Conselho Nacional de Desenvolvimento Científico e Tecnológico / Among various antioxidant therapeutic applications, neuroprotective action is highlighted sinceoxidative stress is recognized as one of the events involved in cell damage which occur in most of neurological disorders, including Parkinson s disease (PD). Consequently, the search for natural and/or synthetic antioxidants which may be effective in the treatment of neurological disorders has grown over the past years. In this context, several studies have shown the antioxidant potential of selenium organic compound diphenyl diselenide (DPDS) in vitro and in vivo, but there are still few studies talking about antioxidant activity of Valeriana officinalis (V. officinalis). Likewise, there is no research on the possible beneficial effects of these agents in models of neurological diseases such as PD using Drosophila melanogaster (D. melanogaster), an specie that has been used with great reliability in the reproduction of dopaminergic dysfunction models. Thus, the objective of this study was to evaluate the in vitro antioxidant activity of V. officinalis, as well as, the effect of supplementation of extract from roots of this plant and DPDS on behavioral and biochemical changes induced by pesticide rotenone exposure in D. melanogaster. As a result, we verify that: ethanolic extract from V. officinalis inhibited the generation of TBARS caused by various pro-oxidants agents in rat s brain homogenate in vitro, diminished deoxyribose degradation and generation of reactive oxygen species (ROS) caused by quinolinic acid (QA); flies exposed to rotenone were significantly lower than control group in behavioral tests of climbind and open-field (number of crossings and immobility time) and higher incidence of mortality. V. officinalis treatment was effective in reducing these effects, except against the decrease in number of crossings. Exposure to rotenone decreased in flies cell viability and non protein thiol content, but V. officinalis treatment normalized to the control levels. Rotenone increased mRNA expression on superoxide dismutase (SOD), catalase (CAT) and tyrosine hydroxilase (TH) enzymes, which were restored by treatment with V. officinalis; DPDS supplementation was not effective in offering protection against locomotor and biochemical alterations induced by rotenone. In addition, DPDS per se induced an increase in ROS production and decreased survival rate of flies. In general, data showed that V. officinalis may be a promising neuroprotective agent, since it was effective in reducing the oxidative damage caused by different neurotoxic agents and toxic effects caused by rotenone exposure. Thus, the use of this plant extract may be beneficial in reducing neurological disorders associated to the oxidative stress. In relation to the use of DPDS, further studies aimed at the concentrations used are necessary, given that the concentration tested in this work did not offer protection against rotenone damage effects, DPDS potentiated the effect of this pesticide on mortality and exhibited toxic effects per se. Overall, these results contribute to the advancement of research focused on the toxicology and pharmacology of natural and synthetic products and screening for agent that provide neuroprotection and may be promising to assist in the treatment of neurodegenerative diseases, including PD. / Dentre as várias aplicações terapêuticas dos antioxidantes, destaca-se a ação neuroprotetora, uma vez que, o estresse oxidativo (EO) é reconhecido como um dos eventos envolvidos nos danos celulares que ocorrem na maioria das doenças neurológicas, incluindo a doença de Parkinson (DP). Consequentemente, a procura por antioxidantes naturais e/ou sintéticos que possam ser eficazes no tratamento de distúrbios neurológicos tem crescido muito ao longo dos últimos anos. Neste contexto, vários trabalhos têm evidenciado o potencial antioxidante do composto orgânico de selênio disseleneto de difenila (DPDS) in vitro e in vivo; mas, ainda, são escassos estudos acerca da atividade antioxidante da planta Valeriana officinalis (V. officinalis). Da mesma forma, ainda não há pesquisas sobre os possíveis efeitos benéficos desses agentes em modelos de doenças neurológicas, como a DP, utilizando a Drosophila melanogaster (D. melanogaster), uma espécie que vem sendo usada com bastante confiabilidade na reprodução de modelos de disfunção dopaminérgica. Assim, o objetivo deste trabalho foi avaliar a atividade antioxidante in vitro da V. officinalis, bem como, os efeitos oriundos da suplementação com o extrato