Le MGDG (monogalactosyldiacylglycerol) et le DGDG (digalactosyldiacylglycerol) sont les lipides les plus abondants des membranes du chloroplaste. Ils sont synthétisés exclusivement dans l'enveloppe plastidiale par l'action des MGDG synthases (MGD1, MGD2 et MGD3) et des DGDG synthases (DGD1 et DGD2). Les galactolipides sont essentiels pour la structuration des photosystèmes et la biogenèse des thylacoïdes. En carence de phosphate, les galactolipides deviennent une source de lipides pour composer certaines membranes en dehors du chloroplaste. Suivant une stratégie de criblage pharmacologique à haut débit, une nouvelle molécule appelée galvestine-1 a pu être identifiée et caractérisée comme un inhibiteur des MGDG synthases. La galvestine-1 agit par compétition avec le diacylglycérol. Cet outil moléculaire permet donc de perturber le système complet constitué par l'ensemble des réactions de synthèses, de conversions et de trafics lipidiques, aboutissant à cet état stable que nous appelons homéostasie des lipides. Le but de cette thèse est de mettre en évidence, à l'aide de la galvestine-1, de nouveaux acteurs ou nouvelles voies permettant l'établissement de l'homéostasie lipidique à l'échelle de la cellule végétale. Pour cela, j'ai réalisé un criblage de mutants EMS (ethyl methanesulfonate) dans le but d'isoler des mutants résistants à la galvestine-1 et d'identifier les gènes mutés conférant cette résistance. Des données transcriptomiques (Affymetrix genome array genechip, ATH1) d'Arabidopsis thaliana traité en présence de galvestine-1 ont par ailleurs été obtenues avant le début des travaux de thèse. Ces données ont permis de cibler des gènes dont l'expression variait et possiblement impliqués dans l'homéostasie lipidique. En parallèle de l'approche sans a priori, j'ai donc réalisé une étude suivant une stratégie de gènes candidats sur ALA10, un gène codant pour une flippase putative, sur-exprimé après traitement à la galvestine-1 et en carence de phosphate. Le second volet de cette thèse vise donc à comprendre la relation entre l'expression d'ALA10 et les gènes impliqués dans la synthèse des galactolipides chez la plante. / MGDG (monogalactosyldiacylglycerol) and DGDG (digalactosyldiacylglycerol) are the most abundant membrane lipids of the chloroplast. They are synthesized exclusively in the chloroplast envelope by the action of MGDG synthases (MGD1, MGD2 and MGD3) and DGDG synthases (DGD1 and DGD2). Galactolipids are known to be essential for the structure (and function) of the photosystems and for the biogenesis of thylakoids. In phosphate deprivation, galactolipids become a source of lipid for other cell membranes, outside the chloroplast. Based on a high throughput chemical screen, a new molecule called galvestine-1 has been identified and characterized as an inhibitor of MGDG synthases. Galvestine-1 competes with the binding of the diacylglycerol substrate to MGDs. This molecular tool can be used to disturb the system comprising all lipid biosynthesis reactions, conversions, and lipid trafficking, responsible for the membrane lipid steady state observed at the whole cell level, or membrane lipid homeostasis. Perturbation of the system occurs at the level of MGDG synthases. The aim of this thesis is to use the effect of galvestine-1 to identify new actors or new pathways involved in the control of lipid homeostasis in plant cells. To this purpose, I designed and performed a screening of a collection of EMS (ethyl methanesulfonate) mutants, in order to isolate galvestine-1-resistant mutants and to identify mutated genes conferring this resistance. Transcriptomic data (Affymetrix genome array genechip, ATH1) of Arabidopsis thaliana treated in the presence of galvestine-1 had been obtained prior to the PhD project. These data were used to identify genes whose expression varied and possibly involved in lipid homeostasis. Based on a complementary candidate gene approach, I focused on Ala10, a putative flippase, which gene is over-expressed after treatment with galvestine-1 and following phosphate deprivation. The purpose of this second part of this thesis is to understand the relationship between the expression of ALA10 and genes involved in galactolipid synthesis in plants.

The budding yeast Saccharomyces cerevisiae has been an invaluable model organism in contributing to the current understanding of cellular biology, owing mainly to its highly tractable genetic system and the completion of its genome sequencing in 1996. Indeed, these bolstered the development of novel methods that have provided great insights into genetic and protein networks in human cells. With the large collection of datasets, S. cerevisiae also became an ideal platform for investigating the mechanism of action of novel compounds. The first part of my thesis uses a validated chemogenomic assay to investigate the mechanism of action of structurally related novel DNA-damaging agents, delineating valuable structure-activity relationship in the process. The second part describes the development of a method that uses drug-induced wild-type growth dynamic to characterize novel compounds, which, in combination with the chemogenomic assay, may complement existing high throughput screening experiments to improve the current drug development process.

The budding yeast Saccharomyces cerevisiae has been an invaluable model organism in contributing to the current understanding of cellular biology, owing mainly to its highly tractable genetic system and the completion of its genome sequencing in 1996. Indeed, these bolstered the development of novel methods that have provided great insights into genetic and protein networks in human cells. With the large collection of datasets, S. cerevisiae also became an ideal platform for investigating the mechanism of action of novel compounds. The first part of my thesis uses a validated chemogenomic assay to investigate the mechanism of action of structurally related novel DNA-damaging agents, delineating valuable structure-activity relationship in the process. The second part describes the development of a method that uses drug-induced wild-type growth dynamic to characterize novel compounds, which, in combination with the chemogenomic assay, may complement existing high throughput screening experiments to improve the current drug development process.