L’interaction symbiotique légumineuses-rhizobium nécessite l'infection des racines de la plante par les bactéries et l’initiation de divisions cellulaires dans le cortex racinaire.Les cytokinines sont des hormones végétales agissant via une signalisation par phosphotransfert qui conduit à l’activation de Régulateurs de Réponse de type B (RRBs), des facteurs de transcription régulant l'expression des gènes de réponse primaire aux cytokinines. Une étude phylogénétique menée sur plusieurs espèces de légumineuses a révélé une expansion génique de la famille des RRBs et l’apparition de formes non-canoniques de ces facteurs de transcription. Chez Medicago truncatula,MtRRB3 est le RRB le plus fortement exprimé dans les racines et les nodosités et est impliqué dans la nodulation. En effet, les plantes dont l’expression de MtRRB3 a été réduite par ARNi ainsi que des mutants rrb3 présentent une diminution significative du nombre de nodosités formées. De plus, l’expression de gènes associés à la nodulation, tels que "Nodulation Signalling Pathway 2" (MtNSP2) et "Cell Cycle Switch 52A"(MtCCS52A), est réduite en réponse aux cytokinines dans ces mutants. Des fusions transcriptionnelles avec le rapporteur GUS montrent que MtRRB3, MtNSP2 et MtCCS52Aprésentent un profil d’expression spatiale largement chevauchant dans les racines et lesnodosités. Des expériences de ChIP-qPCR et de trans-activation en protoplastes indiquent par ailleurs que MtRRB3 peut respectivement interagir avec et activer les promoteurs des gènesMtNSP2 et MtCCS52A. Cette thèse a donc permis d’établir des mécanismes moléculaires impliqués dans les régulations transcriptionnelles médiées par les cytokinines lors de la mise en place des nodosités symbiotiques fixatrices d’azote. / The legume-rhizobium interaction requires the infection of plant roots by rhizobia and the initiation of cell divisions in the root cortex. Cytokinins, a class of plant hormones acts trough a phosphotranfert signalling leading to the activation of Type-B Response Regulators(RRBs) which are transcription factors regulating the expression of cytokinins primary response genes. Phylogenetic analyses carried out indifferent legume species genomes showed anexpansion of the RRB genes family associated toan increase in non-canonical RRBs. In Medicago truncatula nodules, MtRRB3 is the most expressed RRB in roots and nodules. MtRRB3 islinked to nodulation as MtRRB3 RNAi silencedplants as well as rrb3 mutants display asignificant decrease of nodule number. Inaddition, the expression of the nodulation related genes Nodulation Signalling Pathway 2(MtNSP2) and Cell Cycle Switch 52A(MtCCS52A) is reduced in response to cytokininsin rrb3 mutants. The expression pattern of apMtRRB3-GUS fusion overlaps with thepMtNSP2-GUS and pMtCCS52A-GUS fusions in roots and nodules. Finally, ChIP-qPCR and protoplast trans-activation experiments showed that MtRRB3 can respectively interacts with and activate MtNSP2 and MtCCS52A promoters. This thesis have thus established molecular mechanisms associated to transcriptional regulations mediated by cytokinins during the legume symbiotic nitrogen-fixing nodulation.

In recent times, endocrine disrupting chemicals (EDCs) have been associated with the rise in neurodevelopmental disorders such as autism, attention deficit hyperactivity disorder (ADHD) and decreased intelligence quotient (IQ) in children. This effect is suspected to be induced at pre-/peri-natal development, via an alteration in hormonal signaling, thus interfering with neuronal differentiation, with subsequent effect on normal brain development and function in exposed children. This issue increases the need for chemical screening for potential developmental neurotoxicity (DNT) effect. The current available EDC induced DNT test guideline is based on in-vivo testing that requires animal use. Here, a multipotent neural progenitor cell line, the C17.2 cell-line, generated from neural stem cells of the external germinal layer of mouse cerebellum, with potential to differentiate to neurons or astrocytes, is introduced for in-vitro EDC induced DNT testing. This project focused on optimizing the C17.2 cell-line for the detection of EDC-induced DNT with emphasis on the disruption of the oestrogen, androgen, and thyroid hormone systems. It aimed at validating the involvement of oestrogen, androgen, and thyroid hormone on molecular and cellular endpoints relevant for the differentiation of the C17.2 cells.  Herein, the cells were exposed to the hormonal agonist and antagonist at a range of concentrations for a 10-day differentiation period. After exposure, LDH, viability assay and morphological changes (percentage of neurons in culture and neurite outgrowth) were evaluated. The results showed no morphological changes induced by androgen receptor (AR) agonist/antagonist at relevant physiological concentrations. The thyroid receptor (TR) agonist and antagonist on the other hand showed a response in the form of increased neurite outgrowth in relation to the negative control at a concentration range of 40-200 nM and 40 nM respectively. The oestrogen receptor (ER) antagonist at 100 nM also increased percentage neuron in culture. Additionally, in-silico analysis of microarray and RNA sequencing data were used to map out target genes regulated by ER, AR and TR and involved in neurodevelopment. With this approach, 29 marker genes were identified. Validation of the marker genes by means of gene expression (qPCR) was carried out, ER and TR agonist/antagonist were observed to modulate the expression of examined genes. In summary, the model could not be established for detecting EDC induced DNT via androgenic and oestrogenic pathway, while it is a promising model for identifying DNT induced by thyroid hormone signalling disruption.