O fenômeno da tolerância ao lipopolissacarídeo ocorre quando animais expostos a pequenas doses de LPS tornam-se hiporresponsivos a doses letais posteriores. Tendo em vista que a capacidade do hospedeiro de tolerar a presença de agentes patogênicos é uma estratégia do organismo humano e que reduz o impacto negativo durante a sepse, o objetivo do estudo foi investigar se a tolerância ao lipopolissacarídeo resulta em efeitos benéficos na atividade das enzimas antioxidantes e investigar possíveis diminuição da peroxidação lipídica, durante o estresse oxidativo. Após verificarmos a atividade das enzimas antioxidantes concluímos que tolerância ao lipopolissacarídeo tem um papel importante no equilíbrio redox, onde houve aumento da expressão de superóxido dismutase (SOD), nos animais tolerantes submetidos a ligadura e punção cecal, defendendo o hospedeiro de forma que o mesmo não respondesse de forma exacerbada nas exposições subsequentes a produtos de microrganismos patogênicos / Sepsis has recently been classified as a fatal organic dysfunction caused by dysregulation of the immune response to a particular infection. It is a serious condition that can cause failure of one or multiple organs. It represents the main cause of death in patients treated in an intensive care unit (ICU). Most registered cases of sepsis are due to gram-negative bacteria. Lipopolysaccharide (LPS), a component of the outer membrane of the bacterium, triggers a cascade of activities of the immune system. This response to LPS produces several inflammatory mediators and can generate oxidative stress also known as redox imbalance through the release of reactive oxygen species (EROS). Considering that the host\'s ability to tolerate the presence of pathogens is a strategy of the human organism and reduces the negative impact of the disease, we propose to investigate whether endotoxemia results in beneficial changes, maintaining the redox balance during the sepsis period. After verifying the relative mRNA expression and protein quantification, we conclude that endotoxemia caused by lipopolysaccharide plays an important role in the immunoregulation of the inflammatory response defending the host so that it does not respond exacerbatedly in subsequent exposures to products of pathogenic microorganisms

A atividade contrátil no músculo esquelético é um estímulo potente na indução de alterações no metabolismo de glicose e ácidos graxos. Por sua vez, a contração muscular também aumenta a produção de espécies reativas de oxigênio (EROs). O objetivo do presente trabalho foi avaliar o efeito da remoção de EROs, induzida pelo tratamento com o antioxidante N-Acetilcisteína, na captação de glicose, atividades de enzimas glicolíticas e do ciclo de Krebs, produção de lactato, oxidação mitocondrial de ácidos graxos, expressão dos genes do transportador de glicose 4 (GLUT-4), hexoquinase II (HKII), fosfofrutoquinase 1 (PFK-1), carnitina palmitoil transferase 1 (CPT-1) e citrato sintase (CS) em células musculares esqueléticas durante contrações induzidas por eletroestimulação in vitro. Com esse estudo, demonstrou-se que as EROs atuam como sinalizadores das respostas metabólicas mediadas pela atividade contrátil e que a sinalização redox regula o metabolismo de glicose e ácidos graxos em células musculares esqueléticas. / Contractile activity is a potent stimulus for induce changes in glucose and fatty acid metabolism in skeletal muscle. During muscle contraction, the production of reactive oxygen species (ROS) is increased. The purpose of this study was evaluate the effect of removal of ROS, induced by treatment with the antioxidant N-Acetylcysteine, on glucose uptake, activities of glycolytic and TCA cycle enzymes, lactate production, mitochondrial fatty acid oxidation, gene expression of glucose transporter 4 (GLUT-4), hexokinase II (HKII), phosphofructokinase 1 (PFK-1), carnitine palmitoyl transferase 1 (CPT-1) and citrate synthase (CS) mediated by moderate contractions induced by electrical stimulation in vitro. In this study we demonstrated that ROS act as signaling molecules on contraction-mediated metabolic responses and that redox signaling regulates glucose and fatty acid metabolism in skeletal muscle cells.

O nim (Azadirachta indica) é conhecido na Ásia devido a várias propriedades biológicas conhecidas desde a antigüidade. Os estudos referentes à ação inseticida dessa planta restringem-se a análise de seus mecanismos de ação sobre insetos e também de seus efeitos sobre trabalhadores rurais que fazem uso de produtos a base de nim; não havendo, na literatura pesquisada, trabalhos relativos aos impactos causados sobre o sistema vegetal. As plantas, assim como outros organismos, possuem a capacidade de se defenderem contra ataque de patógenos. Uma das respostas desencadeadas pelo reconhecimento do patógeno pelas células vegetais é a reação de hipersensibilidade (RH), que envolve a morte imediata das células do sítio primário de infecção, oferecendo resistência ao crescimento do patógeno. A RH é caracterizada pela necrose dos tecidos onde primeiro se manifestou a infecção, e este processo de morte celular programada envolve uma série de sinais que ainda não estão completamente elucidados. Neste trabalho, foram estabelecidas as condições do meio de cultura de células de Rubus fruticosus para os estudos com extrato de sementes de nim, avaliado o efeito elicitor deste sobre a cultura. Foram obtidos extratos hidroalcoólicos E1 e E2 e suas respectivas frações lioflizadas, L1 e L2. Estes extratos apresentaram maior teor de açúcares e lipídeos em sua composição e revelaram potencial antioxidante. Detectou-se a presença de AZA-A em L1 e L2, por meio de CLAE, cujos teores foram de 5,03 e 1,1 mg/mL, respectivamente, com tempo de retenção em torno de 9,5 minutos, confirmado por meio de análises via espectrometria de massas. O extrato L2 foi fracionado nas frações L2 inicial e AZA2. