissue. Compared to Red oak, the relative calcium level in Bur oak roots exposed to increased salinity remained elevated, while an opposite trend was observed in leaf tissue. This was in contrast to nitrogen and potassium, the relative level of which was higher in Red oak leaves grown in the presence of NaCl. The better performance of Bur oak root tissue under salinity conditions was ascribed to structural modifications of the root system with maturation of casparian bands and suberinization occurring closer to the root tip. These structures are known to act as barriers enhancing ion selectivity. Collectively this study demonstrates that relative to Red oak, Bur oak is more tolerant to NaCl induced salinity conditions. / February 2017

Introdução Estudos indicam que antioxidantes presentes naturalmente em alguns alimentos são capazes de atuar como protetores dos organismos vivos frente aos danos causados pelo estresse oxidativo em macromoléculas como lipídios, proteínas e em DNA. O guaraná (Paullinia cupana), planta originária da Amazônia, contém elevadas concentrações de taninos e cafeína, compostos com comprovada atividade antioxidante. Apesar do aumento no consumo de guaraná e de estudos associando seus efeitos benéficos à saúde, há poucas informações sobre suas propriedades antioxidantes in vivo. Objetivos: avaliar o efeito do consumo de bebida a base guaraná em pó em humanos. Métodos - In vitro: amostras de guaraná em pó foram analisadas para determinação da composição proximal; conteúdo de compostos fenólicos totais (Folin-Ciocalteau) e atividade antioxidante pelo ensaio DPPH foram determinados em amostras extraídas com água, metanol, etanol 60 por cento e acetona 35 por cento. In vivo e ex vivo: amostras de sangue de voluntários saudáveis (n=12) foram coletadas em jejum (J1) e 1h após o consumo da bebida com guaraná em pó foram coletadas novamente amostrar de sangue (G1). Após 15 dias da ingestão diária da bebida foram realizadas duas novas coletas, uma em jejum (J15) e outra após a primeira hora de consumo da bebida (G15). Foi avaliada a resistência da LDL à oxidação ex vivo iniciada com cobre pelo ensaio de dienos conjugados. O perfil antioxidante total (TAS) e a capacidade de absorbância de radical oxigênio (ORAC) foram determinados no plasma dos voluntários. Ensaio Cometa foi realizado para verificar danos oxidativos ao DNA em linfócitos dos voluntários. A atividade das enzimas Superóxido Dismutase (SOD), Catalase (Cat) e Glutationa Peroxidase (GPx) foi determinada em eritrócitos. Os resultados das diferentes análises foram apresentados com média e desvio-padrão. Foram utilizados ANOVA e teste de Tukey para verificar se há diferença no teor de compostos fenólicos totais e na atividade antioxidante das amostras extraídas com diferentes solventes. As verificações de aderência à curva normal foram realizadas pelo teste de KolmogorovSmirnov. As comparações das variáveis de distribuição normal para as amostras pareadas foram baseadas no teste t de Student. Para todas as inferências foi utilizado o nível de significância menor ou igual a 5 por cento. Para todos estes cálculos estatísticos foi utilizado o programa SPSS versão 16.0 for Windows. Resultados: Foi observado aumento significativo no lag time de oxidação da LDL tanto após uma hora do consumo da primeira dose de guaraná em pó quanto após uma hora do consumo no 15º dia de intervenção (G1>J1, G15>J15; p<0,05). O consumo de uma única dose de guaraná aumentou significativamente a atividade da Cat nos tempos G1 e G15 (p < 0,05) e da GPx no tempo G15 (p<0,05). Após intervenção com doses repetidas durante 15 dias houve aumento significativo da atividade da Cat e da GPx no jejum do último dia de intervenção quando comparado ao jejum do baseline (J15>J1; p<0,05). O consumo de guaraná não influenciou a atividade da SOD, tampouco o TAS (p>0,05). O consumo da bebida apresentou efeito agudo sobre o ORAC, uma vez que houve aumento significativo desse parâmetro tanto após uma hora do consumo da primeira dose de guaraná em pó quanto após uma hora do consumo no 15º dia de intervenção (G1>J1, G15>J15; p<0,05). O ORAC no plasma dos voluntários e avaliação de danos oxidativos ao DNA com desafio por peróxido de hidrogênio apresentaram o mesmo comportamento da resistência da LDL à oxidação: apenas efeito agudo foi observado pelo consumo da bebida (G1>J1, G15>J15; p<0,05). Sugere-se que o fracionamento da dose de guaraná em pó seja mais eficiente do que o consumo de uma única dose no dia para a manutenção da concentração dos compostos fenólicos no plasma a fim de promover efeitos pelo consumo de doses repetidas. Os efeitos antioxidantes pelo consumo de uma única dose de guaraná em pó parecem se extender além do tempo de depuração dos compostos fenólicos no plasma. São necessárias ainda novas pesquisas a fim de avaliar a dose e o tempo de intervenção para que sejam observados efeitos em humanos pela ingestão de doses repetidas de guaraná / Introduction Studies indicate that antioxidants found naturally in some foods are capable of acting as protectors of living organisms against oxidative stress in macromolecules such as lipids and proteins and in DNA. Guarana (Paullinia cupana), a plant from Amazonia, contains high concentrations of tannins and caffeine, compounds with proven antioxidant activity. Despite the increase in consumption of guarana and studies linking their beneficial health