La maladie de Parkinson (PD) est considérée comme la deuxième maladie neurodégénérative la plus fréquente. L'examen post-mortem de patients parkinsoniens et des modèles physiologiques d’études de la maladie de Parkinson suggèrent la participation de la mort cellulaire programmée, l'inflammation et l'autophagie dues au stress oxydatif, à des mutations ou l’agrégation de protéines au sein des neurones DA. Les aptamères peptidiques sont de petites protéines combinatoires, consistitués d’une plateforme (dans notre cas, la thiorédoxine humaine, hTRX) et une boucle variable insérée dans le domaine actif de hTRX. Deux aptamères peptidiques ont été identifiés par la sélection fonctionnelle. L’aptamère peptide 32 (Apta-32) ,est spécifique liant deux paralogues T32 impliqués dans le processus d'endocytose. L’aptamère peptidique 34(Apta-34) lie à une cible "T34", une protéine pro-apoptotique ayant un rôle dans la voie apoptotique provenant du noyau. Le travail de cette thèse visait à étudier la fonction anti-apoptotique de nos deux aptamères peptidiques dans deux modèles d’étude de la maladie de Parkinson: un modèle cellulaire (in vitro) et un modèle transgénique D. melanogaster (in vivo). Deux toxines majeures ont été appliquées dans ce travail, 6-hydroxindopamine (6-OHDA) et le paraquat, un pesticide couramment utilisé. Nos observations montrent que la drosophile exprimant Apta-32 dans tous les neurones ont montré une meilleure résistance après 48h de traitement avec le paraquat comparé à deux autre aptamères peptidiques, Apta-34 et Apta-TRX (sans boucle de contrôle variable). Une autre étude a révélé un défaut dans la phagocytose des corps apoptotiques au cours du développement embryonnaire de la drosophile exprimant Apta-32 dans les macrophages, ce qui suggère qu’Apta-32 pourrait participer à et peut-être interférer avec le processus de l’autophygie, et que Apta-32 pourrait protéger contre l'autophagie induite par paraquat dans les neurones. / Parkinson’s disease is considered as the second most common neurodegenerative disease. Although the cause of the progressive cell loss of PD remains unclear to date, programmed cell death, inflammation and autophagy due to oxidative stress, gene mutations or protein aggregations within DA neuron have been suggested as potential causes. Peptide aptamers are small combinatorial proteins, with a variable loop inserted into a scaffold protein, human thioredoxin, hTRX. They are used to facilitate dissection of signaling networks by modulating specific protein interactions and functions. Two peptide aptamers were identified by functional selection which inhibit Bax-dependent cell death in mammalian models. One peptide aptamer (Apta-32) is binding two paralogues involved in endocytotic trafficking T32. The second peptide aptamer (Apta-34) is binding to a target "T34", a pro-apoptotic protein mediating apoptosis emanating from the nucleus. The work of my PhD thesis aimed to investigate the anti-apoptotic function of our two peptide aptamers in different PD models including cell model (in vitro), brain tissue slice and D. melanogaster (in vivo) ; in particular their impact on neuron survival after exposure to specific toxins. Two major toxins were applied in this work, 6-hydroxindopamine (6-OHDA) and Paraquat, a commonly used pesticide. Our observations indicated that Drosophila expressing Apta-32 in all neurons showed more resistance 48h after treatment with Paraquat, compared to drosophila expressing Apta-34 or TRX. Another study revealed a defect in phagocytosis of apoptotic bodies in drosophila embryo’s expressing Apta-32 in macrophage, suggesting Apta-32 could be involved in, and perhaps interfere with, the process of autophagy. This suggests that Apta-32 could protect against paraquat induced autophagy in neurons.

Dans cette thèse, nous présentons la détection électrochimique des herbicides, c'est-à-dire le paraquat et l'isoproturon dans des échantillons aqueux. Leur utilisation intensive est une source de contamination de l'environnement et leur toxicité constitue une menace pour la santé. La détection électrochimique est une technique prometteuse et avantageuse par rapport aux méthodes de détection conventionnelles en raison de ses propriétés telles que l'analyse rapide, la facilité d'utilisation, la rentabilité et la sensibilité élevée résultant de la modification de l'électrode de travail. Ici, nous avons modifié les électrodes modifiées avec des films minces de silice mésoporeuse pour agir comme capteurs d'herbicide. Ces électrodes ont été modifiées par un processus d'auto-assemblage assisté par électrochimie, un processus bien établi pour la modification des électrodes par notre groupe. Dans la première partie, l'adhérence du film de silice mésoporeux aux électrodes de carbone a été améliorée à l'aide d'une amine primaire qui a agi comme colle moléculaire pour une meilleure fixation de ces films à la surface des électrodes. Dans la partie suivante, ces électrodes modifiées ont été utilisées pour la détection électrochimique des herbicides susmentionnés. Les électrodes modifiées ont montré une sensibilité accrue et une limite de détection basse par rapport aux électrodes non modifiées. L'effet des différents paramètres de la solution ainsi que l'épaisseur du film et la géométrie de l'électrode ont également été étudiés et ont un impact critique sur la sensibilité du système / In this thesis, we present the electrochemical detection of herbicides i.e. paraquat and isoproturon in aqueous samples. These herbicides are used worldwide extensively for weed control in different crops. Their intensive use is a source of environmental contamination and their toxicity is a threat to Human health. Electrochemical sensing is a promising and advantageous technique as compared to conventional detection methods due to its properties such as rapid analysis, ease of operation, cost effectiveness and high sensitivity as a result of working electrode modification. Here, we modified electrodes modified with mesoporous silica thin films to act as herbicide sensors. These electrodes were modified by electrochemically assisted self-assembly process, a well-established process for electrode modification by our group. In the first part adhesion of mesoporous silica film at carbon electrodes was improved with the help of a primary amine which acted as molecular glue for better attachment of these films at electrodes surface. In the next part these modified electrodes were used for electrochemical detection of above stated herbicides. Modified electrodes showed enhanced sensitivity and low limit of detection as compared to unmodified ones. Effect of different solution parameters as well as film thickness and electrode geometry was also studied and found to have critical impact on sensitivity of the system

