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Les fibres amyloïdes sont souvent à l’origine de nombreuses maladies dégénératives telles que la maladie d’Alzheimer ou la maladie de Parkinson. La formation de ces plaques insolubles est due à une agrégation anormale de protéines. Les études structurales et biologiques des amyloïdes sont hautement complexes du fait de leur organisation sous forme de superstructures unidirectionnelles composées d’une infinité d’unités peptidiques ou protéiques, mais aussi à cause de leur hétérogénéité conformationnelle et polymorphique. Au cours de ces différents travaux de thèse en collaboration avec différents laboratoires d’analyses structurales, nous avons développé plusieurs outils de synthèse tant pour la formation de différents polymorphes de fibres amyloïdes que pour la formation d’espèces oligomériques de tailles conséquentes qui sont un challenge du point de vue synthétique et méthodologique mais aussi pour leur caractérisation. Ces différentes avancées permettront de mieux comprendre les mécanismes de formation de fibres amyloïdes et de préparer des échantillons homogènes pour les analyses structurales et biologiques. L’étude de modifications chimiques telles que la N-méthylation ou les polypeptides D est également un enjeu important pour l’élucidation des interactions protéine-protéine vis-à-vis des structures amyloïdogéniques et ainsi permettre l’élaboration de nouveaux composés inhibant la formation de plaques amyloïdes. / Amyloid fibrils are associated with many human disorders including Alzheimer’s or Parkinson’s diseases. The formation of insoluble plaques is the result of protein misfolding and aggregation due to abnormal conformational isomerization of the involved protein. The structural and biological studies of amyloids are highly complex. In this thesis, we report on the development of different synthetic methodologies for the preparation of distinct amyloid fibril polymorphs as homogeneous samples for structural and biological studies. We also synthesized covalently-tethered oligomers composed of nine copies of an amyloidogenic peptide segment, where we were able to control the self-assembly of the structure by insertion of N-methylated amino-acids and to obtain monomeric oligomers mimicking a cross section of an amyloid fibril. We also report on the chiral recognition of L-peptides and L-proteins towards corresponding D-enantiomers during amyloid formation. Moreover, we studied various N-methylated peptide analogues to suppress amyloid growth. Overall, the results obtained in this thesis pave the way towards rational design of peptide-based inhibitors and diagnostics against amyloid propagation.

La spectrométrie de masse est devenue un outil indispensable pour la recherche en protéomique, notamment grâce au développement récent de nouveaux spectromètres de masse comme l’Orbitrap et de nouvelles méthodes de dissociation. La stratégie « bottom-up » (analyse des mélanges de peptides protéolytiques) est la plus utilisée par son efficacement et sa simplicité par rapport à la stratégie top-down (analyse des peptides plus longs ou des protéines intactes), mais cette dernière permet une caractérisation plus complète des isoformes de protéines et des modifications post-traductionnelles.Les méthodes de dissociation utilisant des photons, comme la photodissociation dans le domaine ultra-violet (UVPD) et la dissociation multiphotonique infrarouge (IRMPD), ont reçu une grande attention comme approches alternatives aux méthodes de dissociation par collision. L'absorption du photon UV à haute énergie peut être « diluée » sur l'ensemble du peptide ou de la protéine et provoque une fragmentation étendue du squelette peptidique (liaisons C-C), tandis que les photons IR à faible énergie augmentent progressivement l'énergie interne et dissocient préférentiellement les liaisons amide (C-N) les plus labiles.Cette thèse est centrée sur le développement de méthodes et les applications pour une caractérisation structurale de biomolécules par des méthodes d'activation utilisant des photons. L'intérêt de combiner des photons infrarouges à faible énergie et des photons UV à haute énergie dans un spectromètre de masse Orbitrap, pour la caractérisation de petites protéines, a été évalué. En outre, la dissociation infrarouge multiphotonique a été implémentée dans un piège à ions électrostatique afin d’étendre les méthodes de fragmentation aux macromolécules de très haut poids moléculaires dans le domaine mégadalton. L'une des principales avancées de cette thèse a été d'adapter ces méthodes de spectrométrie de masse aux objets biomoléculaires, allant des petits peptides (dans la gamme de masse de kilodalton) à des fibres de protéines entières (dans la gamme de masse de mégadalton) / The structural characterization of proteins often required them to be fragmented into small units containing only few amino acids. In bottom-up approach, proteins are cleaved into small peptides by enzyme then these peptides are subjected to further fragmentation in a collision cell of a tandem mass