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.

Beim neonatalen Atemnotsyndrom ist die Beatmung der Frühgeborenen mit hohen Sauerstoffpartialdrücken eine oft lebensrettende Therapie. Durch diese therapeutische Hyperoxie entstehen allerdings vermehrt reaktive Sauerstoffspezies, die an der Pathogenese verschiedener Lungenerkrankungen beteiligt sind. Ziel dieser Arbeit ist es, Modelle für oxidativen Stress in der Lunge zu entwickeln und die Mechanismen der oxidativen Schädigung näher zu charakterisieren. Folgende Modelle werden untersucht: 1. Untersuchung von isolierten Typ-II-Zellen aus Hyperoxie-exponierten bzw. Kontroll-Ratten (in vivo) und 2. Kultivierung isolierter Typ-II-Zellen in Gegenwart von H2O2 (in vitro). Wir zeigen in dieser Arbeit, das erst die Kultivierung von Typ-II-Zellen unter Basalbedingungen zu einem starken Anstieg der Expression von Hitzeschockproteinen (HSP) führt, wohingegen frisch isolierte Zellen keine HSP exprimieren. In Übereinstimmung mit der neueren Literatur schließen wir daraus, dass allein die basalen Zellkulturbedingungen für die Zellen ein Stressfaktor darstellen können. Unter 44-stündiger Hyperoxie steigt die Konzentration der PAF-ähnlichen Oxidationsprodukte von Phospholipiden (PAF-RC) lediglich im Blutplasma an, bleibt dagegen in der Lungenlavage unverändert. PAF-RC wirken über den Rezeptor des Thrombozyten-aktivierenden Faktors (PAF) proinflammatorisch und werden durch die PAF- Acetylhydrolase (PAF-AH) abgebaut. Unter Hyperoxie ist die PAF-AH-Aktivität im Blutplasma unverändert, in der Lavage jedoch auf ein Drittel vermindert. Wir nehmen daher an, dass die hyperoxische Lungenschädigung weder durch eine verminderte PAF-AH- Aktivität noch durch die direkte Peroxidation von Surfactantlipiden in den Alveolen vermittelt wird. Wahrscheinlich wird die pulmonale Sauerstofftoxizität durch die Lipidperoxidation im Blutgefäßsystem oder –plasma verursacht. Unter H2O2-Stress werden in den Typ-II-Zellen zum einen ungesättigte Surfactantlipide oxidiert, zum anderen wird die Aufnahme von Palmitinsäure in die Zelle und die Synthese von Phosphatidylcholin gehemmt. Der entscheidende Schritt ist dabei vermutlich die oxidative Hemmung eines Schlüsselenzyms der Phospholipidsynthese, der Glycerol-3- Phosphat-Acyltransferase (G3PAT). Die Konzentration der zellulären Antioxidantien Vitamin E und Glutathion ist scheinbar zu gering, um die G3PAT und die zellulären Lipide vor der v.a. initial sehr hohen H2O2-Belastung zu schützen. / Ventilation of preterm babies with infant respiratory distress syndrome (IRDS) using high oxygen pressures is often a life-saving therapy. A severe side effect of this therapeutic hyperoxia though, is the formation of reactive oxygen species that are involved in the pathogenesis of different lung diseases. This thesis is designed to develop models for oxidative stress in the lung and to further characterise the mechanisms of oxidative damage. The following models have been examined: 1. Examination of isolated type II cells from hyperoxia-exposed and control rats (in vivo). 2. Cultivation of isolated type II cells in the presence of H2O2 (in vitro). In this thesis we show that only by cultivating type II cells under basal conditions the expression of heat shock proteins (HSP) is strongly activated, whereas freshly isolated cells do not express HSP. We conclude in accordance with the newer literature, that the basal cell culture conditions alone can represent a stress factor for the cells. Under 44 hours of hyperoxia the concentration of the PAF-like oxidation products of phospholipids (PAF-RC) rises only in blood plasma, but not in the lung lavage. PAF- RC act proinflammatory via the platelet-activating factor (PAF) receptor and are degraded by the PAF-Acetylhydrolase (PAF-AH). Hyperoxia does not affect the PAF- AH-activity in blood plasma, but decreases it to one third in the lavage. According to these findings we assume that neither the decreased PAF-AH activity nor the direct peroxidation of surfactant lipids in the alveoli cause hyperoxic lung injury. More likely the pulmonary surfactant toxicity is caused by the lipid peroxidation in the blood vessel system or blood plasma. Under H2O2 stress unsaturated surfactant lipids in the type II cells are oxidated on the one hand, on the other hand the palmitic acid uptake in the cells as well as the phosphatidylcholine synthesis is inhibited. The oxidative inhibition of glycerol-3- phosphate-acyltransferase (GPAT), the key enzyme of phospholipid synthesis, is supposed to be the crucial step. It seems that the concentration of the antioxidants vitamin E and glutathione is not sufficient enough to protect the GPAT and the cellular lipids from the especially initially high concentrations of H2O2.