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e destas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. O extrato L2, nas concentrações de 0,1; 0,5; 1 e 5 mg/mL, e suas frações AZA2 e L2 inicial nas proporções do extrato L2 nestas concentrações, elicitaram células de Rubus fruticosus. As células de Rubus fruticosus (1,8g) foram incubadas em tampão citrato de sódio contendo o extrato L2 e as frações L2 inicial e AZA2, separadamente, até a concentração de 5 mg/mL, por 1h, em temperatura ambiente. Após este período, os compostos fenólicos, proteínas e açúcares redutores foram determinados no meio extracelular e intracelular por métodos colorimétricos. O efeito destas frações e do extrato L2, na produção de EROs em células intactas de Rubus fruticosus, foi analisado usando a sonda diacetato 2,7-diclorofluoresceína (LEE et al., 1999; MURATA et al., 2001). Os resultados obtidos indicam que AZA isolada não teve efeito sobre respostas de defesa. A fração L2 inicial teve aumento de fenólicos intracelulares, de açúcares redutores extracelulares e diminuição de EROs, com o aumento da concentração do elicitor, indicando potencial antioxidante e mecanismo de defesa. O extrato L2 também demonstrou potencial antioxidante e protetor das células com o aumento da concentração do elicitor, além de possuir ação inseticida. / The neem (Azadirachta indica) is known in Asia due to their several biological properties. The studies on the insecticide action of neem extracts have only been restrict to the insect mechanisms and their effects on rural workers; studies on the impact in the vegetable system are not available. The plants, like the other organisms, have the ability to self-defend against attack of patogens. The hypersensitive response (RH), a type of programmed cell death (PCD) in plants, is triggered by plant cells when they recognize the patogen, and is characterized by necrosis of tissues in the local region surrounding the infection; the signals involved are still not completely elucidated. The present study evaluated the effects of neem extracts in Rubus fruticosus cell. The powdered seeds were submitted to two consecutive extractions with ethanol:water (1:1, v/v) at room temperature for 10 minutes, yielding E1 and E2 fractions. The solvent was evaporated and the aqueous extracts were concentrated and lyophilized, resulting in two samples, L1 and L2. They are used for analyses by high performance liquid chromatograph (HPLC) in C-18 column (4.6 x 250 mm), with acetonitrile-water (4:6 v/v) as mobile phase, flow rate 1 mL/min, monitored at 214 nm. The principal compound of this fraction was azadirachtin (5.03 and 1.1 mg/mL, respectively), the retention time was 9.5 min; it was confirmed using mass spectrophotometry. The L2 extract was partially fractionated by high performance liquid chromatograph (HPLC) in semi preparative C-18 column. The main fractions, analyzed by colorimetric methods, ESI-MS, were L2 initial and AZA2. The Rubus fruticosus cells (18-21 days; 1.8 g) were incubated in sodium citrate buffer containing L2, L2 initial ans AZA2 at concentrations up to 5 mg/mL, for 1h, at room temperature. After this period, the phenolic compounds, proteins and reducing sugar were determined in the extracellular and intracellular medium by colorimetric methods. Also, the effects of these fractions over the production of reactive oxygen species (ROS) in intact cell of Rubus fruticosus, was analyzed using 2,7-dichloro-fluorescein diacetate. AZA2 had no effect on the defense response. The initial L2 fraction increased the phenolic compounds in the intracellular medium and the reducing sugars in the extracellular medium. The same fraction showed an inhibitory effect on ROS and also increased the concentration of the elicitor. These results indicate the antioxidant potential and protector effect of the L2 initial. The L2 extract also demonstrated antioxidant and protective potential of cells with the increase of the elicitor concentration. Therefore, in parallel with its insecticide action, the neem extract contributes to the self-defense ability of the plants.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-05-09T19:13:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeito neuroprotetor de Anacardium Microcapum-duke em dano induzido por 6-ohda em córtex cerebral de Pintainhos.pdf: 1865633 bytes, checksum: 3c3083e33b14387c66dd69ed3da41472 (MD5) / Made available in DSpace on 2017-05-09T19:13:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Efeito neuroprotetor de Anacardium Microcapum-duke em dano induzido por 6-ohda em córtex cerebral de Pintainhos.pdf: 1865633 bytes, checksum: 3c3083e33b14387c66dd69ed3da41472 (MD5) Previous issue date: 2017-04-05 / A Doença de Parkinson (DP) é uma doença degenerativa, crônica e progressiva, acomete o sistema nervoso central e é responsável pela degeneração dos neurônios dopaminérgicos. Sabese que alterações genéticas e ambientais como exposição a agroquímicos, estresse oxidativo e disfunção mitocondrial estão associados à progressão da doença. A neurotoxina 6hidroxidopamina (6-OHDA), é um análogo estrutural da catecolamina dopamina, servindo como um modelo de neurotoxicidade por mecanismos semelhantes ao observado na DP. O mecanismo de citotoxicidade atribuído a 6-OHDA está diretamente ligado com a produção de espécies reativas de oxigênio (EROs) oriundas da inibição da respiração mitocondrial e sua auto-oxidação. A busca por terapias alternativas como os antioxidantes tem crescido ao longo dos anos, buscando atenuar a progressão da DP através de compostos bioativos de plantas. Neste estudo buscou-se avaliar o efeito neuroprotetor de Anacardium microcarpum frente ao dano induzido pela neurotoxina 6-OHDA em fatias corticais de pintainhos. Fatias foram incubada por 2h na presença da neurotoxina e diferentes concentrações do extrato hidroalcoólico (AMHE) e frações acetato de etila (AMEAF) e metanólica (AMMF) de A. microcarpum. AMHE, AMMF e AMEAF (1-1000 µg/mL) não apresentaram citotoxicidade per se nas fatias corticais. AMMF e AMEAF restauraram a queda da viabilidade induzida por 6OHDA (500 µM) a partir da concentração de 100 µg/mL, sendo a AMMF nesta mesma concentração, capaz de reverter a peroxidação lipídica causada pelo composto. 