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / With the purpose to develop environmentally appropriate materials, scales of the Piau fish and sodium alginate were used as precursor materials for the preparation of new ecomaterials which were used in the removal of the herbicide Paraquat and ions Co(II) and Cu(II). The hybrid material obtained from the reaction between the polysaccharide and scales was designated as ALG/ESC, and the hybrid ALG/ESC-CaCl2 was obtained from the crosslinking of the ALG/ESC with calcium chloride. The materials were characterized by FTIR, TG, SEM, XRD and determination of the point of zero charge (PZC). Changes in the peak observed in the FTIR spectrum of the ALG/ESC confirmed the existence of chemical interactions between Ca(II) of the hydroxyapatite present in fish scales, and the carboxylate groups of the sodium alginate. In the spectrum of ALG/ESC-CaCl2, bands were observed to be considerably narrower, due to crosslinking with the Ca(II) ions and tension of the carboxylate group that increases to accommodate the coordination structure around the Ca(II). It was observed an increase in sample thermal stability of the ALG/ESC-CaCl2 when compared to ALG/ESC, indicating the formation of the "egg box" structure. The SEM micrographs exhibited the presence of hydroxyapatite crystals on the surface of the ALG/ESC, and a homogeneous surface on the ALG/ESC-CaCl2 beads. Comparison between the X-ray diffraction profiles of the precursors (pure scales, ESC, and pure alginate, ALG) and those of the ALG/ESC and ALG/ESC-CaCl2 indicated that changes occurred due to incorporation of the hydroxyapatite structure in the alginate. From the PZC values of the materials, it was suggested that, in a general way, the interactions were controlled by electrostatic attractions between negatively charged surface groups and the cationic paraquat, or free Co(II) and Cu(II) ions, that are present in the solution, or between species of cobalt and copper in their anionic form and the surface groups that presented themselves as positively charged in the investigated pH range. From adsorption experiments carried out using the technique of batch with samples of solutions of the herbicide Paraquat and salts of copper and cobalt, the amount of adsorbed species per gram of adsorbent, Nf was determined. The values of Nf showed the highest affinity of the hybrid ALG/ESC-CaCl2 for the herbicide and the Co(II) and Co(II) ions. The adsorption rates were evaluated using the pseudo-first-order, pseudo-second-order and intraparticle diffusion models. It was found that the data did present a good fit to the traditional intraparticle diffusion model. The data obtained from this model suggested that processes, other than simple intraparticle diffusion, may be occurring simultaneously. / Buscando desenvolver materiais ambientalmente adequados, foram utilizadas escamas do peixe Piau e Alginato de Sódio como materiais precursores para preparação de novos ecomateriais que foram utilizados na remoção do herbicida Paraquat e de íons Co(II) e Cu(II). O material híbrido obtido da reação entre o polissacarídeo e as escamas foi designado como ALG/ESC, sendo o híbrido ALG/ESC-CaCl2 obtido a partir da reticulação do ALG/ESC com Cloreto de Cálcio. Os materiais foram caracterizados por FTIR, TG, MEV, DRX e determinação do ponto de carga zero (PCZ). No espectro de FTIR do ALG/ESC deslocamentos de picos para regiões de maiores comprimentos de onda indicaram a existência da interação química entre íons Ca(II) da hidroxiapatita, presentes na escama de peixe, e os grupos carboxilato do Alginato de Sódio. No espectro do ALG/ESC-CaCl2 foram observadas bandas consideravelmente mais estreitas, devido à ligação cruzada com os íons Ca(II) e pela tensão do grupo carboxilato que aumenta para acomodar a estrutura de coordenação em torno do Ca(II). O material ALG/ESC-CaCl2, quando comparado com o ALG/ESC, apresentou-se mais estável termicamente, indicando a formação da estrutura caixa de ovos . As micrografias evidenciaram a presença de cristais de hidroxiapatita na superfície das esferas de ALG/ESC, e uma superfície homogênea nas esferas ALG/ESC-CaCl2. Foram observadas mudanças nos padrões de difração de raios X, quando comparados aos dos precursores (escamas puras, ESC, e Alginato puro, ALG) que ocorreram devido à incorporação da hidroxiapatita na estrutura do Alginato. A partir dos valores do PCZ dos materiais sugeriu-se que, de um modo geral, as interações ocorrem através de atrações eletrostáticas entre grupos superficiais carregados negativamente e o Paraquat catiônico, ou íons Co(II) e Cu(II) livres que se encontram presentes na solução ou ainda entre espécies de cobalto e cobre na sua forma aniônica e os grupos superficiais que apresentavam-se carregados positivamente na faixa de pH investigada. Através de experimentos de adsorção realizados usando a técnica de batelada com amostras de soluções do herbicida Paraquat e de sais de cobre e cobalto, determinou-se a quantidade de espécie adsorvida por grama de adsorvente, Nf. Os valores de Nf indicaram que o híbrido ALG/ESC-CaCl2 apresentou maior afinidade pelo herbicida e pelos íons Co(II) e Co(II). As taxas de adsorção foram investigadas através dos modelos de pseudo-primeira-ordem, pseudo-segunda-ordem e de difusão intrapartícula. Verificou-se que os dados obtidos não se ajustaram aos modelos cinéticos de pseudo-primeira-ordem e pseudo-segunda-ordem, com exceção dos dados referentes à interação entre os íons Cu(II) e as esferas ALG/ESC-CaCl2. O modelo de difusão intrapartícula ofereceu uma boa correlação. Os dados obtidos através deste modelo sugeriram que os processos de interação são controlados não apenas por difusão intrapartícula, mas também outros processos ocorrem concomitantemente.