No description available.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Os estresses salino e osmÃtico sÃo responsÃveis por perdas significativas na produÃÃo agrÃcola, particularmente nas regiÃes semiÃridas. Nessas condiÃÃes, a raiz à o ÃrgÃo da planta que sofre os primeiros efeitos e à responsÃvel pela percepÃÃo e sinalizaÃÃo bioquÃmica dos estresses. Em folhas, as peroxidases do ascorbato do citosol sÃo as principais isoformas envolvidas com a proteÃÃo antioxidativa contra o excesso de H2O2 e sÃo tambÃm relacionadas na sinalizaÃÃo em condiÃÃes de estresses. Entretanto, esses mecanismos de aÃÃo em raÃzes sÃo pouco conhecidos. O objetivo deste estudo foi testar a hipÃtese de que as APXs citosÃlicas sÃo essenciais para a proteÃÃo antioxidativa de raÃzes de arroz expostas Ãs condiÃÃes de estresse salino e osmÃtico. Para isso, plantas transgÃnicas silenciadas nas duas isoformas de APXs citosÃlicas (APX1/2) e plantas nÃo transformadas (45 dias de idade) foram expostas a duas condiÃÃes de estresse: (1) NaCl e manitol em concentraÃÃes iso-osmÃticas (-0,62 MPa) durante oito dias e (2) manitol 268 mM (-0,62 MPa) por dois dias. Em plantas silenciadas, a quantidade de transcritos (RNAs) de OsAPX1 e OsAPX2 foi reduzida em 90% enquanto que a abundÃncia das duas proteÃnas mensurada por western blotting nÃo foi detectÃvel. A atividade total de APX foi diminuÃda em 66% em comparaÃÃo com as NTs na condiÃÃo controle, evidenciando que o silenciamento foi efetivo nas raÃzes. Como consequÃncia da deficiÃncia das APX1/2, o nÃvel de H2O2 foi aumentado em 51% comparado com as NTs. Ambos os estresses afetaram de modo similar o crescimento da raiz e parte aÃrea das APX1/2, em comparaÃÃo com as plantas nÃo transformadas (NT). Os valores de danos de membrana nas raÃzes foram aumentados na mesma intensidade nos dois genÃtipos e nos dois tipos de estresses, indicando que as APX1/2 apresentaram mesma sensibilidade aos estresses estudados. Nas plantas NT, as quantidades de transcritos de OsAPX1 e OsAPX2 foram aumentadas discretamente por NaCl e manitol enquanto que a atividade de APX foi aumentada somente pelo NaCl. As plantas APX1/2 mostraram a mesma tendÃncia das NTs quanto a expressÃo e atividade de APX. O aumento na quantidade relativa dos transcritos das outras isoformas de APX, principalmente de OsAPX3, OsAPX5 e OsAPX8, em ambos os genÃtipos sob estresse salino, foi correlacionado com o aumento da atividade da APX. AlÃm disso, outras peroxidases (GPX e GPOD) apresentaram a mesma tendÃncia de aumento de atividade apenas sob estresse salino. Diferentemente, manitol induziu um aumento proeminente na atividade de catalase nas plantas NT enquanto que nas APX1/2 essa enzima jà apresentava atividade aumentada antes do estresse e permaneceu no mesmo nÃvel. As concentraÃÃes de H2O2 foram aumentadas intensamente por manitol e reduzidas na presenÃa de NaCl. O nÃvel de TBARS (indicador de peroxidaÃÃo lipÃdica) foi mantido inalterado na presenÃa dos dois estresses e nos dois tipos de plantas. Os resultados deste estudo, quando analisados em conjunto, mostram que raÃzes de arroz expostas aos estresses salino e osmÃtico exibiram respostas complexas em termos de metabolismo redox. Aparentemente, as duas APXs citosÃlicas nÃo sÃo essenciais para a proteÃÃo antioxidativa, uma vez que as plantas mutantes apresentaram uma performance fisiolÃgica semelhante as plantas NT. As respostas aos dois fatores de estresse, NaCl e manitol, foram contrastantes nos dois genÃtipos, sugerindo que diferentes mecanismos de proteÃÃo antioxidante foram acionados para cada tipo de estresse. / Os estresses salino e osmÃtico sÃo responsÃveis por perdas significativas na produÃÃo agrÃcola, particularmente nas regiÃes semiÃridas. Nessas condiÃÃes, a raiz à o ÃrgÃo da planta que sofre os primeiros efeitos e à responsÃvel pela percepÃÃo e sinalizaÃÃo bioquÃmica dos estresses. Em folhas, as peroxidases do ascorbato do citosol sÃo as principais isoformas envolvidas com a proteÃÃo antioxidativa contra o excesso de H2O2 e sÃo tambÃm relacionadas na sinalizaÃÃo em condiÃÃes de estresses. Entretanto, esses mecanismos de aÃÃo em raÃzes sÃo pouco conhecidos. O objetivo deste estudo foi testar a hipÃtese de que as APXs citosÃlicas sÃo essenciais para a proteÃÃo antioxidativa de raÃzes de arroz expostas Ãs condiÃÃes de estresse salino e osmÃtico. Para isso, plantas transgÃnicas silenciadas nas duas isoformas de APXs citosÃlicas (APX1/2) e plantas nÃo transformadas (45 dias de idade) foram expostas a duas condiÃÃes de estresse: (1) NaCl e manitol em concentraÃÃes iso-osmÃticas (-0,62 MPa) durante oito dias e (2) manitol 268 mM (-0,62 MPa) por dois dias. Em plantas silenciadas, a quantidade de transcritos (RNAs) de OsAPX1 e OsAPX2 foi reduzida em 90% enquanto que a abundÃncia das duas proteÃnas mensurada por western blotting nÃo foi detectÃvel. A atividade total de APX foi diminuÃda em 66% em comparaÃÃo com as NTs na condiÃÃo controle, evidenciando que o silenciamento foi efetivo nas raÃzes. Como consequÃncia da deficiÃncia das APX1/2, o nÃvel de H2O2 foi aumentado em 51% comparado com as NTs. Ambos os estresses afetaram de modo similar o crescimento da raiz e parte aÃrea das APX1/2, em comparaÃÃo com as plantas nÃo transformadas (NT). Os valores de danos de membrana nas raÃzes foram aumentados na mesma intensidade nos dois genÃtipos e nos dois tipos de estresses, indicando que as APX1/2 apresentaram mesma sensibilidade aos estresses estudados. Nas plantas NT, as quantidades de transcritos de OsAPX1 e OsAPX2 foram aumentadas discretamente por NaCl e manitol enquanto que a atividade de APX foi aumentada somente pelo NaCl. As plantas APX1/2 