L’inflammation joue un rôle clé dans de nombreuses pathologies, telles que les maladies inflammatoires chroniques, les désordres métaboliques et le cancer. L’un de ses médiateurs le plus puissant est l’interleukine-1β (IL-1β), qui est une cytokine pro-inflammatoire participant à tous les stades de l’inflammation et de l’immunité. Son activation est régulée par un complexe multi-protéique nommé inflammasome, dont la caspase-1 active en découlant est responsable du clivage et de la maturation de l’IL-1β. Huit types d’inflammasomes activant et clivant la pro-caspase-1 ont été identifiés et contiennent tous la protéine ASC (apoptosis-associated speck-like protein containing a CARD). Les inflammasomes partagent un signal intracellulaire commun et le mécanisme menant à leur assemblage et leur activation n’est pas totalement élucidé. L’utilisation de la lignée cellulaire humaine monocytaire, THP-1, différenciée en macrophages grâce à un ester de phorbol, le TPA, a permis la mise en place d’un modèle d’étude de modulateurs de l’inflammasome en conditions stériles. Ce modèle a permis l’étude des mécanismes impliqués suite à des signaux issus de l’inflammation chronique, tels que l’ATP et les espèces réactives de l’oxygène (ROS). Ce travail montre qu’il existe une synergie entre ATP et ROS, qui agissent grâce à une boucle d’activation impliquant probablement plusieurs inflammasomes, dont NLRP3. Des donneurs de NO connus (trinitrine et isosorbide dinitrate) ou nouveau (dérivé de purine) ont montré une activité anti-inflammatoire. D’autres composés ont été identifiés comme de potentiels inhibiteurs d’inflammasome (extraits de dattes et dérivé de purine portant un acide lipoïque) / Inflammation has a pivotal role in several disorders, such as chronic inflammatory diseases, metabolic disorders and cancer. Interleukin-1β (IL-1β) is one of the most powerful mediators in this mechanism. IL-1β is a pro-inflammatory cytokine which is implicated in every step of inflammation and immunity. IL-1β is regulated by a multi-protein complex named inflammasome, in which active caspase-1 is responsible for IL-1β processing and maturation. Eight inflammasomes processing and activating pro-caspase-1 has been identified and all contain the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Inflammasomes share a common intracellular signal and the mechanism for their structural assembly and activation is not totally clear. The human monocytic cell line, THP-1, has been used and cells have been differentiated in macrophages by the phorbol ester, TPA. Based on this, a cellular pattern has been established for studying inflammasome modulators under sterile conditions. This pattern allowed the study of implicated mechanisms in chronic inflammatory signals, such as ATP and reactive oxygen species (ROS). This study shows that ATP and ROS synergize for activating inflammasomes and NLRP3 and act in an autocrine loop. Stimulation with others natural or synthetic compounds has been realized for characterization of their inflammatory activity. Therapeutic NO donors (trinitrin and isosorbide dinitrate) and a new NO donor (purine nitrate ester) have been characterized for their anti-inflammatory activity. Others compounds from a new category of inflammasome inhibitors have been identified (date fruit extracts and lipoic acid purine derivative)

GONDIM, Franklin Aragão. Efeitos do peróxido de hidrogênio sobre a germinação e na aclimatação de plantas de milho à salinidade, Fortaleza - CE, 2008. 116 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2008. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-20T12:26:03Z No. of bitstreams: 1 2008_dis_fagondim.pdf: 2614140 bytes, checksum: 6acc0bd93625516278dbfcd0d8714f5d (MD5) / Approved for entry into archive by Nádja Goes (nmoraissoares@gmail.com) on 2016-05-20T12:38:05Z (GMT) No. of bitstreams: 1 2008_dis_fagondim.pdf: 2614140 bytes, checksum: 6acc0bd93625516278dbfcd0d8714f5d (MD5) / Made available in DSpace on 2016-05-20T12:38:05Z (GMT). No. of bitstreams: 1 2008_dis_fagondim.pdf: 2614140 bytes, checksum: 6acc0bd93625516278dbfcd0d8714f5d (MD5) Previous issue date: 2008 / The aim of this work was to evaluate the effects of the hydrogen peroxide (H2O2) on germination and acclimation of maize plants subject to the saline stress, in order to better understand the physiological and biochemical mechanisms involved. In the three experiments the triple hybrid of maize (Zea mays L) BRS 3003 was used. In the first experiment, the effects of H2O2 on germination of the