6-OHDA aumentou a atividade de GST e TrxR e diminuiu a atividade da GPx, além de diminuir os níveis de GSH total e aumentar a razão GSH/GSSG. Tais efeitos não foram observados na presença de AMME. Ainda, 6-OHDA inibiu o complexo I da cadeia respiratória mitocondrial, sendo que a fração não reverteu este efeito. Além disso, a auto-oxidação de 6-OHDA não foi revertida pela planta. A fosforilação de p38, JNK1/2, ERK1/2 e AKT bem como clivagem de PARP foi avaliada frente ao tratamento com neurotoxina e fração metanólica. A fração não levou a aumentos significativos na fosforilação das MAPKs bem como na expressão destas, também não levou à clivagem da proteína PARP. Entretanto, na presença de fração e 6-OHDA houve aumento significativo na fosforilação de ERK1/2 sem alterar sua expressão. Averiguou-se o envolvimento de ERK1/2 e AKT, proteínas envolvidas nos mecanismos de sobrevivência, na neurotoxicidade induzida por 6-OHDA através do uso de inibidores. Observou-se que na presença de inibidor, o extrato não foi capaz de proteger contra o dano promovido por 6-OHDA. Nossos resultados sugerem que o extrato tem ação antioxidante contra o estresse oxidativo decorrente da inibição da respiração mitocondrial e auto-oxidação da neurotoxina, sem no entanto interferir nestes processos. Além disso, sugere-se que as proteínas anti-apoptóticas ERK1/2 e AKT estejam envolvidas no efeito neuroprotetor da fração por mecanismos ainda não conhecidos. Nossos dados mostram pela primeira vez a ação neuroprotetora de A. microcarpum frente ao dano neuronal induzido pela 6-OHDA em fatias cerebrais e / Parkinson's disease (PD) is a degenerative, chronic and progressive disease, which affects the central nervous system and it is responsible for degeneration of dopaminergic neurons. It is known that genetic and environmental factors such as exposure to agrochemical, oxidative stress and mitochondrial dysfunction are associated with progression of the disease. 6hydroxydopamine (6-OHDA) is a structural analogue of catecholamine dopamine, used as a model of neurotoxicity by similar mechanism in PD. The mechanism of cytotoxicity attributed to 6-OHDA is linked to the production of reactive oxygen species (ROS) from inhibition of mitochondrial respiration and its autoxidation. The search for alternative therapies such as antioxidants has grown over the years, seeking to mitigate the progress of PD through bioactive plant compounds. This study aimed to evaluate the neuroprotective effect of Anacardium microcarpum on the damage induced by neurotoxin 6-OHDA in cortical slices of chicks. Slices were incubated for 2 h in the presence of neurotoxin and different concentrations of hydroalcoholic extract (AMHE) and ethyl acetate (AMEAF) and methanol (AMMF) fractions of A. microcarpum. AMHE, AMMF and AMEAF (1-1000 μg/mL) did not show cytotoxicity per se in the cortical slices. AMMF and AMEAF restored the drop in viability caused by 6OHDA (500 μM) from the concentration of 100 μg/mL. 6-OHDA increased GST and TrxR activity while GPx activity and total GSH levels was decreased with an augmented ratio GSH / GSSG. These effects were not observed in the presence of AMMF and 6-OHDA. Furthermore, 6-OHDA inhibited the complex I of the mitochondrial respiratory chain but this effect was not reversed by fraction as well as the self-oxidation of 6-OHDA was not avoided by the plant. Phosphorylation of p38, JNK1/2, ERK1/2 and AKT as well as PARP cleavage was evaluated against treatment with neurotoxin and methanolic fraction. The methanolic fraction and 6OHDA did not alter phosphorylation of MAPKs, as well as expression of these proteins, not did it result in the cleavage of the PARP protein in the time studies. However, in the presence of fraction and 6-OHDA there was a significant increase in ERK1/2 phosphorylation without altering its expression. The involvement of ERK1/2 and AKT proteins in protective mechanism of fraction was analyzed through the use of inhibitors. It was observed that in presence of inhibitor, the extract was not able to protect against the damage promoted by 6-OHDA. Our results suggest that the extract presented antioxidant action against oxidative stress resulted from the inhibition of mitochondrial respiration and neurotoxin autoxidation. In addition, it is suggested that antiapoptotic proteins ERK1/2 and AKT are involved in neuroprotective effect of the fraction. Our data show for the first time a neuroprotective action of A. microcarpum against neuronal damage induced by 6-OHDA in cerebral slices and highlights the potential of this plant as a source of bioactive compounds with therapeutic potential

Defeitos peroxissomais estão associados a diversas doenças complexas. O peroxissomo é responsável pela beta-oxidação de ácidos graxos, quando é gerado peróxido de hidrogênio. A catalase A, de ocorrência peroxissomal, é frequentemente considerada a única defesa antioxidante dessa organela, porém, em diversos organismos, a ausência dessa enzima não acarreta uma alteração fenotípica clara. Em Saccharomyces cerevisiae, linhagens mutantes deficientes em catalase A (Δcta1) apresentam viabilidade muito similar à linhagem selvagem correspondente. Trabalhamos com a hipótese de que peroxidases baseadas em cisteína compensam a ausência de catalase A, contribuindo para a detoxificação de peróxidos provenientes do peroxissomo. De fato, linhagens com os genes para as peroxirredoxinas Ahp1 e Tsa1 nocauteados mostraram-se mais sensíveis a hidroperóxido de terc-butila (tBHP) em comparação a linhagem selvagem. A linhagem de levedura deficiente nas cinco peroxirredoxinas (prxΔ) mostrou-se ainda mais sensível a tBHP. Em relação ao estresse induzido por peróxido de hidrogênio, a prxΔ apresentou maior sensibilidade do que as linhagens selvagem e mutantes com deleções simples, apesar da presença de catalases (peroxissomal e citossólica). Esses dados estão de acordo com resultados obtidos no nosso grupo demonstrando um aumento da expressão de genes referentes às peroxirredoxinas Ahp1, Prx1 e Tsa2 em células Δ cta1 crescidas em condições de alta atividade peroxissomal (oleato), indicando uma cooperação entre catalase e peroxirredoxinas na proteção antioxidante. A peroxirredoxina