da raiz desta planta e com DPDS sobre alterações comportamentais e bioquímicas induzidas pela exposição ao pesticida rotenona em D. melanogaster. Como resultados, verificamos que o extrato etanólico de V. officinalis inibiu a geração de TBARS causada por diferentes agentes pró-oxidantes, em homogeneizado de tecido cerebral de rato in vitro; diminuiu a degradação da desoxirribose e a geração de espécies reativas de oxigênio (ERO), causada pelo ácido quinolínico (AQ); as moscas expostas à rotenona tiveram um desempenho significativamente inferior ao grupo controle nos testes comportamentais de escalada e campo-aberto (número de cruzamentos e tempo de imobilidade), bem como, uma maior incidência de mortalidade. O tratamento com V. officinalis foi eficaz em reduzir esses efeitos, exceto frente à diminuição do número de cruzamentos. A exposição à rotenona diminui a viabilidade celular e o conteúdo de tiol protéico das moscas, que foi normalizada aos níveis do controle pelo tratamento com V. officinalis. A rotenona aumentou a expressão de mRNA das enzimas Superóxido dismutase (SOD), Catalase (CAT) e Tirosina hidroxilase (TH) quando comparado ao grupo controle e a alteração observada na expressão da SOD e CAT foi restaurada pelo tratamento com V. officinalis; a suplementação com DPDS não foi eficaz em oferecer proteção contra as alterações locomotoras e bioquímicas induzidas por rotenona. Além disso, o DPDS induziu per se um aumento na produção de ERO e uma diminuição na taxa de sobrevivência das moscas. De forma geral, os dados obtidos mostram que a V. officinalis pode ser considerada um agente neuroprotetor promissor, uma vez que foi eficaz em reduzir os danos oxidativos causados por diferentes pró-oxidantes e os efeitos tóxicos causados pela exposição à rotenona. Assim, o uso do extrato desta planta pode ser benéfico na redução de complicações neurológicas associadas ao EO. Com relação ao uso do DPDS, mais estudos voltados para as concentrações utilizadas são necessários, tendo em vista que, na concentração testada neste modelo experimental, o mesmo não ofereceu proteção contra os efeitos danosos da rotenona, potencializou o efeito do pesticida sobre a taxa de mortalidade e exibiu efeitos tóxicos per se. De forma geral, esses resultados contribuem para o avanço das pesquisas voltadas para a toxicologia e farmacologia de produtos naturais e sintéticos e para a triagem de agentes que ofereçam neuroproteção e possam ser promissores para auxiliar na terapêutica de doenças neurodegenerativas, incluindo a DP.

Made available in DSpace on 2014-06-11T19:22:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-16Bitstream added on 2014-06-13T20:29:22Z : No. of bitstreams: 1 okamoto_dn_me_sjrp.pdf: 676452 bytes, checksum: 7d8aa473e5171e8c8a4187d9499700da (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A presença de resíduos químicos no ambiente afeta todo o ecossistema. Uma das técnicas empregadas para o estudo dos efeitos tóxicos dos resíduos químicos é o biomonitoramento, que utiliza como modelos organismos vivos, entre eles os insetos. Um dos gêneros utilizados é a Drosophila, em particular a espécie D. melanogaster de distribuição cosmopolita. É um organismo geneticamente bem explorado, de fácil manutenção e rápido ciclo biológico, que após o seqüenciamento completo de seu genoma revelou homologia de 60% de seus genes com os genes de doenças humanas. Entre as substâncias químicas que foram investigadas neste trabalho, incluem-se três metais tóxicos (alumínio, cromo e chumbo), um corante (azul Coomassie brilliant G250) e o produto sintético acrilamida. A análise foi realizada em concentrações baixas (50µM) com ênfase na característica acumulativa de cada produto químico durante dez gerações de exposição. As análises, fundamentadas na biologia do inseto, avaliaram sua produtividade, alterações morfológicas, viabilidade das diversas fases do desenvolvimento, o peso dos insetos, o comportamento sexual e a atividade enzimática da carboxilesterase. Foi realizado também um experimento de produtividade comparativo com duas linhagens, uma massal e outra isofêmea. A exposição à acrilamida, aumentou a produtividade na primeira exposição, e também reduziu a viabilidade de ovos para adultos nas gerações F3 e F10. Por sua vez, entre os metais, o chumbo, afetou a viabilidade de ovos em pupas na décima geração e apresentou alterações no tempo de pré-cópula e de cópula. A exposição ao cloreto de alumínio, revelou na primeira e na quinta geração aumento do tempo de pré-cópula, e na décima geração aumento do tempo de cópula. O dicromato de potássio...

