Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-18T18:12:36Z No. of bitstreams: 1 Stífani Machado Araujo.pdf: 2701104 bytes, checksum: db0f252b8e5b648fd9a2f147be16081a (MD5) / Made available in DSpace on 2016-04-18T18:12:36Z (GMT). No. of bitstreams: 1 Stífani Machado Araujo.pdf: 2701104 bytes, checksum: db0f252b8e5b648fd9a2f147be16081a (MD5) Previous issue date: 2016-02-19 / Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum no mundo, afetando cerca de 1% dos adultos com mais de 60 anos. A DP está relacionada com a degeneração de neurônios dopaminérgicos principais componentes da substância negra cerebral, concomitantemente, com a disfunção do complexo I mitocondrial e o estresse oxidativo que desempenham um papel crucial na patogênese desta doença. A rotenona (ROT) é um pesticida natural e muito utilizado para induzir fenótipo de DP em modelos animais, por ser lipofílico pode atravessar facilmente a barreira hematoencefálica causando disfunção do complexo I mitocondrial e possível morte de neurônios dopaminérgicos. Dentre as várias aplicações terapêuticas dos antioxidantes, ressalta-se sua ação neuroprotetora, uma vez que o sistema nervoso central exibe uma maior vulnerabilidade e susceptibilidade aos insultos oxidativos, o γ-orizanol (ORY) é um produto natural composto por uma mistura de ésteres de ácido ferúlico extraídos a partir do óleo de farelo de arroz, e é bem descrito na literatura por possuir propriedades antioxidantes. Assim, o objetivo deste trabalho foi investigar um possível efeito neuroprotetor do γ-orizanol sobre alterações comportamentais e bioquímicas causadas pela exposição crônica de Drosophila melanogaster a rotenona. A mosca da fruta Drosophila melanogaster, é uma espécie alternativa à utilização de modelos mamíferos que vem sendo usada com bastante confiabilidade na reprodução de modelos de disfunção dopaminérgica. As moscas (de ambos os sexos) com idades compreendidas entre 1 a 5 dias de idade foram divididos em quatro grupos de 50 moscas cada um: (1) de controle, (2) ORY 25 μM, (3) ROT 500 μM, (4) ORY 25 μM + ROT 500 μM. As moscas foram concomitantemente expostos a uma dieta contendo ROT e ORY durante 7 dias de acordo com os seus respectivos grupos. Após o tratamento foram feitas as analises comportamentais e bioquimicas. Como resultados, verificamos que o ORY ofereceu proteção contra as alterações locomotoras causadas por ROT, além de prevenir a mortalidade induzida por rotenona, protegeu contra geração de estresse oxidativo e disfunções mitocondriais além de otimizar as defesas antioxidantes celulares. Nossas descobertas apontam uma restauração dos déficits colinérgicos, e nos níveis de dopamina fornecida por pelo tratamento com ORY. Em conclusão, os presentes resultados mostram que ORY é eficaz na redução da toxicidade induzida ROT em Drosophila melanogaster, o que mostrou uma ação neuroprotetora, possivelmente devido à presença dos componentes de antioxidantes tais como o ácido ferúlico. / Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, affecting about 1% of adults over 60 years. The DP is linked to the degeneration of dopaminergic neurons main components of the substantia nigra brain, concurrently with mitochondrial complex I dysfunction and oxidative stress play a crucial role in the pathogenesis of this disease. The rotenone (ROT) is a natural pesticide, and used to induce PD phenotype in animal models, being lipophilic can easily cross the blood-brain barrier dysfunction causing mitochondrial complex I and possible death of dopaminergic neurons. Among the various therapeutic applications of antioxidants, it emphasizes its neuroprotective action, as the central nervous system displays a greater vulnerability and susceptibility to oxidative insults, the γ-oryzanol (ORY) is a natural product composed of a mixture of esters ferulic acid derived from rice bran oil, and is well described in the literature to possess antioxidant properties. The objective of this study was to investigate a possible neuroprotective effect of ORY on behavioral and biochemical changes caused by chronic exposure of Drosophila melanogaster the rotenone. The fly fruit Drosophila melanogaster, is an alternative species to the use of mammalian models that have been used quite reliable reproduction of dopaminergic dysfunction models. The flies (male and female) between the ages of 1 to 5 days of age were divided into four groups of 50 flies each: (1) control, (2) ORY 25 μM, (3) ROT 500 μM ( 4) ORY 25 μM + ROT 500 μM. The flies were simultaneously exposed to a diet containing ROT and ORY for 7 days according to their respective groups. After treatment were made behavioral and biochemical analysis. As a result, we find that the ORY offered protection against locomotor changes caused by ROT, and to prevent the mortality induced by rotenone, protected against the generation of oxidative stress and mitochondrial dysfunction and optimize cellular antioxidant defenses. Our findings point to a restoration of cholinergic deficits, and dopamine levels provided by the treatment ORY. In conclusion, the present results show that ORY is effective in reducing the ROT induced toxicity in Drosophila melanogaster, which showed a neuroprotective effect, possibly due to the presence of antioxidants components such as ferulic acid.