Ahp1 pode apresentar localização organelar (possivelmente mitocondrial ou peroxissomal), o que sugere que Ahp1 pode ser um componente relevante da defesa contra oxidantes provenientes do peroxissomo. No entanto, a linhagem Δ ahp1, normalmente sensível a peróxido orgânico, apresentou ganho de resistência na ausência de atividade de catalase (com a adição de ATZ e na linhagem duplo-mutante Δcta1/ahp1), indicando a existência de uma via antioxidante compensatória induzida pela ausência de catalase A. A construção das linhagens duplo-mutantes Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δ cta1/prx1 e Δcta1/dot5 foi realizada com o objetivo de investigar mecanismos compensatórios entre enzimas que podem proteger a levedura contra os oxidantes provenientes do peroxissomo. Para tanto, foram realizados ensaios de viabilidade comparativa em condições de alta atividade peroxissomal. Além disso, os níveis comparativos de proteínas carboniladas foram analisados nessas linhagens. Os resultados indicaram maior sensibilidade a peróxido e maiores níveis de danos oxidativos na linhagem Δcta1/tsa2, apontando a peroxirredoxina Tsa2 como candidata a importante componente da via antioxidante de compensação à ausência de catalase A. Nesses ensaios, também foram utilizadas a linhagem quíntupla mutante (prxΔ) e uma linhagem deficiente nas cinco peroxirredoxinas e três glutationa peroxidases - deficiente em oito tiól-peroxidases baseadas em cisteína (Δ8). A comparação das linhagens prxΔ e Δ8 com as linhagens selvagem, simples-mutantes e duplo-mutantes evidenciou a importância das peroxirredoxinas na defesa antioxidante da célula e o fato das tiól-peroxidases serem imprescindíveis em condições de estresse oxidativo. Ao examinar a expressão gênica de TSA2 em células crescidas em oleato, foi verificada a indução do gene na ausência de catalase A, em condição basal. Os resultados obtidos indicam a existência de uma eficiente via de defesa antioxidante, na qual estão envolvidas tiól-peroxidases, que compensa a ausência de catalase A na célula e que protege leveduras contra estresse induzido tanto por peróxido de hidrogênio como peróxido orgânico. A peroxirredoxina Tsa2 parece estar envolvida na via compensatória à ausência de catalase peroxissomal através de um mecanismo ainda não esclarecido / Defects in peroxisomes are associated with several complex diseases. Beta-oxidation of fatty acids takes place in these organelles, with the concomitant generation of hydrogen peroxide. Generally, it is assumed that peroxisomal catalase is the enzyme responsible for degradation of hydrogen peroxide, but in several organisms, deletion of its gene results in no clear phenotype. In Saccharomyces cerevisiae, catalase A- null (Δcta1) mutant strains exhibit very similar viability levels when compared with the corresponding wild-type strain. We hypothesized here that Cys-based peroxidases compensate the absence of catalase A, contributing to the detoxification of peroxides derived from the peroxisome. Indeed, null mutante strains for the peroxiredoxins Ahp1 and Tsa1 displayed increased sensitivity for tert-butylhydroperoxide (tBHP) in comparison to the wild type strain. Furthermore, a mutant strain whose five genes for peroxiredoxins were interrupted (prxΔ) was even more sensitive to tBHP. In regards to hydrogen peroxide insult, the prxΔ strain was more susceptible to oxidative stress than the single mutant and wild-type strains, despite the activity of catalases. These data are in agreement with previous results from our group demonstrating increased expression of genes encoding the three peroxiredoxin enzymes: Ahp1, Prx1 and Tsa2 in Δcta1 cells at high peroxisomal activity (media containing oleate). Indeed, a yeast strain deleted of all five peroxiredoxin genes is more sensitive to peroxides than the corresponding wild type cells. These results indicated that catalase and peroxiredoxins cooperate to protect yeast in conditions of high fatty acid intake. There are evidences of an organellar location of Ahp1 (possible peroxisomal or mitochondrial), suggesting it could be a relevant component of antioxidant defense relative to the insult derived from the peroxisome. Nonetheless, the ahp1-null strain (Δahp1), which is usually sensitive to organic peroxide, displayed a gain of resistance in the absence of catalase activity (in the presence of ATZ and in the double-mutant strain Δcta1/ahp1), indicating the existance of a compensatory antioxidant pathway induced in the absence of catalase A. The double-mutant strains Δcta1/ahp1, Δcta1/tsa1, Δcta1/tsa2, Δcta1/prx1 and Δcta1/dot5 were developed in order to elucidate the identity of the enzymes that cooperate to protect yeast against oxidative insult derived from the peroxisome. To this end, comparative viability assays in conditions of high peroxisomal activity were realised, as well as assays in comparative total protein carbonyl levels. Among the double-mutant strains, Δcta1/tsa2 displayed higher sensibility to peroxide and higher levels of oxidative damage, suggesting that the peroxiredoxin Tsa2 may be an important component in the antioxidant pathway that compensates the lack of catalase A. In addition, a quintuple mutant strain, lacking all peroxiredoxins, and a mutant strain lacking all eight Cys-based, thiol peroxidases were used in these assays. The comparison of these strains with the wild-type, single-mutant and double-mutant strains demonstrated the importance of peroxiredoxins in the cellular antioxidant defence and that thiol-peroxidases are vital in conditions of oxidative stress. The expression of the TSA2 was induced in the absence of catalase A in cells grown in oleate and with no exogenous oxidants. The results suggest the existence of an efficient pathway of antioxidant defense, involving thiol-peroxidases, which compensates the absence of catalase A in the cell and protects yeast against oxidative stress induced by both hydrogen peroxide and organic peroxide. The peroxiredoxin Tsa2 may be involved in the antioxidant pathway that compensates the absence of peroxisomal catalase through an unknown mechanism.