mostraram a mesma tendÃncia das NTs quanto a expressÃo e atividade de APX. O aumento na quantidade relativa dos transcritos das outras isoformas de APX, principalmente de OsAPX3, OsAPX5 e OsAPX8, em ambos os genÃtipos sob estresse salino, foi correlacionado com o aumento da atividade da APX. AlÃm disso, outras peroxidases (GPX e GPOD) apresentaram a mesma tendÃncia de aumento de atividade apenas sob estresse salino. Diferentemente, manitol induziu um aumento proeminente na atividade de catalase nas plantas NT enquanto que nas APX1/2 essa enzima jà apresentava atividade aumentada antes do estresse e permaneceu no mesmo nÃvel. As concentraÃÃes de H2O2 foram aumentadas intensamente por manitol e reduzidas na presenÃa de NaCl. O nÃvel de TBARS (indicador de peroxidaÃÃo lipÃdica) foi mantido inalterado na presenÃa dos dois estresses e nos dois tipos de plantas. Os resultados deste estudo, quando analisados em conjunto, mostram que raÃzes de arroz expostas aos estresses salino e osmÃtico exibiram respostas complexas em termos de metabolismo redox. Aparentemente, as duas APXs citosÃlicas nÃo sÃo essenciais para a proteÃÃo antioxidativa, uma vez que as plantas mutantes apresentaram uma performance fisiolÃgica semelhante as plantas NT. As respostas aos dois fatores de estresse, NaCl e manitol, foram contrastantes nos dois genÃtipos, sugerindo que diferentes mecanismos de proteÃÃo antioxidante foram acionados para cada tipo de estresse. / Salt and osmotic stresses are responsible for significant losses in agriculture, particularly in semiarid regions. In such conditions, roots are the first plant organ in contact with the stress and are responsible for perception and signaling. In leaves, cytosolic ascorbate peroxidases (APX) are the main isoforms involved with antioxidative defence against H2O2 excess and signaling under stressful conditions. Nevertheless, such metabolic mechanisms in roots are still unknown. The aim of this study was to test the hypothesis that cytosolic APX isoforms are essential to antioxidant protection in rice roots exposed to salt and osmotic stresses. To test this hypothesis, rice mutants double silenced for cytosolic APXs (APX1/2) and non-transformed plants, both with 45 day-old, were submitted to two stressful treatments: (1) NaCl and mannitol in iso-osmotic concentrations (-0.62MPa) for eight days and (2) mannitol 268 mM (-0.62MPa) for two days. In mutant plants, OsAPX1 and OsAPX2 transcript amounts (RNAs) were reduced by 90% whereas both protein abundance measured by Western blotting were not detectable. Under control conditions, total APX activity was reduced by 66% in comparison with NT plants, showing that the silencing was effective in roots. In APX1/2 roots, H2O2 level was increased by 51% as a consequence of APX1/2 silencing. Both stresses affected similarly root and shoot growing compared with NT. Membrane damage was increased at the same level in both genotypes and in both stresses, showing that APX1/2 were as sensible as NT plants. In NT roots, OsAPX1 and OsAPX2 transcript amounts was slightly increased by NaCl and mannitol whereas only NaCl increased APX activity. Under salt stress, both genotypes increased other APX isoforms, especially OsAPX3, OsAPX5 and OsAPX8. These increases were correlated with the increased APX activity. In addition, other peroxidases (GPX and GPOD) displayed the same trend as APX, increasing their activity in response to NaCl. On the other hand, mannitol induced a prominent increase in catalase activity in NT plants, while in APX1/2 plants CAT activity did not changed. The H2O2 content was increased in both genotypes exposed to mannitol treatments, and was reduced for NaCl. TBARS level was not altered in the presence of both stresses for both genotypes. This study shows that rice plants exposed to salt or osmotic stresses display complex responses regarding to redox metabolism. Apparently, both cytosolic APXs are not essential to antioxidative protection, since mutant plants presented similar physiological performance to NT plants. The responses to both stresses, NaCl and mannitol, were contrasting for both genotypes, suggesting that different mechanisms of antioxidative protection were triggered for each different stress condition. / Salt and osmotic stresses are responsible for significant losses in agriculture, particularly in semiarid regions. In such conditions, roots are the first plant organ in contact with the stress and are responsible for perception and signaling. In leaves, cytosolic ascorbate peroxidases (APX) are the main isoforms involved with antioxidative defence against H2O2 excess and signaling under stressful conditions. Nevertheless, such metabolic mechanisms in roots are still unknown. The aim of this study was to test the hypothesis that cytosolic APX isoforms are essential to antioxidant protection in rice roots exposed to salt and osmotic stresses. To test this hypothesis, rice mutants double silenced for cytosolic APXs (APX1/2) and non-transformed plants, both with 45 day-old, were submitted to two stressful treatments: (1) NaCl and mannitol in iso-osmotic concentrations (-0.62MPa) for eight days and (2) mannitol 268 mM (-0.62MPa) for two days. In mutant plants, OsAPX1 and OsAPX2 transcript amounts (RNAs) were reduced by 90% whereas both protein abundance measured by Western blotting were not detectable. Under control conditions, total APX activity was reduced by 66% in comparison with NT plants, showing that the silencing was effective in roots. In APX1/2 roots, H2O2 level was increased by 51% as a consequence of APX1/2 silencing. Both stresses affected similarly root and shoot growing compared