maize seeds were evaluated; in the second, the effects of pre-treating by soaking maize seeds in H2O2 solution on the activities of antioxidative enzymes and isoenzymes; and as, the effects of the pre-treatment of maize seeds with H2O2 on acclimation of the plants to salinity and the possible mechanisms involved with this process. In the first experiment, which was carried out in a growth room, H2O2 accelerated the germination rate of maize seeds at 100 mM, but, not at 500 mM. In the second experiment, also carried out in growth room, it was observed that the pre-treatment of the seeds induced a pronounced increase in the activities of the enzymes ascorbate peroxidase (APX) and catalase (CAT), after 30 h of soaking in H2O2. It was also observed that the activity of the Guaiacol peroxidase (GPX) was smaller in the seeds soaked in H2O2 for 12, 24, 30, 36 and 42 h, in relation to those soaked in distilled water (control). However, H2O2 treatment for 48 h showed no significant differences as compared with control. The superoxide dismutase (SOD) activity was not affected by the pre-treatment of the seeds, except for the 24 h treatment. In the seeds, it was detected only one isoform of CAT and six of SOD. The pre-treatment of the seeds did not cause great changes in those isoforms, except for the intensity of the band of activity of CAT visualized in the polyacrylamide gel, which was very superior to that of the control, when the seeds were soaked by 36 and 48 h with H2O2. The increases in the activities of APX and, especially, of CAT, could be associated with the acceleration of the germination process. In the third experiment, which was carried out initially at growth room and, later, at the glasshouse, maize seeds were pre-treated for 36 h by soaking in solution of H2O2 100 mM or in distilled water. Those seeds were germinated on filter paper moistened with nutrient solution in the presence or absence of NaCl 80 mM, in a growth room. After six days, the seedling were transferred to the glasshouse and cultivated in trays containing only nutrient solution (control treatment) or nutrient solution with NaCl at 80 mM. Plants were harvest with 6, 11 and 16 days old. The results showed the pre-treatment of the seeds with H2O2 induced acclimation of the plants to the salinity. It decreased the deleterious effects of salt stress on the growth (biomass production and leaf area) of maize. This fact was associated with a higher efficiency of the antioxidative system of plants pre-treated with H2O2. CAT was the most important among the H2O2 scavenging enzymes in leaves, but, its activity was strongly reduced by salinity in plants 6 and 11 days old, however, this effect was totally reverted in the stressed plants originated from seeds pre-treated with H2O2. On the other hand, in the roots of plants submitted to saline stress, the activity of SOD was stimulated by the pre-treatment of the seeds with H2O2, in the three periods of harvest. In general, salinity reduced the photosynthetic parameters (stomatal conductance, net CO2 assimilation rate, transpiration and intracellular CO2 concentration) and the H2O2 pre-treatment of seeds was not capable to revert that effect. In terms of osmotic adjustment, the contents of organic solutes were not positively correlated to the process of acclimation to salt stress of the plants pre-treated with H2O2 to the salinity. However, the smallest values of the Na+/K+ ratio in roots and in leaves were found for the pre-treated plants submitted to salinity, when compared to those originated from of seeds pre-treated with water (control) and submitted to that same treatment, and it may also be a responsible factor for the acclimation of the maize plants to the salinity. / Este trabalho teve como objetivo avaliar os efeitos do peróxido de hidrogênio (H2O2) sobre a germinação e a aclimatação de plantas de milho ao estresse salino, estudando os mecanismos fisiológicos e bioquímicos envolvidos. Nos experimentos, em número de três, foi utilizado o híbrido triplo de milho (Zea mays L), o BRS 3003. No primeiro experimento, foram avaliados os efeitos do H2O2 na germinação das sementes de milho; no segundo, foram avaliados os efeitos do pré-tratamento de embebição das sementes de milho com H2O2 nas atividades das enzimas e isoenzimas antioxidativas e, no terceiro, foram avaliados os efeitos do pré-tratamento de sementes de milho com H2O2 sobre a aclimatação das plantas de milho à salinidade e os mecanismos possivelmente envolvidos. No primeiro experimento, o qual