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

Made available in DSpace on 2015-04-20T12:31:48Z (GMT). No. of bitstreams: 1 Marcio Rodrigo Alecio.pdf: 5329646 bytes, checksum: 74fcb177f7eeca1140502ad6646743e5 (MD5) Previous issue date: 2012-08-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The abundance, potential insecticide and evidence of synergism among the major constituents of timbo extracts (Derris and Deguelia) and the need to control of Cerotoma tingomarianus Bechyné (Coleoptera: Chrysomelidae) and Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) stimulated the realization of this study, which aimed to evaluate the biological effects of timbo extracts and its purified fractions compared to rotenone, as well as combined in different concentrations, on adults of C. tingomarianus and S. frugiperda larvae. Samples timbo roots were collected of plants in the Acre and Amazonas states, and obtained extracts by hot extraction of dry material method. The timbo extracts were grouped into six distinct groups and the bioassays were carried out by means of topical contact, contact by contaminated surface (filter-paper) and intake of contaminated diet in a completely randomized design. The mortality values of two insect species were submitted to Probit analysis to determine the LC50, LD50 and LT50. The other variables were subjected to variance analysis, when possible, using the Scott-Knott test (P> 0.05) for grouping treatment means. The intoxication by intake of contaminated diet/leaves is the most effective mean of contact for adults of C. tingomarianus and S. frugiperda larvae. The toxicity of timbo species extracts is variable and is related to the chemical composition of the extracts, as well as at form of exposure and the target insect species. Rotenone alone was not toxic to adult C. tingomarianus and showed low toxicity to S. frugiperda larvae, enhancing their biological effects on the insects/larvae when associated with the fractions containing timbo extracts, constituent A, constituent B and deguelin. The toxicity of timbo extracts for C. tingomarianus adult may be related to the combined action of several constituents. Extracts containing high levels of rotenone and deguelin are indicators of toxicity to S. frugiperda larvae. The association the constituent A with constituent B probably causes mortalities in the pre-pupal and pupal weight of S. frugiperda. The extract of timbo G1 and G3 showed the highest toxicity to insects and can be considered as the most promising for the development of biotechnology products for the control of C. tingomarianus adults and S. frugiperda larvae. / A abundância, o potencial inseticida e indícios de sinergismo entre os constituintes majoritários de extratos de timbó (Derris e Deguelia) e a necessidade de controle de Cerotoma tingomarianus Bechyné (Coleoptera: Chrysomelidae) e de Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) estimularam a realização desta pesquisa, que objetivou avaliar os efeitos biológicos de extratos de timbó e de suas frações comparados à rotenona purificada, isoladamente e combinados em diferentes concentrações, sobre adultos de C. tingomarianus e de lagartas de S. frugiperda. Foram coletadas amostras de raízes de plantas de timbó nos estados do Acre e do Amazonas e obtidos extratos a quente utilizando clorofórmio como solvente. Os extratos das plantas de timbó foram agrupados em seis grupos distintos e os bioensaios foram desenvolvidos pelas vias de intoxicação de contato tópico, de contato por superfície contaminada e por ingestão alimentar, com delineamento experimental inteiramente casualizado. Os valores de mortalidade das duas espécies de insetos foram submetidos à análise de Probit para determinação das CL50, DL50 e TL50. As demais variáveis foram submetidas à análise de variância, quando possível, utilizando-se o teste de Scott-Knott (P > 0,05) para agrupamento das médias dos tratamentos. A intoxicação por ingestão alimentar é a via de contato mais efetiva para adultos de C. tingomarianus e lagartas de S. frugiperda. A toxicidade dos timbós é variável e está relacionada à composição química dos extratos, a forma de exposição e a espécie de inseto alvo. A rotenona utilizada isoladamente não foi tóxica para adultos de C. tingomarianus e apresentou baixa toxicidade para lagartas de S. frugiperda, potencializando seus efeitos biológicos sobre as lagartas quando associada com as frações dos extratos de timbó contendo deguelina e os constituintes A e B. A toxicidade dos extratos de timbó para adultos de C. tingomarianus pode estar relacionada à atuação conjunta de diversos constituintes. Extratos com elevados teores de rotenona e deguelina são indicadores de toxicidade para lagartas de S. frugiperda. A associação do constituinte A com o constituinte B provavelmente provoca mortalidades na fase pré-pupa e o peso de pupa de S. frugiperda pode ser reduzido pela atuação conjunta de vários constituintes dos extratos de timbó. O constituinte A pode atuar como antagonista de mortalidade de lagartas, provocar deformações e afetar a viabilidade de adultos de S. frugiperda. Os extratos de timbó G1 e G3 apresentam a maior toxicidade para os insetos e podem ser considerados como os mais promissores para o desenvolvimento de produtos biotecnológicos para o controle de adultos de C. tingomarianus e de lagartas de S. frugiperda.

