Les nanoparticules (NP) métalliques occupent une place de plus en plus importante, tant dans les procédésindustriels que dans la recherche biomédicale. Néanmoins, les données sur leur toxicité potentielle pour lesorganismes vivants restent insuffisantes, notamment à l’échelle moléculaire. Des exemples historiques montrentque certaines pathologies, comme la silicose et l’asbestose, peuvent être engendrées par l’exposition chroniqueà des particules inorganiques (silice et fibres d’amiante). Dans les deux cas, la réponse des macrophagesalvéolaires est en grande partie responsable de la maladie. Les macrophages sont en effet la première ligne dedéfense (immunité innée) contre toute attaque exogène, pathogène ou non, du fait de leurs fortes capacitésphagocytaires et de leurs propriétés inflammatoires. Ainsi, les objectifs de ces travaux étaient d’étudier les effetsde deux types de NP métalliques (Cu/CuO et ZnO) sur deux lignées cellulaires de macrophages murins. Cetteétude a été réalisée par une approche protéomique basée sur l’électrophorèse 2D et la spectrométrie de masse,permettant d’accéder de façon reproductible à des données mécanistiques. Les données obtenues ont étévalidées par plusieurs approches ciblées de biologie cellulaire, sur les lignées ainsi que sur des macrophagesprimaires. Nos résultats montrent que les deux types de NP engendrent des réponses différentes, bien que leurdegré élevé de cytotoxicité soit similaire. Les NP de cuivre induisent un stress oxydant et une réponsemitochondriale intenses, associées à de fortes perturbations de la phagocytose et de la production de certainsmédiateurs de l’inflammation. Cette toxicité semble être essentiellement liée aux propriétés redox du cuivre, etest spécifique de la forme nanoparticulaire. A l’inverse, le zinc induit des réponses modérées sur les mêmesprocessus, n’affectant pas a priori le rôle immunitaire des macrophages. Cette toxicité n’est pas non plusspécifique des NP, les ions ayant des effets très étendus, liés à leurs interactions avec de nombreuses protéines,perturbant leur fonctionnement normal jusqu’à induire la mort cellulaire. L’ensemble de ces résultatspermettent une meilleure compréhension des mécanismes moléculaires expliquant la toxicité de ces NP. / Metallic nanoparticles (NPs) are more and more widely used, from industrial processes to biomedical research.However, data on their potential toxicity towards organisms are still lacking, especially regarding molecularmechanisms. It has been proven that some inorganic particles can lead to diseases when tissues are chronicallyexposed. In the case of pulmonary silicosis and asbestosis, induced by silica particles and asbestos fibers, chronicinflammation through alveolar macrophages is responsible for the disease. Indeed, macrophages are the firstdefense against exogenous attacks, like pathogens or inorganic compounds, which are eliminated throughphagocytosis and inflammatory processes that are part of the innate immune response. Thus, this study aimedat analyzing the molecular effects of both copper- and zinc-based NPs (Cu/CuO and ZnO) on murinemacrophages cell lines. To this end, a reproducible proteomic-based approach using 2D electrophoresis andmass spectrometry was used. The proteomic data were validated using targeted approaches on both cell linesand primary macrophages. Our results show that both NPs exert similar high cytotoxicity, but the molecularresponses are markedly different. Copper-based NPs strongly induce oxidative stress as well as alterations inmitochondrial metabolism, phagocytosis, and inflammatory mediators’ production. These effects seem to bemostly related to the redox properties of copper, and are specific to the NP form. Conversely, zinc inducedlimited effects on the same processes, thus leading to no significant alterations in macrophages’ immunefunctions. These effects are not NP-specific, since Zn2+ ions seem to exert most of them, probably due to theirability to interact with numbers of proteins, slightly altering their normal functions, and eventually leading onlyto cell death without prior functional alterations. This study allowed us to highlight some molecular mechanismsof both NP’s toxicity.