effects, there is little information on its antioxidant properties in vivo. Objectives: investigate the effects of guarana consumption in humans. Methods: In vitro: guarana powder samples were analyzed for proximal composition; content of total phenolics (Folin- Ciocalteau) and antioxidant activity by DPPH assay were determined in samples extracted with water, methanol, ethanol 60 per cent and acetone 35 per cent. In vivo and ex vivo: blood samples from healthy volunteers (n = 12) were collected at a twelve-hour overnight fast (J1) and 1h after consumption of the drink with guarana powder the second blood sample was collected (G1). After 15 days of daily ingestion of the drink other two samples were collected: a twelve-hour overnight fast (J15) and again after the first hour of drink consumption (G15). The resistance of LDL to ex vivo oxidation initiated by copper was evaluated. The total antioxidant status (TAS) and the oxygen radical absorbance capacity (ORAC) were determined in plasma of volunteers. Comet assay was conducted to determine oxidative DNA damage in lymphocytes of volunteers. The activity of superoxide dismutase (SOD), catalase (Cat) and glutathione peroxidase (GPx) was determined in erythrocytes. ANOVA and test of Tukey were used to verify if there was significant differences in total phenolic contents and antioxidant activity of samples extracted with different solvents. The verification of adherence to the normal curve were performed by the Kolmogorov-Smirnov test. Comparisons of variables of normal distribution for the paired samples were based on t test of Student. For all inferences the significance level was less than or equal to 5 per cent. For all these calculations the statistical program SPSS version 16.0 for Windows was used. Results: Significant increase in lag time of LDL oxidation was observed, both after one hour of consumption of the first dose of guarana powder and after one hour of consumption in the 15th day of intervention (G1> J1, G15> J15, p <0.05). The consumption of a single dose of guarana significantly increased the activity of Cat in the times G1 and G15 (p<0.05). After intervention with repeated doses during 15 days, there was a significantly increase in the activity of Cat and GPx in fasting of the last day intervention compared to baseline fasting (J15> J1, p<0.05). The consumption of guarana didnt influence the activity of SOD neither the TAS (p>0.05). The ORAC in plasma of volunteers and assessment of oxidative damage to DNA challenge with hydrogen peroxide showed the same behavior of the resistance of LDL to oxidation: only acute effect was observed by the consumption of the drink (G1> J1, G15> J15, p<0.05). It is suggested that the fractionation of the dose of guarana powder is more efficient than the consumption of a single dose on day to maintain the concentration of phenolic compounds in plasma to promote the consumption effects of repeated doses. The antioxidant effects by consumption of a single dose of guarana powder seem to extend beyond the time of clearance of phenolic compounds in plasma. New reserches are needed in order to evaluate the dose and duration of intervention for being observed effects in humans by ingestion of repeated doses of guarana

O alumínio é limitante à atividade agrícola em todo o mundo. Nos solos ácidos a disponibilidade de Al aumenta. Estes solos constituem a maioria dos solos do mundo e dois terços dos solos brasileiros. O problema da acidez do solo e da toxicidade por Al é altamente significativo para as perdas na produtividade agrícola e florestal. Para se ter Al disponível, primeiramente tem que se ter condições de pH baixo. O primeiro sintoma causado pela toxicidade por Al é a inibição no alongamento do sistema radicular. Existem trabalhos vinculando a inibição a alterações nos processos de divisão e expansão celular. Embora os mecanismos de toxicidade e resistência ao Al não estejam totalmente elucidados, admite-se que em algumas plantas, a quelação do Al por ácidos orgânicos é um dos mecanismos que confere resistência das células ao Al, assim como em outras plantas a elevação do pH da rizosfera, por compostos liberados pelo sistema radicular, atua na queda da disponibilidade do Al na solução do solo. Porém, existem outras alternativas que vêm sendo propostas na literatura como possíveis mecanismos de resistência das plantas ao Al, principalmente ao nível celular e molecular. Alterações nas composições lipídica e protéica da membrana plasmática, assim como na sua estrutura física; ativação do sistema antioxidante celular; alterações na sinalização celular e de atividade dos canais de troca da membrana plasmática vêm sendo estudados como possíveis contribuintes para os mecanismos de resistência ao Al. A sensibilidade celular ao Al depende do seu estágio de desenvolvimento. As células sensíveis ao Al acumulam o metal, enquanto que as resistentes acumulam muito pouco. Foi constatado em nosso trabalho que as células sensíveis ao Al também são sensíveis ao baixo pH. As células sensíveis não conseguem recuperar seu crescimento e sua viabilidade celular após a exposição ao Al ou ao baixo pH.A sacarose ou manitol conferiram proteção às células quanto ao acúmulo de Al. Isso fez com que a viabilidade mantivesse-se em níveis próximos ao controle (pH5,6) e a cultura conseguisse recuperar seu crescimento e viabilidade após a exposição ao Al e ao