Ubiquitin is an essential 76 amino acid protein which can be conjugated to lysine residues on a variety of substrates via its C-terminal diglycine motif. This conjugation allows the protein to act as a molecular tag in a range of processes, including regulation of chromatin compaction, signalling cascades and DNA repair. In addition, ubiquitin moieties are capable of forming chains through the successive conjugation to lysine residues within ubiquitin itself. One of the most well characterized functions of ubiquitin is its role in protein quality control and degradation. Tetra-ubiquitin chains, most commonly through a lysine-48 linkage, are responsible for directing proteins to the 26S proteasome for degradation. This process is of importance both in the removal of miss-folded proteins, and in the regulated destruction of specific targets, such as the cyclins. The 90kDa AAA-ATPase Cdc48/p97/VCP is an essential protein that forms a hexameric complex, which interacts with a wide variety of ubiquitinated substrates. The specificity of Cdc48 is modulated by a series of different cofactors, which together allow Cdc48 to operate in several different contexts, from removal of misfolded proteins from the ER, to regulating securin stability. The role of two Cdc48 cofactors, Ubx4 and Ubx5, was studied in an attempt to dissect their function and to determine how they may modulate the function of Cdc48. Neither protein was found to be essential, as knockouts of either were found to be viable with no major defect in growth rate. The work also describes the findings of a yeast two-hybrid screen to identify potential substrates for both cofactors. Delivery of ubiquitinated proteins to the proteasome is mediated by shuttling factors, which are able to bind to both ubiquitin and the proteasome, and hence mediate the interaction between both. The shuttling factor Dph1 binds ubiquitin via a C-terminal UBA domain, while its N-terminal UBL domain mediates its interaction with the proteasome. This work identified a novel interaction between the Sti1 domains of Dph1 and the N-terminal region of a mitochondrial localized AAA-ATPase, homologous to the Saccaromyces cerevisiae protein Msp1. In addition, cell fractionation experiments revealed the presence of Dph1 at the mitochondria. This interaction provides hints that Mlp1 may be involved in the removal of ubiquitinated proteins from the mitochondria, and their delivery to the proteasome. The thesis begins to try and attempt to identify possible substrates of this proposed mitochondria associated degradation pathway, and looks for ways in which the hypothesis may be tested.

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Les populations sont soumises à des pressions sélectives qui agissent sur certains traits entraînant une divergence phénotypique. L'évolution des morphologies adaptatives est souvent liée avec des changements de structures préexistantes. Les insectes semi-Aquatiques ont subi une croissance de pattes exagérée qui est associée à leur adaptation et locomotion efficace à la surface de l'eau. Cette croissance excessive a facilitée l'exploitation de l'habitat aquatique restreint pour les espèces terrestres apparentées. En outre, le groupe dérivé des gerris a subi des modifications supplémentaires au niveau des pattes, de sorte que la deuxième patte (P2) est plus longue que la troisième patte (P3). Ce plan d'organisation inversé par rapport à celui des espèces terrestres, est associé à la spécialisation pour une vie sur l'eau. Les gerris ont acquis un mode de locomotion dérivée qui consiste à ramer par des mouvements simultanés de leurs P2 et des mouvements plus subtils de leurs P3 pour s'orienter. La structure et la croissance des pattes des insectes semi-Aquatiques sont réalisées durant l'embryogenèse. En effet, la nymphe qui éclot possède des pattes fonctionnelles. Il a été démontré que le facteur de transcription Hox, Ubx, est impliqué dans cette inversion du plan des pattes. Cependant, les mécanismes génétiques responsables de ces adaptations restent toujours obscurs. La thèse présentée examine ces questions à travers deux axes : premièrement, déterminer les gènes et les voies de signalisation responsables du développement et de la croissance remarquable des pattes ; deuxièmement, étudier le rôle du gène Hox impliqué dans l'inversion du plan des pattes caractéristique des gerris / Populations are faced with selective pressures that act on certain traits resulting in phenotypic divergence. The evolution of adaptive morphological traits is often associated with changes in pre-Existing structures. In semiaquatic insects, a dramatic growth of thoracic appendages is associated with their adaptation and efficient locomotion on the water surface. This particular leg allometry facilitated the exploitation of aquatic habitats, a restricted niche for their terrestrial relatives; and hence opens a new array of ecological opportunities. Additionally, the derived group of water striders has undergone further appendage modification, such that T2-Legs are longer than T3-Legs, a ground plan associated with the specialization to open water. Water striders have evolved a derived mode of locomotion through rowing on water. They move their mid-Legs in simultaneous sweeping strokes for propulsion, and move their hind-Legs in fine movements for orientation. Leg specification and elongation in semiaquatic insects happens during early embryogenesis as the newly hatching nymphs emerge with functional legs. The Hox transcription factor Ubx was found to be implicated in the reversal in leg ground plan. Nonetheless, the genetic mechanisms underlying these leg adaptive changes are still poorly understood. The presented thesis investigates these questions through two main goals: first, to uncover the genes and pathways implicated in the development and dramatic elongation of the legs; second, to examine the dynamics of Hox control responsible for the reversal in leg ground plan characteristic of water striders