Food additives are used by the food industry in order to enhance the aroma, flavor and texture of foods. Of all the additives used colors are the most genotoxic. The azo dyes, derived from oil, used by industries, are the tartrazine and red #40. The SMART wing test was used to verify the possible genotoxic effects of colors in somatic cells of Drosophila melanogaster. For this evaluation were used larvae of 48 hours, descendants of the Standard Cross (ST) and High Bioactivation (HB) that who were treated with tartrazine in concentrations 1.5, 2.5 and 3.0 mg / mL and 5, 10 and 20 mg / mL for the red #40. The results showed significant increase of spots in the ST descendants for tartrazine and HB for the red #40. We concluded that the artificial colors tartrazine and red #40 are genotoxic in the doses used for the study. / Os aditivos alimentares são utilizados pela indústria alimentícia com o intuito de melhorar o aroma, o sabor e a textura dos alimentos. De todos os aditivos utilizados os corantes são os mais genotóxicos. Os corantes azóicos, derivados do petróleo, utilizados pelas indústrias, são o tartrazina e vermelho 40. O teste SMART de asa foi utilizado para verificar os possíveis efeitos genotóxicos destes corantes em células somáticas de Drosophila melanogaster. Para esta avaliação foram utilizadas larvas de 48 horas, descendentes do cruzamento padrão (ST) e alta bioativação (HB) que foram tratadas com tartrazina nas concentrações 1,5, 2,5 e 3,0 mg/mL e 5, 10 e 20 mg/mL para o vermelho 40. Os resultados mostraram aumento significativo de manchas nos descendentes ST para tartrazina e HB para o vermelho 40. Concluímos que os corantes artificiais tartrazina e vermelho 40 são genotóxicos nas doses utilizadas para o estudo. / Mestre em Genética e Bioquímica

A velocidade específica de respiração (QO2) é um parâmetro fundamental para entender-se o metabolismo e o estado fisiológico celular, fornecendo informações úteis para o processo e controle em biorreatores. Neste trabalho, cultivou-se diferentes células de insetos em ambiente controlado medindo-se o QO2 e concentração crítica de oxigênio (Ccrít). Foram utilizadas nos ensaios células de insetos Spodoptera frugiperda (Sf9) não infectadas e células de Drosophila melanogaster (S2) selvagem e recombinantes, utilizadas na expressão de diferentes proteínas. Todas as experiências foram realizadas em biorreator Inceltech com volume de trabalho de 1L, mantido a temperatura de 28ºC, agitação de 100 rpm e oxigênio dissolvido (OD) a 40% da saturação de ar, com difusão por membrana de silicone com mistura gasosa (O2 e N2) e vazão gasosa constante. Foi utilizado meio de cultura Sf900II sem soro fetal bovino. O QO2 foi medido pelo método dinâmico e pelo balanço de oxigênio na fase líquida. Neste trabalho foi implementado um novo processo durante o método dinâmico para interromper completamente a transferência gasosa durante a execução deste método. Implementou-se também uma metodologia para medição de Ccrít. Chegou-se a concentrações máximas celulares (Xm), velocidades máximas específicas de respiração (QO2) na fase exponencial e Ccrít, conforme segue: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfectadas para expressão de GPV): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfectadas para expressão de EGFP): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfectadas para expressão de HBsAg): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. Conclui-se que as linhagens selvagens e transfectadas de S2 possuem entre si uma atividade respiratória diferente e também que as novas metodologias implantadas verificaram-se satisfatoriamente. / Specific respiration rate (QO2) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure QO2 and critical oxygen concentration (Ccrit). Wild Spodoptera frugiperda (Sf9) and wild and transfected Drosophila melanogaster S2 cells (able to produce different proteins) were used. All experiments were performed in 1-liter working volume Inceltech bioreactor, maintaining temperature controlled at 28ºC, agitation rate at 100 rpm, and dissolved oxygen (DO) at 40% of air saturation, through membrane diffusion of mixed gases (O2 and N2) at constant total flow rate. SF900II serum free medium was used. QO2 was measured through dynamic method and oxygen mass balance in the liquid phase. In this work a new process was implemented during the dynamic method to interrupt completely the oxygen transfer during the execution of this method. It was also implemented a methodology for measurement of Ccrít (determined when DO reduces its decay rate, without oxygen transfer). Maximum cell concentration (Xm), maximum specific respiration rate (QO2) in the exponential phase and Ccrít were reached, as follows: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfected for GPV expression): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfected for EGFP expression): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfected for HbsAg expression): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. From these results, it can be concluded that the studied cell lines have different respiration activity and the new developed methodologies behave satisfactorily.