baixo pH. O efeito protetor não foi devido ao caráter energético da sacarose, pois o manitol não é metabolizado pelas células BY-2 e os resultados foram semelhantes quando se usou sacarose ou manitol, nas mesmas concentrações. Sabe-se que o Al aumenta a peroxidação lipídica e a oxidação protéica da membrana plasmática, pela geração de EAO?s, desencadeando o processo de estresse oxidativo na célula. Em nosso estudo, nas células sensíveis houve peroxidação dos lipídios, ativação do sistema de enzimas antioxidantes, como SOD, GST, GR, CAT e APX, alteração nos níveis de carboidratos e alteração no perfil protéico de frações enriquecidas de membrana plasmática, obtido por eletroforese 2D. O mesmo comportamento foi verificado em células sensíveis tratadas a baixo pH. Pode-se concluir que o sistema antioxidante celular foi ativado na presença de baixo pH ou Al, pela ocorrência de peroxidação lipídica, que gera maiores concentrações de H2O2 nas células sensíveis (fase log). E que existem diferenças no perfil protéico de células tratadas com Al em relação a células mantidas sob condições de cultivo, tanto em presença de spots como em expressão diferencial. Porém estas diferenças necessitam ser melhores exploradas. A peroxidação lipídica é um bom indicador da sensibilidade celular ao Al e ao baixo pH, assim como a ativação do sistema antioxidante e a geração do peróxido de hidrogênio. Poderiam ser realizados experimentos no tempo, medindo-se o acúmulo de Al e relacionando-o aos níveis de peroxidação lipídica, atividade das enzimas antioxidantes e geração do peróxido, para que pudéssemos indicar talvez um processo que se iniciasse antes que outro, ou mesmo que decaísse antes do outro. Assim como um monitoramento das condições de oxidação protéica na presença de Al. / Aluminum limits crop production in all over the world. In acid solis the Al disponibility is larger. Acid soils compose the major part of the brazillian soils. The problem of acidity and Al toxicity results in losses of productivity in agriculture and forestry. The first symptom of Al toxicity is inhibition of root growth. There is many studies that indicate relations between the inhibition of root growth and cell division and expansion alterations. The mechanisms of Al toxicity and resistance aren?t completely understood in plants. The resistance mechanism of Al chelation by organic acid is one of the mechanisms accept, like the elevation of the rizosphere pH by substances exsudated by the root system. Other possible mechanisms that are being mentionated are the alterations in plasma membrane composition and structure, antioxidant cell system activation, alterations in cell signal and alterations in the membrane channels activity. Aluminum cell sensibility depends of the status cellular. The cells that are sensible to Al, are in the log phase of growth and accumulate the metal, whereas the resistant cells do not accumulate and were in the stationary phase of growth. In our work, we observed that the sensible cells are sensible to low pH too. The sensible cells don?t recover their growth rate and cellular viability after the treatment exposition. Sucrose or mannitol confers cellular protection against the Al. The cellular viability was high (next to the control, pH5,6) and the cell culture recovery their growth and viability after the Al or low pH exposition. The protective effect don?t occurs in response to the energetic role of sucrose, because cells treated with mannitol showed the same results and the mannitol did not metabolizated by tobacco BY-2 cells. Al induces lipid peroxidation and protein oxidation in plasma membrane, by the ROS generation promoting the oxidative stress. We found that sensible Al cells showed lipid peroxidation, H2O2 generation, antioxidant enzymes activation (SOD, le carbohydrate levels and protein profile alterations by 2D electrophoresis. The same responses were observed in the pH sensible cells, at log phase of growth. This differences should be more explored. We concluded that the lipid peroxidation is an indicator of sensitivity to Al and low pH, like the antioxidant enzymes activities and the H2O2 generation. Studies should be done with the Al accumulated in time, measuring the activities of antioxidant system and the lipid peroxidation with the objective to indicated what process could start firstly

O capim tanzânia (Panicum maximum cv. Tanzânia) é promissor para uso em fitorremediação, devido ao seu sistema radicular extenso, boa adaptação a variados ambientes e alto rendimento de biomassa quando bem nutrido. O enxofre faz parte de compostos essenciais ao sistema antioxidante das plantas. A toxicidade causada por alta disponibilidade de metais, como o zinco, no meio de cultivo é um problema ambiental crescente. Objetivou-se avaliar o efeito do enxofre em amenizar o estresse causado pelo excesso de zinco no capim tanzânia. O experimento foi conduzido em casa de vegetação em Piracicaba - SP, durante o verão, em delineamento de blocos casualizados em esquema fatorial fracionado, com as combinações de doses de enxofre (mmol L-1) e zinco (&mu;mol L-1): 0,1/0,7; 0,1/500; 0,1/3000; 1,0/250; 1,0/1000; 1,9/0,7; 1,9/500; 1,9/3000; 2,8/250; 2,8/1000; 3,7/0,7; 3,7/500 e 3,7/3000. Foram avaliadas as seguintes variáveis: componentes de produção (perfilhos, folhas, área foliar, massa de parte aérea e de raízes), concentrações dos nutrientes (N, S, Ca, Mg, Cu, Fe, Mn e Zn), valor SPAD, atividades de enzimas do sistema antioxidante (GR, GPOX, APX, CAT e SOD) e concentração de prolina. A toxicidade de zinco no primeiro crescimento do capim limitou a produção de massa seca e o número total de folhas, enquanto no segundo crescimento também ocorreu limitação na área foliar e no número total de perfilhos. A concentração de enxofre na planta aumentou em função do fornecimento de enxofre e do excesso de zinco no meio de cultivo. O excesso de zinco na planta provocou desequilíbrio na nutrição da planta com os micronutrientes cobre, ferro e manganês. Os indicadores do estresse oxidativo foram sensíveis para detectar a toxicidade do zinco no capim. As reduções na concentração de prolina e nas atividades de enzimas do sistema antioxidante demonstraram o efeito do enxofre em aliviar o estresse por toxicidade de zinco no capim tanzânia. / Tanzania guinea grass (Panicum maximum cv. Tanzania) is promising for use in phytoremediation due to its extensive root system, good adaptation to diverse environments, and high biomass production when well nourished. Sulfur is part of essential compounds to the plant\'s antioxidant system. Toxicity caused by high availability of metals, such as zinc, in the growth medium is a growing environmental problem. The objective was to evaluate the effect of sulfur in mitigating the stress caused by zinc excess in tanzania guinea grass. The experiment was carried out in a greenhouse at Piracicaba, SP, during the summer, in a randomized block design with rates of sulfur (mmol L-1) and zinc (&mu;mol L-1) of 1/0.7; 0.1/500; 0.1/3000; 1.0/250; 1.0/1000; 1.9/0.7; 1.9/500; 1.9/3000; 2.8/250; 2.8/1000; 3.7/0.7; 3.7/500 and 3.7/3000. The response variables evaluated were plant production components (number of tillers, number of leaves, leaf area, and shoots and roots dry matter), nutrient concentrations (N, S, Ca, Mg, Cu, Fe, Mn and Zn), SPAD value, activities of the antioxidant enzymes (GR, GPOX, APX, CAT and SOD) and proline concentration. Zinc toxicity in the first growth of the grass limited the dry mass production and the total number of leaves, while in the second growth also there was limitation in the leaf area and the total number of tillers. The concentration of sulfur in the plant increased as a function of sulfur supply and excess zinc in the growth medium. Excess zinc in the plant caused imbalance in plant nutrition for the micronutrients copper, iron and manganese. The indicators of oxidative stress were sensitive to detect grass toxicity by zinc. Reductions in proline concentration and enzyme activities of the antioxidant system demonstrated the effect of sulfur in alleviating the stress of zinc toxicity in tanzania guinea grass.

Introdução: O aumento do consumo de frutas e hortaliças está associado à redução do risco de ocorrência de doenças crônicas não transmissíveis. Este efeito protetor tem sido atribuído particularmente à presença de vários compostos bioativos como compostos fenólicos e organosulfurados, além de fitosteróis presentes no alho que podem contribuir com os efeitos antioxidante e hipolipemiante. Porém, o processamento do alho pode acarretar mudanças na quantidade e na efetividade dos compostos bioativos. Este trabalho teve como objetivo avaliar se a cocção e a fritura do alho reduziram as concentrações de compostos bioativos, o potencial antioxidante in vitro e in vivo em hamsters hipercolesterolemizados. Métodos: In vitro - foram determinados nos alhos cru, frito e cozido: a) composição centesimal (proteínas, lipídios, cinzas, carboidratos, fibra alimentar solúvel e insolúvel); b) perfil de ácidos graxos; c) teor de fenólicos totais; d) teor de quercetina, miricetina e apigenina; e) fitosteróis; f) alicina; g) teor de cobre, zinco e selênio; h) produtos intermediários da reação de Maillard; i) potencial antioxidante utilizando os testes ORAC (Oxygen radical absorbance capacity), Rancimat® e o sistema -caroteno/ácido linoléico. In vivo - hamsters machos foram distribuidos em 5 grupos com 10 animais em cada grupo. 1 - controle; 2 - hipercolesterolêmico; 3- hipercolesterolêmico e alho cru; grupo 4 - hipercolesterolêmico e alho cozido; grupo 5 - hipercolesterolêmico e alho frito. Os animais foram eutanasiados após 4 semanas de estudo para análises do plasma e do tecido hepático. No plasma foi determinado o potencial antioxidante pelo teste ORAC, o perfil lipídico (colesterol total e frações e triacilgliceróis) e verificado a atividade das enzimas aspartato aminotransferase (AST) e alanina aminotransferase (ALT). No tecido hepático foram avaliadas a atividade das enzimas hepáticas (glutationa peroxidase, catalase e superóxido dismutase) e o potencial antioxidante utilizando dois métodos, ORAC e ensaio cometa. Resultados: In vitro - O teor de fibras totais para o alho cru foi de 10,0por cento (71,6por cento é solúvel e 28,4por cento é insolúvel). O alto conteúdo de ácidos graxos trans no alho frito (14,9por cento ) é devido ao processo de fritura com 50por cento de gordura vegetal hidrogenada. A cocção não alterou o teor dos minerais analisados. O teor de compostos fenólicos nas amostras de alho variou de 4,2 a 187,7 mg EAG/100g (base seca), dependendo do solvente (água, água/metanol, etanol ou acetona) e do método de extração utilizados. A fritura diminuiu os teores de quercetina e alicina em torno de 24por cento e 87por cento , respectivamente. Os