Neste trabalho sintetizaram-se novos complexos imínicos e diimínicos de Cu(II) derivados da isatina, um indol endógeno, que foram então extensivamente caracterizados por análise elementar, medidas de condutividade molar, técnicas espectroscópicas: IV, UV/Vis e EPR, e por espectrometria de massa, ESI-MS. Estes compostos apresentaram equilíbrio ceto-enólico em solução, variando a geometria ao redor do íon Cu(II), ao se alterar o pH do meio, indo de tetraédrico em meio ácido à tetragonal em meio básico. Para a elucidação do papel do cobre no mecanismo de oxidação de carboidratos pelo oxigênio molecular, realizaram-se estudos cinéticos tendo como catalisadores estes complexos de cobre. Determinou-se a uma lei cinética global mais abrangente para estas reações, incluindo uma etapa dependente do cobre (a concentrações bem baixas), seguida de outra independente do metal. As etapas no mecanismo proposto, sob condições de pseudo-primeira ordem, combinam transferência eletrônica intramolecular, com provável redução do íon de cobre pelo substrato, levando a espécies muito reativas (OH•-, O2•-, H2O2, CO2•-), responsáveis pelo processo de iniciação e propagação, e a formação de produtos carbonílicos de cadeia curta (glicolato, glicerato e formiato). Para o estudo da interação metal-carboidrato, importante para se entender melhor o papel do cobre frente a este ligante biológico, complexos de Cu(II) com monossacarídeos foram sintetizados e caracterizados por análise elementar e termogravimétrica, medidas de condutividade molar, espectroscopias UV/Vis, IV e EPR, além da espectroscopia Raman, através da qual investigou-se o modo de ligação do carboidrato ao metal em cada um destes complexos. Foram ainda preparados e caracterizados por análise elementar, medidas de condutividade molar, espectroscopias UV/Vis, IV, EPR, ESI-MS e CD dois novos complexos imínicos quirais de Cu(II), com ligantes do tipo aminocarboidrato, e que também foram utilizados para estudos biológicos. Estudos de atividade biológica foram feitos in vitro, usando as linhagens celulares tumorais promonocítica sanguínea U937 e neuroblastoma SH-SY5Y, com os complexos imínicos de cobre, principalmente aqueles derivados da isatina. As células tratadas com os complexos mais ativos sofreram apoptose, verificada por ensaios citofluorimétricos, em que os complexos agiram em diferentes fases do ciclo celular de cada linhagem (fase G1, S ou G2/M). Através da técnica citofluorimétrica foi observada também a geração de radicais livres na célula e, através de ensaios imunológicos, determinou-se a quantidade de proteínas citoplasmáticas carboniladas e glicosiladas, geradas após os tratamentos com os compostos, em diferentes tempos de incubação. De uma maneira geral, estes estudos indicaram modulação da atividade biológica, com um comportamento antiproliferativo muito diferente, indo de baixa eficácia até alta eficiência. Dentre os compostos mais ativos, pode ser observada uma especificidade diferente, tanto com relação ao tipo de célula quanto ao seu modo de ação, evidenciando sua potencial aplicação como agentes antitumorais. / In this work, novel imine and diimine copper(II) complexes with ligands derived from isatin, an endogenous indol, were synthesized, and extensively characterized by elemental analysis, conductivity measurements, spectroscopic techniques (IR, UV/Vis, and EPR), and electrospray mass spectrometry (ESI-MS/MS). These compounds showed a keto-enolic equilibrium in solution, with variations in the geometry around the copper ion with increasing pH, varying from a more tetrahedral configuration in acidic medium to a tetragonal one in basic solution. In order to elucidate the role of copper in the carbohydrate oxidation by molecular oxygen, kinetic studies were performed using these complexes as catalysts. A more comprehensive global rate law was determined for this process, including a copper-dependent pathway (at very low concentrations), in addition to an independent one, both influenced by alkaline medium. The proposed mechanism, under pseudo-first order conditions, combine intramolecular electronic transfer with reduction of the copper ion by the substrate, leading to the formation of intermediary reactive species (OH•-, O2•-, H2O2, CO2•-), responsible for initiation and propagation steps, and of short chain carbonylic products (glycolate, glycerate and formiate ions). To better understanding metal-carbohydrate interactions, copper(II) complexes with simple monosacharides were isolated, and characterized by elemental and thermogravimetric analyses, and spectroscopic techniques (IR, UV/Vis, and EPR), besides Raman spectroscopy, used to investigate the binding mode of the carbohydrate moiety to the copper ion, in each one of these complexes. Additionally, two novel chiral imine copper(II) complexes, derived from aminocarbohydrate ligands, were prepared and characterized by elemental analysis, conductivity measurements, and spectroscopic techniques (IR, UV/Vis, EPR, ESI-MS and CD), and one of them was also used in biological studies. Biological activity studies were carried out with the imine and diimine copper(II) complexes derived from isatin, verifying antiproliferative effect toward some tumor cell lines (promonocite U937 and neuroblastoma SH-SY5Y). Cells treated with the most active complexes were committed by the apoptotic program, as verified by citofluorimetric assays, with the complexes interfering the cell cycle in different ways (G1, G2/M or S phase. Formation of free radicals was detected, and citoplasmatic carbonylated and glycosilated proteins inside the treated cells were determined by imunologic assays. In conclusion, these studies indicated modulation of the biological activity by the imine ligand in the copper(II) complexes, with very different antiproliferative behavior, going from undetectable activity to high efficacy. Among the most active compounds, a different specificity and action mode in both cell type could be observed, evidencing their potential application as antitumoral agents.