foi realizado em sala da germinação, observou-se que o H2O2 na concentração de 100 mM acelerou o processo de germinação das sementes de milho, o mesmo não ocorrendo na concentração de 500 mM. No segundo experimento, o qual também foi realizado em sala de germinação, observou-se que o pré-tratamento das sementes induziu forte aumento nas atividades das enzimas peroxidase do ascorbato (APX) e catalase (CAT), desde o tempo de embebição de 30 h das sementes com H2O2. Já com relação à peroxidase do guaiacol (GPX), observou-se que a atividade dessa enzima foi menor nas sementes embebidas com H2O2 nos tempos de 12, 24, 30, 36 e 42 h, em relação àquelas embebidas em água destilada (controle), porém, nas pré-tratadas por um tempo de 48 h não foram observadas diferenças significativas entre os tratamentos. A dismutase do superóxido (SOD), por sua vez, não foi afetada pelo pré-tratamento das sementes, exceto no tratamento de embebição das sementes com H2O2 por 24 h. Nas sementes, foi detectada apenas uma isoenzima de CAT e seis de SOD. O pré-tratamento das sementes não provocou alterações nessas isoformas, exceto com relação à intensidade da banda de atividade da CAT visualizada no gel de poliacrilamida, que se mostrou muito superior àquela do controle, quando as sementes foram embebidas por 36 e 48 h com H2O2. É possível que os aumentos nas atividades da APX e, especialmente, da CAT, tenham sido responsáveis pela aceleração do processo de germinação. No terceiro experimento, o qual foi conduzido inicialmente em Sala de germinação e, em seguida, em casa de vegetação, foram utilizadas sementes de milho prétratadas por 36 h de embebição em solução de H2O2 a 100 mM ou em água destilada. Essas sementes foram postas para germinar em folhas de papel de filtro umedecidas com solução nutritiva em presença ou ausência de NaCl a 80 mM em sala de germinação. Decorridos seis dias, as plântulas foram transferidas para a casa de vegetação e cultivadas hidroponicamente em presença ou ausência de NaCl a 80 mM, sendo feitas coletas das plantas aos 6, 11 e 16 dias de idade. Como resultado, observou-se que o pré-tratamento das sementes com H2O2 induziu a aclimatação das plantas à salinidade, reduzindo parcialmente os efeitos deletérios da salinidade na produção de matéria e na área foliar. Esse resultado pode ser atribuído, pelo menos em parte, a uma maior eficiência do sistema antioxidativo das plantas oriundas de sementes pré-tratadas com H2O2. A CAT, que se mostrou a principal enzima eliminadora de H2O2, teve sua atividade nas folhas fortemente reduzida pela salinidade, nas plantas com seis dias de idade, sendo este efeito totalmente revertido nas plantas provenientes de sementes pré-tratadas com H2O2. Por outro lado, nas raízes das plantas submetidas ao estresse salino, a atividade da SOD foi estimulada pelo pré-tratamento das sementes com H2O2, nos três tempos de coleta. De modo geral, a salinidade reduziu os parâmetros fotossintéticos (condutância estomática, transpiração, fotossíntese e concentração interna de CO2) e o pré-tratamento das sementes não foi capaz de reverter esse efeito. Não foi possível estabelecer-se uma correlação precisa entre os teores de solutos orgânicos e o processo de aclimatação das plantas pré-tratadas com H2O2 à salinidade, em termos de ajustamento osmótico. No entanto, os menores valores da razão Na+/K+ nas raízes e, especialmente, nas folhas das plantas pré-tratadas e submetidas à salinidade, em relação àquelas oriundas de sementes pré-tratadas com água (controle) e submetidas a esse mesmo tratamento, aos 16 dias de idade, pode também ter sido um fator responsável, pelo menos em parte, pela aclimatação das plantas de milho à salinidade.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Este trabalho teve como objetivo avaliar os efeitos do prÃ-tratamento de pulverizaÃÃo foliar das plantas de milho com perÃxido de hidrogÃnio (H2O2) sobre a aclimataÃÃo ao estresse salino, estudando os mecanismos fisiolÃgicos e bioquÃmicos envolvidos. A presente tese foi dividida em trÃs experimentos independentes que resultaram na produÃÃo de trÃs capÃtulos, cada um correspondendo a um artigo cientÃfico. Os experimentos foram conduzidos em casa de vegetaÃÃo, sob condiÃÃes hidropÃnicas, utilizando o hÃbrido triplo de milho (Zea mays L), BRS 3003. Oito dias apÃs a semeadura, as plÃntulas foram pulverizadas com Ãgua destilada (controle) ou soluÃÃo aquosa de H2O2 na concentraÃÃo de 10 mM e, 48 h apÃs o inÃcio da pulverizaÃÃo, foram submetidas ao tratamento com NaCl a 80 mM. No primeiro