fitosteróis -sitosterol e campesterol estão presentes em todas as amostras, sendo que o alho frito apresentou os maiores teores destes compostos em relação aos alhos cru e cozido, além de apresentar stigmasterol. A fritura foi o processamento térmico que contribuiu com os maiores valores de produtos intermediários da reação de Maillard. O potencial antioxidante pelo teste ORAC (extratos etanólicos, metanol/água e acetona) reduziu com o processamento do alho, sendo que a redução foi maior para a fritura. A inibição da oxidação lipídica foi melhor nos extratos metanol/água. In vivo - O grupo de animais que recebeu ração hiperlipemiante e alho cru teve menor ganho de peso em relação aos grupos que receberam alho frito ou cozido. Os alhos cru e cozido foram eficazes na redução de lipídios no plasma dos hamsters. O potencial antioxidante, avaliado pelos testes ORAC e ensaio cometa, dos grupos hipercolesterolemizados suplementados com alho cru ou cozido apresentaram valores superiores em relação ao grupo hipercolesterolemizado não suplementado. Houve aumento da atividade das enzimas antioxidantes catalase, glutationa peroxidase e superóxido dismutase para todos os grupos suplementados com alho. Em todos os grupos estudados não ocorreram danos estruturais ou funcionais no tecido hepático. Conclusões: Os resultados corroboram com o esperado e sugerem que os alhos cru e cozido podem ocasionar benefícios à saúde, haja vista que estes produtos possuem compostos bioativos, efeito hipolipemiante e apresentaram alto potencial antioxidante no plasma e no tecido hepático, além do aumento da atividade de enzimas antioxidantes que estão envolvidas em mecanimos de proteção à saúde / Introduction: The increased consumption of fruits and vegetables is associated with reduced risks of chronic diseases. This protective effect has been attributed particularly to the presence of several bioactive compounds such as phenolic and organosulfur compounds, likewise phytosterols present in garlic that may contribute to the antioxidant and lipid-lowering effects. However, the processing of garlic can cause changes in the quantity and effectiveness of bioactive compounds. This study aimed to evaluate whether the cooking and frying of garlic reduced the bioactive compounds concentrations, the antioxidant potential in vitro and in vivo in hypercholesterolemic hamsters. Methods: In vitro - were determined in raw garlic, fried and boiling: a) composition (protein, fat, ash, carbohydrates, dietary fiber, soluble and insoluble), b) fatty acid profile, c) total phenolic content, d ) content of quercetin, myricetin and apigenin, e) phytosterols, f) allicin, g) content of copper, zinc and selenium, h) Maillard reaction products, i) antioxidant potential using the ORAC test (Oxygen radical absorbance capacity), Rancimat® and system -caroteno/ácido linoleic. In vivo - male hamsters were divided into five groups with 10 animals each. 1 - control, 2 hypercholesterolemic, 3 - hypercholesterolemic and raw garlic, 4 - hypercholesterolemic and boiling garlic, group 5 - hypercholesterolemic and fried garlic. Samples of blood and liver were collected after a 4-week experimental period. In plasma were determined the antioxidant potential by the ORAC assay, the lipid profile (total cholesterol and fractions and triacylglycerols) and verified the activity of enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In liver tissue were evaluated the activity of liver enzymes (glutathione peroxidase, catalase and superoxide dismutase) and the antioxidant potential using two methods, ORAC and comet assay. Results: In vitro - The content of dietary fiber for raw garlic was 10.0per cent (71.6per cent is soluble and 28.4per cent is insoluble). The high content of trans fatty acids in fried garlic (14.9per cent ) is due to the frying process with 50per cent hydrogenated vegetable fat. The cooking did not alter the content of the minerals analyzed. The content of phenolic compounds in garlic samples ranged from 4.2 to 187.7 mg EAG/100g (dry matter), depending on the solvent (water, water / methanol, ethanol or acetone) and the extraction method used. Frying decreased the content of quercetin and allicin around 24per cent and 87per cent respectively. The phytosterols -sitosterol and campesterol are present in all samples, and the fried garlic showed the highest levels of these compounds in relation to raw and boiling garlic, besides presenting stigmasterol. Frying was the heat processing that contributed to the higher values of products of the Maillard reaction. The antioxidant potential by the ORAC assay (ethanol extracts, methanol/water and acetone) was reduced with the processing of garlic, and the reduction was greater for frying. The inhibition of lipid oxidation was better in methanol/water extracts. In vivo - The group of animals that received ration hyperlipidemic and raw garlic had less weight gain compared with groups that received garlic fried or boiling. Raw and boiling garlic were effective in reducing lipids in hamsters plasma. The antioxidant potential (measured by the ORAC and comet assay tests) of the groups hypercholesterolemic supplemented with raw or boiling garlic had higher values than the not supplemented group hypercholesterolemic. There was increased activity of antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase in all groups supplemented with garlic. In all groups there was no structural or functional damage in liver tissue. Conclusions: These results corroborate the expected and suggest that the raw and boiling garlic may lead to health benefits, given that these products have bioactive compounds, hypolipidemic effect and showed a high antioxidant potential in plasma and liver tissue, in addition to increased activity of antioxidant enzymes that are involved in mechanisms of health protection