with NT. Membrane damage was increased at the same level in both genotypes and in both stresses, showing that APX1/2 were as sensible as NT plants. In NT roots, OsAPX1 and OsAPX2 transcript amounts was slightly increased by NaCl and mannitol whereas only NaCl increased APX activity. Under salt stress, both genotypes increased other APX isoforms, especially OsAPX3, OsAPX5 and OsAPX8. These increases were correlated with the increased APX activity. In addition, other peroxidases (GPX and GPOD) displayed the same trend as APX, increasing their activity in response to NaCl. On the other hand, mannitol induced a prominent increase in catalase activity in NT plants, while in APX1/2 plants CAT activity did not changed. The H2O2 content was increased in both genotypes exposed to mannitol treatments, and was reduced for NaCl. TBARS level was not altered in the presence of both stresses for both genotypes. This study shows that rice plants exposed to salt or osmotic stresses display complex responses regarding to redox metabolism. Apparently, both cytosolic APXs are not essential to antioxidative protection, since mutant plants presented similar physiological performance to NT plants. The responses to both stresses, NaCl and mannitol, were contrasting for both genotypes, suggesting that different mechanisms of antioxidative protection were triggered for each different stress condition.

Células de Nicotiana tabacum cv BY-2 foram tratadas por cinco dias com 0,075 e 0,750 mM de NiCl2. A relação entre a toxidade do níquel (Ni) e as reações oxidativas foram estudadas nas células durante a acumulação do metal. A atividade da superóxido dismutase não se alterou na presença do Ni. Entretanto, as atividades da catalase e da guaiacol peroxidase aumentaram às 36 e 72h depois do tratamento com o metal. As atividades da glutationa redutase, da glutationa-Stransferase e da ascorbato peroxidase aumentaram nas primeiras horas do tratamento. A peroxidação lipídica da membrana aumentou somente às 24h do tratamento com o metal. Os resultados sugerem que a desordem oxidativa é resultante dos efeitos da toxidade do Ni nas células de Nicotiana tabacum cv BY-2. / Células de Nicotiana tabacum cv BY-2 foram tratadas por cinco dias com 0,075 e 0,750 mM de NiCl2. A relação entre a toxidade do níquel (Ni) e as reações oxidativas foram estudadas nas células durante a acumulação do metal. A atividade da superóxido dismutase não se alterou na presença do Ni. Entretanto, as atividades da catalase e da guaiacol peroxidase aumentaram às 36 e 72h depois do tratamento com o metal. As atividades da glutationa redutase, da glutationa-Stransferase e da ascorbato peroxidase aumentaram nas primeiras horas do tratamento. A peroxidação lipídica da membrana aumentou somente às 24h do tratamento com o metal. Os resultados sugerem que a desordem oxidativa é resultante dos efeitos da toxidade do Ni nas células de Nicotiana tabacum cv BY-2.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-20T17:04:51Z No. of bitstreams: 1 Manuela Maria Cavalcante Granja.pdf: 696391 bytes, checksum: 1cfe62663cfe614a373e837e03fde09b (MD5) / Made available in DSpace on 2017-02-20T17:04:51Z (GMT). No. of bitstreams: 1 Manuela Maria Cavalcante Granja.pdf: 696391 bytes, checksum: 1cfe62663cfe614a373e837e03fde09b (MD5) Previous issue date: 2010-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Sugarcane (Saccharum officinarum) is a grass considered moderately sensitive to salinity. Although traditionally cultivated in the soil of the Atlantic Coast and the Northeast, the cultivation of sugarcane is expanding to semi-arid regions. The poor quality of irrigation water, high rates of evaporation and low rainfall, combined with other factors contribute to the process of soil salinization in semi-arid. For the development and selection of tolerant genotypes, it is necessary to understand the physiological mechanisms with which plants face the salt stress. Techniques for in vitro plant tissue for studies of physiology and biochemistry of plants under stress are important tools to allow control and uniformity of culture conditions. In this study, two genotypes of sugarcane (RB931011 and RB872552) developed within the Program of Genetic Improvement of Sugarcane of rides, for the Northeast region (RIDESA) were evaluated for sudden and gradual action of salt stress-induced by adding NaCl to the culture medium during in vitro development of plants. The varieties of sugarcane were subjected to three concentrations of NaCl (0 mM, 50 mM and 100 mM) in a gradual and sudden. At 35 days of experiment, enzyme activities (CAT, POX, APX, PPO), levels of sodium (Na+), potassium (K+), soluble protein content and free proline were recorded. It was possible to observe differences in responses of varieties depending on the condition of induced salt stress, shock or gradual, rather than depending on the concentration of NaCl in the culture medium. The stress response is therefore not only conditioned by salt concentration, but by way of exposure to salt. / A cana-de-açúcar (Saccharum officinarum) é uma gramínea considerada moderadamente sensível à salinidade. Embora tradicionalmente cultivada nas zonas de Mata úmida e Litoral da região Nordeste, o cultivo da cana-de-açúcar vem se expandindo também para regiões semi-áridas. A má qualidade da água de irrigação, altas taxas de evaporação e baixa precipitação pluviométrica, aliada a outros fatores contribuem para o processo de salinização dos solos do semi–árido. Para o desenvolvimento e seleção de genótipos tolerantes, é necessário conhecer os mecanismos fisiológicos com os quais as plantas enfrentam o estresse