Este estudo foi realizado com o objetivo de identificar genótipos de feijão Phaseolus vulgaris resistentes aos carunchos Acanthoscelides obtectus e Zabrotes subfasciatus, bem como avaliar o efeito associado desses genótipos resistentes com inseticidas de origem vegetal. Para tanto, foram utilizados acessos de P. vulgaris do Banco de Germoplasma do Instituto Agronômico de Campinas e inseticidas comerciais de origem botânica. No screening inicial, amostras dos genótipos foram infestadas com cada uma das espécies de bruquídeos separadamente, avaliando-se o número de insetos emergidos aos 50 dias após a infestação. Dos 49 genótipos testados contra A. obtecus, não houve emergência naqueles com números de acesso 525, 584 e 615, podendo ser considerados os mais resistentes. Em relação a Z. subfasciatus, os genótipos com números de acesso 2, 35, 251, 570, 583, 584, 610, 621, 634, 816, 818 e 819 se mostraram mais resistentes entre os 185 avaliados. Destes, os genótipos portadores de arcelina 583, 584, 816, 818 e 819, além de 570 e 610, foram selecionados como os mais promissores para os testes subseqüentes, juntamente com a variedade Bolinha, que foi utilizada como controle de suscetibilidade. Não foi observada correlação entre as características morfoagronômicas dos genótipos de P. vulgaris e a sua resistência às espécies de bruquídeos avaliadas, indicando que a resistência a estas pragas não está associada às características da flor, vagem, semente e fenologia dos genótipos. A massa de mil sementes, que é indicativa da origem dos genótipos, foi um dos descritores analisados, mostrando também que a resistência de P. vulgaris a A. obtectus e a Z. subfasciatus não está relacionada à origem dos genótipos. Em testes de livre escolha e de confinamento, avaliou-se o efeito dos genótipos selecionados no screening, juntamente com a variedade Bolinha, sobre o comportamento e biologia de Z. subfasciatus. Verificou-se que a avaliação da preferência de Z. subfasciatus por genótipos de P. vulgaris em teste de livre escolha pode ser feita com 1 dia após a infestação e que \'Bolinha\', apesar de ser suscetível a Z. subfasciatus e favorecer o seu desenvolvimento, apresenta antixenose para oviposição em relação à praga. Nos testes realizados, os genótipos contendo arcelina tenderam a ser mais resistentes que os demais sem essa proteína, sendo que os seus efeitos sobre Z. subfasciatus incluíram o aumento da mortalidade no período de desenvolvimento, alongamento desse período e redução do peso de adultos emergidos, mantendo-se, de certa forma, estáveis ao longo de duas gerações da praga. A resistência conferida pela arcelina revelou ser do tipo antibiose, tendo como causas a impropriedade nutricional e a ação no metabolismo do inseto. Com relação aos inseticidas botânicos, foram testados 3 produtos comerciais, sendo 2 à base de azadiractina e um à base de rotenona. Entre estes, o produto que mais afetou o desenvolvimento de Z. subfasciatus foi NeemPro®, derivado de nim (Azadirachta indica), o qual apresentou efeito ovicida e prolongou a duração do período de desenvolvimento de Z. subfasciatus. Frente a isto, avaliou-se o efeito associado de NeemPro® com o genótipo resistente portador de arcelina 818 sobre alguns parâmetros biológicos de Z. subfasciatus. Verificou-se que os efeitos mais severos sobre Z. subfasciatus foram causados pelo genótipo resistente, independentemente do inseticida à base de nim e que o uso associado de ambos não provoca efeito aditivo ou sinérgico, não sendo recomendado para o manejo de Z. subfasciatus. / This research was carried out to identify Phaseolus vulgaris genotypes resistant to the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, as well as to evaluate the effect of these genotypes in association with botanical insecticides. To reach this objective, P. vulgaris accessions from the Germplasm Bank of Instituto Agronômico de Campinas and commercial insecticides from botanical origin were tested. In the initial screening, samples of bean genotypes were infested with the weevil species separately and the number of adults emerged at the 50th day after infestation was evaluated. There was no A. obtectus emergence in genotypes 525, 584 and 615, among the 49 ones screened against this pest. In relation to Z. subfasciatus, genotypes with accession numbers 2, 35, 251, 570, 583, 584, 610, 621, 634, 816, 818 and 819 showed themselves resistant among 185 screened ones. The arcelin-containing genotypes 583, 584, 816, 818 and 819, plus 570 and 610 (both lacking this protein), were selected as the most promising for additional evaluations. Bolinha variety was also used as the susceptible standard. No correlation between morpho-agronomical characteristics of the P. vulgaris genotypes and their resistance to the weevils was observed, indicating that resistance to these two pests is not associated to genotypes