Aurora-A (AurA) est une sérine/thréonine kinase jouant un rôle majeur dans le cycle cellulaire. Elle est connue pour son rôle oncogène et les compagnies pharmaceutiques développent des inhibiteurs ciblant son activité kinase. Cependant, il a été montré chez différentes espèces qu’Aurora-A possède des rôles indépendants de son activité kinase et agit également comme suppresseur de tumeur quand son activité kinase est altérée. Ceci pose donc un problème dans le développement des inhibiteurs car cibler l’activité kinase d’Aurora-A pour traiter le cancer pourrait mener à l’effet inverse. Pour résoudre ce dilemme, j’ai étudié en détail les phénotypes de mutants AurA nul et hypomorphe chez Drosophila melanogaster. J’ai étudié à la fois les défauts de développement en me basant sur le temps de pupation des larves et le rôle de suppresseur de tumeur en me basant sur les neuroblastes du cerveau central. Dans ce modèle, une caractéristique des suppresseurs de tumeur est leur capacité à induire la formation de neuroblastes supplémentaires dans le cerveau central conduisant à une surcroissance du cerveau. Chez les mutants AurA, la taille du cerveau est plus petite jusqu’à 96h de développement larvaire. Cependant, la pupation arrivant normalement entre 96h et 120h de développement larvaire est retardée chez le mutant et les larves ont une taille plus importante. Chez les mutants en retard de pupation le cerveau devient plus gros que ceux du contrôle. Le cerveau des mutants AurA a une importante augmentation du nombre de cellules positives pour Deadpan, un marqueur spécifique des neuroblastes et ce, avant que le cerveau des mutants AurA devienne plus grand que celui du contrôle. De plus, les disques imaginaux d’ailes et la glande annulaire sont clairement plus petits que ceux du contrôle à 96h de développement larvaire et les larves mutantes atteignent les stades L2 et L3 plus tôt. En conclusion, les mutants AurA montrent 1) une avance dans leur développement précoce certainement reliée au défaut de croissance de la glande annulaire ; 2) un retard de pupation ressemblant à celui observé en cas de défauts dans la voie de l’ecdysone, certainement dû à des défauts de croissance des disques imaginaux d’ailes ; 3) une surcroissance du cerveau à mettre en lien à la fois avec une augmentation du nombre de pseudo-neuroblastes et avec le retard de pupation. / Aurora-A (AurA) is a major kinase playing various roles in cell cycle. It’s a well-known oncogene and companies are developing drugs inhibiting its kinase activity. However, it has been shown in different species that AurA can have a kinase independent role or act as a tumor suppressor when its kinase activity is altered. This represents a problem for drugs development as inhibiting AurA kinase activity only could lead to life threatening phenotypes. To address this dilemma, we carefully deciphered phenotypes of AurA null and AurA hypomorph mutants in Drosophila melanogaster using the pupation as readout for development timing and larval central brain neuroblasts as model for tumorigenic study. One readout to define a tumor suppressor in this model is a brain overgrowth phenotype associated to central brain neuroblasts over-proliferation. In AurA mutants, brain size appears slightly smaller until 96h of larval development. However, pupation occurring normally between 96 and 120h of larval development is delayed in AurA mutants and larvae have an increased size. In this “delayed” mutant larvae, brains are eventually bigger than wild-type controls. Furthermore, AurA mutant central brains show a huge increased number of cells positive for deadpan, a marker of neuroblast identity, even before the appearance of brain over-growth phenotype. Additionally, wing discs and ring glands are clearly smaller in AurA mutants at 96h compared to control and mutant larvae reach L2 and L3 developmental stage earlier than control. In conclusion, AurA mutants have: 1) a precocious developmental advance certainly related to ring gland growth defect; 2) a pupation delay which resembles Ecdysone pathway timing defects certainly due to wing discs growth defect; 3) an enlarged brains phenotype due to an increased of the number of neuroblast-like cells and the pupation delay.