Evidências apontam para o envolvimento da peroxidação lipídica em diversas patologias. Os hidroperóxidos de lipídios (LOOH) são os produtos primários da peroxidação lipídica e sua decomposição resulta em produtos de maior reatividade e toxicidade, como os radicais peroxila. Esses radicais desempenham papel importante na propagação da peroxidação lipídica e também podem gerar oxigênio molecular singlete (1O2) por meio da combinação de dois radicais peroxila. Neste trabalho investigamos a possibilidade dos LOOH, em particular dos hidroperóxidos de ácido linoléico (LAOOH), de servirem como fonte 1O2 na presença de oxidantes de relevância biológica como metais, peroxinitrito ou ácido hipocloroso. A formação de 1O2 foi claramente demonstrada na reação de LAOOH com esses oxidantes pelas detecções (i) da emissão bimolecular na região espectral do vermelho (λ>570 nm), (ii) da emissão monomolecular no infravermelho-próximo (λ=1270 nm), (iii) do espectro de emissão no infravermelho, e (iv) da intensificação e supressão da luminescência na presença de D2O e azida, respectivamente. Além disso, os mecanismos de reação foram estudados utilizando LAOOH marcados com oxigênio-18 (LA18O18OH) e captadores químicos específicos para 1O2 aliada à tecnica de detecção por HPLC acoplada à espectrometria de massa. Os resultados mostraram a formação de 1O2 marcado [18(1O2) ] na reação de LA18O18OH com os três oxidantes, revelando que os átomos de oxigênio do 1O2 são derivados do hidroperóxido. Em conjunto, as evidências obtidas levam à conclusão de que os LOOH podem servir como fontes potenciais de 1O2 em sistemas biológicos em situações onde haja a coexistência de LOOH e metais, peroxinitrito ou ácido hipocloroso. / Evidences point to the involvement of lipid peroxidation in several diseases. Lipid hydroperoxides (LOOH) are the primary products of lipid peroxidation and their decomposition generates more reactive and toxic compounds, such as peroxyl radicals. These radicals play an important role in the propagation of lipid peroxidation and may also generate singlet molecular oxygen (1O2) by the combination of two peroxyl radicals. In this study we have investigated the possibility of LOOH, in particular linoleic acid hydroperoxide (LAOOH), to be a source of 1O2 in the presence of biologically relevant oxidants such as, metal ions, peroxynitrite or hypochlorous acid. The formation of 1O2 was clearly demonstrated in the reaction of LAOOH with all the three tested oxidants by detecting: (i) the dimol light emission in the red spectral region (λ>570 nm), (ii) the monomol light emission in the near-infrared region (λ=1270 nm), (iii) the infrared light emission spectrum, and (iv) the enhancing effect of deuterium oxide and the quenching effect of azide on light emission. Furthermore, the mechanism was studied using LAOOH labeled with 18-oxygen isotope (LA18O18OH) and specific 1O2 chemical traps in combination with HPLC coupled to mass spectrometry detection. The results have showed the formation of 18-oxygen labeled 1O2 [18(1O2) ] in the reaction of LA18O18OH with the three oxidants, indicating that oxygen atoms in 1O2 are derived from the hydroperoxide. Altogether, the obtained evidences lead to the conclusion that LOOH may serve as a potential source of 1O2 in biological systems, in situations where LOOH can interact with metals, peroxynitrite or hypochlorous acid.

by Wai Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 123-157). / Abstracts in English and Chinese. / Acknowledegment --- p.iv / Abstract --- p.v / List of Abbreviations --- p.ix / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Parkinson's Disease (PD) --- p.1 / Chapter 1.1.1 --- Epidemiology --- p.1 / Chapter 1.1.2 --- Neuropathology --- p.2 / Chapter 1.1.3 --- Clinical Symptoms --- p.3 / Chapter 1.1.4 --- Treatment --- p.6 / Chapter 1.2 --- Proposed Mechanisms of Neurodegeneration in PD --- p.11 / Chapter 1.2.1 --- Oxidative Stress --- p.11 / Chapter 1.2.2 --- Mitochondrial Dysfunction --- p.13 / Chapter 1.2.3 --- Genetic Factors --- p.15 / Chapter 1.2.4 --- Environmental Factors --- p.17 / Chapter 1.2.5 --- Ageing --- p.20 / Chapter 1.3 --- "1-Methy-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) as a PD Model" --- p.22 / Chapter 1.3.1 --- Discovery of MPTP --- p.22 / Chapter 1.3.2 --- The Mechanisms of MPTP-induced Neurotoxicity --- p.23 / Chapter 1.4 --- Antioxidants in the Central Nervous System --- p.26 / Chapter 1.4.1 --- Superoxide Dismutase --- p.26 / Chapter 1.4.2 --- Glutathione --- p.27 / Chapter 1.5 --- Metallothioneins (MTs) --- p.29 / Chapter 1.5.1 --- Characteristics of MTs --- p.29 / Chapter 1.5.2 --- Functions of Astrocytes --- p.31 / Chapter 1.6 --- Astrocytes --- p.34 / Chapter 1.6.1 --- Characteristics of Astrocytes --- p.34 / Chapter 1.6.2 --- Functions of Astrocytes --- p.35 / Chapter 1.6.3 --- Role of Astrocytes in Parkinson's Disease --- p.39 / Chapter 1.7 --- Aim of Project --- p.41 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- In Vitro Study --- p.44 / Chapter 2.1.1 --- Astrocyte Cultures --- p.44 / Chapter 2.1.2 --- Treatment Regimen --- p.46 / Chapter 2.1.2.1 --- 1 -methyl-4-phenyl-pyridinium (MPP+) Treatment --- p.46 / Chapter 2.1.2.2 --- Induction of Metallothioneins (MTs) and Glutathione (GSH) --- p.46 / Chapter 2.1.2.2.1 --- Northern Blot Analysis --- p.47 / Chapter 2.1.2.2.2 --- Immunocytochemical