Cerca de 20 % dos casos familiares de esclerose lateral amiotrófica (ELAf) são causados por mutações na enzima Cu,Zn-superóxido dismutase (SOD1). Inicialmente se supôs que as enzimas mutantes teriam a atividade SOD comprometida, entretanto isto não foi comprovado. Atualmente, considera-se que as enzimas mutantes adquiram propriedades tóxicas. Quais seriam estas propriedades e como levariam à degeneração do neurônio motor são questões ainda não respondidas. Neste trabalho, comparamos neuroblastomas humanos transfectados com SOD1 G93A associada à ELAf (SH-SY5YG93A), e SOD1 selvagem (SH-SY5YWT) com células parentais (SH-SY5Y) em relação ao crescimento, viabilidade, produção basal de oxidantes, atividades SOD e peroxidásica e modificações estruturais da SOD. As células transfectadas apresentaram aumento na taxa de crescimento e na produção basal de oxidantes. As células SH-SY5YWT e SH-SY5YG93A mantiveram a expressão de SOD1 e atividade consistente com o aumento esperado de duas vezes, em estágios iniciais de cultura. A atividade peroxidásica do homogenato da célula SH-SY5YG93A foi maior. Após quatro semanas, a linhagem SH-SY5YG93A manteve a expressão de SOD1, mas as atividades dismutásica e peroxidásica diminuíram. A expressão de SOD1 aumentou a proporção de formas alteradas de SOD1, como enzima reduzida, multímeros formados por ponte dissulfeto e formas insolúveis em detergente, particularmente na linhagem SH-SY5YG93A. Entre estas formas insolúveis, identificamos um dímero covalente de SOD. Estas formas alteradas provavelmente são responsáveis pela ativação do proteassomo e estresse do retículo endoplasmático, verificados nas células transfectadas. Concluindo, a superexpressão da SOD1 foi suficiente para elevar as formas imaturas e oligomerizadas de SOD1 e a oxidação basal, e a mutação G93A ressaltou estes processos. / Some familial ALS (fALS) are caused by mutations in the Cu,Zn-superoxide dismutase enzyme (SOD1). It was thought that the mutated enzymes would have impaired SOD activity, but this has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a new toxic function. What this new toxic function is and how it relates to the degeneration of motor neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) in regard to growth, viability, basal oxidant production, SOD and peroxidase activities, and SOD forms. Transfected cells presented increased growth rate and basal oxidant production. SH-SY5YWT and SH-SY5YG93A cells in early culture stage showed SOD expression and activity consistent with the expected two-fold increase; SH-SY5YWT homogenates showed increased peroxidase activity. After four weeks, SH-SY5YG93A maintained SOD1 expression levels but peroxidase and dismutase activities were lower. SOD1 expression increased the levels of altered SOD1 forms such as the reduced enzyme, disulfide multimers and detergent-insoluble forms, particularly in SH-SY5YG93A cells. Among the insoluble forms a covalent SOD dimer was identified. These altered SOD forms are probably responsible for proteasome activation and endoplasmatic reticulum stress response verified in transfected cells. In conclusion, SOD1 over-expression was sufficient to increase intracellular immature and oligomerized SOD1 forms and basal oxidation and the G93A mutation enhanced these processes.

