Slits and their Roundabout (Robo) receptors were identified based on their role in regulating axon guidance, but are known to play multiple roles in development, including regulating heart development and myoblast migration. There are 3 vertebrate Slits (Slit1 – 3) and 4 Robos (Robo1 – 4), and previous work has demonstrated expression of Slit and Robo family members in and around developing joints where their function is unclear. Mutations in human Robo3 have been linked to degenerative joint disorders, such as scoliosis and rheumatoid arthritis. Misregulation of other members of the Slit/Robo signalling pathway is also reported in cells from arthritic joints. This suggests that Slit/Robo signalling is required for normal joint development and/or maintenance, though our understanding of their roles in these processes is rudimentary. The central question of my thesis is to determine the role/s of Slit/Robo signalling in limb and joint development. In situ hybridisation confirmed strong expression of Slits and Robos throughout mouse limb and joint development, though no expression of Slit1 or Robo3 was detected. Analysis of Slit1/2, Slit3 and Robo1 mutant (loss-of-function) mice revealed normal limb development, however misexpression of dominant-negative Robo2 during chicken limb development caused shortening of cartilage elements. To begin to identify molecular changes that may compensate for the loss of Slit/Robo signalling I demonstrated members of the Sema3/PlexinA/Nrp axon guidance family are expressed in patterns comparable to those of Robo1, Robo2 and Slit3. I discovered that PlexinA1 is downregulated in Slit3 mutant mouse limbs. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. Previous work has linked Slit/Robo signalling to development of degenerative joint disorders, and I propose some hypotheses as to how Slit/Robo signalling could cause bone and joint defects.

In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below.I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate.Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate in response to any stalled ribosome.I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.

L'expression génétique est un processus cellulaire fondamental réglé de manière ne. Les promoteurs inducibles perme ent de perturber l'expression génétique en changeant l'expression d'une protéine par rapport à son niveau physiologique de référence. Ce e propriété en fait un outil incontournable pour décrypter le fonctionnement des processus biologiques via la comparaison du comportement de la cellule sous divers niveaux d'induction. Toutefois, une limite actuelle à l'utilisation des promoteurs inducibles provient de la difficulté à appliquer des perturbations précises et dynamiques. Les deux obstacles principaux étant: (i) la variabilité intercellulaire ainsi qu'à la nature aléatoire de l'expression génétique qui limite la précision de la perturbation appliquée. (ii) la difficulté à prèdire quantitativement le comporteement des systèmes biologiques sur les longues periodes requises pour des objectifs d'expression variables dans le temps. Or des perturbations précises et changeant dans le temps perme ent d'obtenir de riches informations sur la dynamique d'un système biologique. Est présenté ici une plate-forme de contrôle temps réel en boucle fermée qui permet le contrôle quantitatif sur une longue durée de l'expression génétique chez la levure. Ce e plateforme utilise la microscopie par uorescence pour suivre l'expression génétique, un système micro uidique pour interagir avec l'environnement cellulaire ainsi qu'un logiciel perme ant l'analyse d'image en temps réel et le calcul de la stratégie de contrôle à appliquer. Ce système permet le contrôle de l'expression d'un gène chez la levure, tant au niveau d'un population cellulaire qu'au niveau de la cellule seule et ceci pour un objectif d'expression constant ou dépendant du temps. Le système de réponse au chocs hyper-osmotiques de la levure S. cerevisiae (HOG pathway) a été utilisé pourin uencer l'expression génétique. Toutefois, la possibilité d'utiliser un autre système d'induction sans profondes modi cations de la plate-forme est démontrée. De surcroît au développement de ce e plate-forme est également ici démontré la possibilité de contrôler le système HOG. A n de comprendre la dynamique cellulaire et de pouvoir la quanti er, il est nécessaire de pouvoir appliquer des perturbations précises. La plate-forme de contrôle de l'expression génétique présentée ici permet de perturber avec précision le niveau d'expression d'une protéine et représente donc une contribution majeure dans ce e direction.