A fumaça do cigarro é composta por mais de 4.700 substâncias, muitas das quais tóxicas. O sistema nervoso central (SNC) possui poucas defesas antioxidantes, além de ser rico em lipídeos facilmente oxidáveis e de conter alto teor de metais de transição envolvidos na formação de radicais livres. Existem evidências de que a fumaça do cigarro causa alterações nas enzimas antioxidantes em roedores adultos, mas pouco se sabe sobre os efeitos do tabaco no SNC ainda em desenvolvimento. Assim, este trabalho tem como objetivo avaliar os possíveis efeitos da fumaça do cigarro no SNC de camundongos jovens. Camundongos BALB/c foram expostos a uma mistura de fumaça central e lateral do cigarro (Souza Cruz - Derby Vermelho) dentro de uma câmara de polipropileno acoplada a um sistema Venturi. Além da exposição aguda, realizada no 18º dia de vida, os animais foram expostos a partir do 5º dia de vida por 13 ou 35 dias, durante 2 horas por dia, 7 dias por semana. Os animais foram então eutanasiados por deslocamento cervical imediatamente ou três horas após a última exposição e foram realizadas as determinações das atividades enzimáticas da glutationa peroxidase, glutationa redutase, glutationa S-transferase, além da quantificação de malonaldeído (MDA) e 3-nitrotirosina no cerebelo, córtex frontal, estriado, hipocampo e hipotálamo. Nossos resultados indicam que o SNC em desenvolvimento é susceptível à fumaça do cigarro. Foram detectadas alterações nas enzimas antioxidantes em diferentes estruturas encefálicas imediatamente após a última exposição sugerindo diferença de sensibilidade das diferentes áreas à fumaça do cigarro. Contudo, essas perturbações não são persistentes, uma vez que a maior parte delas desapareceu três horas após a exposição. O cerebelo parece ser a estrutura mais resistente enquanto o córtex frontal e o estriado, as mais sensíveis. No córtex frontal as alterações enzimáticas são mais persistentes e houve aumento de MDA no grupo agudo e eutanásia 3 horas após a exposição, enquanto no cerebelo, estriado e hipocampo houve aumento de MDA no grupo agudo e eutanásia imediatamente após a exposição. Esses resultados sugerem uma resposta mais tardia do córtex frontal e uma possível adaptação dos tecidos ao xenobiótico. É importante destacar que a eutanásia realizada imediatamente após a exposição crônica à fumaça do cigarro não reflete somente o efeito da última exposição, já que tanto o padrão encontrado na alteração das enzimas quanto na determinação de MDA foi diferente do observado após exposição aguda. / Cigarette\'s smoke is composed of more than 4.700 substances, many of them are toxic. The central nervous system (CNS) has few antioxidant defenses, is rich in easily oxidable lipids and contains high levels of transition metals which are involved in the formation of free radicals. There are evidences that tobacco smoke causes changes in the antioxidant enzymes in adult rodents, but little is known about its effects during CNS development. Thus, the aim of this work was to evaluate the possible effects of cigarettes smoke in the CNS of young mice. BALB/c mice were exposed to a mixture of mainstream and sidestream smoke of cigarette (Souza Cruz Red Derby) in a polypropylene chamber attached to a Venturi system. Besides acute exposure, performed at the 18th day of life, the animals were exposed from the 5th day of life for 13 or 35 days, 2 hours a day, 7 days in a week. The animals were then euthanized by cervical dislocation, immediately or three hours after the last exposure. The determinations of enzymatic activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase, and the quantification of malonaldehyde (MDA) and 3-nitrotyrosine in cerebellum, frontal cortex, striatum, hippocampus and hypothalamus were performed. Our results indicate that the CNS in development is susceptible to cigarettes smoke. Alterations in antioxidant enzymes in different brain structures were detected immediately after the last exposure suggesting differences in sensitivity of different areas to cigarette\'s smoke. However, these disturbances are not persistent, as most of them disappeared three hours after exposure. Cerebellum seems to be the more resistant structure, while frontal cortex and striatum the most sensitive. Enzyme changes in frontal cortex were more persistent and there was an increase of MDA only in the acute group and euthanasia 3 hours after exposure, whereas in cerebellum, striatum and hippocampus, MDA increased only in acute group and immediately euthanasia after exposure. These results suggest a delayed response of the frontal cortex and a possible adaptation of tissues to xenobiotics. It is important to point out that euthanasia performed immediately after chronic exposure to cigarette smoke not only reflect the effect of the last exposure, since the pattern found in the modification of enzymes and in the determination of MDA was different from that observed after acute exposure.