salino. Técnicas de cultivo in vitro para estudos da fisiologia e bioquímica de plantas, em condições de estresse, são importantes ferramentas por permitirem o controle e homogeneidade das condições de cultivo. No presente trabalho, dois genótipos de cana-de-açúcar (RB931011 e RB872552) desenvolvidos pelo Programa de Melhoramento Genético da Cana-de-Açúcar da RIDESA, para a região Nordeste, foram submetidos ao estresse salino in vitro mediante o acréscimo de NaCl ao meio de cultura sob uma ação gradativa e súbita para avaliar o comportamento fisiológico e bioquímico dessas plantas. As variedades de cana-de-açúcar foram submetidas a duas concentrações de NaCl (56mM, 112mM e o controle) de forma gradativa e repentina. Aos 35 dias de experimento, atividades enzimáticas da Catalase (CAT), Peroxidase (POX), Ascorbato peroxidase (APX), Polifenoloxidase (PPO), teores de sódio (Na+), potássio (K+), concentração de proteínas solúveis e prolina livre foram determinadas. Foi possível observar diferenças nas respostas das variedades em função da condição de indução do estresse salino, gradativo ou por choque, mais do que em função das concentrações de NaCl no meio de cultura. A resposta ao estresse é, portanto, condicionada não só pela concentração dos sais, mas pela forma que a planta é exposta ao meio salino.

Thesis (M.Sc. (Biochemistry )) --University of Limpopo, 2007 / Refer to document / Medical Research Council (MRC), National Research Foundation (NRF) and University of Limpopo

Lead (Pb) is one of the most commonly occurring, highly persistent and widely distributed heavy metal contaminants in the environment. It has a tendency to bioaccumulate in animals and plants, and potentially, it is able to enter the human food chain where it poses a hazard to public health. Generally, conventional remediation technologies applied to decontaminate heavy metals from groundwater and soils are very costly. Hence, phytoremediation has emerged as an ecologically friendly and economically attractive technology that uses green plants to clean up heavy metal contaminated sites. However, a lack of knowledge of the biological processes associated with plant responses to Pb (e.g. Pb uptake, accumulation, translocation, and tolerance) has been a bottleneck for the application of Pb phytoremediation in the field. A model genetic system of higher plants, Arabidopsis thaliana, was selected to further examine the physiological, biochemical and molecular events occuring in plants under Pb stress. The overall aim of this project was to obtain a better understanding of plant responses to Pb contaminants in the early developmental stages of A. thaliana seedlings. This research encompassed the physiological responses of A. thaliana seedlings to Pb exposure, monitoring their antioxidative defence systems, and investigating the participation of annexin 1 in the response to Pb-mediated oxidative stress. This research also assessed the protective effect of nitric oxide on Pb-induced toxicity of A. thaliana seedlings and it isolated a putative Pb tolerant mutant from an EMS-mutagenized M2 population. A multiexperimental approach was adopted to achieve these objectives. A. thaliana seedlings were grown on modified Huang & Cunningham (1996) nutrient solution containing 0.8% (w/v) agar, with and without Pb(NO3)2, under controlled conditions. A. thaliana seedlings were insensitive to Pb during seed germination. In treatments with up to 200 μM Pb(NO₃)₂, morphological changes and inhibition of root growth were observed in the 7-d-old seedlings. A tolerance index revealed that Pb(NO₃)₂ concentration of 75 μM and higher brought about more than 50% root growth inhibition. Pb was predominantly retained in the roots. Analysis using a graphite furnace atomic absorption spectroscopy indicated that the level of Pb accumulation in A. thaliana roots was greatly dependent on the Pb(NO₃)₂ concentrations, but only a small fraction of the accumulated Pb was translocated to the shoots (18 - 43%). Transmission electron microscopy analysis showed that Pb was mainly immobilized in the cell walls and intercellular spaces. This was interpreted as a mechanism that minimizes the entry of Pb into cells and interference with cellular functions. Pb that gained entry into the cytoplasm was sequestered into the vacuoles. The toxicity of Pb in the cytosol of A. thaliana seedlings was studied by measuring the H₂O₂ and lipid hydroperoxide levels using a microplate reader. When the Pb(NO₃)₂ concentration in the growth medium was 100 μM, the 7-d-old seedlings contained 2.2-fold higher H₂O₂ and 9.6-fold higher lipid hydroperoxide than the control without Pb(NO₃)₂. This was followed by an up-regulation of the activity of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), and general peroxidase (POD) by 2.1-, 3.2-, 2.3-, 1.8- and 4.6-fold, respectively, compared with the control. Pb toxicity is known to trigger oxidative stress, but A. thaliana seedlings appeared to be capable of activating cell rescue, defending themselves against harmful oxidative stress and also acclimating to Pb. Data from physiological and biochemical analysis indicate that a combination of avoidance and tolerance mechanisms exists in Pb-treated A. thaliana seedlings to maintain the essential cellular metabolism for survival. Real-time reverse-transcription polymerase chain reaction was used to show the involvement of AnnAt1 in the response of 7-d-old A. thaliana seedlings to a high threshold concentration of Pb. When the seedlings were treated with 100 μM Pb(NO₃)₂, AnnAt1 message levels were up-regulated by 2.12-fold. Pb-mediated oxidative stress may be a component of