Made available in DSpace on 2016-08-10T10:38:40Z (GMT). No. of bitstreams: 1 ALEANDRO GERALDO ALVES.pdf: 687501 bytes, checksum: 815caf3ef15e76a3ef410c769760c097 (MD5) Previous issue date: 2011-08-11 / Amyotrophic lateral sclerosis (ALS) is a multifactorial disease that affects motor neurons. In most cases, the disease is sporadic, however, 5 to 10% of patients have a familial history (FALS). Among patients with FALS, 12 to 23% present with mutations in the SOD1 gene. Objectives: To present a systematic review about the mutations described in SOD1 gene in patients with FALS. Methods: The databases used in this study included PubMed, ISI Web of Science and Cochrane Library Virtual Health. After reading the abstracts, 71 articles were selected and systematically reviewed on this study. Results: The largest number of publications was found in 1997, and Japan was the country with the majority of published studies on the subject, with 23 articles. The majority of the mutations were described in éxons four and five of SOD1 gene, and A4V, I113T, I144F, D90A and L38V were the most commonly mutation described. More than 156 mutations in the SOD1 gene have been cataloged in patients with ALS-F and these data are deposited in ALS GENETICS ONLINE DATABASE, a database that contains specific information on mutations associated with amyotrophic lateral sclerosis. However, the articles reviewed in this study described 103 mutations. Conclusions: Several mutations in the SOD1 gene have been described in patients with ALS-F, however, the relationship between such mutations and the pathogenesis of ALS-F remains unclear, as well as the relationship between mutations and disease progression. Further studies are necessary in order to better explain such relationship. / A esclerose amiotrófica lateral (EAL) é uma doença multifatorial que afeta os neurônios motores. Na maioria dos casos, a doença é esporádica, entretanto, 5 a 10% dos pacientes apresentam história familiar (EAL-F). Dentre os pacientes com EAL-F, 12 a 23% apresentam mutações no gene SOD1. O objetivo deste trabalho foi realizar uma revisão sistemática acerca das mutações descritas no gene SOD1 em pacientes com EAL-F. As bases de dados consultadas incluíram Pubmed, ISI Web of Science e Cochrane Biblioteca Virtual em Saúde. Após a revisão dos resumos, 71 artigos foram selecionados descrevendo mutações no gene SOD1 em pacientes com EAL-F. O ano que apresentou o maior número de publicações foi 1997 e o Japão foi o país que mais publicou sobre o assunto, aparecendo em 23 artigos. O maior número de mutações foi descrito nos éxons 4 e 5 do gene SOD1 e as mutações A4V, I113T, I144F, D90A e L38V foram as mais comumente citadas. Até o momento 156 mutações no gene SOD1 já foram catalogadas em pacientes com EAL-F e esses dados encontram-se depositados no ALS ONLINE GENETICS DATABASE, um banco de dados que contém informações específicas sobre mutações associadas à esclerose amiotrófica lateral. Entretanto, os artigos revisados neste estudo descrevem 103 destas mutações. As causas relacionadas às mutações no gene SOD1 permanecem incertas, assim como a relação entre tais mutações e a evolução da doença, portanto, muito ainda deve ser estudado acerca desse tema.

À ce jour, nos connaissances sur les propriétés structurales et fonctionnelles des métalloprotéines sont essentiellement basées sur des structures résolues par des méthodes de diffraction à rayons X appliquées à des échantillons monocristallins. Cependant, certaines protéines ne cristallisent pas ou cristallisent sous une forme qui n’est pas manipulable ou compatible avec des techniques des diffraction, et même si une structure à très haute résolution est disponible, la nature de l’ion métallique, sa géométrie de coordination ou son état d’oxydation restent souvent indéterminés.La Résonance Magnétique Nucléaire en rotation à l’angle magique (MAS NMR) est une technique très performante pour l’étude de systèmes biologiques et pour la caractérisation de la structure du site actif des métalloprotéines paramagnétiques, mais son application à l’analyse des noyaux proches d’un site paramagnétique est limitée à cause de la résolution et de la sensibilité faibles.L’objectif de cette thèse a été de développer des méthodes RMN basées sur des