trabalho, foram estudados os efeitos da aplicaÃÃo foliar de H2O2 no crescimento e nos teores de solutos orgÃnicos e inorgÃnicos de plantas de milho crescendo sob condiÃÃes salinas. Verificou-se que o prÃ-tratamento de pulverizaÃÃo das plantas de milho com H2O2 induziu aclimataÃÃo das plantas de milho ao estresse salino, revertendo parcialmente os efeitos deletÃrios da salinidade no crescimento. Este efeito foi atribuÃdo, pelo menos em parte, a um maior acÃmulo de proteÃnas solÃveis, carboidratos solÃveis e NO3-, bem como a um menor acÃmulo de Ãons tÃxicos (Na+ e Cl-) nas folhas. O segundo trabalho avaliou os efeitos da aplicaÃÃo foliar de H2O2 no crescimento, na atividade das enzimas antioxidativas, na peroxidaÃÃo dos lipÃdios (teores de malondialdeÃdo-MDA) e na expressÃo da enzima catalase (CAT) em plantas de milho sob condiÃÃes de estresse salino. Constatou-se que a salinidade reduziu o crescimento das plantas e que a aplicaÃÃo foliar de H2O2 minimizou este efeito. Observou-se tambÃm que as enzimas antioxidativas estudadas (catalase, peroxidase do guaiacol, perdoxidase do ascorbato e dismutase do superÃxido) tiveram suas atividades aumentadas pela aplicaÃÃo foliar de H2O2. A CAT se mostrou a principal enzima responsiva ao H2O2 e seu aumento de atividade parace estar relacionado à regulaÃÃo da expressÃo gÃnica. Sob condiÃÃes salinas, a menor peroxidaÃÃo de lipÃdios foi encontrada nas plantas que apresentaram maiores atividades da CAT. De modo geral, concluiu-se que a pulverizaÃÃo foliar das plantas de milho com H2O2 foi capaz de reduzir os efeitos deletÃrios da salinidade no crescimento das plantas e na peroxidaÃÃo dos lipÃdios. Essas respostas podem ser atribuÃdas, pelo menos em parte, à capacidade do H2O2 de induzir aumento na atividade e/ou expressÃo das enzimas antioxidativas, especialmente a CAT. O terceiro trabalho analisou os efeitos da aplicaÃÃo foliar de H2O2 na Ãrea foliar, nos teores relativos de clorofila, nos teores relativos de Ãgua, nas trocas gasosas e nos teores de H2O2, ascorbato e glutationa de plantas de milho crescendo sob condiÃÃes salinas. De modo geral, a salinidade reduziu a Ãrea foliar, os teores relativos de clorofila e os teores relativos de Ãgua e a pulverizaÃÃo foliar com H2O2 foi eficaz em minimizar esse efeito. A salinidade reduziu os parÃmetros fotossintÃticos (condutÃncia estomÃtica, transpiraÃÃo, fotossÃntese e concentraÃÃo interna de CO2) e o prÃ-tratamento de pulverizaÃÃo das plantas com H2O2 foi capaz de reverter parcialmente esse efeito. Os teores de H2O2 foram aumentados pela salinidade tanto nas folhas como nas raÃzes e a pulverizaÃÃo foliar com H2O2 mostrou-se eficiente em reduzir este efeito, sem, contudo, alterar o estado redox dos antioxidantes analisados (ascorbato e glutationa). / This study evaluated the effects of H2O2 leaf spraying pretreatment on the maize plant acclimation to salt stress, studying the physiological and biochemical mechanisms involved. The present thesis was divided into three independent experiments that resulted in three chapters, each one corresponding to a scientific article. The experiments were conducted under hydroponic conditions and maintained in greenhouse, the plant model used was triple hybrid of maize (Zea mays L.), BRS 3003. Eight days after sowing (DAS), the seedlings were sprayed with 10 mM H2O2 solution or distilled water (as a control). Forty-eight hours after the spraying beginning, the seedlings were subjected to treatment with NaCl at 80 mM. In the first study, we analyzed the effects of H2O2 leaf spraying pretreatment on the growth and on the levels of organic and inorganic solutes in maize plants under salt stress. It was observed that H2O2 leaf spraying pretreatment promoted plant acclimation to salt stress, reducing the deleterious effects of salinity on the maize growth. This effect can be attributed, at least partially, to a great production of proteins, and soluble carbohydrates and NO3- as well as lower levels of Cl- and Na+ in leaves. The second study evaluated the effects of H2O2 leaf spraying pretreatment on growth, antioxidative enzymes activity, lipid peroxidation (levels of malondialdehyde - MDA) and on the catalase expression (CAT) in maize plants under salt stress. It was observed that salinity reduced maize seedling growth when compared to control conditions, and H2O2 foliar spraying was effective in minimizing this effect. Analysis of the antioxidative enzymes (catalase, guaiacol peroxidase, ascorbate peroxidase and superoxide dismutase) revealed that H2O2 