Ein Schlüsselbefund der Parkinson-Krankheit auf zellulärer Ebene ist das Auftreten von Protein-Einschlusskörperchen, sogenannten Lewykörperchen. Der Hauptbestandteil dieser Lewykörperchen ist pathologisch aggregiertes, fibrilläres α-Synuklein, ein Protein, welches Einfluss auf präsynaptische Vesikel, Protein- und Enzymfunktionen sowie den Dopaminstoffwechsel und den axonalen Transport hat. Bis heute ungeklärt ist die Ursache der Aggregation des Proteins. Zahlreiche Forschungsaktivitäten werden in diese Richtung unternommen. Die pathologischen Mechanismen, die zur abnormen Aggregation von α-Synuklein führen, bleiben noch weitgehend unbekannt. Ein großer Teil der Literatur unterstützt die Hypothese, dass α-Synuklein bei Punktmutationen oder erhöhter Expression anfällig für Aggregationen ist und damit Neuronen geschädigt werden. Die Einzelheiten dieses sukzessiven Aggregationsprozesses und die Mechanismen, die dabei letztlich den Zelltod verursachen, bleiben unklar. Alterungsprozesse und Umweltfaktoren sind entscheidende Risikofaktoren. Obwohl es immer mehr Hinweise gibt, dass α-Synuklein-Aggregate eine wichtige pathophysiologische Rolle spielen, wird bisher noch unzulänglich verstanden, wie die für die dopaminergen Neurone toxische Wirkung entfaltet wird. Als gesicherter Pathomechanismus der Degeneration der dopaminergen Nervenzellen gilt ein erhöhter oxidativer Stress. Es wird vermutet, dass er zur Aggregation des α-Synukleins beitragen kann. Ziel dieser vorgelegten Arbeit ist es, durch die Verwendung eines geeigneten Zellkulturmodells zur Aufklärung der beschriebenen pathologischen Mechanismen beizutragen. In dieser Studie wurden zwei artifizielle Modellsubstanzen in einer dopaminergen Primärzellkultur eingesetzt, die Pestizide Rotenon und Paraquat, um die pathologischen Verhältnisse in der Substantia nigra zu simulieren. Sie erzeugen oxidativen Stress durch Hemmung der mitochondrialen Atmungskette bzw. Redoxreaktionen mit molekularem Sauerstoff, was zum dopaminergen Zelltod führt. Im Rahmen dieser Studie gelang es, beide Parkinson-Zellkulturmodelle anhand der Lokalisierung, des Aggregationsverhaltens, des Einflusses auf die Mikroglia-Aktivierung sowie des Abbaus von α-Synuklein näher zu charakterisieren. Hierzu wurde α-Synuklein durch Fluoreszenzfärbung, Westernblot und Immunpräzipitation analysiert. Eine kurzzeitige Behandlung mit hoch konzentrierten Toxinen löst eine akute Degeneration dopaminerger Neurone aus, die so nicht der des Idiopathischen Parkinsons entspricht. Beim IPS erfolgt die Degeneration über Jahre hinweg. Um auch den Einfluss der zellulären Alterung auf die α-Synuklein-Aggregation zu zeigen, wurden die in dieser Arbeit verwendeten Zellkulturen über 46 Tage kultiviert und die Pestizid-Konzentration so eingestellt, dass etwa 25-50 % der dopaminergen Neurone absterben. Es konnte gezeigt werden, dass es nach chronischer Behandlung mit dem jeweiligen Pestizid zu den verschiedenen Zeitpunkten Unterschiede in der Lokalisation sowie in der Konformation von α-Synuklein gibt. Die zum Vergleich nur bis zum Tag 11 kultivierten Zellkulturen zeigten nach kurzer Behandlung mit hochkonzentrierter Toxin-Menge eine Ansammlung von α-Synuklein im Soma, aber keine Auswanderung und Lokalisierung in und an den Neuriten, wie es nach chronischer Rotenon-Behandlung beobachtet werden konnte. Bei den Rotenon-behandelten Zellen ist ein Prozess der Verlagerung und Anhäufung des α-Synuklein aus dem Soma in die Neuriten bereits ab einem früheren Zeitpunkt zu beobachten als in den Kontrollen, welche sich aber mit zunehmendem Alter ähnlich verhalten. Außerdem konnte in den Kulturen, besonders bei den Rotenon-behandelten dopaminergen Neuronen, ein punktförmiges Verteilungsmuster und größere Ansammlungen des Proteins in den Neuriten beobachtet werden, was für eine aggregierte Form des α-Synukleins spricht. Diese Aggregate ließen sich durch die Proteo-Aggreosom-Färbung nachweisen. Auch der Nachweis von am Serin 129 phosphoryliertem α-Synuklein in diesen größeren Ansammlungen gilt als Zeichen für eine aggregierte Form. Die Beobachtung, dass α-Synuklein mit zunehmendem Kulturalter aus dem Soma in die Peripherie austritt, konnte bei der chronischen Paraquat-Behandlung so nicht getätigt werden. Unter Paraquat-Behandlung war keine Herauf-Regulierung der Gesamt-α-Synuklein Expression in der Kultur zu beobachten, wie dies sowohl bei Kontrolle als auch bei Rotenon der Fall ist. Wir vermuten im Paraquat-Modell, dass die sich im Soma ansammelnde Form von α-Synuklein durch vermehrte Einschleusung in den Zellkern zur Toxizität beitragen kann. Auch eine Interaktion von α-Synuklein mit der Zellmembran könnte unter Paraquat-Einfluss zum dopaminergen Zelltod beitragen. Durch Immunpräzipitation und Fluoreszenz-Doppelfärbung von α-Synuklein und Ubiquitin konnten wir den Abbau des fehlgefalteten α-Synukleins in der Zelle durch Ubiquitinierung nachweisen. Unter Rotenon ist ab DIV 14 eine starke Kolokalisation von α-Synuklein und Ubiquitin zu erkennen, die im Verlauf der Kultur nachlässt und nur noch in punktförmigen Aggregaten außerhalb der Zelle zu sehen ist. Unter Paraquat zeigte sich während der gesamten Kultivierung Polyubiquitinierung und Kolokalisation der beiden Proteine in der gesamten Zelle. Die Aggregationsform von α-Synuklein scheint das Proteinabbausystem durch Ubiquitinierung zu beeinflussen, da wir davon ausgehen, dass es sich bei der α-Synuklein-Konformation zu verschiedenen Zeitpunkten in den Paraquat-behandelten dopaminergen Neuronen nicht um die Aggregationsform handelt, die wir unter Rotenon beobachten konnten.