hautes fréquences de rotation (60-111 kHz MAS) pour faire face à ces problématiques. Un répertoire de séquences d’impulsion pour la détection et l’attribution des noyaux à proximité d’un centre paramagnétique est proposé, et à l’aide de méthodes de calculs de pointes, les données expérimentales acquises sont converties en contraintes structurales afin de déterminer la géométrie du site actif à l’échelle atomique. Cette approche est validée avec l’analyse de sites actifs de deux protéines microcristallines contenants différents ions paramagnétiques : Fe, Cu et Co. Ensuite, des données préliminaires sur un transporteur membranaire d’ions métalliques divalents non cristalline sont présentées.Les méthodes analytiques présentées ici constituent un ensemble d’outils indispensable pour l’élucidation de la structure et la fonction des sites métalliques de systèmes macromoléculaires biologiques. / Most of our understanding of metalloproteins derives from atomic or molecular structures obtained from diffraction methods on single crystal samples. However, not all proteins are amenable for diffraction studies, and even when a highly-resolved structure is available, often the nature of the metal ion, its coordination geometry or its oxidation state are not determined. The aim of the present thesis is the investigation of structural properties of metal sites in paramagnetic metalloproteins by Magic-Angle Spinning Nuclear Magnetic Resonance (MAS NMR). MAS NMR is a powerful technique for the investigation of biological systems, and may represent a direct probe of the structure at the active site of paramagnetic metalloproteins. However, it suffers from limited sensitivity and resolution when applied to nuclei close to a paramagnetic center.In this thesis, we address these limitations by developing NMR methods based on ultra-fast (60-111 kHz) MAS rates. A “toolkit” of suitably designed pulse sequences is built for the detection and the assignment of nuclei in close proximity of a paramagnetic center. State-of-the-art computational techniques are also employed to convert the experimental data into structural restraints for obtaining atomic-resolution geometries of active sites. We benchmark this approach with the study of Fe, Cu and Co sites in two microcrystalline proteins, and we also provide preliminary data on a non-diffracting divalent metal ion transporter in lipid membranes. We anticipate that the techniques described here are an essential tool to elucidate many currently unanswered questions about structure and function of metal sites in structural biology.

Made available in DSpace on 2014-12-17T15:47:00Z (GMT). No. of bitstreams: 1 PlacidoASN_TESE.pdf: 5575988 bytes, checksum: b29fc882ea45700b3c27d134527a79ed (MD5) Previous issue date: 2012-12-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / This thesis presents ⇡SOD-M (Policy-based Service Oriented Development Methodology), a methodology for modeling reliable service-based applications using policies. It proposes a model driven method with: (i) a set of meta-models for representing non-functional constraints associated to service-based applications, starting from an use case model until a service composition model; (ii) a platform providing guidelines for expressing the composition and the policies; (iii) model-to-model and model-to-text transformation rules for semi-automatizing the implementation of reliable service-based applications; and (iv) an environment that implements these meta-models and rules, and enables the application of ⇡SOD-M. This thesis also presents a classification and nomenclature for non-functional requirements for developing service-oriented applications. Our approach is intended to add value to the development of service-oriented applications that have quality requirements needs. This work uses concepts from the service-oriented development, non-functional requirements design and model-driven delevopment areas to propose a solution that minimizes the problem of reliable service modeling. Some examples are developed as proof of concepts / Esta tese apresenta ⇡SOD-M (Policy-based Service Oriented Development Methodology), uma metodologia para a modelagem de aplica??es orientadas a servi?os a qual usa Pol?ticas de qualidade. O trabalho prop?e um m?todo