AnnAt1 gene expression. AnnAt1 potentially could be invoked to reduce the toxic effects of Pb stress by acting as ROS and/or Ca²⁺ signals, as a membrane protector, in detoxification of excessive ROS, or in sequestration of Pb. Pb stress symptoms were less evident in seedlings pre-treated with 1 mM sodium nitroprusside (SNP), a nitric oxide (NO) donor. The present study found that exogenous NO did not alter Pb transport into the plants or efflux pumping of Pb at the plasma membrane. However, NO conferred protection to 7-d-old A. thaliana seedlings primarily by acting as an antioxidant or a signal for actions to scavenge excessive ROS level. The application of exogenous NO before subjecting to 100 μM Pb(NO₃)₂ decreased H₂O₂ back to its original level, and reduced 50% lipid hydroperoxide in the Pb-treated seedlings. As a result, the antioxidative enzyme activities in Pb-exposed seedlings pre-treated with SNP were 23 - 45% lower than those without SNP pre-treatment. Less antioxidative enzyme activities were probably needed to counteract the reduced amount of Pb-induced ROS in A. thaliana seedlings. A post-germination procedure involving prolonged exposure to 150 μM Pb(NO₃)₂ was developed to screen an EMS-mutagenized M2 population of A. thaliana. Potential Pb tolerant mutants were selected based on the ability to grow with their roots penetrating into the medium and maintain purple-green leaves without wilting. A minority of the survivors appeared to go into a resting stage and they seemed to have altered transporters that prevented Pb from entering the cells. Only one putative Pb mutant (M3-1) was recovered from the rescue and set seeds. The M₄ generation of this putative Pb mutant was re-screened for phenotypic confirmation and to determine the regulation of AnnAt1. The 7-d-old putative Pb mutant seemed to display enhanced root and shoot growth in the presence of 150 μM Pb(NO₃)₂ compared to the wild-type seedlings. The transcript level of AnnAt1 in this putative Pb tolerant mutant increased by 2.19-fold when exposed to 150 μM Pb(NO₃)₂.

Der Mastitis-Metritis-Agalaktie-Komplex (MMAK) zählt in der Schweineproduktion zu den wichtigsten Puerperalerkrankungen der Sau. Es existieren verschiedene Prophylaxe- und Therapiekonzepte. Diese können jedoch nicht verhindern, dass es zu wirtschaftlichen Verlusten durch erhöhte Ferkelmortalität und herabgesetzte Fruchtbarkeitsleistung der Sauen kommt. Ziel dieser Untersuchung war es zu ermitteln, wie sich der antioxidative und Stoffwechselstatus von Sauen eines Betriebes mit vermehrtem Auftreten vom MMAK im peripartalen Zeitraum verhält und ob Unterschiede zwischen gesunden und am MMAK erkrankten Sauen bestehen. Des Weiteren wurde geprüft, ob Unterschiede zwischen Jung- und Altsauen existieren und ob er antioxidative Status saisonalen Schwankungen unterliegt. Versuchsanordnung: Im Versuchszeitraum von September 2004 bis Oktober 2005 wurden insgesamt 67 zu Beginn der Untersuchung klinisch gesunden Sauen im peripartalen Zeitraum je vier Blutproben entnommen: (1.) eine Woche vor dem Abferkeln, (2.) 1. Tag nach der Abferkelung, (3.) 8. Tag nach der Abferkelung und (4.) 15. Tag nach der Abferkelung. Die Blutentnahme erfolgte jahreszeitlich gestaffelt. Es wurden die Trolox equivalent antioxidant capacity (TEAC), die Antioxidant capacity of water soluble substances (ACW), die Aktivitäten der Superoxid-Dismutase (SOD), der Glutathion-Peroxidase (GPX), die Selen- und Vitamin E-Konzentration sowie ausgesuchte klinisch-chemische Parameter bestimmt. Parallel dazu wurden Futtermittelproben und eine Brunnenwasserprobe im Labor der Thüringer Landesanstalt für Landwirtschaft untersucht. Die Beurteilung der Sauengesundheit erfolgte nach den Kriterien Fieber, vaginaler Ausfluss, Mastitis und/oder Agalaktie. Es wurde zwischen Jungsau (inklusive 2. Wurf) und Altsau (ab dem 3. Wurf) unterschieden. Ergebnisse: Die TEAC-Konzentration lag bei den am MMAK erkrankten Sauen eine Woche vor dem Abferkeln mit 196 (1. Quartil: 192; 3. Quartil: 224) µmol/l signifikant unter der Konzentration bei den gesunden Sauen mit 246 (192; 275) µmol/l. Bei der folgenden Messung am ersten Tag nach der Abferkelung kam es bei den am MMAK erkrankten Sauen zu einem signifikanten Anstieg der TEAC-Konzentration auf 239 (191; 268) µmol/l. Bei den gesunden Sauen unterlag die TEAC-Konzentration im Untersuchungszeitraum nicht signifikanten Schwankungen. Die ACW-Konzentration lag in fast 70 % der Proben unterhalb der Nachweisgrenze. Die SOD-Aktivität bei den am MMAK erkrankten Sauen lag eine Woche vor dem Abferkeln mit 2725 (2479; 2936) U/g Hb signifikant höher, als die Aktivität bei den gesunden Sauen mit 2366 (2158; 2663) U/g Hb. Bei den gesunden Sauen nahm die Aktivität der SOD bis zum achten Tag nach der Abferkelung signifikant zu und sank am 15. Tag nach der Abferkelung signifikant ab. Bei den am MMAK erkrankten Tieren unterlag die SOD-Aktivität von einer Woche vor dem Abferkeln bis zum achten Tag nach der Abferkelung nicht signifikanten Schwankungen, am 15. Tag nach der Abferkelung sank sie signifikant. Die GPX-Aktivität lag bei den am MMAK erkrankten Sauen eine Woche vor dem Abferkeln mit 267 (207; 298) U/g Hb signifikant niedriger als bei den gesunden Sauen mit 286 (255; 316) U/g Hb. Bei der anschließenden Untersuchung am ersten Tag nach der Abferkelung sank die GPX-Aktivität bei den am MMAK erkrankten Sauen auf 239 (178; 277) U/g Hb signifikant ab, im weiteren Untersuchungsverlauf blieb sie auf einem Niveau. Die Selenkonzentration stieg bei den gesunden und am MMAK erkrankten Sauen während der Untersuchung signifikant an. Die CK- und AST-Aktivitäten stiegen bei den am MMAK erkrankten