Ein Schlüsselbefund der Parkinson-Krankheit auf zellulärer Ebene ist das Auftreten von Protein-Einschlusskörperchen, sogenannten Lewykörperchen. Der Hauptbestandteil dieser Lewykörperchen ist pathologisch aggregiertes, fibrilläres α-Synuklein, ein Protein, welches Einfluss auf präsynaptische Vesikel, Protein- und Enzymfunktionen sowie den Dopaminstoffwechsel und den axonalen Transport hat. Bis heute ungeklärt ist die Ursache der Aggregation des Proteins. Zahlreiche Forschungsaktivitäten werden in diese Richtung unternommen. Die pathologischen Mechanismen, die zur abnormen Aggregation von α-Synuklein führen, bleiben noch weitgehend unbekannt. Ein großer Teil der Literatur unterstützt die Hypothese, dass α-Synuklein bei Punktmutationen oder erhöhter Expression anfällig für Aggregationen ist und damit Neuronen geschädigt werden. Die Einzelheiten dieses sukzessiven Aggregationsprozesses und die Mechanismen, die dabei letztlich den Zelltod verursachen, bleiben unklar. Alterungsprozesse und Umweltfaktoren sind entscheidende Risikofaktoren. Obwohl es immer mehr Hinweise gibt, dass α-Synuklein-Aggregate eine wichtige pathophysiologische Rolle spielen, wird bisher noch unzulänglich verstanden, wie die für die dopaminergen Neurone toxische Wirkung entfaltet wird. Als gesicherter Pathomechanismus der Degeneration der dopaminergen Nervenzellen gilt ein erhöhter oxidativer Stress. Es wird vermutet, dass er zur Aggregation des α-Synukleins beitragen kann. Ziel dieser vorgelegten Arbeit ist es, durch die Verwendung eines geeigneten Zellkulturmodells zur Aufklärung der beschriebenen pathologischen Mechanismen beizutragen. In dieser Studie wurden zwei artifizielle Modellsubstanzen in einer dopaminergen Primärzellkultur eingesetzt, die Pestizide Rotenon und Paraquat, um die pathologischen Verhältnisse in der Substantia nigra zu simulieren. Sie erzeugen oxidativen Stress durch Hemmung der mitochondrialen Atmungskette bzw. Redoxreaktionen mit molekularem Sauerstoff, was zum dopaminergen Zelltod führt. Im Rahmen dieser Studie gelang es, beide Parkinson-Zellkulturmodelle anhand der Lokalisierung, des Aggregationsverhaltens, des Einflusses auf die Mikroglia-Aktivierung sowie des Abbaus von α-Synuklein näher zu charakterisieren. Hierzu wurde α-Synuklein durch Fluoreszenzfärbung, Westernblot und Immunpräzipitation analysiert. Eine kurzzeitige Behandlung mit hoch konzentrierten Toxinen löst eine akute Degeneration dopaminerger Neurone aus, die so nicht der des Idiopathischen Parkinsons entspricht. Beim IPS erfolgt die Degeneration über Jahre hinweg. Um auch den Einfluss der zellulären Alterung auf die α-Synuklein-Aggregation zu zeigen, wurden die in dieser Arbeit verwendeten Zellkulturen über 46 Tage kultiviert und die Pestizid-Konzentration so eingestellt, dass etwa 25-50 % der dopaminergen Neurone absterben. Es konnte gezeigt werden, dass es nach chronischer Behandlung mit dem jeweiligen Pestizid zu den verschiedenen Zeitpunkten Unterschiede in der Lokalisation sowie in der Konformation von α-Synuklein gibt. Die zum Vergleich nur bis zum Tag 11 kultivierten Zellkulturen zeigten nach kurzer Behandlung mit hochkonzentrierter Toxin-Menge eine Ansammlung von α-Synuklein im Soma, aber keine Auswanderung und Lokalisierung in und an den Neuriten, wie es nach chronischer Rotenon-Behandlung beobachtet werden konnte. Bei den Rotenon-behandelten Zellen ist ein Prozess der Verlagerung und Anhäufung des α-Synuklein aus dem Soma in die Neuriten bereits ab einem früheren Zeitpunkt zu beobachten als in den Kontrollen, welche sich aber mit zunehmendem Alter ähnlich verhalten. Außerdem konnte in den Kulturen, besonders bei den Rotenon-behandelten dopaminergen Neuronen, ein punktförmiges Verteilungsmuster und größere Ansammlungen des Proteins in den Neuriten beobachtet werden, was für eine aggregierte Form des α-Synukleins spricht. Diese Aggregate ließen sich durch die Proteo-Aggreosom-Färbung nachweisen. Auch der Nachweis von am Serin 129 phosphoryliertem α-Synuklein in diesen größeren Ansammlungen gilt als Zeichen für eine aggregierte Form. Die Beobachtung, dass α-Synuklein mit zunehmendem Kulturalter aus dem Soma in die Peripherie austritt, konnte bei der chronischen Paraquat-Behandlung so nicht getätigt werden. Unter Paraquat-Behandlung war keine Herauf-Regulierung der Gesamt-α-Synuklein Expression in der Kultur zu beobachten, wie dies sowohl bei Kontrolle als auch bei Rotenon der Fall ist. Wir vermuten im Paraquat-Modell, dass die sich im Soma ansammelnde Form von α-Synuklein durch vermehrte Einschleusung in den Zellkern zur Toxizität beitragen kann. Auch eine Interaktion von α-Synuklein mit der Zellmembran könnte unter Paraquat-Einfluss zum dopaminergen Zelltod beitragen. Durch Immunpräzipitation und Fluoreszenz-Doppelfärbung von α-Synuklein und Ubiquitin konnten wir den Abbau des fehlgefalteten α-Synukleins in der Zelle durch Ubiquitinierung nachweisen. Unter Rotenon ist ab DIV 14 eine starke Kolokalisation von α-Synuklein und Ubiquitin zu erkennen, die im Verlauf der Kultur nachlässt und nur noch in punktförmigen Aggregaten außerhalb der Zelle zu sehen ist. Unter Paraquat zeigte sich während der gesamten Kultivierung Polyubiquitinierung und Kolokalisation der beiden Proteine in der gesamten Zelle. Die Aggregationsform von α-Synuklein scheint das Proteinabbausystem durch Ubiquitinierung zu beeinflussen, da wir davon ausgehen, dass es sich bei der α-Synuklein-Konformation zu verschiedenen Zeitpunkten in den Paraquat-behandelten dopaminergen Neuronen nicht um die Aggregationsform handelt, die wir unter Rotenon beobachten konnten.