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Honey is a complex mixture produced by honey bees from the nectar, which is used by its sweetening properties, as well as by its human health benefits. The aim of this study was to characterize, for the first time in literature, the Brazilian Pampa Biome honey in terms of quality parameters and determination of its antioxidant properties in vitro and in vivo. A total of 10 honey samples were tested for physicalchemical parameters, as moisture, free acidity, reducing sugars, hydroxymethylfurfural (HMF) levels, among others. The antioxidant activity (in vitro), as evaluated by total phenolics, flavonoid content, FRAP and DPPH-ABTS scavenging activity. Drosophila melanogaster (in vivo) were exposed to oxidative stress induced by Iron (Fe) and Paraquat (PQ) in different treatment protocols, in the presence or absence of honey. The survivorship and locomotor activity (negative geotaxis) were analyzed. Possible glycemic alterations on honey diet were also evaluated. The results of physicochemical analysis indicated that all honeys were in accordance with the standards established by the Brazilian law, in which follows international standards. All honey samples showed significant antioxidant activity in vitro. Flies treated with honey showed increased lifespan and were protected against oxidative stress induced by Fe and PQ in the different treatment protocols (48h and 7 days), in regards of mortality and locomotor deficits. Despite the high sugar content of honey, glucose content were unchanged in honey fed flies, when compared to flies fed on honey-equivalent amounts of sugars. This study demonstrates that Brazilian Pampa Biome honey has a high quality level, a significant antioxidant activity in vitro and a protective potential against oxidative stress (in vivo). It has been also demonstrated that Drosophila melanogaster could be a valid model for studies with honey, as well as the usage of this natural product as an alternative in the therapy of oxidative stress-associated diseases. / O mel é uma mistura complexa produzida por abelhas melíferas a partir do néctar, sendo um produto bastante utilizado pelas suas propriedades edulcorantes, bem como pelos seus benefícios para a saúde humana. O objetivo deste estudo foi caracterizar, pela primeira vez na literatura, o mel Bioma Pampa Brasileiro em termos de parâmetros de qualidade e determinação de suas propriedades antioxidantes in vitro e in vivo. Um total de 10 amostras de méis foram testadas quanto os parâmetros físico-químicos, como a umidade, acidez livre, açúcares redutores, níveis de hidroximetilfurfural (HMF), entre outros. Na atividade antioxidante (in vitro), foram avaliados os compostos fenólicos totais, teor de flavonóides, FRAP e atividade sequestradora de radicais (DPPH e ABTS). Drosophila melanogaster (in vivo) foram expostas a estresse oxidativo induzido por Ferro (Fe) e Paraquat (PQ) em diferentes protocolos de tratamento, na presença ou ausência de mel. Foram analisadas a sobrevivência e a atividade locomotora (geotaxia negativa). Possíveis alterações glicêmicas na dieta de mel também foram avaliadas. Os resultados das análises físico-químicas indicaram que todos os méis estavam de acordo com os padrões estabelecidos pela legislação Brasileira, que segue os padrões internacionais. Todas as amostras de mel mostraram atividade antioxidante significativa in vitro. Moscas tratadas com mel mostraram aumento da expectativa de vida e foram protegidos contra o estresse oxidativo induzido por Fe e PQ nos diferentes protocolos de tratamento (48 horas e 7 dias) em relação a déficit locomotores e mortalidade. Apesar do alto teor de açúcar do mel, moscas alimentadas com este não tiveram seus níveis de glicose alterados, quando comparado com moscas alimentadas com quantidades de açúcares em quantidades equivalentes ao mel. Este estudo demonstra que o mel do Bioma Pampa Brasileiro tem um alto nível de qualidade, uma atividade antioxidante in vitro significativa e um potencial de proteção contra o estresse oxidativo (in vivo). Também está demonstrado que a Drosophila melanogaster pode ser um modelo válido para estudos com mel, bem como a utilização deste produto natural, como uma alternativa para a terapia de doenças associadas ao estresse oxidativo.