orientado a modelos para desenvolvimento de aplica??es confi?veis. ⇡SOD-M consiste de: (i) um conjunto de meta-modelos para representa??o de requisitos n?o-funcionais associados a servi?os nos diferentes n?veis de modelagem, a partir de um modelo de caso de uso at? um modelo de composi??o de servi?o, (ii) um meta-modelo de plataforma espec?fica que representa a especifica??o das composi?oes e as pol?ticas, (iii) regras de transforma??o model-to-model e model-to-text para semi-automatizar a implementa??o de composi?oes de servi?os confi?veis, e (iv) um ambiente que implementa estes meta-modelos e regras, representando assim aspectos transversais e limita??es associadas a servi?os, que devem ser respeitados. Esta tese tamb?m apresenta uma classifica??o e nomenclatura de requisitos n?o-funcionais para o desenvolvimento de aplica??es orientadas a servi?os. Nossa abordagem visa agregar valor ao desenvolvimento de aplica??es orientadas a servi?os que t?m necessidades de garantias de requisitos de qualidade. Este trabalho utiliza conceitos das ?reas de desenvolvimento orientado a servi?os, design de requisitos n?o-funcionais e desenvolvimento dirigido a modelos para propor uma solu??o que minimiza o problema de modelagem de servi?os web confi?veis

L'objectif de ce travail est de mettre au point des méthodes pour détecter, localiser, quantifier et suivre l'évolution de l'endommagement dans les câbles courts, tels que les suspentes des ponts suspendus, à partir de leurs réponses vibratoires. Afin de modéliser ces câbles, un modèle linéaire 1D de poutre d'Euler Bernoulli avec tension est utilisé. Ce modèle permet de modéliser une large gamme de structures, allant de la corde vibrante à la poutre sans tension. Pour le câble, l'endommagement est introduit dans l'équation vibratoire par des modifications locales de la masse linéique et de la rigidité en flexion et par un changement global de la tension. De plus, pour introduire une "fissure" dans l'équation vibratoire d'une poutre, la modification de la rigidité peut être remplacée par un ressort de rotation au niveau de la fissure. Pour ces deux modèles d'introduction d'endommagements, une estimation analytique au premier ordre des variations des paramètres modaux en fonction des modifications est établie. Grâce aux estimations analytiques obtenues pour la variation relative des fréquences en fonctions des modifications physiques, nous développons des techniques de localisation pour deux cas d'étude : deux essais seuls correspondants à deux états (sain et endommagé) et une série d'essais (plusieurs essais de l'état sain à l'état endommagé). Pour ce second cas, une autre méthode de détection et de localisation utilisant cette fois la SVD est proposée. Les méthodes proposées sont testées sur des données numériques et sur des données expérimentales existant dans la littérature ou effectuées pendant la thèse / The objective of this work is to develop methods to detect, localize, quantify and follow the evolution of the damage in short cables, such as suspenders of the suspension bridges, using their vibratory responses. To simulate these cables, a 1D Euler Bernoulli beam linear model with tension is used. This model allows to study a wide range of structures from the vibrating string to the beam without tension. For cables, damage is introduced into the vibratory equation by local changes of the linear density and the bending stiffness and a global change in the tension. To introduce a crack in the vibrating beam equation, the change in the rigidity may be replaced by a pinned joint at the location ofthe crack. For both these models, a first order analytical estimation of the variation of modal parameters due to theses changes is established. Using these analytical estimations of the relative frequency variations in functions of the physical changes, we develop methods of localization for two cases : only two tests corresponding to two states (healthy and damaged) and a series of tests (several tests on the healthy state and several tests on the damaged state). For the second case, we propose another method of detection and localization which uses the SVD tool . These methods are tested on numerical data and experimental data from literature or from tests performed during the phD.

