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Université Lille I Université Lille I SCIENCES ET TECHNOLOGIES THÈSE Pour l’obtention du grade de DOCTEUR DE L’UNIVERSITE DE LILLE I Formation Doctorale: Ecotoxicologie Ecole Doctorale: Sciences de la Matière, du Rayonnement et de l’Environnement Intérêts et validation de marqueurs précoces de génotoxicité environnementale Présentée et soutenue publiquement par Fabien BERNARD Le 6 décembre 2013 Devant le jury composé de : Pr. Alain LEPRETRE, Université de Lille 1 Président du jury Dr. Carole COSSU-LEGUILLE, Université de Lorraine Rapporteur Dr. Séverine PARIS-PALACIOS, Université Reims Champagne-Ardennes Rapporteur Pr. François LEBOULENGER, Université du Havre Examinateur Dr. Anissa LOUNES-HADJ SAHRAOUI, Université du Littoral Côte d’Opale Examinatrice Dr. Franck MAROT, Ingénieur ADEME Examinateur Dr. Annabelle DERAM, Université de Lille 2 Co-directrice de thèse Pr. Franck VANDENBULCKE, Université de Lille 1 Directeur de thèse REMERCIEMENTS Tout d’abord je tiens à remercier toutes les personnes qui ont pris part d’une manière ou d’une autre au bon déroulement de cette thèse. Je tiens à exprimer toute ma gratitude aux personnes qui m’ont permis de bénéficier d’un financement ADEME / Région Nord/Pas-de-Calais pour réaliser cette thèse et plus particulièrement M. Franck MAROT, ingénieur ADEME. Je tiens à remercier en tout premier lieu le Pr. Franck VANDENBULCKE et la Dr. Annabelle DERAM, qui ont co-dirigé cette thèse dans la continuité de mon stage de Master 2. Je remercie ces deux personnes du fond du cœur pour m’avoir proposé ce projet de thèse. Vous avez cru en mes compétences et en mes qualités personnelles. Je vous remercie aussi pour votre soutien et votre disponibilité tout au long de ces trois années ainsi que pour votre grande aide pour la rédaction de ce mémoire. Ce fut un honneur et un très grand plaisir de partager ces trois années avec vous. Je tiens à remercier le Pr. Isam SHAHROUR, directeur du Laboratoire de Génie Civil et géo- Environnement (Université de Lille 1), le Pr. Alain LEPRETRE, directeur de l’équipe Ecologie Numérique et Ecotoxicologie (Université de Lille 1) et le Pr. Régis COURTECUISSE, directeur du Laboratoire des Sciences Végétales et Fongiques (Université de Lille 2) qui m’ont accueilli dans leur laboratoire dans un cadre propice pour men er à bien ce travail. Mes remerciements vont également vers le Dr Claude COCQUERELLE, grâce à qui j’ai beaucoup appris en biologie moléculaire, pour ses nombreux conseils, ses remarques toujours pertinentes, son soutien et sa bonne humeur au quotidien. Mes remerciements s’adressent également au Dr. Carole COSSU-LEGUILLE et au Dr. Séverine PARIS-PALACIOS qui me font l’honneur de juger ce travail. Je tiens également à remercier le Pr. François LEBOULENGER (Université du Havre) et le Dr. Anissa LOUNES-HADJ SAHRAOUI (Université du Littoral Côte d’Opale) qui ont accepté d’examiner ce travail. Je voudrais également remercier les membres du jury du comité de suivi de thèse le Dr. Anissa LOUNES-HADJ SAHRAOUI, de l’Unité de Chimie Environnementale et Interactions sur le Vivant (Université du Littoral Côte d’Opale) et le Dr. Marc BONNARD, du 1 Laboratoire d’Ecologie-Ecotoxicologie (Université de Reims Champagne-Ardennes) pour leurs discussions, leurs conseils et leur intérêt pour ce travail de thèse. Mes remerciements vont également vers Mme Cécile GODE, assistante ingénieur au Laboratoire de Génétique et Evolution des Populations Végétales (Université de Lille 1) et Mme Valérie MONTEL, assistante ingénieur au Département de Neurosciences et de Physiologie/Endocrinologie (Université de Lille1), pour leur disponibilité et pour l’utilisation du LightCycler ® 480. Je tiens à remercier Christophe (Technicien) du Laboratoire de Toxicologie/Génopathies (Centre de Biologie et Patholologie du Centre Hospitalier Régional Universitaire de Lille) pour l’utilisation du NanoDrop. Mes remerciements les plus sincères s’adressent également à l’équipe du Laboratoire des Sciences Végétales et Fongiques (Université de Lille 2) pour leur accueil durant mes séjours «test des comètes». Merci beaucoup à Damien CUNY pour son aide et ses conseils lors du test des comètes, à Inès DEVRED pour son aide précieuse pour le tamisage des sols et sa bonne humeur au quotidien et à Stéphanie QUARRE, Julie LECLERCQ et Florent OCCELI pour leur aide dans l’entretien et la collecte des échantillons. Je tiens à remercier du fond du cœur le Dr. Sylvain DUMEZ (Ingénieur de Recherche) pour sa disponibilité, sa bonne humeur au quotidien et ses nombreux conseils en biologie moléculaire et lors de la rédaction des articles. Un grand merci à toi Sylvain, j’ai appris beaucoup de nouvelles méthodes grâce à toi. Je tiens à remercier vivement le Dr. Franck BRULLE pour ses nombreux conseils en biologie moléculaire et pour la rédaction des articles ainsi que pour sa disponibilité et sa bonne humeur au quotidien. J’ai été très honoré de pouvoir travailler de nouveau avec toi et d’avoir acquis des compétences supplémentaires grâce à toi. Mes remerciements les plus sincères et chaleureux aux membres de l’Equipe d’Ecologie Numérique et d’Ecotoxicologie (Laboratoire de Génie Civil et géo-Environnement, Université de Lille 1) pour leur soutien, le Dr. Sébastien LEMIERE, le Dr. Claude COCQUERELLE, la Dr. Céline PERNIN, le Dr. Sylvain DEMUYNCK, le Dr. Fabien GRUMIAUX, Régine LEROUX (adjointe technique) et Brigitte MACQUART (secrétaire et gestionnaire). Je tiens aussi à remercier Marion DELATTRE qui a grandement contribué aux expérimentations. 