Métodos analíticos empregando técnicas eletroquímicas foram desenvolvidos para o monitoramento qualitativo e quantitativo de pesticidas carbamatos. O sensor utilizado no desenvolvimento dos métodos foi o eletrodo de diamante dopado com boro pré-tratado catodicamente. No desenvolvimento do método qualitativo, foi possível discriminar e classificar cinco pesticidas carbamatos (aldicarb, carbaril, carbofurano, metomil e propoxur) com a ajuda de ferramentas quimiométricas, tais como análise de componentes principais e do algoritmo dos k-vizinhos mais próximos. Nesse método, os valores de corrente, obtidos por voltametria de onda quadrada, para os pesticidas foram utilizados como dados de entrada nas ferramentas quimiométricas. Um método eletroquímico, utilizando voltametria de pulso diferencial, para a quantificação do pesticida propoxur foi desenvolvido. Após a otimização dos parâmetros da técnica e das condições experimentais, construiu-se uma curva analítica de 1,66 a 155 µmol L-1 (R2 = 0,9935) e limite de detecção estimado de 0,50 µmol L-1. A exatidão do método foi avaliada por adição e recuperação em amostras de águas naturais, onde se alcançou níveis de recuperação na faixa de 86,7 a 103%. Para o pesticida pirimicarbe, foi realizado um amplo estudo visando elucidar o comportamento eletroquímico de oxidação desse composto, o qual foi suportado por dados de espectrometria de massas. Na faixa de pH estudada (2 a 8), o pirimicarbe apresentou três sinais de oxidação irreversíveis, sendo os dois primeiros dependentes do pH. Por outro lado, em experimento realizado em meio orgânico, evidenciou a presença de apenas um sinal de oxidação para o pesticida. Também foi desenvolvido um método eletroquímico por voltametria de pulso diferencial para a quantificação do pirimicarbe. Os parâmetros da técnica de voltametria de pulso diferencial foram otimizados utilizando planejamento experimental. A curva analítica apresentou faixa de trabalho de 2,00 a 219 µmol L1-1 (R2 = 0,9982) e limite de detecção estimado de 1,24 µmol L-1. O método de adição e recuperação juntamente com calibração externa foram utilizados para avaliação da exatidão do método em amostras de águas naturais, obtendo-se recuperações entre 88,6 e 96,3%. Adicionalmente, um método para a quantificação do pesticida paraquate foi desenvolvida utilizando a técnica de voltametria de onda quadrada. O pesticida apresentou dois sinais de redução reversíveis e com magnitude de corrente semelhantes, portanto, sendo possível utilizar qualquer um dos dois sinais para sua quantificação. Com a otimização dos parâmetros da técnica e das condições experimentais, foi possível obter uma curva analítica na faixa de 0,800 a 167 µmol L-1 (R2 = 0,9990) e limite de detecção estimado em 70 nmol L1-1. O método foi aplicado em amostras de saliva humana e águas naturais e níveis de recuperação na faixa de 83,0 a 105% foram alcançados. Vale ressaltar que todos os métodos analíticos aqui propostos apresentaram simplicidade, confiança e podem ser considerados adequados tanto para análises de rotina quanto para aplicações em campo, devido à portabilidade apresentada pelos métodos eletroquímicos. / Electroanalytical methods were developed for the qualitative and quantitative monitoring of carbamate pesticides. The sensor used for the development of the proposed methods was the boron-doped diamond electrode cathodically pre-treated. In the development of the qualitative method, it was possible to discriminate and classify five carbamate pesticides (aldicarb, carbaryl, carbofuran, methomyl and propoxur) using chemometric tools such as principal component analysis and the k-nearest neighbors algorithm. In qualitative approach, current values obtained by square-wave voltammetry for pesticides were used as input data of the chemometric tools. An electrochemical method, using differential pulse voltammetry, for the quantification of pesticide propoxur was also developed. At the best conditions (parameters of the technique and the experimental conditions), an analytical curve from 1.66 to 155 µmol L-1 (R2 = 0.9935) and an estimated detection limit of 0.50 µmol L-1 were obtained. The accuracy of the method was evaluated by addition and recovery approach in natural waters samples and recovery levels ranging from 86.7 to 103% were reached. For the pesticide pirimicarb, a study was carried out to elucidate the electrochemical oxidation behavior of this compound, which was supported by mass spectrometry data. In the pH range (2 to 8) studied, the pirimicarb shown three irreversible oxidation signals, the first two were pH-dependent. On the other hand, in an experiment carried out in organic medium, it exhibited the presence of only one oxidation signal for the pesticide. An electrochemical method was also developed by differential pulse voltammetry for the quantification of pirimicarb. The parameters of the differential pulse voltammetry technique were optimized using experimental