Cerca de 20 % dos casos familiares de esclerose lateral amiotrófica (ELAf) são causados por mutações na enzima Cu,Zn-superóxido dismutase (SOD1). Inicialmente se supôs que as enzimas mutantes teriam a atividade SOD comprometida, entretanto isto não foi comprovado. Atualmente, considera-se que as enzimas mutantes adquiram propriedades tóxicas. Quais seriam estas propriedades e como levariam à degeneração do neurônio motor são questões ainda não respondidas. Neste trabalho, comparamos neuroblastomas humanos transfectados com SOD1 G93A associada à ELAf (SH-SY5YG93A), e SOD1 selvagem (SH-SY5YWT) com células parentais (SH-SY5Y) em relação ao crescimento, viabilidade, produção basal de oxidantes, atividades SOD e peroxidásica e modificações estruturais da SOD. As células transfectadas apresentaram aumento na taxa de crescimento e na produção basal de oxidantes. As células SH-SY5YWT e SH-SY5YG93A mantiveram a expressão de SOD1 e atividade consistente com o aumento esperado de duas vezes, em estágios iniciais de cultura. A atividade peroxidásica do homogenato da célula SH-SY5YG93A foi maior. Após quatro semanas, a linhagem SH-SY5YG93A manteve a expressão de SOD1, mas as atividades dismutásica e peroxidásica diminuíram. A expressão de SOD1 aumentou a proporção de formas alteradas de SOD1, como enzima reduzida, multímeros formados por ponte dissulfeto e formas insolúveis em detergente, particularmente na linhagem SH-SY5YG93A. Entre estas formas insolúveis, identificamos um dímero covalente de SOD. Estas formas alteradas provavelmente são responsáveis pela ativação do proteassomo e estresse do retículo endoplasmático, verificados nas células transfectadas. Concluindo, a superexpressão da SOD1 foi suficiente para elevar as formas imaturas e oligomerizadas de SOD1 e a oxidação basal, e a mutação G93A ressaltou estes processos. / Some familial ALS (fALS) are caused by mutations in the Cu,Zn-superoxide dismutase enzyme (SOD1). It was thought that the mutated enzymes would have impaired SOD activity, but this has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a new toxic function. What this new toxic function is and how it relates to the degeneration of motor neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) in regard to growth, viability, basal oxidant production, SOD and peroxidase activities, and SOD forms. Transfected cells presented increased growth rate and basal oxidant production. SH-SY5YWT and SH-SY5YG93A cells in early culture stage showed SOD expression and activity consistent with the expected two-fold increase; SH-SY5YWT homogenates showed increased peroxidase activity. After four weeks, SH-SY5YG93A maintained SOD1 expression levels but peroxidase and dismutase activities were lower. SOD1 expression increased the levels of altered SOD1 forms such as the reduced enzyme, disulfide multimers and detergent-insoluble forms, particularly in SH-SY5YG93A cells. Among the insoluble forms a covalent SOD dimer was identified. These altered SOD forms are probably responsible for proteasome activation and endoplasmatic reticulum stress response verified in transfected cells. In conclusion, SOD1 over-expression was sufficient to increase intracellular immature and oligomerized SOD1 forms and basal oxidation and the G93A mutation enhanced these processes.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-25T16:04:47Z No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) / Made available in DSpace on 2016-05-25T16:04:47Z (GMT). No. of bitstreams: 1 Nicole Cristine Rigonato de Oliveira.pdf: 1240943 bytes, checksum: 14fa67b6692555f3bc76fb1f34186220 (MD5) Previous issue date: 2014-12-19 / The acute respiratory distress syndrome (ARDS) is a disease characterized by respiratory failure due to an inflammatory response, which has high morbidity and mortality. Several cytokines seem to orchestrate the acute and chronic processes, mainly mediated by toll like receptors (TLRs). The literature demonstrates that aerobic exercise (AE) is capable of decreasing the secretion of pro-inflammatory cytokines in the lungs mediated increased release of interleukin 10 (IL-10), and AE modulate expression of TLRs and antioxidant enzymes. The objective of this study was to evaluate whether the anti-inflammatory effects of AE in an experimental model of intra and extrapulmonary ARDS are mediated by IL-10. For this, the animals were subjected to physical training on a treadmill, moderate for 4 weeks. 