Gesteigerter oxidativer Stress ist ein typisches Zeichen in der Pathogenese der Herzinsuffi-zienz. Anhand von 2 Rattenmodellen sollen charakteristische Veränderungen des enzymatischen antioxidativen Systems, repräsentiert durch die Glutathionperoxidase (GSH-Px) und Superoxiddismutase (SOD) sowie Änderungen im Hitzeschockproteinsystem (Hsp) untersucht werden, für das stellvertretend Hsp25 und Hsp72 bestimmt wurden. Daneben wurde die Lipidperoxidation durch die Konzentration von Thiobarbitursäure-reaktiven Substanzen (TBARS) quantifiziert. Im ersten Rattenmodell, der Hypertonie-induzierten Herzinsuffizienz durch permanente Renin-Angiotensin-System-Aktivierung in doppelt transgenen Ratten konnte eine linksventrikuläre Hypertrophie mit CK-Isoenzym-Shift mit vermehrter Expression von CK-MB und -BB beobachtet werden. Es kam in beiden Ventrikeln der transgenen Tiere zu einer Zunahme der Lipidperoxidation begleitet von einer Erniedrigung der Serum-Vitamin E-Konzentration durch Verbrauch. SOD und Hsp72 blieben unverändert. Durch die biventrikuläre Zunahme der GSH-Px-Aktivität bei linksventrikulär erhöhtem Hsp25-Gehalt konnte die Toleranz gegenüber Hypoxie/Reoxygenierungsstress erhöht werden. In der 2. Teilstudie - der Myokardinfarkt-induzierten Herzinsuffizienz - wurden die Parameter oxidativer Schädigung und antioxidativen Schutzes im Akutstadium (14-16h) und 3, 6 und 9 Wochen nach experimentellem Infarkt (MI) bestimmt. In der Akutphase zeigten sich in beiden Ventri-keln und im Papillarmuskel gesteigerte GSH-Px- und SOD-Aktivitäten, ohne dass kardiale Hypertrophie vorlag. Hsp72 und Hsp25 waren während der Akutphase nach MI im Papillar-muskel und im linken Ventrikel erhöht. In der Folge resultiert eine verbesserte kontraktile Funktion bei experimentellem Hypoxie/Reoxygenierungsstress. Damit gelang es erstmals eine Selbstprotektion des Myokards während eines akuten MI nachzuweisen. Erst ab der 6. Woche trat kardiale Hypertrophie auf, begleitet vom charakteristischen CK-Isoenzym-Shift. Während die SOD und die Hitzeschockproteine nach der akuten Phase auf das Niveau der Kontrollen absanken, blieb die hohe GSH-Px-Aktivität im linken Ventrikel über den gesamten Untersuchungszeitraum bestehen, bei zunehmender Toleranz des Herzens gegenüber Hypoxie/Reoxygenierungsstress. Die vorgelegten tierexperimentellen Untersuchungen zeigen, dass das Herz sowohl unter akut als auch unter chronisch gesteigertem oxidativen Stress Mechanismen zur Selbstprotektion aktivieren kann, die eine Prävention bzw. Minimierung von Schädigungsreaktionen durch Sauerstoffradikale ermöglichen. / Elevated oxidative stress is typical in the pathogenesis of heart failure. Characteristical changes in antioxidant enzyme status, represented by glutathionperoxidase (GSH-Px) and superoxide dismutase (SOD), and changes in heat shock protein status (Hsp), denoted by Hsp25 and Hsp72, have been revealed in two different rat-models. Lipidperoxidation was quantified by the concentration of thiobarbituric acid reactive substances (TBARS). In the first model of heart failure caused by permanent activation of renin-angiotensin system in double transgenic rat, leftventricular hypertrophy accompanied by a shift of creatine kinase (CK) isoenzyme pattern to higher concentration of fetale CK-MB an -BB-isoforms was found. Higher TBARS concentrations and lower alpha-tocopherol levels caused by consumption have been measured. SOD and Hsp72 remained unchanged. The tolerance against experimental hypoxia/reoxygenation was improved by higher levels of GSH-Px and Hsp25 in both right and left ventricular tissue. In the second study of heart failure caused by experimental myocardial infarction (MI) the parameters of oxidant demolishing and antioxidant defence were evaluated under acute condi-tions (14-16h) and 3, 6 and 9 weeks after infarction. In the acute period higher activities of GSH-Px and SOD in non-hypertrophied left and right ventricular tissue and papillary muscle have been reported. Leftventricular and papillary muscle Hsp25 and Hsp72 content showed higher levels 14-16 hours after MI compared with controls, improving the contractile function in hypoxia/reoxygenation experiments. These findings suggest for the first time a myocardial self-protection after acute myocardial infarction. 6 and 9 weeks after MI leftventricular hyper-trophy occurred attended by the characteristic CK-isoenzymeshift. While SOD-activity and Hsp-content decreased to the levels of the controls, GSH-Px remained on higher altitude in leftventricular tissue in all examined periods after MI. This was accompanied by better toler-ance against hypoxia/reoxygenation stress. These findings of experimental-induced heart failure show the activation of myocardial self protecting mechanisms under acute and chronic oxidative stress conditions, minimizing or even preventing demolishing reactions of oxygen radicals.