Staining for MTs --- p.48 / Chapter 2.1.2.2.3 --- GSH Assay --- p.49 / Chapter 2.1.2.3 --- Iron Chelation --- p.51 / Chapter 2.1.2.4 --- Combined Pretreatment --- p.51 / Chapter 2.1.3 --- Lactate Dehydrogenase (LDH) Assay --- p.51 / Chapter 2.1.4 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) Assay" --- p.53 / Chapter 2.1.5 --- Reactive Oxygen Species (ROS) Assay --- p.55 / Chapter 2.1.6 --- Protein Assay --- p.56 / Chapter 2.1.7 --- Statistics --- p.57 / Chapter 2.2 --- In Vivo Study --- p.57 / Chapter 2.2.1 --- "Administration of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)" --- p.57 / Chapter 2.2.2 --- Tyrosine Hydroxylase (TH) Immunocytochemical Staining --- p.58 / Chapter 2.2.3 --- DAT Receptor Binding Assay --- p.59 / Chapter 2.2.4 --- Dopamine (DA) and DA metabolites - High Performance Liquid Chromatography (HPLC) --- p.60 / Chapter 2.2.5 --- Statistics --- p.61 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- In Vitro Study --- p.62 / Chapter 3.1.1. --- Induction of Metallothioneins (MTs) in Astrocytes with Zinc Sulfate (ZnS04) --- p.62 / Chapter 3.1.1.1 --- Immunocytochemical changes --- p.62 / Chapter 3.1.1.2 --- Northern Blot Analysis --- p.62 / Chapter 3.1.1.3 --- The Effects of ZnSO4 Pretreatment on 1 -methyl-4-phenyl- pyridinium (MPP+)-treated Astrocytes --- p.63 / Chapter 3.1.1.3.1 --- Lactate Dehydrogenase (LDH) Activities --- p.63 / Chapter 3.1.1.3.2 --- "3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl- tetrazolium bromide (MTT) Activities" --- p.67 / Chapter 3.1.1.3.3 --- Reactive Oxygen Species (ROS) Production --- p.71 / Chapter 3.1.2 --- The Effects of NAc Pretreatment on MPP+-treated Astrocytes --- p.75 / Chapter 3.1.2.1 --- Glutathione (GSH) levels --- p.75 / Chapter 3.1.2.2 --- LDH Activities --- p.77 / Chapter 3.1.2.3 --- MTT Activities --- p.80 / Chapter 3.1.2.4 --- ROS Production --- p.83 / Chapter 3.1.3 --- The Effects of Deferoxamine on MPP+-treated Astrocytes --- p.87 / Chapter 3.1.3.1 --- LDH Activities --- p.87 / Chapter 3.1.3.2 --- ROS Production --- p.89 / Chapter 3.1.4 --- The Effects of ZnSO4 and NAc Combined Treatment on MPP+-treated Astrocytes --- p.92 / Chapter 3.1.4.1 --- LDH Activities --- p.92 / Chapter 3.1.4.2 --- ROS Production --- p.95 / Chapter 3.2 --- "Effects of 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on MT-I, -II Knock-out Mice" --- p.99 / Chapter 3.2.1 --- The Effects of MPTP on Substantia Nigral (SN) Cell Loss --- p.99 / Chapter 3.2.2 --- The Effects of MPTP on Striatal (ST) and SN Dopamine Transporter (DAT) Binding --- p.99 / Chapter 3.2.3 --- The Effects of MPTP on ST Dopamine (DA) Metabolites --- p.100 / Chapter CHAPTER FOUR: --- DISCUSSION AND CONCLUSION --- p.102 / REFERENCES --- p.123

Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

La maladie coronaire d’origine ischémique reste l’une des principales causes de mortalité dans le monde industrialisé.Le traitement de l’ischémie aiguë du myocarde est entré dans une nouvelle ère où la mortalité peut être diminuée de moitié en utilisant des procédures qui permettent un retour rapide du débit sanguin dans la zone ischémique du myocarde: la revascularisation.Cette reperfusion entraîne des complications appelées lésions de la reperfusion qui ont été décrites pour la 1ère fois par Jennings et al., en 1960. Le développement de stratégies cardioprotectrices associées à la reperfusion constitue un besoin majeur en clinique afin d’améliorer la fonction myocardique, de diminuer l'incidence des arythmies, de retarder l'apparition de la mort cardiomyocytaire et de limiter la taille de l’infarctus du myocarde lors de l'ischémie/reperfusion (I/R).La découverte de 2 formes principales de mécanismes cardioprotecteurs endogènes, a encouragé la recherche de nouveaux moyens pharmacologiques capables de protéger le myocarde ischémié/reperfusé et a développé nos connaissances sur les bases moléculaires des lésions et de la survie cellulaire au cours des processus d’I/R.L’étude des mécanismes responsables de l’induction de la mort cellulaire a permis de mettre en évidence le rôle joué par la mitochondrie et l’augmentation de la perméabilité de ses membranes, induite par la formation/ouverture d’un pore au niveau des points de contacts entre les membranes mitochondriales;ce pore a été appelé « pore de transition de la perméabilité mitochondriale » (mPTP).L’inhibition de l’ouverture de ce pore apparaît comme une stratégie privilégiée pour protéger le myocarde.Des études ont montré que les espèces réactives d’oxygène (EROs) jouent un rôle majeur dans les lésions de l’I/R et dans l’ouverture du mPTP.Il existe peu d’informations claires sur le seuil et la période de production (ischémie et/ou reperfusion) des EROs qui conduisent à l’ouverture du mPTP.Nous avons mis au point un modèle cellulaire d’hypoxie/réoxygénation (H/R) afin d’établir une relation causale entre la production d’EROs, l’ouverture du mPTP et la mort cellulaire tout en explorant le rôle de différents types d’EROs.Ce modèle d’H/R nous a permis de mesurer en temps réel et simultanément la production des EROs, l’ouverture du mPTP et la mort cellulaire.Nous avons montré que la production des EROs débute pendant la période d’hypoxie et qu’elle est directement liée à l’augmentation du temps d’hypoxie.Cette production d’EROs à l’hypoxie, plus particulièrement de radicaux hydroxyles et de peroxyde d’hydrogène, a