Over 100 years ago, X-ray crystallography gave scientists a window to the atomic world with varied applications in biology, chemistry and physics among other subjects. Macromolecular crystallography is now considered an essential tool for solving the three-dimensional structure of proteins and understanding their physiological role at the atomic level. As crystal growth remains a bottleneck in crystallography, various other techniques are often employed to help understand the protein structure and function. These methods range from simple analysis of the protein sequence to experiments such as dynamic light scattering, isothermal titration calorimetry, activity assays, and analytical ultracentrifugation. The additional knowledge gained about proteins from these methods can then assist in the modification of the protein to facilitate its crystallisation. The structural biology of proteins belonging to two diverse families; Galectins and Sirtuins, both involved in the regulation of cancer, was studied in this thesis. Galectins are evolutionarily conserved and ubiquitously present animal lectins with a high affinity for -galactose containing oligosaccharides. To date, 15 mammalian galectins have been identified. Their involvement in cell–cell and cell–matrix interactions has highlighted their importance in signal transduction and other intracellular processes. Human Galectin-7 (hGal-7) is a 16 kDa prototype galectin which is involved in the stimulation and development of cancer. The crystal structure of native hGal-7 and its complex with galactose and lactose have been reported. In this study, cross-linking of hGal-7 by glycodendrons and the resulting clustering and lattice formation have been studied. For this purpose, the high resolution X-ray structures of hGal-7 in complex with carbohydrate-based multivalent dendrons have been elucidated and analysed. Also discussed in this thesis are preliminary binding affinity results obtained using isothermal calorimetry. Supramolecular assembly formation was also assessed using dynamic light scattering. These experiments reveal how multivalent glycodendendrons interact with and form cross-links with hGal-7 molecules. Understanding how these dendrimeric compounds interact with hGal-7 would help in the design of new tools to investigate the recognition of multivalent carbohydrates by lectins and their resulting role in aggregation processes in tumour embolisation and survival. Sirtuins are NAD+-dependent deacylases that are involved in the regulation of diverse biological functions such as ageing, metabolism and stress resistance, in normal cellular physiology. Their role in ageing and ageing-related diseases, including cancer and neurodegenerative diseases among others, has received much attention and sirtuins have been extensively studied to help in extension of human lifespan. Seven members of the sirtuin family (SIRT1-7) are known, which are diversely sub-localised in the cell. A myriad of questions regarding their deacylation activity, their interplay and their role in various diseases still remain unanswered. In this study, structural biology techniques have been used to understand the role of SIRT1, SIRT2 and SIRT7. The cloning, expression, purification and crystallisation of these sirtuins are presented in this thesis. Various supporting techniques used to confirm the identity and activity of the proteins are also discussed. A brief discussion of the methods that can be employed to overcome various barriers in structural biology is also presented in this thesis. Elucidating the structure of full length sirtuins would help in the development of highly selective modulators of sirtuins to aid in the understanding of their role in ageing and ageing-related diseases.

Les galectines forment une famille de lectines solubles impliquées dans de multiples processus. Elles sont caractérisées par la présence d’un domaine de liaison aux carbohydrates conservé au cours de l’évolution et une affinité particulière pour les β-galactosides. Au cours de ce projet doctoral, nous nous sommes intéressés à galectine-7, une lectine exprimée spécifiquement dans les épithéliums pluristratifiés, comme l’épiderme. Grâce aux modèles de souris invalidées pour galectine-7 ou surexprimant galectine-7, notre équipe a précédemment montré que cette protéine était impliquée dans l’adhérence entre les cellules de l’épiderme et dans la migration collective, deux processus clés de la progression tumorale et de la cicatrisation épidermique. Cependant, les mécanismes moléculaires sous-jacents restent à élucider.En combinant différentes approches, nous avons pu déterminer que le retard de migration observé en absence de galectine-7 pouvait s’expliquer, du moins en partie, par une diminution de la coordination et du comportement collectif des kératinocytes en migration. De plus, nos données montrent que galectine-7 interagit directement avec le domaine extracellulaire de la E-cadhérine, un des composants majeurs des jonctions adhérentes et une protéine clé dans la migration collective. De façon surprenante, cette interaction ne fait pas intervenir de groupements carbohydrates. Tentant de préciser le rôle de galectine-7 au niveau des jonctions adhérentes, nous avons identifié une nouvelle fonction de galectine-7 dans la stabilisation de la E-cadhérine à la membrane plasmique. De manière intéressante, l’augmentation du renouvellement de la E-cadhérine à la membrane plasmique causée par l’extinction de galectine-7 est également couplée à une baisse de la force de l’adhérence intercellulaire. Enfin, nos expériences indiquent que cette nouvelle fonction de galectine-7 requiert une activité lectine fonctionnelle, suggérant l’implication d’un autre acteur glycosylé dans ce mécanisme de régulation de la E-cadhérine par galectine-7.En conclusion, ce doctorat a permis de préciser le rôle de galectine-7 dans la migration collective et de découvrir une fonction non-décrite auparavant de galectine-7 dans la régulation de la dynamique de la E-cadhérine. Cette modulation de la E-cadhérine par galectine-7 pourrait permettre à la cellule de s’adapter aux perturbations de l’environnement, comme c’est le cas au cours de la migration collective. En effet, les galectines étant des molécules avec une capacité de redistribution rapide, ce sont de bons candidats pour créer des réponses adaptatives / Galectins composed a family of soluble lectins implicated in multiple processes. They are characterized by the presence of a carbohydrate recognition domain evolutionary conserved and an affinity for β-galactosides containing sugars. During this thesis, we focused on a protein called galectine-7 whose expression is restricted to stratified epithelia such as the epidermis. Using mouse models with altered expression of galectin-7, our team previously showed that this protein participates in intercellular adhesion and collective cell migration, two key processes in tumour progression and epidermal wound healing. However, the underlying mechanisms remain to be elucidated. Combining different approaches, we discovered that the migration delay observed in the absence of galectin-7 during wound healing could be explained, at least in part, by a reduction of cell coordination and collective cell behaviour of migrating keratinocytes. Moreover, our data showed that galectin-7 directly interact with E-cadherin, a key component of adherent junctions and a major player in collective migration. Surprisingly, this binding did not involve carbohydrate groups. Aiming to precise the role of galectin-7 at adherent junctions, we identified a new function of galectin-7 in the stabilisation of E-cadherin at the plasma membrane. Interestingly, the increased of E-cadherin turnover caused by galectin-7 extinction is also associated to a decreased of the strength of adherent junctions-based intercellular adhesion. Eventually, our experiments indicated that this previously unknown function of galectin-7 required a functional lectin activity, suggesting the involvement of an additional glycosylated actor in this regulation mechanism of E-cadherin dynamics by galectin-7.In conclusion, this thesis allowed to precise the role of galectin-7 in collective cell migration and revealed a novel function of galectin-7 in the regulation of the E-cadherin stability at the plasma membrane. This modulatory effect of galectin-7 on E-cadherin could provide the cell a possible adaptive response to environmental perturbations, as during collective cell migration. Indeed, galectins, because they exhibit rapid redistribution capacities, are good candidates to create adaptive responses