FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O presente trabalho teve como objetivo estudar o efeito do armazenamento dos frutos de seis clones de aceroleira (BRS 152, BRS 235, BRS 236, BRS 237, BRS 238 e II47/1) à temperatura abaixo da temperatura mÃnima de seguranÃa (TMS), analisando os efeitos na qualidade dos frutos, no conteÃdo de compostos bioativos e na atividade enzimÃtica. Os frutos foram colhidos no inÃcio do estÃdio de maturaÃÃo, no campo experimental da Embrapa AgroindÃstria Tropical, na cidade de Pacajus, sendo acondicionados em bandejas de plÃstico envolvidas com filme de PVC, as quais foram armazenadas em cÃmara fria Ãs temperaturas de 4 e 8 ÂC por atà 12 dias. A cada trÃs dias foram retiradas amostras (bandejas contendo cerca de 180 g de frutos) para realizaÃÃo das anÃlises, sendo os frutos, em seguida, submetidos a um processamento em multiprocessador com peneira de 1 mm para obtenÃÃo da polpa. O delineamento experimental foi o inteiramente casualizado, em esquema fatorial 6 x 5, com trÃs repetiÃÃes, sendo o primeiro fator os seis clones de aceroleira e o segundo os tempos de armazenamento dos frutos (0, 3, 6, 9 e 12 dias). Os dados foram submetidos à anÃlise de variÃncia e, quando a interaÃÃo entre os dois fatores foi significativa, eles foram submetidos à anÃlise de regressÃo; quando nÃo houve interaÃÃo significativa foi realizado o teste de Tukey a 5%. Para avaliar a qualidade dos frutos, foram analisadas as caracterÃsticas fÃsicas (firmeza, perda de massa e cor) e fÃsico-quÃmicas e quÃmicas (pH, sÃlidos solÃveis, acidez titulÃvel e aÃÃcares solÃveis totais). Como resultado, foi observado que tais caracterÃsticas dos frutos acondicionados a 4 e 8 ÂC foram praticamente as mesmas, exceto com relaÃÃo à perda de massa que foi 31% menor a 4 ÂC. Portanto, de modo geral, o armazenamento dos frutos a 4 ÂC nÃo comprometeu os parÃmetros de qualidade dos frutos, quando comparados com aqueles armazenados na TMS para a acerola (8 ÂC). Com relaÃÃo ao conteÃdo dos compostos bioativos (Ãcido ascÃrbico, carotenoides e antocianinas) dos frutos armazenados a 4 ÂC, de modo geral, ele nÃo diferiu muito daquele dos frutos armazenados na temperatura de 8 ÂC. Houve uma tendÃncia de queda nos teores de Ãcido ascÃrbico durante o armazenamento nas duas temperaturas, resultado tambÃm observado para os teores de antocianinas a 8 ÂC. Por outro lado, os de carotenoides apresentaram tendÃncia de aumento à 4 ÂC, mas nÃo variaram com o tempo de armazenamento a 8 ÂC. O clone II47/1 foi o que apresentou frutos com maiores teores de Ãcido ascÃrbico, carotenoides e antocianinas nas duas temperaturas de armazenamento. De modo geral, as enzimas oxidativas de fenÃis (polifenoloxidase e peroxidase do guaiacol) dos frutos dos clones armazenados a 4 e 8 ÂC apresentaram comportamentos semelhantes durante o acondicionamento refrigerado. O estudo com as enzimas antioxidativas (dismutase do superÃxido, catalase e peroxidase do ascorbato) revelou que os frutos armazenados a 4 ÂC apresentaram, respectivamente, valores de atividade da dismutase do superÃxido e da catalase 263% e 37%, em mÃdia, maiores que aqueles armazenados na TMS para a acerola (8 ÂC). Tais resultados sugerem que a 4 ÂC os frutos estÃo sob estresse oxidativo. A catalase foi a principal enzima removedora de perÃxido de hidrogÃnio nos frutos, tendo sua atividade intensificada apÃs o 6 dia de armazenamento. Os resultados sugerem que os clones II47/1 e o BRS 152 sÃo resistentes as condiÃÃes de armazenamento nas temperaturas 4 e 8 ÂC. / The present work aimed to study the effect of fruit storage of six clones of acerola (BRS 152 BRS 235 BRS 236 BRS 237 BRS 238 and II47/1) at temperature below the temperature minimum security (TMS), analyzing the effects on fruit quality, the content of bioactive compounds and enzyme activity. The fruits were harvested at the beginning of ripening stage on the experimental field of Embrapa Tropical, in the city of Pacajus being packed in plastic trays involved with PVC film, which were stored in cold storage at temperatures of 4 ÂC and 8 ÂC for up to 12 days. Every three days samples were withdrawn (tray containing about 180 g of fruit) for carrying out analyzes, and the fruits are then subjected to a processing multiprocessor 1 mm screen to obtain the pulp. The experimental design was completely randomized in factorial scheme 6 x 5 with three replications, with the first factor corresponding to six clones of acerola and the second one corresponding to the five times of fruit storage (0, 3, 6, 9 and 12 days). The data were subjected to analysis of variance and, when the interaction between the two factors was significant, they were subjected to regression analysis; when there was no significant interaction, the means were compared by Tukeyâs test at 5% of probability. To assess the quality of fruits, it was analyzed their physical (strength, weight loss and color) and physico-chemical and chemical characteristics (pH, soluble solids, titratable acidity and total soluble sugars). As a result, it was observed that the characteristics of the fruit stored at 4 ÂC to 8 ÂC were practically the same, except with respect to mass loss, which was 31% lower at 4 ÂC. Therefore, in general, the fruit storage at 4 ÂC did not affect the quality parameters of fruits, compared to those stored in the TMS to the acerola (8 ÂC). Regarding the content of bioactive compounds (ascorbic acid, carotenoids and anthocyanins), the fruit stored at 4 ÂC, generally did not differ from that of the fruit stored at 8 ÂC. There was a trend towards a decrease in the ascorbic acid content during storage at both temperatures, a result also observed for the anthocyanins to 8 ÂC. On the other hand, the carotenoid content increased at 4 ÂC and did not change with time of storage at 8 ÂC. The fruits of clone II47/1 had the higher contents of ascorbic acid, carotenoids and anthocyanins in the two storage temperatures. In general the phenol oxidizing enzymes (polyphenol oxidase and guaiacol peroxidase) of fruit clones stored at 4 and 8 ÂC had similar behavior during cold storage, and the fruits of clones II47/1 and BRS 152 had the higher enzyme activity during storage at both temperatures The study about the antioxidant enzymes (superoxide dismutase catalase and ascorbate peroxidase) revealed that fruits stored at 4 ÂC showed respectively activity values of superoxide dismutase and catalase 263% and 37% on average higher than those stored at TMS for acerola (8 ÂC) These results suggest that the fruits storaged at 4 ÂC are under oxidative stress Catalase was the main hydrogen peroxide removing enzyme in fruit which activity increased after 6 days of storage The results suggest that clones II47 / 1 and BRS 152 are resistant storage conditions at temperatures 4 and 8 ÂC

Adipositas und das polyzystische Ovarsyndrom (PCOS) sind häufig Gründe für Anovulation, Infertilität und unerfüllten Kinderwunsch. Sowohl Adipositas als auch das PCOS können zu einem Ungleichgewicht zwischen Anti- und Prooxidanzien im menschlichen Körper führen. Durch Übergewicht der Prooxidanzien ensteht oxidativer Stress. Reaktive Sauerstoffspezies (reactive oxygen species, ROS) fallen vermehrt an und oxidieren Lipoproteine zu „oxidized low density lipoproteins“ (oxLDL). Durch Bindung von oxLDL an den „lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1)“ wird Apoptose und Autophagie induziert. Wir vermuten, dass sich diese Prozesse auch in