24 hours after the last physical test, animals received LPS by intratracheally (it) (10ug / animal) or intraperitoneally (ip) (100ug / animal). After 24 hours, the animals were assessed for the number of cells and pro- and anti-inflammatory cytokines in bronchoalveolar lavage fluid (BAL) and serum were the number of neutrophils in the lung parenchyma and expression of TLR4, TLR7, SOD, ânion and QL in lung homogenates. AE reduced accumulation of neutrophils in the lung parenchyma, so it LPS and LPS ip (p <0.01) and BAL (p <0.01). AE attenuate the levels of proinflammatory cytokines in BAL and serum (p <0.05), as measured by ELISA. AE had levels of anti-inflammatory cytokine IL-10 increased in the serum and BAL (p <0.05). AE also reduced the expression of TLR4 in lung homogenates (p <0.05) and increased the expression of TLR7 in group LPS + AE evaluated by western blotting (p <0.05). The results for the antioxidant enzyme SOD showed a significant increase in the groups submitted to AE. It follows that the impact of reduced LPS-induced ARDS, regardless of etiology, and these effects appear to be mediated by modulation of the AE secretion of anti-inflammatory cytokines, especially IL-10, modulating TLR4 and TLR7 and increased expression of SOD (p <0.05). / A síndrome do desconforto respiratório agudo (SDRA) é uma doença caracterizada pela insuficiência respiratória decorrente a uma resposta inflamatória, que apresenta alta morbi-mortalidade. Diversas citocinas parecem orquestrar os processos agudo e crônico, mediados principalmente por receptores toll like (TLRs). A literatura demonstra que o exercício aeróbio (EA) é capaz de diminuir a secreção de citocinas pró-inflamatórias nos pulmões, mediado pelo aumento da liberação de interleucina 10 (IL-10), além de o EA modular a expressão de TLRs e enzimas antioxidantes. Assim, o objetivo desse estudo foi avaliar se os efeitos anti-inflamatórios do EA em modelo experimental da SDRA intra e extrapulmonar são mediados por IL-10. Para isso, os animais foram submetidos ao treinamento físico em esteira, de intensidade moderada, durante 4 semanas. Após 24 horas ao último teste físico, os animais receberam LPS por via intra-traqueal (it) (10ug/animal) ou por via intra-peritoneal (ip) (100ug/animal). Após 24 horas, os animais foram avaliados para o número de células e de citocinas pró e anti-inflamatórias no fluído do lavado broncoalveolar (LBA) e no soro, número de neutrófilos no parênquima pulmonar e expressão do TLR4, TLR7, SOD, ânion e QL nos homogenatos de pulmão. EA reduziu a acumulação de neutrófilos no parênquima pulmonar, tanto LPS it e LPS ip (p <0,01) e no LBA (p <0,01). EA atenuou os níveis de citocinas pró-inflamatórias no LBA e soro (p <0,05), avaliada por ELISA. EA teve os níveis da citocina anti-inflamatória IL-10 aumentados no soro e LBA (p <0,05). EA também reduziu a expressão de TLR4 nos homogenatos de pulmão (p <0,05) e aumentou a expressão de TLR7 no grupo de LPS + EA avaliada por western blotting (p <0,05). Os resultados para a enzima antioxidante SOD apresentou aumento significante nos grupos submetidos ao EA. Conclui-se que EA reduziu o impacto de SDRA induzida por LPS, independente da etiologia e tais efeitos parecem estar mediados pela modulação do EA na secreção de citocinas anti-inflamatórias, principalmente da IL-10, na modulação de TLR4 e TLR7 e no aumento da expressão de SOD (p<0,05).