flower, pod and seed characters or plant phenology. The mass of 1000 seeds, which indicates the origin of genotypes, was one of the used descriptors, showing that P. vulgaris resistance to A. obtectus and Z. subfasciatus is not related to genotypes origin too. In free- and no-choice tests, it was evaluated the effect of the screened genotypes on Z. subfasciatus behavior and biology, compared to \'Bolinha\'. It was verified that in free-choice tests, the evaluation of Z. subfasciatus preference for P. vulgaris genotypes can be done 1 day after infestation. Despite being susceptible to Z. subfasciatus and supporting its development, \'Bolinha\' holds antixenosis for oviposition in relation to the pest. In the bioassays carried out, genotypes containing arcelin tended to be more resistant than those lacking this protein and their effects on Z. subfasciatus include increasing of the mortality in the developmental period, enlargement of this period and reduction in adult weight, also being stable during two pest generations. The resistance provided by arcelin revealed itself to be antibiosis, by acting as antinutrients and also as antimetabolics. In relation to botanical insecticides, 3 commercial products, 2 of them based on azadirachtin and 1 based on rotenone, were evaluated. The insecticide NeemPro®, extracted from neem (Azadirachta indica) was the only one significantly causing ovicidal effect and enlarging Z. subfasciatus developmental period. Based on these results, the associated effect of NeemPro® and the resistant arcelin-containing P. vulgaris genotype 818 on some Z. subfasciatus biological parameters was evaluated. It was verified that the most severe effects on Z. subfasciatus were caused by the resistant bean genotype, independently of the neem based insecticide. The associated use of these two control methods no results in additive or synergistc effect and is not recommended for the management of Z. subfasciatus.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-06-23T17:23:05Z No. of bitstreams: 1 2004 - Jos? Roberto Pererira.pdf: 1063290 bytes, checksum: ac86f4bd5958ba416f5739fd8e9c3de5 (MD5) / Made available in DSpace on 2017-06-23T17:23:05Z (GMT). No. of bitstreams: 1 2004 - Jos? Roberto Pererira.pdf: 1063290 bytes, checksum: ac86f4bd5958ba416f5739fd8e9c3de5 (MD5) Previous issue date: 2004-05-25 / Laboratory and field trials were performed to evaluate the efficiency of Timb? root extract (Dahlstedtia pentaphylla) (Taub). Burk. (Leguminosae, Papilionoidae, Millettiedae) on samples of Boophilus microplus (Canestrini, 1887) in bovines of the Para?ba Valley region ? S?o Paulo, Brasil. The ?in vitro? trials were carried out using immersion of engorged females, and ?in vivo? on bovines in the field by means of artificial infestation. The standard dilution was made from one part of root powder and three parts of ethanol this was considered the 100% solution. To determine the more efficient solvent to extract rotenone from the root, laboratory trials were conducted on B. microplus engorged females with in three solvents: water, ethanol a.p. and acetone a.p. The laboratory trials permitted the calculation of lethal doses at 90% (LD90) and 50% (LD50) for larvae between seventh and fourtheenth days of application and efficient concentrations at 90% (EC90) and 50% (EC50) on strain local and Mozo with B. microplus engorged females. For the larvae of the local strain (Para?ba Valley Regional Hub) LD50 calculated was 1:31,37 mL and LD90 was 1:85,24 mL. The EC90 for engorged females, of the same strain was of 1:10,19 mL and the EC50 1:34,94 mL. For the engorged females on the hypersersitive strain Mozo the EC90 was 1:23,91 mL and the EC50 1:60,46 mL. The mortality of engorged females in relation to the different kinds of solvents, was analyzed. In the field, the best results (76,10% of control) were obtained three days after application of the product extracted in ethanol, in 1:10, on animals. Then gradually between the seventh and fourteenth days the products lost efficiency, there was no significant difference between treatments and the control group after 21 days. / Foram realizados testes laboratoriais e de campo para avaliar a efici?ncia do extrato de ra?zes da planta Dahlstedtia pentaphylla (Taub.) Burk., (Leguminosae, Papilionoideae, Millettiae) sobre amostras de Boophilus microplus ( Canestrini, 1887 ) de bovinos da regi?o do Vale do Para?ba e da cepa sens?vel Mozo. Os testes foram efetuados ?in vitro?, pela t?cnica de imers?o de tel?ginas e ?in vivo?, sobre bovinos no campo, ap?s infesta??o artificial. As dilui??es foram obtidas a partir da extra??o de roten?ides utilizando?se uma parte de p? das ra?zes da planta para tr?s de etanol, sendo considerada padr?o 100%. Visando determinar o solvente mais eficiente para a extra??o de roten?ides das ra?zes, conduziram-se testes laboratoriais sobre tele?ginas de