Au cours d’une infection virale, la survie des cellules dépend d’informations adéquatement distribuées, reçues et traitées, permettant l’établissement d’une réponse antivirale performante. La communication cellulaire est donc essentielle pour permettre la propagation de signaux immuns protecteurs à tout l’organisme.Chez les insectes, la principale réponse antivirale est l’ARN interférent (ARNi), activé lors de la détection d’ARN double brin (ARNdb) d’origine virale. Le mécanisme antiviral de l’ARNi peut être cellulaire ou systémique. Dans la première catégorie, la régulation de l’expression génique est limitée à la cellule dans laquelle l’ARNdb est produit, alors que dans la seconde, cette même régulation s’effectue dans des cellules distinctes de celles produisant l’ARNdb. Chez les insectes, l’ARNi systémique reste très peu décrit.Ma thèse explore le rôle de la protéine de drosophile CG4572/DORA, dans les mécanismes permettant l’établissement de l’ARNi systémique. J’ai également cherché la nature des signaux déclencheurs de cette réponse antivirale. Nous montrons l’existence de deux mécanismes de communication cellulaire permettant la propagation de signaux antiviraux: des vésicules extracellulaires et des nanotubes. Nous mettons en évidence que des vésicules contenant DORA et des fragments d’ARN viraux peuvent se propager dans les mouches en leur conférant une protection antivirale spécifique. Nous montrons également pour la première fois la présence de nanotubes membranaires qui contiennent des protéines de la machinerie ARNi ainsi que DORA.Les mécanismes que nous proposons sont pour la première fois associés à la réponse antivirale chez Drosophila melanogaster. / During viral infection, cell survival will depend on adequately giving, receiving and processing information to establish an efficient antiviral immune response. Cellular communication is therefore essential to allow the propagation of immune signals that will confer protection to the entire organism.The major antiviral defense in insects is the RNA interference (RNAi) mechanism that is activated by detection of viral double-stranded RNA (dsRNA). The antiviral RNAi mechanism can be divided in cell- and non-cell- autonomous. In cell-autonomous RNAi, the silencing process is limited to the cell in which the viral dsRNA is produced. In non-cell-autonomous (systemic) RNAi, the interfering effect occurs in cells different from where the viral dsRNA was produced. In insects the systemic RNAi response remains poorly characterized. My PhD explores the role of the Drosophila CG4572/DORA protein in the establishment of systemic antiviral RNAi. It also investigates the nature of immune signals that trigger the antiviral response. I provide evidence for the existence of two different mechanisms of cell-cell communication that allow the spread of the immune signal: extracellular vesicles and tunneling nanotubes. I describe that DORA-positive extracellular vesicles carry fragments of viral RNAs that can spread and confer specific antiviral protection in flies. I also present the characterization of tunneling nanotubes (TNTs) containing components of the RNAi machinery, DORA and dsRNA and I hypothesize on the use of TNTs in the spread of the immune signal.Both mechanisms of cell-to-cell communication are coupled for the first time to the antiviral response in Drosophila melanogaster.

Chez les insectes, la voie des petits ARNs interférants (siARN) joue un rôle majeur dans la réponse antivirale. Ces dernières années, il a été montré que les petits ARNs interagissant avec les protéines PIWI (piARNs) sont impliqués dans la défense des moustiques face aux infections arbovirales. Le but de mon travail de thèse fut de caractériser l'implication de la voie des piARNs dans la réponse antivirale de la Drosophila melanogaster, utilisée ici comme un organisme modèle. Dans un premier temps, j’ai demontré qu’à la suite d’une infection virale, la survie et le titre viral chez les drosophiles mutées pour les protéines Piwi, Aubergine, Argonaute-3 et Zucchini, ne présente aucune différence avec les données observées pour les drosophiles sauvages. Ensuite, via l’utilisation de virus provoquant une infection aigue, persistante ou se transmettant de manière verticale par les cellules germinales, j’ai montré l’absence de production de piARNs viraux durant l’infection chez les drosophiles adultes. Finalement, l’utilisation de mes données de séquencage m’a permis d’observer la production de piARNs dérivant d’un gène codant une protéine impliquée dans la réponse antivirale. Suggérant ainsi un rôle hypothétique des piARNs dans la régulation de l’immunité de l’hôte durant l’infection virale.Mes travaux visent à améliorer la compréhension de la réponse immunitaire antivirale chez l’insecte. Je montre que la fonction antivirale de la voie des piARN dépend plus de la biologie de l’hôte et du virus que de la réponse antivirale en elle-même. / In insects, the small interfering RNA (siRNA) pathway is the major antiviral response. In recent years, the piwi-interacting RNA (piRNA) pathway has been also implicated in antiviral defense in mosquitoes infected with arboviruses. The aim of my thesis was to characterize the involvement of the piRNA pathway in antiviral defense in Drosophila melanogaster. I first showed that following virus infection, the survival and viral titers of Piwi, Aubergine, Argonaute-3, and Zucchini mutant flies were similar to those of wild type flies. Then, by studying an array of viruses that infect the fruit fly acutely or persistently or are vertically transmitted through the germ line, I showed that no viral piRNAs are produced during infection in adult Drosophila melanogaster. Finally, using the next generation sequencing data generated during viral infections, I showed the presence of piRNAs derived from protein coding gene and suggested their potential role in regulating the immune status of the host during viral infection.This work improves the current understanding of the antiviral response in insects. It shows that, in contrast to what was observed in mosquitoes, the piRNA pathway is not directly implicated in antiviral defence in adult Drosphila melanogaster and that viral piRNAs production depends on the biology of the host–virus combination rather than being part of a general antiviral process.

