α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.

Les enjeux écologiques liés au réchauffement climatique, et plus généralement la lutte contre la pollution, ont occasionné une révolution sans précédent dans le domaine des transports. De nombreuses recherches portant sur l’identification de voies d’amélioration du rendement mécanique des moteurs à combustion interne ont été menées au cours de ces dernières décennies. Dans les moteurs Diesel, le contact Segments-Piston-Chemise (SPC) représente à lui seul près de 40 % des pertes d’énergie par frottement mécanique totales du moteur. Ce travail de thèse s’inscrit dans le cadre général de la lubrification des moteurs Diesel en présence de suies et s’intéresse plus particulièrement au poste SPC pour des contacts Diamond-Like Carbon (DLC) lubrifiés. Ce travail de thèse s’appuie sur des outils de tribométrie originaux pour reproduire les cinématiques particulières des contacts impliqués au niveau de la segmentation. Cette thèse s’attache à identifier l’influence d’un lubrifiant vieilli en fonctionnement sur les mécanismes de lubrification et les mécanismes de frottement associés de couches minces dures de type DLC, en balayant l’ensemble des régimes de lubrification pour des conditions stationnaires et transitoires. Les revêtements DLC développés dans le cadre de ce travail de thèse ont permis de diminuer significativement le frottement limite en conditions stationnaires et transitoires. La déstructuration du lubrifiant via la formation d’agrégats, générés par le passage des suies, ou par une annulation temporaire de la vitesse d’entraînement représentative des cinématiques de contact observées en zone SPC, sont gouvernés par le triptyque, lubrifiant, surface et cinématique de contact. Enfin, l’analyse de la réponse tribologique de l’interface lubrifiée en conditions stationnaires et transitoires permet à la modélisation théorique du frottement lors d’un cycle complet de glissement à vitesses variables. / Ecological issues related to global warming, and more generally the reduction of pollution, have lead to a major revolution in the field of transport. Considerable research work has been carried out during the past decades in order to improve the mechanical efficiency of internal combustion engines. In Diesel engines, almost 40 % of total engine energy losses due to mechanical friction occur in the Piston rings-Piston-Cylinder contact (PPC). The overall framework of this PhD thesis is Diesel engine lubrication in presence of soot and this work focuses more particularly on Diamond-Like Carbon (DLC) lubricated contacts for PPC region. Unique tribometry tools are used to reproduce the particular contact kinematics involved in the piston assembly. This thesis aims to identify the influence of an aged lubricant on the lubrication and friction mechanisms of DLC hard coatings for all lubrication regimes in steady-state and transient conditions. DLC coatings developed during this thesis significantly reduce the boundary friction in steady-state and transient conditions. The lubricant destructuring due to aggregate formation, generated by the passage of soot, or by a temporary vanishing of the entrainment speed, are governed by the triplet, lubricant, surface and contact kinematics. Finally, the analysis of the tribological response of the lubricated interface in steady-state and transient conditions leads to the theoretical modeling of the friction during a complete cycle of sliding at variable velocities.