B. microplus em tr?s solventes: ?gua, etanol p.a e acetona p.a. Os testes laboratoriais permitiram calcular a Dose Letal 90% (DL90) e DL50 para larvas com idade entre sete e 21 dias e a Concentra??o Eficaz 90% (CE90) e CE50 sobre tele?ginas de B. microplus das cepas local e Mozo. Para as larvas da cepa local (Polo Regional do Vale do Para?ba) a DL50 calculada foi de 1: 231,37 mL e DL90 1: 85,24 mL. A CE90 para as tele?ginas, da mesma cepa, foi de 1:10,19 mL e a CE50 1: 34,94 mL. Para tele?ginas da cepa sens?vel Mozo a CE90 foi de 1: 23,91 mL e a CE50, 1: 60,46 mL. Analisou-se tamb?m a mortalidade das tele?ginas frente aos diferentes solventes. No campo os melhores resultados obtidos (76,10% de controle) foram obtidos tr?s dias ap?s a aplica??o do produto extra?do em etanol, na dilui??o 1: 10, sobre os animais. A partir da? gradualmente nos dias sete e quatorze os produtos foram perdendo efici?ncia, n?o apresentando mais diferen?a significativa entre os tratamentos e o grupo controle no dia +21.

Rotenone is a commercially available pesticide with a variety of industrial applications. However, occupational exposure to rotenone has been implicated in the development of Parkinson’s disease. To explore the mechanism of dopamine neuron death secondary to rotenone exposure, the zebrafish was used as a live animal screening tool for environmentally-induced Parkinson’s disease. After testing a variety of small molecule compounds on embryonic zebrafish for their potential to cause dopamine neuron loss, we identified that rotenone exposure induces a bradykinetic dopamine neuron loss phenotype. This phenotype was characterized by decreased locomotion, sensory insensitivity, and a transient but marked decrease in the number of dopamine neurons in embryos exposed to 100nM rotenone, with a concomitant decrease in dopamine transporter mRNA levels. The dopamine neuron deficits were observed in the subpallium, pretectum, olfactory bulb, but these losses were most pronounced in the ventral diencephalon after rotenone exposure. Rotenone damages the mitochondria, generating reactive oxygen species (ROS), and subsequently induces ROS-mediated apoptosis in these dopamine neurons. The rotenone-induced dopamine neuron loss and locomotion phenotypes could be partially rescued in zebrafish larvae with ascorbic acid co-treatment during rotenone exposure. Adults raised from zebrafish embryos exposed to rotenone did not show any deficits to their dopamine neuron distribution, but did show anxiety-like behaviors and upregulation of dopamine receptor D1 mRNA levels. These results suggest that rotenone exposure can cause dopamine neuron death through ROS- mediated apoptosis, and supports an environmental cause of Parkinson’s disease.
 La roténone est un pesticide disponible dans le commerce et utilisé pour une variété d’usages industriels. Cependant, l’exposition à la roténone a été impliquée dans le développement de la maladie de Parkinson. Afin d’explorer le mécanisme menant à la perte de neurones dopaminergiques suite à une exposition à la roténone, le poisson-zèbre (Danio rerio) a été utilisée comme modèle animal du développement de la maladie de Parkinson attribuable à des causes environnementales. Après avoir testé une série de petites molécules sur des embryons de poisson-zèbre pour déterminer leur capacité à causer une perte de neurones dopaminergiques, nous avons identifié la roténone comme causant un phénotype de perte de neurones associée à une bradykinésie. Ce phénotype était caractérisé par une perte de la locomotion, une insensibilité sensorielle, et une diminution transitoire mais marquée du nombre de neurones dopaminergiques chez des embryons exposés à une concentration de roténone de 100nM. Ceci coïncidait avec une réduction des niveaux d’expression du gène du transporteur de la dopamine (dat). Des pertes de neurones dopaminergiques furent observés dans le sub-pallium, le pré-tectum et le bulbe olfactif mais étaient plus prononcées dans le diencéphale ventral. La roténone cause des dommages aux mitochondries en générant des dérivés réactifs de l’oxygène (ROS) et, subséquemment, induit une apoptose attribuable aux ROS dans les neurones dopaminergiques. La perte de neurones dopaminergiques due à la roténone ainsi que les déficits locomoteurs ont pu être partiellement empêchés par un co-traitement à l’acide ascorbique. Les poissons adultes ayant été exposés à la roténone au stade embryonnaire ne montrèrent pas de déficits quant à la distribution des neurones dopaminergiques mais présentaient des comportements indiquant un plus haut niveau d’anxiété ainsi qu’une augmentation des ARNm du récepteur D1 de la dopamine. Ces résultats suggèrent que l’exposition à la roténone peut causer la mort des neurones dopaminergiques via une apoptose médiée par les ROS et pourrait constituer une cause environnemental de la maladie de Parkinson.