Les acides gras (AGs) sont des molécules nécessaires pour la survie des êtres vivants car ils participent à de nombreuses voies métaboliques et constituent un réservoir énergétique important. Malgré le lien établi entre les AGs et certaines maladies humaines (cancers, obésité, maladies cardiovasculaires), les mécanismes sous-jacents à la perception de ces composés ont été, jusqu’à présent, très peu explorés, en particulier chez les invertébrés. C'est l’une des raisons pour lesquelles, nous avons étudié, lors de notre thèse, la perception des AGs à longue chaine (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3), en utilisant l’espèce modèle Drosophila melanogaster. Nous avons aussi tenté de caractériser quelques facteurs biologiques sous-jacents à la modulation de cette réponse. Nos observations comportementales, effectuées sur des individus ou des groupes issus des deux lignées sauvages Dijon2000 (Di2) et Canton-S, ont permis de démontrer que les larves étaient capables de distinguer ces AGs. En particulier, nous avons observé que les larves sont attirées par les AGs insaturés et fortement repoussées et/ou stressés par les AGs saturés. Ces derniers déclenchent d’ailleurs une forte réaction comportementale chez les groupes de larves qui montrent un phénomène durable d’agrégation. Une autre raison pour effectuer cette étude vient de la proximité structurale des AGs et des hydrocarbures cuticulaires qui servent de phéromones sexuelles chez les adultes. Ainsi, nous avons testé la réponse de mutants pour les gènes desat1 et CheB42a, qui sont impliqués dans la perception et/ou la discrimination de ces phéromones. Les larves mutantes desat1 ne montrent pas de comportement d’agrégation, ce qui suggère une altération de leur perception ou de leur réaction sur les AGs saturés. Nous avons ciblé des perturbations de desat1 dans divers tissus en combinant différentes lignées pilotes (correspondant aux différentes régions régulatrices de desat1) avec un transgène exprimant l’ARN interférent de desat1. Nous avons découvert qu’une région régulatrice ciblant l’ARN interférent dans une partie du cerveau perturbe la réponse aux AGs saturés, ce qui est cohérent avec le résultat précédent. Les larves portant le transgène CheB42a-Gal4 montrent des défauts plus subtils et néanmoins une répulsion moins forte vis-à-vis des AGs saturés. Nous avons tiré profit du temps court de génération de la drosophile pour réaliser deux expériences de sélection sur la lignée Di2, pendant environ 50 générations. Dans un premier temps, nous avons sélectionné, génération après génération, les larves montrant des réponses atténuées envers le C18:0, et nous avons observé, après 20 générations, une plus faible tendance à éviter cet AG. Dans un deuxième temps, nous avons élevé la lignée Di2 sur un milieu appauvri en AGs et nous avons noté que les larves ainsi élevées perçoivent anormalement le C18:3. Afin de vérifier si la préférence envers les AGs pouvait changer au cours de la vie, nous avons testé la réponse des adultes. Un test réalisé dans un olfactomètre, et visant à mesurer la réponse olfactive, montre que les adultes sont repoussés par le C18:3, et indifférents au C18:0. Un test de réponse gustative (répression de l’extension du proboscis) montre que les AGs insaturés répriment plus fortement la réponse que les AGs saturés. En conclusion, au cours de notre thèse, nous avons pu démontrer que les préférences envers les AGs saturés et insaturés varient au cours de la vie de la drosophile, peut-être en relation avec leurs besoins nutritifs. Les AGs semblent être perçus par olfaction, gustation, et peut être par mécanosensation. / Fatty-acids (FAs) are crucial for animal survival and reproduction since these molecules are involved in many metabolic pathways and constitute a substantial store of energy. Despite the strong relationship established between excessive FA consumption and severe human etiologies (obesity, cancer, vascular diseases), our knowledge of the mechanisms underlying FA perception and preference is limited particularly in invertebrates. This is one of the reasons why we decided to investigate, during our PhD thesis, the perception of long-chain FAs (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3), using