Les insectes présentent une étonnante diversité morphologique dans les organes de vol, et cette évolution a conduit à leur rayonnement au sein du règne animal. L'une des modifications les plus frappantes est la transformation des ailes postérieures en structures d'équilibrage très réduites, appelées haltères. Des travaux pionniers chez la drosophile ont montré que la spécification des haltères est sous le contrôle du gène Hox Ultrabithorax (Ubx). En revanche, la formation des ailes antérieures est décrite pour être indépendante des gènes Hox, mais cette observation est controversée chez d’autres insectes. Au cours de mon doctorat, j'ai réexaminé le rôle des gènes Hox pour la spécification des organes du vol chez la drosophile. Mes travaux montrent que la protéine Hox Antennapedia (Antp) est exprimée à un niveau faible dans des cellules spécifiques du primordium de la marge et est nécessaire à la formation correcte de l’aile adulte. De manière étonnante, Antp peut également fonctionner comme Ubx et former un haltère quand la protéine est exprimée à des niveaux similaires à ceux de Ubx. Ainsi, la formation d’organes de vol divergents chez la drosophile est directement contrôlée par une dose spécifique de protéine Hox et non par une protéine Hox spécifique. Les gènes Hox sont intrinsèquement liés à l'évolution de la diversité morphologique chez les animaux. Par conséquent, le rôle de la dose des protéines Hox a également été testé d'un point de vue évolutif parmi plusieurs lignages d'insectes. Les résultats montrent que la dose de protéines Hox est à peu près la même entre les primordia antérieur et postérieur d’un insecte à quatre ailes comme Bombyx mori. Dans l’ensemble, mes résultats démontrent que la spécification des organes de vol n’est pas un programme Hox-indépendant et que la variation de la dose des protéines Hox est un moyen de modifier la taille et la forme de l’aile, pouvant ultimement aboutir à la création d’un tout nouvel organe d’équilibrage au cours de l’évolution des insectes. Enfin, au cours de mon doctorat, j'ai également participé à plusieurs projets parallèles visant à identifier et à caractériser le rôle des nouveaux cofacteurs des protéines Hox dans différents contextes développementaux, notamment la spécification de l’haltère et la répression de l'autophagie. Ces travaux s'appuient en partie sur la complémentation de fluorescence bimoléculaire (BiFC), une méthode que nous avons récemment couplée à la panoplie d'outils génétiques de la drosophile pour réaliser des criblages d'interactions protéine-protéine à grande échelle in vivo. / Insects display an astonishing array of diversity in flight appendage morphologies and theirevolution led to the catalyzed radiation of insects in the animal kingdom. The first definite modellinking the Hox genes to morphological evolution was demonstrated in Drosophila. One of themost striking modifications is the transformation of hindwings into highly reduced balancingstructures called halteres. Work in Drosophila established that the specification of halteres isunder the control of a single Hox gene, Ultrabithorax (Ubx). In contrast, the formation of forewingshas been described to be Hox-independent. During my Ph.D., I reconsidered the role of Hox genesfor flight appendage specification in Drosophila. I show that the Hox protein Antennapedia (Antp)is expressed at a low level in specific cells of the wing blade primordium and required for theproper formation of the adult wing. Moreover, Antp works like Ubx to form a haltere whenexpressed in the levels of Ubx. Thus, the formation of divergent flight organs in Drosophila is notdependent on a specific Hox protein but on a specific Hox dose.Hox genes are intrinsically linked to the evolution of morphological diversity in animals.Therefore the role of the Hox dose was also tested from an evolutionary point of view amongseveral insect lineages. Results show that the Hox dose is for example pretty much the samebetween the forewing and hindwing primordia of the four-wing insect species Bombyx mori.Altogether, my results demonstrate that the specification of flight appendages is not aHox-independent developmental program and that the variation in the Hox dose is a way tomodify the wing size and shape, ultimately leading to a completely new balancing organ duringinsect evolution.

A major goal of evolutionary developmental biology is to explore mechanisms and events underlying evolution of the myriad body plan morphologies expressed both genetically and phenotypically within the animal kingdom. Arthropods exhibit an astounding array of morphological diversity both within and between representative sub-phyla, thus providing an ideal phylum through which to address questions of body plan innovation and diversification. Major arthropod groups are recognised and defined by the distinct form and number of articulated appendages present along the antero-posterior axis of their segmented bodies. A great deal is known about the developmental genetics of limb development in the model insect Drosophila melanogaster, added to which, much comparative gene expression data and a growing body of functional genetic data is emerging for other arthropod species. Arthropod limb primordia are consistently marked by expression of the homeobox gene Distal-less (Dll), and the focus of this thesis is to compare signalling mediated by early Dll regulatory genes activity along antero-posterior and dorso-ventral embryonic axes during limb specification in Drosophila, with the activity of their orthologs in the widely disparate chelicerate, the spider mite Tetranychus urticae - interpreting new data with that available for other arthropods. Having made a detailed study of spider mite embryonic (and post-embryonic) development, to provide a basis for understanding mRNA transcription and protein activity patterns, I confirmed typical expression of Tetranychus Dll in prosomal limb primordia. I obtained limited results for the candidate antero-posterior positioning genes wingless and engrailed, although one of the two engrailed paralogs I identified is reportedly expressed in posterior segmental compartments, consistent with possible conservation of Engrailed-Wingless interactions in metameric patterning and positive regulation of Dll in arthropod limb specification. In Drosophila, wingless-dependent Dll transcription is restricted along the dorso-ventral axis by dorsal Dpp-mediated and ventral EGFR-mediated signalling gradients. Based on data from Tetranychus and other arthropods, neither dorsal nor ventral signalling regimes appear conserved outside the Drosophila system. Dll suppression in fly abdominal segments occurs due to powerful Hox (Ubx/AbdA) repression of the early Dll cis-regulatory element; this is discussed in relation to the independently evolved limbless chelicerate opisthosoma, informed by hypothetical scenarios of cis (regulatory DNA) and trans (coding sequence) evolution. Given practical difficulties and limitations encountered while working with spider mites, I offer a final assessment of the place of Tetranychus urticae as a non-model, and yet still valuable chelicerate species to consider carrying into the exciting future of evolutionary developmental biology.