Microglia activity has been detected in Parkinson’s disease (PD) post-mortem brains and experimental animal models; however the precise interplay between microglia and dopamine neurons of the SNpc is not well understood. In the blood plasma of PD patients, our laboratory found elevated levels of interferon-gamma (IFN-γ), a proinflammatory cytokine and potent activator of microglia. Given this, we sought to untangle the immune responses relevant to PD in mice, examining IFN-γ’s involvement and signaling mechanism using an inflammatory co-culture model of microglia and midbrain neurons treated with rotenone. By means of RT-PCR, we discovered IFN-γ mRNA transcripts are produced by microglia, and this expression increases upon exposure to rotenone. We delineated IFN-γ’s signaling mechanism in co-cultures using different IFN-γ receptor deficient cells, and showed it engages receptors in an autocrine (not paracrine) manner to further microgliosis and dopamine cell loss. After exploring the innate immune response in a model of PD, we subsequently shifted focus to an in vivo system to better investigate any involvement of the delayed humoral arm of the adaptive immune system. Needing a time appropriate death paradigm, we developed a protracted low dose regimen of MPTP, which elicits dopaminergic cell death after 2 weeks of treatment. Subjected to this paradigm, Rag 2 mutant mice (deficient in both T and B cells) exhibit resistance to dopamine cell loss, microglia activation and motor impairments. Further evidence in support of immune involvement came with the resensitization of Rag2 mice to MPTP after reconstitution with WT splenocytes. Additionally, mice deficient in Fcγ receptors exhibited neuroprotection in our protracted degeneration model. Taken together, these data indicate the innate and humoral arm can modulate the microglial response to dopaminergic degeneration and may participate in Parkinson's disease.

La maladie de Parkinson (MP) est une maladie neurodégénérative caractérisée par trois symptômes moteurs principaux : la bradykinésie, la rigidité et le tremblement de repos. Son diagnostic définitif repose sur l’identification post-mortem d’une importante mort des neurones dopaminergiques de la substance noire (SN) et la présence de corps de Lewy dans les neurones survivants. Cette maladie progresse lentement et les premiers symptômes moteurs n’apparaissent qu’après la dégénérescence de plus de 50% de la SN. Le diagnostic clinique de MP est donc établi tardivement, réduisant ainsi la fenêtre d’action thérapeutique. De plus, les traitements actuels ne soulagent que temporairement les symptômes moteurs. Les défis de la recherche actuelle pour la MP sont donc : 1) d’anticiper le diagnostic de la MP à un stade où la SN est encore suffisamment intacte pour mettre en place des stratégies neuroprotectrices, et 2) d’améliorer les traitements actuels et/ou développer de nouvelles stratégies thérapeutiques pour stopper la progression de la maladie avant que le phénotype moteur ne soit installé. Le stade clinique de la MP est précédé d’une phase prémotrice durant laquelle les patients présentent souvent des symptômes non moteurs tels que l’anosmie, la dépression ou la constipation. Des travaux récents suggèrent que les lésions caractéristiques de la MP pourraient d’abord apparaître dans le système nerveux périphérique puis progresser lentement jusqu’au cerveau. Ces stades précoces de la MP sont cependant mal connus et leurs caractéristiques méritent d’être étudiées dans des modèles expérimentaux appropriés. Ainsi, des études récentes ont montré que la ghréline, un peptide gastro-intestinal, protège les neurones dopaminergiques de la SN contre la mort dans des modèles in vivo et in vitro de syndrome parkinsonien. De plus, dans un modèle animal de syndrome parkinsonien, la ghréline prévient l’aggravation des symptômes gastro intestinaux par la L-DOPA, traitement médicamenteux principal de la MP. Enfin, des altérations des concentrations plasmatiques de ghréline ont également été observées chez les patients aux stades précoces de la maladie. Dans ce contexte, nous avons émis l’hypothèse que la ghréline pourrait jouer un rôle important aux stades précoces de la maladie et donc être utilisée comme biomarqueur et/ou agent neuroprotecteur dans la MP. Ainsi, l’objectif de ma thèse était d’étudier les rôles de la ghréline aux stades précoces de la MP par des approches in vitro et in vivo.La première étape a consisté à déterminer les effets de la ghréline dans des cultures primaires de cellules mésencéphaliques exposées au pesticide roténone, un inhibiteur du complexe I mitochondrial connu pour son association avec la MP. Contrairement aux données de la littérature, nous montrons un effet délétère en fonction de la dose et du temps sur les cellules exposées à la roténone. Nous ne confirmons donc pas l’effet neuroprotecteur de la ghréline dans nos conditions