der Follikelflüssigkeit (FF), dem Milieu der Eizelle, abspielen und zum Absterben reifender Follikel und somit zur Anovulation und Infertilität führen. Das Ziel dieser Arbeit war es, zu untersuchen, welchen Einfluss Adipositas, hormonelle Stimulation und PCOS auf die enzymatischen Antioxidanzien Superoxiddismutase (SOD), Katalase, Glutathionperoxidase (GPx) und Glutathionreduktase (GR) sowie auf den oxLDL-Spiegel haben. Es wurden Serum und FF von Frauen unter IVF (in vitro Fertilisation) -Therapie untersucht, die anhand ihres Body Mass Index (BMI), des Taille-Hüft-Quotienten (T/H-Quotient) sowie des PCOS in vier Gruppen eingeteilt wurden. Die Konzentration an oxLDL als Repräsentant des oxidativen Systems und die Aktivität der Enzyme SOD, Katalase, GPx und GR, Repräsentaten des antioxidativen Systems, wurden im Serum vor Stimulationsbeginn und zum Zeitpunkt der Follikelpunktion sowie in der FF gemessen. Adipöse Frauen mit und ohne PCOS hatten höhere Konzentrationen an oxLDL in der FF als normalgewichtige. Die oxLDL-Konzentrationen der FF waren 1000-fach niedriger als die der Seren. Interessanterweise waren auch die Katalase-Aktivitäten in der FF adipöser Frauen mit und ohne PCOS höher als die der normalgewichtigen. Zusammenfassend lässt sich folgern, dass erhöhte oxLDL-Konzentrationen in der FF von adipösen Frauen, unabhängig vom Vorliegen eines PCOS, mit einer gesteigerten Katalase-Aktivität und einer niedrigeren IVF-Erfolgsrate assoziiert sind.:Inhaltsverzeichnis Bibliographie III Abkürzungsverzeichnis IV 1. Einleitung 1 1.1. Infertilität 1 1.2. PCOS 2 1.2.1. Definition des PCOS 2 1.2.2. PCOS und Adipositas 3 1.3. Oxidativer Stress 4 1.3.1. Reaktive Sauerstoffspezies 4 1.3.2. Superoxiddismutase (SOD) – Schutz vor Superoxidradikalen 5 1.3.3. Katalase – Schutz vor H2O2 5 1.3.4. Glutathionperoxidase (GPx) und Glutathionreduktase (GR) 5 1.4. Oxidativer Stress, „oxidized low density lipoprotein“(oxLDL) und 6 Adipositas 1.5. Oxidativer Stress, antioxidative Enzyme und Infertilität 7 1.6. Ziele der Arbeit 9 2. Material und Methoden 10 2.1. Patientinnen und Material 10 2.2. Methoden 12 2.2.1. Follikelspektrumanalyse – Kontamination mit Blutbestandteilen 12 2.2.2. Proteinbestimmung 12 2.2.3. Bestimmung der SOD-Aktivität 13 2.2.4. Bestimmung der Katalase-Aktivität 14 2.2.5. Bestimmung der GPx-Aktivität 15 2.2.6. Bestimmung der GR-Aktivität 16 2.2.7. Bestimmung der oxLDL-Konzentration mittels ELISA 17 2.3. Statistische Auswertung 18 3. Ergebnisse 19 3.1. Vergleich der Proteinkonzentrationen in Seren und FF 19 3.2. Erhöhte SOD-Aktivität in der FF im Vergleich zum Serum 20 3.3. Erhöhte Katalase-Aktivität bei Adipositas unabhängig vom PCOS-Status 21 3.4. Erhöhte GPx-Aktivität bei adipösen Patientinnen ohne PCOS 22 3.5. Erhöhte GR-Aktivität bei Adipositas unabhängig vom PCOS-Status 23 3.6. Erhöhte oxLDL-Konzentrationen in der FF adipöser Patientinnen 25 unabhängig vom PCOS-Status 3.7. Schwangerschaftsrate der vier Patientengruppen 26 4. Diskussion 28 4.1. Das oxidative System 28 4.1.1. LOX-1 und oxLDL – Stand der Forschung 28 4.1.2. OxLDL – Diskussion der Methode 29 4.1.3. OxLDL – Konzentration im Serum 30 4.1.4. Einfluss der hormonellen Stimulationstherapie auf oxLDL 31 4.1.5. OxLDL – Konzentration in der FF 32 4.1.6. Oxidativer Status - Zusammenfassung 34 4.2. Das antioxidative System 34 4.2.1. SOD-Aktivität im Serum 35 4.2.2. SOD-Aktivität in der FF 35 4.2.2. Katalase, ein klinischer Parameter zur Bestimmung des 36 oxidativen Stress? 4.2.3. Zelltod als Ursache für erhöhte Enzymaktivitäten bei Adipositas? 36 4.2.5. Grenzen der Studie 37 4.3. Schlussfolgerung und Ausblick 38 5. Zusammenfassung 40 6. Literaturverzeichnis 44 7. Anhang 59 7.1. Tabellenverzeichnis 59 7.2. Abbildungsverzeichnis 59 7.3. Eidesstattliche Versicherung 60 7.4. Werdegang 61 7.5. Danksagung 63