Sugarcane (Saccharum spp) is an important commercial crop cultivated widely in tropical and subtropical countries. Primarily sugarcane is used to produce sugar and recently it is proven to be a valuable resource for bioethanol, biodiesel, bioplastic and bioelectricity. Smut is one of the most serious sugarcane disease and occurs in sugarcane fields all over the world. The disease is caused by the biotrophic fungus Sporisorium scitamineum. The fungus induces metabolic changes in the plant leading to the production of a whip-like structure where fungal sporogenesis take place. The objective of this study was to analyse the reactive oxygen species (ROS) production, antioxidant enzymes activity and expression of genes associated with the ROS metabolism in smut susceptible (IAC66-6) and resistant sugarcane genotypes (SP80-3280). In addition, this work assessed the relationship between antioxidant enzymes and sensitivity of S. scitamineum to exogenous hydrogen peroxide (H2O2). This thesis is presented in the format of two chapters (chapters 2 and 3). In the second chapter, the results revealed that there were variations in the antioxidant system as well as in the ROS production in resistant sugarcane genotype, whereas few changes occurred in the susceptible genotype inoculated with S. scitamineum. Microscopic analysis revealed that S. scitamineum teliospore germination and appressorium formation were delayed during early infection in the smut resistant genotype, which coincided with H2O2 accumulation. In chapter 3, the results demonstrated that S. scitamineum is highly resistant to exogenous H2O2. At 2 mM exogenous H2O2 concentration the fungus presented an effective antioxidant system in response to the secondary products of oxidative stress. Furthermore, S. scitamineum when exposed for a long time at 2 mM exogenous H2O2 concentration it can acquire an adaptive response to H2O2. The results obtained in this study contributed to increase the understanding of this very complex interaction between sugarcane and S. scitamineum and it will be helpful toward understanding which aspects are involved in the resistance to S. scitamineum. These informations are important to create strategies for improving smut resistance in sugarcane. / Cana-de-açúcar (Saccharum spp) é uma importante cultura comercial amplamente cultivada em países tropicais e subtropicais. A cana-de-açúcar é principalmente utilizada para produzir açúcar e recentemente é considerada uma valiosa fonte para produção de bioetanol, biodiesel, bioplásticos e bioeletricidade. O carvão é uma das doenças mais graves da cana-de-açúcar e ocorrem em canaviais do mundo inteiro. A doença é causada pelo fungo biotrófico Sporisorium scitamineum. Este fungo induz mudanças metabólicas na planta, levando a formação de uma estrutura chamada chicote, onde ocorre a esporogênese. O objetivo desse estudo foi analisar a produção de espécies reativas de oxigênio (EROs), atividade de enzimas antioxidantes e a expressão de genes associados ao metabolismo de EROs em genótipos de cana-açúcar susceptível (IAC66-6) e resistente (SP80-3280). Além disso, este trabalho avaliou a relação entre as enzimas antioxidantes e sensibilidade de S. scitamineum a peróxido de hidrogênio (H2O2) exógeno. Esta tese está apresentada no formato de 2 capítulos (capítulos 2 e 3). No segundo capítulo, os resultados revelaram que ocorreram alterações no sistema antioxidante, bem como na produção de EROs no genótipo resistente, enquanto que poucas mudanças ocorreram no genótipo susceptível inoculado com S. scitamineum. Análises de microscopia revelaram que a germinação de teliósporos e a formação de apressórios de S. scitamineum atrasou durante o início da infeção no genótipo resistente ao carvão, coincidindo com o acúmulo de H2O2. No capítulo 3, os resultados demonstraram que S. scitamineum é altamente resistente a H2O2 exógeno. O fungo crescendo na concentração de 2 mM de H2O2 apresentou um eficiente sistema antioxidante em resposta a produtos secundários do estresse oxidativo. Além disso, quando S. scitamineum foi exposto a 2 mM de H2O2 exógeno, ele pode adquirir uma resposta adaptativa ao H2O2. Os resultados obtidos neste estudo contribuíram para aumentar o entendimento dessa complexa interação entre cana e S. scitamineum e será útil para a compreensão de quais aspectos estão envolvidos na resistência a este fungo. Estas informações são importantes para criar estratégias para o melhoramento de cana a essa doença.