design. The analytical curve showed a working range from 2.00 to 219 µmol L-1 (R2 = 0.9982) and an estimated detection limit of 1.24 µmol L-1. The addition and recovery approach with external calibration were used to evaluate the accuracy of the method in natural water samples, obtaining recoveries values between 88.6 and 96.3%. In addition, a method for the quantification of the paraquat pesticide was developed using the square-wave voltammetry technique. The pesticide presented two reversible reduction signals with similar current magnitude, therefore, it is possible to use either of the two signals for its quantification. Under optimized parameters of the technique and the experimental conditions, it was possible to obtain an analytical curve in the range from 0.800 to 167 µmol L-1 (R2 = 0.9999) and detection limit estimated of 70 nmol L-1. This method was applied for human saliva and natural water samples and recovery levels ranging from 83.0 to 105% were achieved. It should be noted that all the analytical methods proposed here exhibited simplicity, reliability and can be considered adequate for routine analysis and in-field applications due to the portability characteristics of the electrochemical methods.

Este trabalho teve como objetivo analisar os efeitos fisiológicos do herbicida Paraquat, indutor de estresse oxidativo, em plantas de cana-de-açúcar (Saccharum officinarum L.). A escolha desta espécie como modelo fisiológico deste estudo deveu-se a sua importância econômica para o país, em especial para o Estado de São Paulo. O Paraquat (PQ) é um herbicida de contato que atua especificamente no Fotossistema II bloqueando o fluxo de elétrons e impedindo a redução do NADPH, aceptor final de elétrons. Como consequência, há a formação de radicais instáveis, reativos com a molécula de oxigênio (EROs), os quais causam danos em membranas, proteínas e ácidos nucleicos. A hipótese central foi a de que a atividade de peroxidase de ascorbato é mais sensível ao PQ do que a atividade de superóxido dismutase, nas mesmas condições. O experimento principal consistiu da aplicação, via pulverização foliar, de diferentes doses de PQ (0, 2, 4, 6 e 8 mM de Metil Violágeno, agente ativo), que permaneceu em contato por 24 e 48 horas. Após 18 horas de exposição, observou-se sintomas visuais de toxocidade e uma redução significativa da atividade fotossintética, medida através de parâmetros relacionados com a fluorescência da clorofila a. O nível de peroxidação de lipídeos, indicado pela concentração de TBARS, foi aumentado, indicando danos nas membranas causados pela geração de EROs. Um resultado importante foi o aumento da atividade específica de SOD (Superóxido Dismutase), primeira enzima de defesa contra EROs. Esse aumento foi gradativo com o aumento da dose e do tempo de exposição ao PQ. A isoforma mais evidente foi a SOD Cu/Zn, visualizada por atividade em gel nativo. Esta isoforma está presente preferencialmente em cloroplastos, organela alvo da ação do PQ. É importante ressaltar que mesmo em doses quase letais, a atividade desta enzima manteve-se em níveis semelhantes ao controle. Entretanto, a atividade de APX (Ascorbato Peroxidase), enzima que faz a remoção de radicais H2O2, gerados pela SOD, foi reduzida abruptamnete a partir de 4 mM PQ, apesar de ter sido observado um aumento na dose inicial (2 mM). Este fato sugere que os danos oxidativos em cana-de-açúcar tratadas com PQ podem ser causados pelo excesso de radicais H2O2. Diferente da SOD, a APX é considerada bastante susceptível a certos tipos de estresse ambiental, logo perdendo sua atividade. Analisou-se também a expressão de proteínas por SDS-PAGE e eletroforese 2-D. Em ambos resultados, foram observados diminuição na expressão de algumas proteínas ocasionada pelo tratamento com PQ. Contudo, não foi possível detectar claramente a indução de peptídeos nesse caso, provavelmente devido ao alto grau de degradação das estruturas celulares. O aumento da atividade de SOD em cloroplastos de folhas de cana-de-açúcar tratadas com PQ, deve ser mais estudado, pois esta pode ser uma enzima alvo para programas de melhoramento genético visando obtenção de plantas com resistência ao PQ e a outros herbicidas com modo de ação no sistema fotossintético. / This work had the objective to analyze physiological effects of Paraquat herbicide, an oxidative stress inductor, in sugarcane (Saccharum officinarum L.) plants. The choice to use this species as a physiological model for this study was due its recognized economical importance for our country, in special to the State of Sao Paulo. Paraquat (PQ) is a contact herbicide that acts specifically on Photosystem II by blocking the normal electron flux and the reduction reaction of NADPH, the electron acceptor molecule. As consequence, there is formation of instable radicals and highly reactive oxygen species (ROS), which cause damage to membranes, proteins and nucleic acids. The central hypothesis was that ascorbate peroxidase activity is more sensitive to PQ than the activity of superoxide dismutase in the same conditions. The main experiment consisted on the sprayed of different concentrations of PQ (0, 2, 4, 6 e 8 mM of Metil Violagen) that stayed in leaf contact for 24 and 46 hours. After 18 hours, it was observed visible symptoms of toxicity and the photosynthetic activity decreased, measured by chlorophyll a