spectrometer. However, in top-down approach, proteins can directly be dissociated (without enzyme) into small fragments by collision, electron and photon-driven dissociations. Photon-based activation methods including ultraviolet photodissociation (UVPD) and infrared multiphoton dissociation (IRMPD) have received great attention as an alternative to electron-driven and collision induced dissociation methods. Absorption of the high-energy UV photon is dispersed over the whole peptide or protein and stimulates extensive C?Ca backbone fragmentation while the low-energy IR photons gradually increases the internal energy and thus favorably dissociates the most labile amide (C?N) bonds. This thesis focuses on the method development and applications for characterizing biomolecules by photon-based activation methods. The interest of combining high-energy UV photons and low-energy IR photons in an Orbitrap mass spectrometer, for protein and post-translationally modified peptide characterization, has been evaluated. Moreover, infrared multiphoton dissociation has been implemented in a gated electrostatic ion trap to push forward the limit of fragmentation methods to large megadalton ions. One of the main breakthroughs in this thesis is the ability to adapt these method developments and applications to biomolecular objects ranging from small peptides (in kilodalton mass range) to entire protein fibrils (in megadalton mass range)

Nonlinear optical microscopy is a collection of very powerful imaging techniques. Linear optical microscopes probe the refractive index and absorption, which both stem from the first-order linear electric susceptibility. Especially in biological tissue, the variation in the refractive index is often small and the tissue is, in many cases, transparant. Nonlinear optical microscopes on the other hand probe the nonlinear higher-order susceptibilities, which can be chemically sensitive, leading to the capability to achieve label-free imaging. Nonlinear optical microscopes have been in development for more than thirty years and they are based on numerous nonlinear optical processes. The ones I will concentrate on in this thesis are second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS), and stimulated Raman Scattering (SRS). The first technique is commonly used to image collagen as those molecules have a particularly large second-order nonlinear susceptibility due to their chiral structure. CARS and SRS on the other hand are often used because they resonantly target vibrational resonances in molecules, giving rise to the aforementioned label-free imaging. Deep understanding of the nonlinear imaging process is crucial to the interpretation of the images these techniques produce. Computational tools are exceptionally suited for this task as they allow studying the electromagnetic field anywhere in the sample as well as the far-field, and one can change any of the material properties to study their effect. One such tool is finite-difference time-domain (FDTD) that our group developed for nonlinear optical microscopy simulations. It is a direct discretization of Maxwell's equation. While computationally costly, it does allow any arbitrary shaped sample to be simulated. The sample can have frequency dependent refractive indexes, and also nonlinear media with third-order nonlinearities such as Kerr media and Raman-active media, but also second-order nonlinearities for SHG. The code is designed in such a way that it can run on thousands of CPUs on a wide variety of compute cluster which allows our group to obtain nanoscale resolution. Another computational tool I use is the free-space Green's function solution to the Helmholtz equation, which can be used to calculate the Hertz vector in the frequency domain, both in the near- and far-field, based on the induced nonlinear polarization. The electric field is then calculated from this Hertz vector. This technique is much faster then FDTD and also allows for arbitrary shapes of the nonlinear electric susceptibility in the sample. However, it assumes a homogeneous refractive index throughout the entire spatial domain and requires complete knowledge of the input beam or beams that induce the nonlinear polarization. In this thesis, I use these tools to study the image formation process of various nonlinear optical processes mentioned earlier. For example, I study the effect of an inhomogeneous refractive index on the images produced by these microscopes. In literature the index of refraction is almost always assumed to be homogeneous, because, as mentioned before, the inhomogeneity of the refractive index is often small. However, I show that these small differences in the index of refraction can have a significant effect on the measured far-field intensity signal. For example, in SRS and CARS images, the measured signal can increase by an order of magnitude depending on the index mismatch and structure of the sample. Additionally, significant shifts in perceived position occur. Even nonresonant nonlinear signals can be evoked purely through a mismatch in linear refractive