the model species Drosophila melanogaster. We have also attempted to characterize some of the biological factors involved in the modulation of this response. Our behavioral tests, carried both on individuals and groups of the two wild-type strains Dijon2000 (Di2) and Canton-S, revealed that larvae are able to distinguish these FAs. More precisely, we observed that larvae are attracted by unsaturated FAs and strongly repulsed and/or stressed by saturated FAs. The latter compounds also induce a strong behavioral reaction in groups of larvae resulting into a long-lasting aggregation figure. Another reason to study FAs is their structural proximity with cuticular hydrocarbons which act as sex pheromones in adults. Therefore, we measured the behavioral response of mutants for the desat1 and CheB42a genes, both of which are involved in the perception and/or discrimination of these pheromones. The fact that mutant desat1 larvae do not show aggregation behavior suggests that they have an abnormal perception or reaction to saturated FAs. We then targeted genetic alteration of desat1 using drivers (made with different regulatory regions of desat1) combined with a transgene carrying the interferential RNA of desat1. When targeting a specific brain region, we found that larvae do not properly react to C18:0. This result is coherent with the defect described above. On the other hand, larvae carrying the CheB42a-Gal4 transgene exhibit more subtle defects including a slightly altered reaction to saturated FAs. We also took advantage of the short generation time of D. melanogaster to perform two selection experiments using the parental Di2 strain during 50 generations. First, we selected, generation after generation, larvae showing decreased aversive response to C18:0, and we observed, after 20 generations, a weaker tendency to avoid this FA. Second, we maintained the Di2 strain on a food with a poor FA content, and we noted that resulting larvae show an altered response to C18:3. To check whether FA preference could change during life time, we also measured adult behavior. A first test performed in a olfactometer to assess olfactory response, revealed that flies are repulsed by C18:3, but are indifferent to C18:0. A second test assessing adult gustatory response (with the repression of the proboscis extension reflex) showed that unsaturated FAs repress behavior more strongly than saturated FAs. In summary, during our PhD thesis, we have shown that FA preference can change during the life of D. melanogaster, maybe in relation with different nutritional requirements. FAs seem to be perceived by olfaction, taste and maybe mechanosensation.

Les éléments transposables sont des composants majeurs de la plupart des génomes, et leur impact sur l’évolution des génomes est maintenant bien documenté. Cependant, la manière par laquelle ils participent au transcriptome n’est pas encore clairement établie. En utilisant le génome séquencé de Drosophila melanogaster et les bibliothèques d’EST, nous avons déterminé les insertions d’éléments transposables qui sont transcrites sans équivoque, ainsi que leur localisation dans le génome séquencé de D.melanogaster. Nous montrons que la plupart des familles d’éléments transposables sont transcrites, et nous identifions spécifiquement 69 insertions d’éléments transposables exprimés, dont la moitié réside dans des gènes, la plupart dans des introns et des régions régulatrices 5’UTR. / Transposable elements (TEs) are major components of most genomes, and their impact on genome evolution is now well documented. However, the way they affect the transcriptome is still not clearly established. Using the sequenced genome of Drosophila melanogaster and EST libraries (“Expressed Sequence Tag”, large tags (~500bp) corresponding to subsequences of a transcribed cDNA sequences), we describe here the TE insertions that are unequivocally transcribed, and we have determined their location in the sequenced genome of Drosophila melanogaster. We show that most TE families are transcribed, and we have specifically identified 69 expressed TE insertions, half of which are located inside genes, mostly within introns and 5′UTRs regulatory regions.