Die chronisch ischämische Herzkrankheit und der Myokardinfarkt (MI) sind die häufigsten Gründe für schwere Krankheit und vorzeitigen Tod in den entwickelten Ländern. Langfristig kommt es als Folge des Infarktes zur Kollateralgefäßbildung und zur Entwicklung einer kompensatorischen Herzhypertrophie. Eine Vielzahl von adaptativen Veränderungen in diesem Prozess konnte identifiziert werden. Wir konnten zeigen, dass in der akuten Phase nach MI Kontraktions- und Relaxationsgeschwindigkeit des Myokards erhöht waren. Die Expression der Hitzeschockproteine (HSP) 25 und 72 war verstärkt und korrelierte mit der Relaxationsgeschwindigkeit. In der chronischen Phase nach MI entwickelte sich eine signifikante Herzhypertrophie, die mit verminderter Kontraktions- und Relaxationsgeschwindigkeit einherging. Für die verlangsamte Relaxation war die verminderte Aktivität der Ca2+-ATPase des sarkoplasmatischen Retikulums (SERCA) als entscheidender Faktor anzusehen. Bei transgener Überexpression von Renin / Angiotensinogen ist die Relaxationsgeschwindigkeit des Myokards war wie auch nach MI durch geringere SERCA- Protein Expression vermindert. Die Empfindlichkeit der kontraktilen Funktion gegenüber Sauerstoffmangel und Reoxygenierung war nach MI gegenüber dem Kontrollmyokard geringer. Dafür konnten die verstärkte Expression der antioxidativ wirksamen HSPs und die erhöhte Aktivität der Glutathionperoxidase und der Superoxiddismutase, eine Verschiebung des Kreatinkinase (CK)- Isoenzymmusters und eine verminderte SERCA- Aktivität verantwortlich gemacht werden. Die Repolarisation der Aktionspotentiale der Kardiomyozyten war nach MI gegenüber den Kontrolltieren signifikant verlangsamt. Bereits eine 10-fach geringere artifizielle Dehnung des Gewebes führte nach MI im Vergleich zu Kontrolltieren zum Auftreten von Nachdepolarisationen und Extra-Aktionspotentialen. Ausschließlich in MI ließ sich durch die artifizielle Dehnung Vorhofflimmern auslösen, d.h. nach Myokardinfarkt war der mechano- elektrische Feedback Mechanismus empfindlicher. Die dehnungsinduzierten Veränderungen konnten durch Gadolinium unterdrückt werden, was auf eine Beteiligung von dehnungsaktivierten Ionenkanälen an den beobachteten Phänomenen schließen ließ. Auch kardiale Fibroblasten zeigten nach MI signifikante Änderungen ihrer elektrophysiologischen Eigenschaften, was zur Arrhythmieentstehung beitragen kann. Mittels molekularer Analysen konnten wir zeigen, dass der unter Sauerstoffmangel stabilisierte Transkriptionsfaktor Hif-1alpha in der Lage ist, den Promoter des Wilms' Tumor Suppressor Gens 1 (WT1) direkt transkriptionell zu aktivieren. Das führte zu verstärkter Expression von WT1 in den Herzen nach Myokardinfarkt, und zu verstärkter Expression von WT1 in Herz und Niere bei systemischer normobarer Hypoxie. Die WT1 Expression im Herzen nach MI ließ sich in den Koronargefäßen lokalisieren. Koexpression mit Proliferations- und Vaskulogenesemarkern ließ vermuten, dass WT1 nach MI eine wichtige Rolle für die Neovaskulogenese spielt. Die gewonnenen Ergebnisse tragen zum Verständnis der pathophysiologischen Veränderungen bei kardialer Hypertrophie nach Myokardinfarkt bei und eröffnen möglicherweise langfristig neue therapeutische Ansätze. / Chronic ischemic heart disease and myocardial infarction are the most common causes for morbidity and mortality in industrialized countries. A survived myocardial infarction (MI) results in a long run in collateral formation and the development of cardiac hypertrophy. A variety of adaptive responses in this process had been identified. We could show that in the acute phase after Mi in rats, contraction- and relaxation rates