leaf spraying increased antioxidant enzyme activities. CAT was the most responsive of these enzymes to H2O2, with higher activity since the beginning of the treatment (48 h), while guaiacol peroxidase and ascorbate peroxidase were responsive only at later stages (240 h) of treatment. Increased CAT activity appears linked to gene expression regulation. Lower MDA levels were detected in plants with higher CAT activity, which may result from the protective function of this enzyme. Overall, we can conclude that pretreatment with H2O2 leaf spraying was able to reduce the deleterious salinity effects on seedling growth and lipid peroxidation. These responses could be attributed to the H2O2 ability to induce antioxidant defenses, especially CAT activity. The third study evaluated the effects of H2O2 leaf spraying pretreatment on leaf area, relative chlorophyll content, relative water content, gas exchange and on the H2O2, ascorbate and glutathione contents in salt-stressed maize plants. In general, the salinity reduced leaf area, relative chlorophyll content and relative water content of the maize plants in comparison to the plants that grew under control conditions, moreover H2O2 leaf spraying was effective in minimize this effect. The salt treatment reduced photosynthetic parameters and, the H2O2 leaf spray was able to partially reverse this effect. The H2O2 content was increased by salinity in both, leaves and roots, and H2O2 leaf spray was effective in reducing this negative effect. The H2O2 foliar application did not alter the redox state of the antioxidants studied (ascorbate and glutathione).

AssociaÃÃo TÃcnico-CientÃfica Eng. Paulo de Frontin / nÃo hà / Este trabalho teve como objetivo avaliar os efeitos do perÃxido de hidrogÃnio (H2O2) sobre a germinaÃÃo e a aclimataÃÃo de plantas de milho ao estresse salino, estudando os mecanismos fisiolÃgicos e bioquÃmicos envolvidos. Nos experimentos, em nÃmero de trÃs, foi utilizado o hÃbrido triplo de milho (Zea mays L), o BRS 3003. No primeiro experimento, foram avaliados os efeitos do H2O2 na germinaÃÃo das sementes de milho; no segundo, foram avaliados os efeitos do prÃ-tratamento de embebiÃÃo das sementes de milho com H2O2 nas atividades das enzimas e isoenzimas antioxidativas e, no terceiro, foram avaliados os efeitos do prÃ-tratamento de sementes de milho com H2O2 sobre a aclimataÃÃo das plantas de milho à salinidade e os mecanismos possivelmente envolvidos. No primeiro experimento, o qual foi realizado em sala da germinaÃÃo, observou-se que o H2O2 na concentraÃÃo de 100 mM acelerou o processo de germinaÃÃo das sementes de milho, o mesmo nÃo ocorrendo na concentraÃÃo de 500 mM. No segundo experimento, o qual tambÃm foi realizado em sala de germinaÃÃo, observou-se que o prÃ-tratamento das sementes induziu forte aumento nas atividades das enzimas peroxidase do ascorbato (APX) e catalase (CAT), desde o tempo de embebiÃÃo de 30 h das sementes com H2O2. Jà com relaÃÃo à peroxidase do guaiacol (GPX), observou-se que a atividade dessa enzima foi menor nas sementes embebidas com H2O2 nos tempos de 12, 24, 30, 36 e 42 h, em relaÃÃo Ãquelas embebidas em Ãgua destilada (controle), porÃm, nas prÃ-tratadas por um tempo de 48 h nÃo foram observadas diferenÃas significativas entre os tratamentos. A dismutase do superÃxido (SOD), por sua vez, nÃo foi afetada pelo prÃ-tratamento das sementes, exceto no tratamento de embebiÃÃo das sementes com H2O2 por 24 h. Nas sementes, foi detectada apenas uma isoenzima de CAT e seis de SOD. O prÃ-tratamento das sementes nÃo provocou alteraÃÃes nessas isoformas, exceto com relaÃÃo à intensidade da banda de atividade da CAT visualizada no gel de poliacrilamida, que se mostrou muito superior Ãquela do controle, quando as sementes foram embebidas por 36 e 48 h com H2O2. à possÃvel que os aumentos nas atividades da APX e, especialmente, da CAT, tenham sido responsÃveis pela aceleraÃÃo do processo de germinaÃÃo. No terceiro experimento, o qual foi conduzido inicialmente em Sala de germinaÃÃo e, em seguida, em casa de vegetaÃÃo, foram utilizadas sementes de milho prÃtratadas por 36 h de embebiÃÃo em soluÃÃo de H2O2 a 100 mM ou em Ãgua destilada. Essas sementes foram postas para germinar em folhas de papel de filtro umedecidas com soluÃÃo nutritiva em presenÃa ou ausÃncia de NaCl a 80 mM em sala de germinaÃÃo. Decorridos seis dias, as plÃntulas foram transferidas para a casa de vegetaÃÃo e cultivadas hidroponicamente em presenÃa ou ausÃncia de NaCl a 80 mM, sendo feitas coletas das plantas aos 6, 11 e 16 