fluorescence. Also, the level of lipids peroxidation measured by the increase concentration of TBARS formation, indicate damage to cell membrane caused by the generation of ROS. An important result was the increase of SOD specific activity (superoxide dismutase) first enzyme related to defense against ROS. This gradation increase was correspondent to the exhibition period to PQ. The most evident SOD isoform by native PAGE was the SOD Cu/Zn. This isoform is located preferentially in chloroplasts, organelle target to the action of PQ. It is important to remark that even in almost lethal concentrations of PQ the chloroplast SOD activity still remains expressed beilng responsible for the removal of H2O2 generated by SOD, which was reduced abruptly after 4 mM treatment. Although, it has been observed increase with the initial dose (2 mM). This fact suggests that the oxidative damages in sugarcane treated with PQ can be caused by excess of H2O2. Differently of SOD, this enzyme is considered to be fragile to certain kinds of environmental adversities, loosing its activity Also the protein expression was analyzed by SDSPAGE and 2-D electrophoresis. In both analyses, it was observed decrease of some protein expression caused by PQ. However, it was not possible to clearly detect peptides induction in this case, probably due to the elevated level of degradation of cell structures. The increase of chloroplast SOD activity in sugarcane leaves treated with PQ should be detailed investigated since it can be a molecular target for breeding programs with the goal to obtain plants with an acquired resistance to PQ or others herbicides with model action in the photochemical system.

Métodos analíticos empregando técnicas eletroquímicas foram desenvolvidos para o monitoramento qualitativo e quantitativo de pesticidas carbamatos. O sensor utilizado no desenvolvimento dos métodos foi o eletrodo de diamante dopado com boro pré-tratado catodicamente. No desenvolvimento do método qualitativo, foi possível discriminar e classificar cinco pesticidas carbamatos (aldicarb, carbaril, carbofurano, metomil e propoxur) com a ajuda de ferramentas quimiométricas, tais como análise de componentes principais e do algoritmo dos k-vizinhos mais próximos. Nesse método, os valores de corrente, obtidos por voltametria de onda quadrada, para os pesticidas foram utilizados como dados de entrada nas ferramentas quimiométricas. Um método eletroquímico, utilizando voltametria de pulso diferencial, para a quantificação do pesticida propoxur foi desenvolvido. Após a otimização dos parâmetros da técnica e das condições experimentais, construiu-se uma curva analítica de 1,66 a 155 µmol L-1 (R2 = 0,9935) e limite de detecção estimado de 0,50 µmol L-1. A exatidão do método foi avaliada por adição e recuperação em amostras de águas naturais, onde se alcançou níveis de recuperação na faixa de 86,7 a 103%. Para o pesticida pirimicarbe, foi realizado um amplo estudo visando elucidar o comportamento eletroquímico de oxidação desse composto, o qual foi suportado por dados de espectrometria de massas. Na faixa de pH estudada (2 a 8), o pirimicarbe apresentou três sinais de oxidação irreversíveis, sendo os dois primeiros dependentes do pH. Por outro lado, em experimento realizado em meio orgânico, evidenciou a presença de apenas um sinal de oxidação para o pesticida. Também foi desenvolvido um método eletroquímico por voltametria de pulso diferencial para a quantificação do pirimicarbe. Os parâmetros da técnica de voltametria de pulso diferencial foram otimizados utilizando planejamento experimental. A curva analítica apresentou faixa de trabalho de 2,00 a 219 µmol L1-1 (R2 = 0,9982) e limite de detecção estimado de 1,24 µmol L-1. O método de adição e recuperação juntamente com calibração externa foram utilizados para avaliação da exatidão do método em amostras de águas naturais, obtendo-se recuperações entre 88,6 e 96,3%. Adicionalmente, um método para a quantificação do pesticida paraquate foi desenvolvida utilizando a técnica de voltametria de onda quadrada. O pesticida apresentou dois sinais de redução reversíveis e com magnitude de corrente semelhantes, portanto, sendo possível utilizar qualquer um dos dois sinais para sua quantificação. Com a otimização dos parâmetros da técnica e das condições experimentais, foi possível obter uma curva analítica na faixa de 0,800 a 167 µmol L-1 (R2 = 0,9990) e limite de detecção estimado em 70 nmol L1-1. O método foi aplicado em amostras de saliva humana e águas naturais e níveis de recuperação na faixa de 83,0 a 105% foram alcançados. Vale ressaltar que todos os métodos analíticos aqui propostos apresentaram simplicidade, confiança e podem ser considerados adequados tanto para análises de rotina quanto para aplicações em campo, devido à portabilidade apresentada pelos métodos eletroquímicos. / Electroanalytical methods were developed for the qualitative and quantitative monitoring of carbamate pesticides. The sensor used for the development of the proposed methods was the boron-doped diamond electrode cathodically pre-treated. In the development of the qualitative method, it was possible to discriminate and classify five carbamate pesticides (aldicarb, carbaryl, carbofuran, methomyl and propoxur) using chemometric tools such as principal component analysis and the k-nearest neighbors algorithm. In qualitative