2 Je voudrais remercier plus particulièrement le Dr. Sébastien LEMIERE pour ses nombreux conseils lors de la rédaction des articles, pour son soutien lors des colloques et pour sa bonne humeur au quotidien. Je voudrais également remercier les personnes qui ont été présentes au laboratoire lors de ma thèse: le Pr. Peter ENGELMANN (Université de Pecs, HONGRIE), Levente (Université de Pecs, HONGRIE) et Abby (Université de Lincoln, NOUVELLE-ZELANDE) grâce à qui j’ai pu perfectionner mon anglais; Coralie, Dima, Hanine, Lieselot, Léa (INRA de Versailles) et Agnieszka (Université de Czestochowa, POLOGNE) pour leur bonne humeur au quotidien et leur sympathie. Enfin, c’est pour moi un grand honneur et un très grand plaisir de pouvoir dédier ce travail à mes proches, mes parents (Joël et Claudine), ma sœur (Muriel), Stéphanie (ma femme) et Gioia Noa (ma fille) pour leur énorme soutien. Je tiens à dire un grand merci à Stéphanie et à Gioia Noa pour m’avoir permis d’accomplir ce mémoire dans les meilleures conditions. 3 SOMMAIRE REMERCIEMENTS .................................................................................................................. 1 SOMMAIRE .............................................................................................................................. 4 TABLEAUX .............................................................................................................................. 5 FIGURES ................................................................................................................................... 5 INTRODUCTION ...................................................................................................................... 6 PREMIERE PARTIE ............................................................................................................... 15 I.1) Activités enzymatiques versus expressions géniques .................................................... 16 I.2) Test des comètes .......................................................................................................... 105 I.2.1) Le test des comètes ................................................................................................ 105 I.2.2) Test des comètes chez les Annélides ..................................................................... 106 I.2.3) Test des comètes chez les végétaux supérieurs ..................................................... 111 I.3) Modèles d’étude ........................................................................................................... 114 I.3.1) Eisenia fetida ......................................................................................................... 114 I.3.2) Trifolium repens .................................................................................................... 116 SECONDE PARTIE .............................................................................................................. 120 II.1) Identification de gènes de référence appropriés afin d’obtenir une quantification robuste des niveaux d’expression de gènes chez une espèce donnée. ................................ 122 II.2) Etude au niveau transcriptionnel de protéines impliquées dans le stress oxydatif chez Trifolium repens et Brassica oleracea exposés à du Cd et du Pb ...................................... 156 II.3) Utilisation du test des comètes afin d’évaluer les dommages à l’ADN chez Trifolium repens exposé à des éléments traces métalliques (Cd et Pb). ............................................. 209 II.4) Etude au niveau transcriptionnel de protéines impliquées dans le stress oxydatif et évaluation des dommages à l’ADN (test des comètes) chez Eisenia fetida exposé à du Cd et du Pb ................................................................................................................................... 234 DISCUSSION GENERALE, CONCLUSIONS & PERSPECTIVES ................................... 291 LISTE DES PUBLICATIONS ..............................................................................................
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