expérimentales. En parallèle, nous avons étudié le potentiel de la ghréline en tant que biomarqueur dans un modèle murin de syndrome parkinsonien reproduisant les stades précoces de la maladie après exposition orale chronique à de faibles doses de roténone. Nous avons d’abord validé ce modèle et confirmé le développement des altérations non motrices et l’absence de mort neuronale au sein de la SN après 1.5 mois de ce régime. En revanche, nos résultats ne montrent pas de modification des taux plasmatiques de ghréline chez les souris exposées 1.5 mois à la roténone. Cependant, des facteurs tels que l’anxiété pourraient avoir affecté les taux de ghréline. Ces données devront donc être confirmées avec des animaux stratifiés selon leur niveau d’anxiété et/ou de plus longues expositions. En conclusion, nos résultats interrogent le rôle neuroprotecteur de la ghréline dans la MP et posent les bases pour de futures recherches sur l’implication de cette hormone orexigène dans la MP. / Parkinson’s disease (PD) is the second most frequent neurodegenerative disease in the world. It is characterized by motor symptoms such as bradykinesia, rigidity and resting tremor. Its definite diagnosis relies on the identification of specific neuropathological hallmarks at autopsy including severe neuronal death within the substantia nigra (SN) and the presence of Lewy bodies in the surviving neurons. PD progresses slowly and the first motor symptoms appear when more than 50% of the SN has degenerated. Therefore, the clinical diagnosis is established late in the course of the disease, thus restricting the therapeutic window for clinicians. In addition, the currently available therapeutic options can only temporarily alleviate PD motor symptoms. The challenges of current PD research are: 1) to anticipate the diagnosis and be able to identify the disease as early as possible, when the SN is still intact enough to implement a disease-modifying/neuroprotection strategy to prevent the appearance of motor symptoms, and 2) to improve current medications and/or develop new therapeutic strategies able to stop the disease before the motor phenotype is installed. The decade preceding PD clinical diagnosis is of particular interest since patients often complain about non-motor symptoms such as anosmia, depression or constipation. Moreover, recent evidences suggest that PD-characteristic lesions could first appear in the peripheral nervous system and slowly progress towards the brain. Thus PD earlier stages and their characteristics deserve better investigations using appropriate experimental models. In this regard, recent studies realized in animal and cellular models of advanced parkinsonism have suggested that ghrelin, an orexigenic peptide mainly produced in the stomach, could play a neuroprotective role in PD. Indeed, exposure to ghrelin has shown a protective effect against the neuronal death in animal and cellular models of parkinsonism. In addition, in a rodent model of parkinsonism, ghrelin was shown to alleviate the L-DOPA-induced worsening of gastro-intestinal symptoms, L-DOPA being the current main therapeutic option in PD. Moreover, ghrelin plasma concentrations have shown alterations in early stages of the disease in small cohorts of PD patients. We therefore hypothesized that ghrelin might play an important role in PD early stages and could serve as a biomarker and a neuroprotective agent in PD. In this context, the aim of my PhD was to investigate the roles of ghrelin in PD early stages using both in vitro and in vivo approaches.We first studied the effects of ghrelin in primary mesencephalic cells exposed to the pesticide rotenone, a potent inhibitor of mitochondrial complex I known for its association with PD. Contrary to the data of the literature, we show a dose and time-dependant deleterious effect of ghrelin on mesencephalic cells exposed to rotenone. This does not confirm the neuroprotective potential of ghrelin in our experimental conditions. In parallel, we investigated the potential of ghrelin as a biomarker in a rodent model of parkinsonism mimicking early stages of the disease after chronic oral exposure to low doses of rotenone. We first validated this model in our animal facility and confirmed that mice exposed to such a regimen develop progressive non-motor alterations but no dopaminergic neuronal death in the SN after 1.5 months. Our initial results do not show a modification of plasma ghrelin levels in rotenone-exposed mice at early stages of the pathological condition. However, confounding factors such as anxiety might have altered ghrelin levels. This should therefore be further ascertained in animals stratified for their anxiety levels and/or in longer exposures. In conclusion, these results challenge the suggested role of ghrelin as a disease-modifying agent in PD and set the bases for future investigations of ghrelin in the context of PD.

