Les nanoparticules (NP) de poly(acide lactique) (PLA) sont des vecteurs biodégradables prometteurs pour la vaccination et la délivrance thérapeutique. Cependant leur évaluation in vivo n'est pas toujours couronnée de succès. Un des écueils réside dans la difficulté à suivre la prise en charge cellulaire de ces nanomatériaux à l'échelle d'un organisme, ces NP étant indécelables dans les tissus profonds. L'objectif de cette thèse est de valider l'utilisation des NP de PLA comme plateforme d'imagerie et de vectorisation d'actifs chez Drosophila Melanogaster et d'analyser ainsi le devenir de NP dans un corps entier. Le modèle drosophile a été choisi pour son faible encombrement, sa facilité d'élevage, la diversité des lignées transgéniques et la puissance des outils génétiques à disposition. Il permet également de mener des études mécanistiques in vivo dans un laps de temps restreint. Nous avons évalué in cellulo et in vivo la toxicité de ces NP afin d'établir des conditions optimales expérimentales. Ensuite le potentiel des NP de PLA a été évalué in cellulo sur des cellules de drosophile transfectées transitoirement par un plasmide porteur du gène GFP sous le contrôle du promoteur UAS. Les NP vectorisant le gène gal4 ou la protéine GAL4 permettent de confirmer par simple observation en microscopie à fluorescence l'efficacité de délivrance de molécules actives dans la cellule via la fixation de la protéine GAL4 sur le promoteur UAS. Enfin, ces formulations ont été administrées par voie orale à des drosophiles transgéniques UAS-RFP pour confirmer les résultats précédents in vivo. GAL4 est un outil prometteur pour le suivi indirect de NP dans des organismes transgéniques / The biodegradable NanoParticles (NPs) of PolyLactic Acid) (PLA) are promising vectors for vaccination and therapeutic delivery. However, their in vivo evaluation is not always successful. NPs being undetectable in deep tissues, one of the challenges is the difficulty to follow the cellular uptake of these nanomaterials at the organism level. The aim of this thesis is to validate the use of PLA NPs as an imaging and drug vectorization platform in Drosophila melanogaster, and to analyze their fate in the whole fly body. The Drosophila model was chosen for its small footprint, the ease of breeding, the variety of transgenic lines, and the power of genetic tools available. Tt also allows to carry out in vivo mechanistic studies in a limited time window. We evaluated in cellulo and in vivo toxicity of these NP to determine optimal experimental conditions. Then the potential of PLA NPs was evaluated in cellulo on transiently transfected Drosophila cells by a plasmid carrying the GFP gene under the control of the UAS promoter. A simple observation by fluorescence microscopy of NPs vectorizing the gal4 gene or the GAL4 protein can confirm the effective delivery of active molecules into the cell through the binding of GAL4 protein to the UAS promoter. Finally, these formulations were orally administered to transgenic Drosophila UAS-RFP to confirm the previous in vivo results. GAL4 is a promising tool for indirect monitoring of NPs in transgenic organisms

Soy contains many bioactive molecules known to elicit anti-cancer effects. One such peptide, Lunasin, has been shown to selectively act on newly transformed cells while having no cytotoxic effect on non-tumorigenic or established cancer cell lines. In this study we attempt to understand the developmental effects of Lunasin overexpression in vivo and create reagents that will help us understand Lunasin’s anti tumorigenic effects in an intact organism. cDNA encoding lunasin and EGFP-lunasin were cloned into pUAST and microinjected into Drosophila embryos. Tissue-specific overexpression of EGFP-Lun in the resulting transgenic lines was accomplished by crossing transgenics to various GAL4 driver lines. Progeny were assessed for phenotypic alterations and no phenotypic abnormalities were observed in tissues expressing EGFP-Lunasin, supporting current studies that show Lunasin does not affect normal cells. Previous studies have localized Lunasin to the nuclear compartment. To test if this was the case for EGFP-Lun, subcellular localization of EGFP-Lun was determined via fluorescence microscopy. Salivary glands from EGFP-Lun expressing individuals were dissected, fixed, and mounted in Vectashield® with the nuclear stain, DAPI. Our results demonstrate that EGFP-Lun, like native Lunasin, is localized to the nucleus. Eight transgenic lines were mapped to specific chromosomes and EGFP-Lun transgenic line GEJ1-L2 was balanced in preparation for use in tumor suppression studies. In summary, we have created and characterized transgenic flies capable of overexpressing Lunasin under the control of the GAL4/UAS system. Localization of EGFP-Lunasin to the nucleus and data on the phenotypic consequence of its overexpression in flies is presented. Finally, reagents created as part of this thesis will aid experiments aimed at understanding the effects of Lunasin on benign and invasive tumors.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24758号 / 生博第499号 / 新制||生||66(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 今吉 格, 教授 鈴木 淳, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

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