approach, current values obtained by square-wave voltammetry for pesticides were used as input data of the chemometric tools. An electrochemical method, using differential pulse voltammetry, for the quantification of pesticide propoxur was also developed. At the best conditions (parameters of the technique and the experimental conditions), an analytical curve from 1.66 to 155 µmol L-1 (R2 = 0.9935) and an estimated detection limit of 0.50 µmol L-1 were obtained. The accuracy of the method was evaluated by addition and recovery approach in natural waters samples and recovery levels ranging from 86.7 to 103% were reached. For the pesticide pirimicarb, a study was carried out to elucidate the electrochemical oxidation behavior of this compound, which was supported by mass spectrometry data. In the pH range (2 to 8) studied, the pirimicarb shown three irreversible oxidation signals, the first two were pH-dependent. On the other hand, in an experiment carried out in organic medium, it exhibited the presence of only one oxidation signal for the pesticide. An electrochemical method was also developed by differential pulse voltammetry for the quantification of pirimicarb. The parameters of the differential pulse voltammetry technique were optimized using experimental design. The analytical curve showed a working range from 2.00 to 219 µmol L-1 (R2 = 0.9982) and an estimated detection limit of 1.24 µmol L-1. The addition and recovery approach with external calibration were used to evaluate the accuracy of the method in natural water samples, obtaining recoveries values between 88.6 and 96.3%. In addition, a method for the quantification of the paraquat pesticide was developed using the square-wave voltammetry technique. The pesticide presented two reversible reduction signals with similar current magnitude, therefore, it is possible to use either of the two signals for its quantification. Under optimized parameters of the technique and the experimental conditions, it was possible to obtain an analytical curve in the range from 0.800 to 167 µmol L-1 (R2 = 0.9999) and detection limit estimated of 70 nmol L-1. This method was applied for human saliva and natural water samples and recovery levels ranging from 83.0 to 105% were achieved. It should be noted that all the analytical methods proposed here exhibited simplicity, reliability and can be considered adequate for routine analysis and in-field applications due to the portability characteristics of the electrochemical methods.

La maladie de Parkinson (MP) se caractérise par des troubles moteurs d'évolution progressive, conséquence de la dégénérescence des neurones dopaminergiques de la substance noire. Dans cette maladie, le vieillissement est un facteur de prédisposition majeur. Au cours de ma thèse, j'ai examiné dans un premier temps, les modèles in vivo utilisés au laboratoire pour étudier la MP chez la drosophile, à savoir l'expression de l'α-synucléine et l'intoxication au paraquat. J'ai ainsi contribué à l'identification d'une sous-population de neurones dopaminergiques impliquée dans les effets locomoteurs de l'α-synucléine et à la mise en évidence du rôle d'un récepteur dopaminergique de type D1 dans la neurotoxicité du paraquat. J'ai ensuite montré que l'activité des neurones dopaminergiques accélère la sénescence et diminue la durée de vie des drosophiles en contribuant de manière significative à la production des DRO dans le cerveau. Enfin, nous avons identifié et caractérisé chez la drosophile une protéine homologue de LAMP-2A, le récepteur lysosomal de l'autophagie médiée par les chaperonnes (AMC), et démontré que l'augmentation de la clairance neuronale des protéines cytosoliques par l'expression du LAMP-2A humain ou de son homologue de drosophile a des effets positifs sur le déclin locomoteur lié à l'âge et la résistance aux facteurs de la MP, mais qu'elle n'augmente pas la longévité. Ces résultats suggèrent que l'AMC est un mécanisme conservé chez la drosophile et que son activation protège contre le stress oxydatif et le vieillissement neuronal. / Parkinson's disease (PD) is characterized by progressive motor disorders resulting in dopaminergic neurons degeneration in the substantia nigra. In this pathology, aging is a major predisposing factor. During my thesis, I examined initially, in vivo models used in the laboratory to study the PD in Drosophila, namely the expression of α-synuclein and paraquat. I have contributed to the identification of dopaminergic neurons subpopulation involved in locomotor effects of α-synuclein and highlighting the role of a dopamine D1-like receptor in neurotoxicity of paraquat. I then shown that the activity of dopaminergic neurons accelerated senescence and decreases Drosophila lifespan by contributing significantly to the production of ROS in the brain. Finally, we identified and characterized Drosophila homologous of LAMP-2A protein, involves in autophagy-mediated chaperone (AMC), and demonstrated that the increase in neuronal clearance of cytosolic proteins by the expression human LAMP-2A or its Drosophila homologue has positive effects on the locomotor decline associated with aging and resistance against PD factors, but it does not increase longevity. These results suggest that the CMA is a conserved mechanism in Drosophila and its activation protects against oxidative stress and neuronal aging.