The molecular mechanism responsible for degenerative process in the nigrostriatal dopaminergic system in Parkinson’s disease (PD) remains unknown. One major advance in this field has been the discovery of several genes associated to familial PD, including alpha synuclein, parkin, LRRK2, etc., thereby providing important insight toward basic research approaches. There is an consensus in neurodegenerative research that mitochondria dysfunction, protein degradation dysfunction, aggregation of alpha synuclein to neurotoxic oligomers, oxidative and endoplasmic reticulum stress, and neuroinflammation are involved in degeneration of the neuromelanin-containing dopaminergic neurons that are lost in the disease. An update of the mechanisms relating to neurotoxins that are used to produce preclinical models of Parkinson´s disease is presented. 6-Hydroxydopamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and rotenone have been the most wisely used neurotoxins to delve into mechanisms involved in the loss of dopaminergic neurons containing neuromelanin. Neurotoxins generated from dopamine oxidation during neuromelanin formation are likewise reviewed, as this pathway replicates neurotoxin-induced cellular oxidative stress, inactivation of key proteins related to mitochondria and protein degradation dysfunction, and formation of neurotoxic aggregates of alpha synuclein. This survey of neurotoxin modeling—highlighting newer technologies and implicating a variety of processes and pathways related to mechanisms attending PD—is focused on research studies from 2012 to 2014.

Se evaluó la toxicidad individual y binaria de los plaguicidas metomilo y rotenona en la macrofita Lemna minor (Linnaeus, 1753) para determinar la acción sinérgica o antagónica de una mezcla equitóxica. Los ensayos se realizaron bajo condiciones de laboratorio (T 29 ± 2°C; H 48,6 ± 9,4 %) y los resultados fueron analizados con los programas estadísticos BMDS® y Probit para calcular la concentración efectiva media (CE50) a través de los parámetros Área de la Fronda (AF) y Nuevas Frondas (NF). Los resultados sugieren una mayor precisión al utilizar el Método del Punto de Referencia del programa BMDS® para el cálculo de la concentración efectiva. Adicionalmente, se calculó la concentración total de clorofila (CTC) y peso húmedo (PH) para determinar la inhibición del crecimiento (Ir). Del mismo modo, se realizó una comparación entre los valores NOAEL (nivel de efecto no observado) y LOAEL (nivel bajo de efecto observado) versus los valores de BMDL (límite inferior del punto de referencia) y BMD (nivel de variación en el punto de referencia), respectivamente. El análisis muestra que existe una mayor precisión al emplear los valores BMD y BMDL. La acción fitotóxica varió según el parámetro analizado. La CE50 mostró mayores valores en el parámetro NF (1079,22 mg·L-1 para rotenona; 7147,42 mg·L-1 para metomilo; 597,904 mg·L-1 mezcla binaria: metomilo y 298,93 mg·L-1 mezcla binaria: rotenona) que en el parámetro AF (782,173 mg·L-1 para rotenona; 6919,79 mg·L-1 para metomilo; 629,513 mg·L-1 mezcla binaria: metomilo y 314,754 mg·L-1 mezcla binaria: rotenona). Finalmente, el modelo de concentración-adición mostró que la acción fitotóxica de la mezcla de ambos plaguicidas en Lemna minor tiene un efecto sinérgico. Macrophyte Lemna minor (Linnaeus, 1753) was used to evaluate the phytotoxicity of the individual and mix action of methomyl and rotenone pesticides under laboratory conditions (Temperature 29 ± 2°C; Humidity 48,6 9,4 %). The taken measures to establish half effective concentration (EC50) were the frond area (AF) and production of new fronds (NF). In this study, BMDS® and Probit stadistical data analyses were used to compare the results about effective concentration. Results suggest Benchmark dose method of BMDS® program was more effective than Probit program when EC50 were determined. Additionally, total chlorophyll concentration (CTC) and humidity weight (PH) parameters were calculated to determinate the growth inhibition (Ir). Likewise, NOAEL (no observed adverse effect level) and LOAEL (low observer adverse effect level) were compared with BMDL (lower bench mark dose) and BMD (Bench mark dose), respectively. BMD and BMDL values were more accurate than NOAEL and LOAEL values. Phytotoxic action varied according the parameter. EC50 values of Lemna minor showed poor sensibility to methomyl pesticide with an elevated concentration in both parameters (6919,79 mg•L-1 and 7147,42 mg•L-1 for AF and NF, respectively). Results of rotenone pesticide were lower than methomyl, with 1079,22 mg•L-1 (NF) and 782,173 mg•L-1 (AF). Finally, the mix toxicity was 597,904 mg•L-1 (methomyl: NF), 298,93 mg•L-1 (rotenone: NF), 629,513 mg•L-1 (methomyl: AF) and 314,754 mg•L-1 (rotenone: AF). For synergy calculation, Concentration-Adition value was used, concluding a higher synergism.