Sauen am ersten Tag nach der Abferkelung signifikant an und näherten sich bei den folgenden Untersuchungen wieder der Ausgangsaktivität eine Woche vor der Abferkelung an. Der antioxidative Status von gesunden und am MMAK erkrankten Jung- und Altsauen zeigte signifikant höhere Selenkonzentrationen eine Woche vor dem Abferkeln bei den am MMAK erkrankten Altsauen im Vergleich zu den am MMAK erkrankten Jungsauen, sowie eine signifikant höhere TEAC-Konzentration am 15. Tag nach der Abferkelung bei dem am MMAK erkrankten Altsauen im Vergleich zu den an am MMAK erkrankten Jungsauen. Im jahreszeitlichen Vergleich lagen in erster Linie Parameter der September/Oktober Kontrolle signifikant erniedrigt vor. Wobei mit Ausnahme der Selenkonzentration diese signifikanten Unterschiede nicht während des gesamten Probenzeitraumes bestanden. Schlussfolgerungen: In dieser Untersuchung wurden die größten Unterschiede im antioxidativen Abwehrsystem zwischen den gesunden Sauen und am MMAK erkrankten Sauen eine Woche vor dem Abferkeln sowie am ersten Tag nach der Abferkelung festgestellt. Dies lässt vermuten, dass das antioxidative Abwehrsystem der kranken Sauen schon vor der Abferkelung stärkeren Belastungen ausgesetzt war als das der gesunden Sauen. Im Alters- und jahreszeitlichen Vergleich lagen vereinzelt signifikante Unterschiede im antioxidativen Status vor. / The mastitis, metritis, agalactia syndrome (MMAS) counts among the most prominent diseases of sows in pig breeding. Several prophylactic and therapeutic concepts exist but those cannot prevent economical losses due to an increased mortality of piglets and decreased fertility of sows. This examination aims at showing how the sows antioxidant and metabolic status in farms with increased cases of MMAS during the periparturient period behaves and whether differences between young and elder sows exist and whether the antioxidative status is liable to seasonal fluctuations. Test arrangement: During the period from september 2004 to october 2005, four blood samples of 67 peripartal sows which were clinically healthy at the examination’s outset were taken: (1.) one week before littering, (2.) one day after littering, (3.) 8 days after littering and (4.) 15 days after littering. Blood withdrawal occurred seasonally equated. Trolox equivalent antioxidant capacity (TEAC), antioxidant capacity of water-soluble substances (ACW), activity of superoxide dismutase (SOD) glutathione peroxidase (GPX), concentrations of selenium and vitamin E as well as assorted clinical and chemical parameters were analysed. In addition, feeding stuff and water sample were analysed in the laboratories of the Thüringer Landesanstalt für Landwirtschaft. Appraisal of the sows’ health was based on the criteria fever, vaginal discharge, mastitis and/or agalactia. Young sows (including second litter) an elder sows (third litter and up) were distinguished. Results: Concentration of TEAC in sows afflicted with MMAS one week before littering was at 196 (1. quartile: 192; 3. quartile: 224) µmol/l significantly lower than the concentration in healthy sows at 246 (192; 275) µmol/l. The following measurement taken on the first day after littering showed a significant rise to 239 (191; 268) µmol/l in the concentration of TEAC in sows afflicted with MMAS. Healthy sows TEAC concentration showed no significant fluctuations for the duration of the examination. ACW concentration was below detection limit in almost 70 % of the samples. SOD activity one week before littering in sows afflicted with MMAS was significantly higher at 2725 (2479; 2936) U/g Hb than activity in healthy sows at 2366 (2158; 2663) U/g Hb. SOD activity in healthy sows decreased significantly until the 8 day after littering and decreased significantly on day 15 after littering. No significant fluctuations in SOD activity was detected in sows afflicted with MMAS during the period between one week before littering until 8 day after littering, 15 days after littering it decreased significantly. GPX activity was significantly lower in sows afflicted with MMAS than healthy sows one week before littering at 286 (255; 316) U/g Hb. Following examinations one day after littering showed that GPX activity in sows afflicted with MMAS sank significantly to 239 (178; 277) U/g Hb and held that level during the remainder of the examination period. The concentration of selenium rose significantly in healthy and afflicted sows during the examination. CK and AST activities rose significantly in afflicted sows one day after littering and went back to its basic value of one week before littering. The antioxidant status of healthy and afflicted young and elder sows showed significantly higher selenium concentrations a week before littering in elder sows afflicted with MMAS compared to afflicted younger sows, as well as a significantly higher TEAC concentration on day 15 after littering in elder sows afflicted with MMAS compared to younger afflicted sows. During seasonal comparison, parameters of the september/october group were significantly lower, although it must be considered that with exception of selenium concentrations those significant differences were not detected during the entire examination period. Conclusions: The largest differences in the antioxidant defence system between healthy sows and those afflicted with MMAS were detected on e week before and one day after littering. This leads to assumption that the antioxidant defence of afflicted sows was subjected to more stress before littering than that of healthy sows. Sporadic significant differences of the antioxidant status were detected in age and seasonal comparisons.