Les effets du GSSE (Grape seed and skin extract), extrait de raisin particulièrement riche en antioxydants, ont été étudiés pour prévenir les troubles métaboliques et cardiovasculaires liés à l’obésité. L’obésité est caractérisée par une accumulation ectopique de graisse dans les tissus non adipeux tels que le cerveau. Cette lipotoxicité cérébrale induit une inflammation chronique au niveau du cerveau. Dans ce présent travail, nous avons décrit l’effet anti-obésité du GSSE dans un modèle expérimental d’obésité induite par un régime alimentaire à haute teneur en graisses (HFD) tout en mettant l’accent sur le stress oxydant ainsi que le dysfonctionnement métabolique du cerveau qui n’est pas organe cible de l’obésité. Grâce à ce travail, nous avons développé une approche protéomique quantitative Nano LCMS/MS Label free afin d’identifier les biomarqueurs liés au traitement riche en lipides (HFD) et à la protection apportée au cerveau par le GSSE. Pour ce faire, on a eu recours à un modèle animal afin de mieux comprendre les voies métaboliques potentielles altérées par l’obésité et la protection apportée par le GSSE. Plusieurs protéines ont été identifiées et quantifiées en comparant le protéome cérébral total chez les rats dans les différentes conditions de traitements. On a eu recours à des outils de bioinformatique qui nous ont permis de conclure que ces protéines significativement différenciées sont principalement liées à la voie de la phosphorylation oxydative, de la glycolyse / néoglucogenèse et celle de la signalisation du calcium. Ces résultats ont été confirmés par la mesure de quelques activités enzymatiques métaboliques. Fait intéressant, qu’elles soient sous ou surexprimées par le traitement du HFD, le GSSE corrige l’effet délétère apporté aux différentes protéines et enzymes suite au traitement du HFD. D’autres voies métaboliques cérébrales ont été induites par le GSSE telle que le « HIF signaling pathway ». Ces résultats nous permettent de fournir un élan pour l’investigation thérapeutique du GSSE contre différents désordres métaboliques. / The effects of GSSE (Grape seed and skin extract) extracted from grapes particularly rich in antioxidants have been studied to prevent metabolic and cardiovascular disorders related to obesity. Obesity is characterized by an ectopic accumulation of fat in non-adipose tissues such as the brain. This cerebral lipotoxicity induces chronic inflammation of the brain. In this work, using quantitative proteomic analysis, biochemical and bio-informatic tools allows us to identify actors of metabolic and biological pathways that were disregulated in brain of experimentally-induced obese rats and corrected by GSSE treatment. While our data are consistent with previous findings of obesity-induced brain lipotoxicity, such as enhancement of proteins belonging to the OXPHOS and calcium pathways, they also unveiled novel pathways including the up-regulation of HIF-signaling pathway in HFD brains. In the same context, GSSE abrogated HFD-induced signaling pathway elevation either by modulating several proteins or by inducing some others that were not affected by HFD

Submitted by Franciele Grego Esteves null (francielegrego@gmail.com) on 2017-04-07T15:06:37Z No. of bitstreams: 1 Dissertação de Mestrado p biblioteca.pdf: 6918114 bytes, checksum: e45b8e0514dd2fe63b041ac88e0cd8f7 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: Incluir o número do processo de financiamento nos agradecimentos da dissertação/tese. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-04-17T14:12:17Z (GMT) / Submitted by Franciele Grego Esteves (francielegrego@gmail.com) on 2017-04-20T20:14:39Z No. of bitstreams: 1 Dissertação Mestrado Franciele - Autoarquivamento UNESP.pdf: 6753928 bytes, checksum: 6e5332799980f808f7228f38b18d6a7c (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-04-25T13:55:38Z (GMT) / Submitted by Franciele Grego Esteves (francielegrego@gmail.com) on 2017-04-25T15:04:46Z No. of bitstreams: 1 Dissertação Mestrado Franciele - Autoarquivamento UNESP.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-25T16:05:41Z (GMT) No. of bitstreams: 1 esteves_fg_me_rcla.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) / Made available in DSpace on 2017-04-25T16:05:41Z (GMT). No. of bitstreams: 1 esteves_fg_me_rcla.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) Previous issue date: 2017-03-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A aranha Nephila clavipes pertence ao grupo das aranhas construtoras de teias orbitais, que desenvolveram a capacidade de sintetizar fios adesivos. Esses fios adesivos são encontrados nos círculos centrais destas teias, e apresentam gotículas oleosas contendo vesículas que aprisionam em seu interior soluções de proteínas, peptídeos e muitos compostos de baixa massa molecular. Estudos da análise química dessas gotículas identificaram toxinas, ácidos graxos saturados e até alcaloides. Especula-se que quando um inseto presa é aprisionado pela teia, os ácidos graxos dessas gotículas auxiliam no processo de desestabilização da cutícula do inseto, permitindo a difusão das toxinas para o interior do corpo da presa. Também são relatados alcaloides que atuam como repelentes a predadores em algumas teias, e como inseto-toxinas em outras. A presença dessas moléculas são evidências de que a teia não é uma simples ferramenta para captura mecânica e aprisionamento de presas, mas sim uma complexa estrutura, que parece desempenhar um papel estratégico “ativo” na captura de suas presas. Considerando isso, o objetivo deste estudo foi analisar a ultraestrutura, a disposição das gotículas sobre os fios de seda, e visualizar a presença de vesículas lipídicas extraídas das gotículas da teia orbital da aranha N. clavipes através de microscopia. Além disso explorou-se a riqueza do perfil químico dos compostos de baixas massas moleculares da seda da teia através da cromatografia gasosa bidimensional abrangente acoplada a um detector de massas; e por fim investigar a riqueza