5 pp. / Eight different landscape mulches were tested for their flammability using a propane torch, charcoal briquette, and a cigarette at two different times of the year. Three randomized compete blocks with eight one square meter plots were tested at three locations; Tucson, Prescott, and Flagstaff, Arizona. Each of the mulches was subjected to the heat of a handheld propane torch (15 seconds), a glowing charcoal briquette (five minutes), and a lit cigarette (until burned out). We found that the least dense mulches (pine needles and straw) burned rapidly when subjected to the torch and ignited after the briquette was removed. The medium density mulches (pine bark nuggets and wood chips) had low flame lengths and smoldered. Heavy density mulches (garden compost and shredded bark) only smoldered. The decomposed granite and sod did not ignite or smolder.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T17:23:20Z No. of bitstreams: 1 Suzi Wollenhaupt.pdf: 1934742 bytes, checksum: 05b51e77aeefc91c2211fee9a7cd6131 (MD5) / Made available in DSpace on 2016-04-18T17:23:20Z (GMT). No. of bitstreams: 1 Suzi Wollenhaupt.pdf: 1934742 bytes, checksum: 05b51e77aeefc91c2211fee9a7cd6131 (MD5) Previous issue date: 2013-05-10 / Compostos orgânicos de selênio (Se) e telúrio (Te) apresentam propriedades antioxidantes em muitos modelos de estresse oxidativo. No entanto, devido à complexidade dos modelos de mamíferos, tem sido difícil de determinar as vias moleculares e proteínas específicas que são moduladas em resposta aos tratamentos com esses compostos. Neste contexto, o presente trabalho investigou os efeitos e possíveis mecanismos de ação de uma nova classe de compostos orgânicos de Se e Te chamados Xilofuranosídeos, utilizando como modelo experimental alternativo o nematóide Caenorhabditis elegans (C. elegans). Tal modelo permite fácil manipulação genética, marcação de diversas proteínas com proteína verde fluorescente e análise de toxicidade in vivo e ao vivo. Neste estudo, desafiamos os nematóides ao manganês (Mn), um agente pró-oxidante conhecido, uma vez que evidências apontam que o estresse oxidativo é consequência da sua toxicidade. Utilizando este agente pró-oxidante, investigamos a eficácia do Se e Te-xylofuranosídeos em reverter e/ou proteger os vermes da toxicidade induzida por Mn. Adicionalmente, investigamos um suposto mecanismo de ação. Primeiramente encontramos a dose letal 50% dos compostos, as quais foram de 0,73mM e 0,8mM para os compostos contendo Se e Te, respectivamente. Em concentrações subletais, encontramos que ambos Se e Te-xylofuranosídeo reverteram à mortalidade induzida por Mn, diminuíram a produção de espécies reativas de oxigênio (ERO) e aumentaram a expressão da superóxido dismutase (SOD-3::GFP), indicando que o aumento na sobrevivência está associado com a diminuição do estresse oxidativo. Além disso, observamos que os Se e Te-xylofuranosídeos induzem a translocação nuclear do fator de transcrição DAF-16/FOXO, que no viii verme é conhecido por regular a resposta ao estresse, envelhecimento e metabolismo e tem também como gene alvo a sod-3, corroborando com o aumento na expressão da proteína codificada por este gene. Esses achados sugerem que os Se e Te-xylofuranosídeos atenuam a geração de espécies reativas induzidas por Mn através da regulação da via de sinalização DAF-16/FOXO. / Organoselenium and organotellurium compounds have been reported as antioxidant in several models of oxidative stress. Nevertheless, because of the complexity of mammalian models, it has been difficult to determine the molecular pathways and specific proteins that are modulated in response to treatments with these compounds. In this context, the present study investigated the effects and action mechanisms of a novel class of organic compounds of selenium (Se) and tellurium (Te) called Xylofuranosides, utilizing as an alternative experimental model the nematode Caenorhabditis elegans (C. elegans), that affords easy genetic manipulations, green fluorescent protein tagging and in vivo live analysis of toxicity. In this study, we challenged worms to manganese (Mn), a known pro oxidizing agent, as there is abundant evidence pointing out to oxidative stress in mediating its toxicity. We investigated the efficacy of Se- and Te- xylofuranosides in reversing and/or protecting the worms from Mn-induced toxicity. In addition, we investigated their putative mechanism of action. First, we found the lethal dose 50% (LD50) for the compounds, which were 0.73mM and 0.8mM for Se and Te compound, respectively. This was followed by studies on the ability of the xylofuranosides to afford protection against Mn-induced toxicity. Both Se and Te-xylofuranosides reversed the Mn-lethality, decreased reactive oxygen species (ROS) production and increased the expression of superoxide dismutase (SOD-3), indicating that the increased survival was associated with decreased oxidative stress. Furthermore, we observed that the xylofuranosides induced nuclear translocation of the transcriptional factor DAF-16/FOXO, which in the worm is known to regulate stress responsiveness, aging, metabolism and the expression of SOD-3, as verified in this study. These findings x suggest that xylofuranosides attenuate Mn-induced ROS generation by regulating the DAF-16/FOXO signaling pathway.

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