été directement relié, à l’ouverture du mPTP et à la mort cellulaire lors de l’H/R.Nous avons utilisé ce modèle pour étudier le mécanisme d’action de deux stratégies pharmacologiques cardioprotectrices, un nouveau ligand de la protéine translocatrice mitochondriale (TSPO), le TRO 40303, et l’activation de la voie RISK par la morphine. Nous avons ainsi montré que (1) les propriétés cardioprotectrices du TRO40303 sont associées à une inhibition de l’ouverture du mPTP, ce qui n’avait pas pu être démontré au moyen d’expériences réalisées ex vivo et (2) l’activation de la voie RISK par la morphine, qui aboutit à une limitation de la taille d’infarctus associée à une amélioration des fonctions respiratoires mitochondriales, entraîne également une inhibition de l’ouverture du mPTP et un retard de la mort cellulaire des cardiomyocytes isolés soumis à une H/R.La suite de ce travail sera de rechercher si l’inhibition du stress oxydant peut constituer un mécanisme commun aux deux stratégies pharmacologiques cardioprotectrices en utilisant notre modèle d’H/R.Il serait possible d’étendre notre modèle à des animaux génétiquement modifiés pour appréhender les phénomènes impliqués dans cette activité antioxydante.A plus long terme, il sera nécessaire d’approfondir nos connaissances sur la production d’EROs pendant l’I/R en recherchant plus spécifiquement l’origine de cette production. / Ischemic coronary artery disease remains one of the main causes of mortality in the industrialized countries. The treatment of acute myocardial ischemia entered a new era where mortality can be reduced by 50% using revascularization procedures that allow a rapid return of blood flow to the ischemic area. However, this reperfusion leads to complications known as lethal reperfusion induced injury that have been described for the first time by Jennings et al., in 1960. It became crucial to develop cardioprotective strategies in combination with early reperfusion in order to improve myocardial function, to reduce the incidence of arrhythmias, to delay the onset of cardiomyocytes death and to limit the extension of infarct size following reperfusion. The discovery of two major forms of endogenous cardioprotective mechanisms, which consist of the realization of short cycles of ischemia/reperfusion (I/R) prior to a long period of ischemia (ischemic preconditioning) or before reperfusion after the long period of ischemia (Ischemic Postconditioning), encouraged the search for new pharmacological tools to protect the ischemic myocardium to develop our knowledge on the molecular mechanisms of lethal reperfusion injury and cell survival in the I/R process.The study of cell death mechanisms has highlighted the crucial role of the mitochondria and more specifically the increase in mitochondrial membrane permeability following I/R.One reason for increasing permeability is the formation/opening of a pore at mitochondrial membranes contact sites at reperfusion.This pore has been called "the mitochondrial permeability transition pore" (mPTP). Inhibition of this pore opening has been presented as a main strategy to protect the myocardium.Many studies have shown that reactive oxygen species (ROS) play a major role in I/R injury and mPTP opening, but there is very few information to date about the threshold and the period of ROS production (ischemia and/or reperfusion) that lead to mPTP opening.We designed a cellular model of hypoxia/reoxygenation (H/R) to establish a causal relationship between ROS production, mPTP opening and cell death while exploring the role of different types of ROS.This H/R model used freshly isolated adult rat cardiomyocytes and allowed us to measure online and simultaneously ROS production, mPTP opening and cell death. We have demonstrated that ROS production starts during the period of hypoxia and thisproduction is directly linked to the increase in the duration of hypoxia.This ROS production during hypoxia has been, for the first time, directly related to mPTP opening and cell death following H/R.We used this model to study the mechanism of action of two cardioprotective strategies, a new ligand of the mitochondrial translocator protein (TSPO), TRO 40303 and a RISK (Reperfusion Injury Salvage Kinase) pathway activator, morphine. We have shown that (1) the cardioprotective properties of TRO40303 were associated with inhibition of mPTP opening, a mechanism that could not be demonstrated using ex vivo experiments and (2) morphine that provoked infarct size limitation associated with an improvement of mitochondrial respiratory functions through RISK pathway activation, also inhibited mPTP opening and delayed cell death of isolated cardiomyocytes subjected to H/R.Finally, a question comes into sight whether the inhibition of oxidative stress may be a common mechanism to both cardioprotective pharmacological strategies that we have described using our H/R model. To do this, it would be possible to extend our model to genetically modified animals specifically adapted to understand the phenomena involved in antioxidant activity.On long-term, it will be necessary to develop our knowledge on ROS production during I/R by looking for the origin of this production, more precisely the role of the mitochondria and the effect of other reactive species in order to target the treatment and to develop new cardioprotective strategies.