dias de idade. Como resultado, observou-se que o prÃ-tratamento das sementes com H2O2 induziu a aclimataÃÃo das plantas à salinidade, reduzindo parcialmente os efeitos deletÃrios da salinidade na produÃÃo de matÃria e na Ãrea foliar. Esse resultado pode ser atribuÃdo, pelo menos em parte, a uma maior eficiÃncia do sistema antioxidativo das plantas oriundas de sementes prÃ-tratadas com H2O2. A CAT, que se mostrou a principal enzima eliminadora de H2O2, teve sua atividade nas folhas fortemente reduzida pela salinidade, nas plantas com seis dias de idade, sendo este efeito totalmente revertido nas plantas provenientes de sementes prÃ-tratadas com H2O2. Por outro lado, nas raÃzes das plantas submetidas ao estresse salino, a atividade da SOD foi estimulada pelo prÃ-tratamento das sementes com H2O2, nos trÃs tempos de coleta. De modo geral, a salinidade reduziu os parÃmetros fotossintÃticos (condutÃncia estomÃtica, transpiraÃÃo, fotossÃntese e concentraÃÃo interna de CO2) e o prÃ-tratamento das sementes nÃo foi capaz de reverter esse efeito. NÃo foi possÃvel estabelecer-se uma correlaÃÃo precisa entre os teores de solutos orgÃnicos e o processo de aclimataÃÃo das plantas prÃ-tratadas com H2O2 à salinidade, em termos de ajustamento osmÃtico. No entanto, os menores valores da razÃo Na+/K+ nas raÃzes e, especialmente, nas folhas das plantas prÃ-tratadas e submetidas à salinidade, em relaÃÃo Ãquelas oriundas de sementes prÃ-tratadas com Ãgua (controle) e submetidas a esse mesmo tratamento, aos 16 dias de idade, pode tambÃm ter sido um fator responsÃvel, pelo menos em parte, pela aclimataÃÃo das plantas de milho à salinidade. / The aim of this work was to evaluate the effects of the hydrogen peroxide (H2O2) on germination and acclimation of maize plants subject to the saline stress, in order to better understand the physiological and biochemical mechanisms involved. In the three experiments the triple hybrid of maize (Zea mays L) BRS 3003 was used. In the first experiment, the effects of H2O2 on germination of the maize seeds were evaluated; in the second, the effects of pre-treating by soaking maize seeds in H2O2 solution on the activities of antioxidative enzymes and isoenzymes; and as, the effects of the pre-treatment of maize seeds with H2O2 on acclimation of the plants to salinity and the possible mechanisms involved with this process. In the first experiment, which was carried out in a growth room, H2O2 accelerated the germination rate of maize seeds at 100 mM, but, not at 500 mM. In the second experiment, also carried out in growth room, it was observed that the pre-treatment of the seeds induced a pronounced increase in the activities of the enzymes ascorbate peroxidase (APX) and catalase (CAT), after 30 h of soaking in H2O2. It was also observed that the activity of the Guaiacol peroxidase (GPX) was smaller in the seeds soaked in H2O2 for 12, 24, 30, 36 and 42 h, in relation to those soaked in distilled water (control). However, H2O2 treatment for 48 h showed no significant differences as compared with control. The superoxide dismutase (SOD) activity was not affected by the pre-treatment of the seeds, except for the 24 h treatment. In the seeds, it was detected only one isoform of CAT and six of SOD. The pre-treatment of the seeds did not cause great changes in those isoforms, except for the intensity of the band of activity of CAT visualized in the polyacrylamide gel, which was very superior to that of the control, when the seeds were soaked by 36 and 48 h with H2O2. The increases in the activities of APX and, especially, of CAT, could be associated with the acceleration of the germination process. In the third experiment, which was carried out initially at growth room and, later, at the glasshouse, maize seeds were pre-treated for 36 h by soaking in solution of H2O2 100 mM or in distilled water. Those seeds were germinated on filter paper moistened with nutrient solution in the presence or absence of NaCl 80 mM, in a growth room. After six days, the seedling were transferred to the glasshouse and cultivated in trays containing only nutrient solution (control treatment) or nutrient solution with NaCl at 80 mM. Plants were harvest with 6, 11 and 16 days old. The results showed the pre-treatment of the seeds with H2O2 induced acclimation of the plants to the salinity. It decreased the deleterious effects of salt stress on the growth (biomass production and leaf area) of maize. This fact was associated with a higher efficiency of the antioxidative system of plants pre-treated