Made available in DSpace on 2015-03-26T13:36:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 314133 bytes, checksum: 1d6a5d1c68ecfce7803cec5192e08e88 (MD5) Previous issue date: 2006-06-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tolerance to salt stress was studied in maize seedlings (Zea mays L.) cultivars AGN 3150, BR 106, BR 201, BR 206 and SHS 4040, grown in Hoagland s nutrient solution. Treatments with NaCl 50 and 100 mM led to a decrease in the biomass production and to an increase in the relative electrolyte leakage in the roots and shoots in all cultivars. However, the largest decrease in growth and electrolyte leakage were shown by cultivar BR 106 and the smallest ones by cultivar AGN 3150, lead them to be considered as the most sensitive and the most tolerant to salt stress, respectively. The effect of NaCl 100 mM was investigated by considering some components of the antioxidative metabolism and mitochondrial respiration in roots of the seedlings. The tolerant cultivar showed increased activities of the enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione redutase (GR), as well as increase in ascorbate/dehydroascorbate ratio. The sensitive cultivar showed reduction in catalases (CAT) and GR activities as well as in ascorbate/dehydroascorbate ratio. The lipid peroxidation degree increased in roots and isolated mitochondria, only in the sensitive cultivar. In mitochondria isolated from the tolerant cultivar, NaCl treatment promoted an increase in the state 4 respiration rate and in the alternative pathway capacity. Additionally, salinity promoted a decrease in ADP/O and respiratory control ratios, indicating a lower coupling, likely due to an increase in alternative oxidase (AOX) activity. In sensitive cultivar, total respiration activity and the ADP/O ratio were strongly decreased, however the capacity of the alternative pathway was not altered. The NaCl treatment promoted decrease in the uncoupling protein (UCP) activity in the tolerant cultivar, but an increase it in the sensitive one occurred. These results suggest that the tolerant cultivar exhibits a larger efficiency in the reactive oxygen intermediates scavenging, as compared to the sensitive one. In addition, the high tolerance of cultivar AGN 3150 to the salt stress could be related to its high AOX capacity. A possible contribution of the UCP could not be confirmed from the results observed. / A sensibilidade ao estresse salino foi avaliada em plântulas de milho (Zea mays L.) das cultivares AGN 3150, BR 106, BR 201, BR 206 e SHS 4040, cultivadas em solução nutritiva de Hoagland. Os tratamentos com 50 e 100 mM de NaCl reduziram a produção de biomassa e aumentaram o extravasamento relativo de eletrólitos nas raízes e nas partes aéreas de todas as cultivares estudadas. Porém, os maiores valores de redução no crescimento e de extravasamento de eletrólitos foram apresentados pela cultivar BR 106 e os menores valores observados na cultivar AGN 3150. Portanto, entre as cultivares avaliadas, estas duas foram consideradas, respectivamente, como a mais sensível e a mais tolerante ao estresse salino. Verificou-se, então, o efeito do tratamento com 100 mM de NaCl sobre alguns componentes do metabolismo antioxidativo e sobre a respiração mitocondrial nas raízes de plântulas dessas duas cultivares. A cultivar tolerante apresentou aumento nas atividades das enzimas dismutase do superóxido (SOD), peroxidase do ascorbato (APX) e redutase da glutationa (GR), bem como aumento na razão ascorbato/desidroascorbato. A cultivar sensível apresentou redução na atividade das catalases (CAT) e GR, e também da razão ascorbato/desidroascorbato. O grau de peroxidação de lipídeos aumentou nas raízes e nas mitocôndrias isoladas, apenas na cultivar sensível. Nas mitocôndrias isoladas da cultivar tolerante, o tratamento com NaCl resultou em aumento na taxa respiratória no estado 4 e na capacidade da rota alternativa. Além disso, houve redução nas razões ADP/O e de controle respiratório, indicando um menor grau de acoplamento, possivelmente resultante da maior atividade da oxidase alternativa (AOX) na cultivar tolerante. Na cultivar sensível, a atividade respiratória total e a razão ADP/O foram fortemente reduzidas, porém a participação percentual da rota alternativa não foi alterada. O tratamento com NaCl resultou em redução na atividade da proteína desacopladora (UCP) na cultivar tolerante e em aumento desta atividade na cultivar sensível. Pelos dados obtidos, a maior tolerância da cultivar AGN 3150 está relacionada à sua maior eficiência na remoção de intermediários reativos de oxigênio, além da maior possibilidade de desvio de elétrons pela sua rota alternativa na cadeia respiratória. Entretanto, a contribuição da UCP nos mecanismos de tolerância à salinidade dessa cultivar não foi evidenciada.