of the myocardium are increased. The higher relaxation rate correlates to an increased expression of heat shock proteins. In the chronic phase after MI, with the development of cardiac hypertrophy, contraction and relaxation rates decrease. The decrease in the relaxation rate could be attributed to a reduced activity of the Ca- ATPase of the sarcoplasmic reticulum (SERCA2). Transgenic overexpression of renin / angiotensinogen also resulted in a reduced SERCA2 expression and, consequently, lower relaxation rate. The susceptibility of contractile function to hypoxia - reoxygenation was reduced after MI compared to sham operated control animals. The lower susceptibility to hypoxia - reoxygenation could be attributed to an increased expression of heat shock proteins, higher activities of the antioxidant enzymes glutathionperoxidase and superoxiddismutase, shifts in the isoenzyme distribution of the creatine kinase, and a reduced SERCA2 activity. Repolarization of cardiomyocyte action potentials was found to be delayed after MI. A 10-fold lower artificial stretch of the tissue after MI than after sham operation caused afterdepolarizations and extra action potentials. Higher artificial stretch caused atrial fibrillation only after MI suggesting an intensified mechano-electrical feedback mechanism after MI. Stretch- induced electrical abnormalities could be suppressed by gadolinium suggesting the involvement of stretch-activated ion channels in the electrical abnormalities. Also electrophysiological properties of cardiac fibroblasts were significantly altered after MI, which may contribute to the increased risk for arrhythmia after infarction. Furthermore, we could show that the Hif-1alpha transcription factor, which is stabilized under hypoxic conditions is capable to directly activate the Wilms'' tumor suppressor 1 (WT1) transcriptionally. This leads to an increased expression of WT1 in the heart after MI and in heart and kidneys after systemic hypoxia. After MI, WT1 is expressed mainly in coronary vessels. Co-expression of WT1 with markers of proliferation and vasculogenesis suggests a role of WT1 in neovasculogenesis. These findings contribute to our understanding of pathophysiological alterations in the development of cardiac hypertrophy after MI and may contribute to the development of new therapeutic approaches.

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.

Adaptation to chronic hypoxia activates endogenous signaling cascades, which lead to cardiac protection against acute ischemia/reperfusion (I/R) injury. The molecular mechanism of this phenomenon has not been fully clarified yet. However, it was proved that reactive oxygen species (ROS) take part in cardioprotective signaling pathway inducted by chronic hypoxia. The high level of ROS must be precisely regulated by antioxidative system of a cell. The aim of diploma thesis was to examine the effect of intermittent hypobaric hypoxia (IHH, 7 000 m) on relative amount of antioxidative enzymes (peroxiredoxin 6 - PRX6, thioredoxin 1 and 2 - TRX1 and TRX2, thioredoxin reductase 1 - TRXR1) and also enzymes of iron metabolism (heme oxygenase 1 and 2 - HO1 and HO2, aconitase 1 and 2 - ACO1 and ACO2), which participate in regulation of cell redox state. Moreover, we studied the effect of adaptation to IHH and an antioxidant tempol on relative amount of calcium-independent phospholipase A2 (iPLA2). iPLA2 can remove peroxidized fatty acids from membrane phospholipids. On the other hand, iPLA2 can damage cell in I/R conditions. All enzymes were studied in homogenates from normoxic and IHH adapted rat left ventricular myocardium by Western blot. Adaptation to IHH caused a decrease of PRX6 and on the opposite an increase of...