with H2O2. CAT was the most important among the H2O2 scavenging enzymes in leaves, but, its activity was strongly reduced by salinity in plants 6 and 11 days old, however, this effect was totally reverted in the stressed plants originated from seeds pre-treated with H2O2. On the other hand, in the roots of plants submitted to saline stress, the activity of SOD was stimulated by the pre-treatment of the seeds with H2O2, in the three periods of harvest. In general, salinity reduced the photosynthetic parameters (stomatal conductance, net CO2 assimilation rate, transpiration and intracellular CO2 concentration) and the H2O2 pre-treatment of seeds was not capable to revert that effect. In terms of osmotic adjustment, the contents of organic solutes were not positively correlated to the process of acclimation to salt stress of the plants pre-treated with H2O2 to the salinity. However, the smallest values of the Na+/K+ ratio in roots and in leaves were found for the pre-treated plants submitted to salinity, when compared to those originated from of seeds pre-treated with water (control) and submitted to that same treatment, and it may also be a responsible factor for the acclimation of the maize plants to the salinity.

A racionalização dos recursos hídricos tem sido uma das metas das indústrias em vários setores. Tais metas exigem inovações tecnológicas tanto para novos processos produtivos quanto para novas técnicas de tratamento e reutilização de água na cadeia de produção. Os custos elevados de água industrial no Brasil, particularmente nas regiões metropolitanas, têm estimulado as indústrias nacionais a avaliar as possibilidades de reúso. O objetivo deste trabalho é a aplicação do tratamento de águas de processo contendo polipropileno utilizando radiação ultravioleta e peróxido de hidrogênio, isto é, o sistema UV/H2O2, visando adequá-las para reúso no próprio processo, reduzindo a necessidade de captação de água pré-tratada e de descarte de efluente. A primeira parte do estudo consistiu na realização de experimentos em um sistema fotoquímico de batelada, empregando quatro diferentes correntes efluentes de processo, para a avaliação da viabilidade técnico-econômica do tratamento fotoquímico, bem como para a obtenção de dados referentes à cinética das reações fotoquímicas. Com base nas informações obtidas, na segunda parte do estudo foram realizados experimentos em um sistema fotoquímico contínuo, a fim de obter dados para o aumento de escala para aplicação industrial do processo de tratamento contínuo. Os resultados experimentais indicaram a viabilidade técnica de aplicação do sistema UV/H2O2 utilizando fonte de luz artificial para todas as correntes de processo estudadas, tendo sido alcançados níveis de remoção de matéria orgânica acima de 90%. No entanto, sob o ponto de vista econômico, apenas as correntes com baixo teor de carbono orgânico total dissolvido (COT), entre 6 e 12 mgC L-1, mostraram-se adequadas ao reúso, após o tratamento. / Rationalization of water use has been one of the goals in many industrial activities, and, in particular, in the petrochemical industry. Such goals demand technological innovations in the productive processes and in techniques for treatment and reuse of water in the production chain. The high costs of industrial water, particularly in some metropolitan regions, have stimulated the industries to evaluate the possibilities of water reuse. The objective of this work is to evaluate the feasibility of the UV/H2O2 photochemical process applied to the treatment of process waste water containing polypropylene, aiming at the reuse of the waste water in the as process water in the industrial complex, thus reducing the need for tap water supply and waste water generation rate. The first part of this study consisted of laboratory-scale experiments in a batch photochemical reactor with four different waste water streams to perform the technical and economical feasibility of the photochemical treatment, as well to obtain data on the degradation rate. Based on the results of the first part, the second part of this study consisted of experiments in a continuous photochemical reactor, aimed at obtaining experimental data for reactor scale-up. Experimental results indicate that the UV/H2O2 photodegradation process is able to remove more than 90% of the organic compounds contained in the waste water. However, only waste waters containing relatively low contaminant levels (between 6 and 12 mgC L-1) can be treated at economically favourable costs.

Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Le but de ce travail consistait à étudier les effets d'une activité physique modérée aiguë ou chronique sur le fonctionnement mitochondrial musculaire et hépatique. Nous avons étudié en parallèle la respiration mitochondriale et la production de ROS estimée par la mesure de l'H2O2 en présence de la sonde Amplex Red. Nous avons montré que l'exercice était à l'origine d'une augmentation de la production radicalaire et cela de façon persistante après 2h de récupération dans le muscle mais qui était décalée dans le foie. Cette production apparaît comme tolérée par la mitochondrie dont le statut antioxydant n'est pas affecté. Des mesures en présence de substrats et inhibiteurs spécifiques de la chaîne respiratoire mitochondriale, permettant d'isoler fonctionnellement les complexes de l'oxydation phosphorylante, nous ont permis de révéler des adaptations spécifiques des tissus. La deuxième partie du travail concernait l'étude de l'activité physique chronique modérée qui induit une augmentation de densité mitochondriale à la fois dans le muscle et le foie, associée à des adaptations fonctionnelles. La mitochondrie musculaire semble en effet plus efficace pour extraire des équivalents réduits en provenance des acides gras à travers un processus de slipping métabolique. La mitochondrie hépatique présente une amélioration de rendement d'oxydation en état phosphorylant à partir de substrats du complexe I. Les résultats suggèrent un fonctionnement diminué des mitochondries en réponse à l'exercice compensé par une augmentation du contenu en unités fonctionnelles. Les sites de production de ROS à l'exercice apparaissent tissu spécifique. En effet on observe que l'exercice modifie la production d'H2O2 à partir du complexe III dans le muscle et du I dans le foie. L'utilisation d'oligonucléotides antisens de PGC-1α, destinée à réprimer l'expression protéique de PGC-1α, n'a pas d'influence sur l'augmentation de densité mitochondriale induite par l'entraînement au niveau musculaire mais l'inhibe complètement dans le foie. Des adaptations fonctionnelles liées à l'absence de cette protéine régulatrice, passant par une perturbation de la respiration et de la production de ROS semblent confirmer un rôle majeur de PGC-1α dans les adaptations mitochondriales tissus spécifiques à l'exercice. Ces résultats suggèrent que les ROS pourraient contribuer par rétrocontrôle sur PGC-1α aux adaptations énergétiques induites par l'activité physique.

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence of mortality (i.e., catastrophic failure of liquid cultures). These defects were eliminated when Prochlorococcus was grown in co-culture with a phylogenetically diverse array of helper bacteria. The primary mechanism of helping was enzymatic removal of hydrogen peroxide (HOOH) from the culture medium. Axenic Prochlorococcus cultures were profoundly sensitive to HOOH additions in comparison with reported tolerance levels for all other wild-type aerobic bacteria, but in co-culture their resistance was similar to that of the helpers. Neither is dependence on helpers limited to the laboratory. Sterile-filtered seawater exposed to sunlight accumulated enough HOOH in 24h to kill ecologically relevant cell concentrations of Prochlorococcus. We also refined a method for delivering HOOH at a defined, steady rate using the buffer HEPES to more accurately simulate the steady accumulation of HOOH in natural waters. Even at the lowest production rates that could sustain the in situ HOOH concentration in the ocean, HEPES-generated HOOH was lethal to Prochlorococcus; again, co-culture with helpers prevented this effect. We speculate on the ecological consequences of Prochlorococcus’ dependency on other organisms for survival, as well as the evolutionary forces that have led to this lack of self-sufficiency.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. The specific aim of this project was to study if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells and to do this with dose and time responses in focus. In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 in five different concentrations, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that regardless of dose or time, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. This is a follow-up to an earlier study, and the specific aim of this project was to find out if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells during long-term treatment (up to 48 hours). In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 of the concentration 10-8 M, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that despite of the rather high dose used, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP, regardless of time.