de proteínas presentes na seda da teia e nas glândulas produtoras de seda, através da digestão proteolítica em solução, cromatografia líquida, e espectrometria de massas, ressaltando as possíveis toxinas envolvidas na paralisia de presas. Primeiramente, foi realizado um estudo com microscopia eletrônica de varredura das gotículas depositadas sobre os fios de seda, e com microscopia de luz das vesículas lipídicas que se encontram em suspensão, retidas dentro do conteúdo aquoso das gotículas. Posteriormente na análise da perfilagem química foram encontrados 316 compostos, dentre esses 25 foram identificados a partir de padrões químicos, sendo a maioria hidrocarbonetos saturados e alguns ácidos graxos. Este estudo também demonstrou através de bioesaios de repelência, que alguns dos ácidos graxos e hidrocarbonetos identificados nas gotículas da teia apresentaram potencial como repelentes a formigas invasoras. Enquanto que o ácido palmítico apresentou ação potencial no processo de dissolução/desintegração da camada superficial da cutícula de abelhas, para permitir a passagem das toxinas ao interior do corpo das presas da aranha N. clavipes. Na análise proteômica foram indentificados um total de 2051 proteínas na seda da teia, sendo que 163 dessas proteínas são toxinas. Também foram identificadas um total de 927, 1961, 849, e 860 proteínas nas glândulas agregada, ampulada maior, flageliforme e ampulada menor, respectivamente; sendo que desses totais, 194, 78, 32 e 30 são toxinas pertencentes a cada glândula citada acima, respectivamente. Este estudo sugere que as glândulas de seda, principalmente a glândula agregada, podem sintetizar e depositar sobre a seda da teia toxinas importantes, que são comuns a alguns venenos animais, tornando dessa forma as teias como uma estrutura ativa na captura de presas. A partir dos resultados obtidos foi possível elaborar uma hipótese que relaciona o papel das gotículas, vesículas lipídicas e dos compostos identificados como parte da estratégia química da teia na pré digestão e paralisia da presa. Portanto, este estudo forneceu uma melhor compreensão da química-ecológica da captura de presas pela teia da aranha N. clavipes, além de informações que podem possibilitar o uso desses compostos no desenvolvimento de inseticida-seletivo, ou até mesmo em possíveis aplicações farmacológicas. / Nephila clavipes belongs to the group of orb-weavings spiders that have developed the ability to synthesize adhesive threads. Such adhesive threads are found in the core circles of the orb-webs, and present oily droplets which in turn contain many vesicles in suspension, entrapping solutions of proteins, peptides and many small-molecular mass compounds. Chemical analysis studies of these droplets identified toxins, saturated fatty acids and even alkaloids. It is speculated that when an insect-prey is trapped by the web, the fatty acids of these droplets aid in the process of destabilizing the insect cuticle allowing the diffusion of the toxins into the prey body. Some studies also reported alkaloids that act as repellents to predators in some webs, and as insect-toxins in others. The presence of these molecules is evidence that the web is not a simple tool for mechanical capture and imprisonment of prey; but rather a complex structure that seems to play a strategic "active" role in capturing its prey. Considering this, the aim of this study was to analyze the ultrastructure, the disposition of the droplets on the silk fibers, and to visualize the presence of lipid vesicles extracted from the web’s droplets of N. clavipes spider, by microscopy. In addition, the richness of the chemical profile of the small-molecular mass compounds in the web-silk was explored through comprehensive two-dimensional gas chromatography coupled to a mass detector; and finally to investigate the richness of proteins present in web-silk and silk-producing glands through in solution proteolytic digestion, liquid chromatography, and mass spectrometry, highlighting the possible toxins involved in the prey paralysis. First, was performed scanning electron microscopy study of the droplets deposited on the web-silk and light microscopy of the suspended lipid vesicles retained within the aqueous contents of the droplets. Posteriorly in the profiling chemical analysis were identified 316 compounds, among these 25 were identified from chemical standards, most of them are hydrocarbons saturated and some fatty acids. This study also demonstrated through repellence bioassays, that some of the fatty acids and hydrocarbons identified in the web's droplets presented potential as repellents to invasive ants. While the palmitic acid presented potential action in the process of dissolution / disintegration superficial layer of cuticle bees, to allow the difusion of toxins into the prey body. In the proteomic analysis a total of 2051 proteins were identified in the web-silk, of which 163 of these proteins are toxins. A total of 927, 1961, 849, and 860 proteins were also identified in the aggregate, major ampullate, flagelliform and minor ampullate glands, respectively; and of these totals, 194, 78, 32 and 30 are toxins belonging to each gland mentioned above, respectively. This study suggests that silkproducing glands, especially the aggregate gland, can synthesize and deposit on the silk-web important toxins, which are common to some animal venoms, thereby making the webs an active structure in the prey capture. From the obtained results it was possible to elaborate a hypothesis that relates the role of the droplets, lipid vesicles and the compounds identified as part of the chemical strategy of the web in the pre-digestion and prey paralysis. Thus, this study provided a better understanding of the chemical-ecological prey capture by N. clavipes spider web, in addition to information that may enable the use of these compounds in the development of insecticide-selective or even possible pharmacological applications. / FAPESP: 2011/51684-1 / FAPESP: 2015/14220-8