index. Computational modelling can also help reveal additional detail. As SHG is a coherent process, subwavelength information can be inferred through the phase information. Our experimental collaborators built an interferometric SHG (I-SHG) microscope for exactly that purpose. We used this to image collagen fibrils, which are all aligned in a parallel fashion. However, because collagen fibrils have a chiral molecular structure, they can point either ``up'' or ``down''. Using my Green's function simulations of the SHG imaging process of collagen fibrils, I was able to predict the standard deviation in the measured phase and link it to the orientation of collagen fibrils in the focal spot of the probing laser beam, even though the diameters are far below the minimum resolvable capabilities of the microscope. We found that the ``upwards'' fibrils make up 46--53% of the sample. Even with a normal SHG microscope that does not measures phase, additional subresolution information is obtainable. With our collaborators we measured the ratio of the forward SHG intensity signal to that in the backward direction and with my simulations, we are able to link this to the fibril diameters in collagen tissue. Thus we inferred that the fibril diameter increases as a function of tissue depth. Furthermore, a computational technique called ptychography is able to retrieve phase information without an interferometric reference beam. Additionally, it increases resolution to the theoretical limit, independent of the laser focal spot size, and corrects for distortions in the input beam as well. I have developed this technique for use with nonlinear optical microscopy and was able to show it is a viable alternative to I-SHG by imaging simulated rat tail tendon at the diffraction limit while retrieving the orientation of the fibrils through the phase of the SHG signal. I also implemented the algorithm for CARS, where the phase information can be used to greatly increase the signal-to-noise ratio by reducing the nonresonant background radiation that results from competing nonlinear optical processes. I showed an example of this by imaging a simulated fibroblast cell where the CARS process was tuned to the lipid droplets inside of the cell. I am currently in talk with experimentalists to apply this theoretical technique to experiments as that would further demonstrate the impact of my work. Finally, keeping in theme with the collagen fibrils, I show that the ratio of the forward SHG signal to the backward signal, the F/B ratio, is affected by a mismatch in the refractive index for fibrils larger than 100nm. This measure is an indicator of fibril diameter and thus important for making qualitative predictions. Single fibrils are generally too small to be significantly affected by near-field effects, but the bigger fibrils can be. Fibrils in rat tail tendon have a distribution of fibrils diameters and the large fibrils occur infrequent. However, I found that the large fibrils are largely responsible for the forward as well as backward signal, thus refractive index mismatches still affect the F/B ratio significantly despite their infrequency. The F/B ratio for a collection of fibrils placed in a n=1.47 medium was found to be 31.8±0.7% higher than for those in a n=1.33 medium. Our experimental colleagues have done preliminary measurements on mouse tail tendon where they found an increase of 40±20%, in line with the value of 28.1±0.6% that I found for simulations with mouse tail tendon. In conclusion, the theoretical tools I have used in my thesis have provided me with the ability to study nonlinear optical image formation processes with a level of detail that would be near-impossible to do experimentally. I have used this ability to show how refractive index mismatches, such as those found in biological tissue, can significantly distort the far-field intensity signals. I have shown this for SRS and CARS where the far-field intensity signal appeared an order-of-magnitude larger compared to the same sample without a refractive index mismatch with the background medium. Additionally, shifts in the perceived position of the object under investigation were observed and I showed the presence of a nonresonant background signal in AM-SRS. Likewise I showed that in the SHG imaging of collagen fibrils significant changes in the F/B ratio can occur. All of these effects have important implications as these types of images as biomedical researches rely on the correct interpretation of nonlinear optical microscopy images for both research and diagnostics. Apart from showing the effect of a refractive index mismatch, I have also shown that computation modelling can be used to infer subwavelength features in SHG imaging experiments of collagen fibril such as fibril orientation and fibril diameter. These methods have the potential to aid medical researchers as changes in the structure of collagen are often an early indicator of diseases such as osteoarthritis. Finally, I showed that the ptychography algorithm I developed for nonlinear optical microscopy is able to retrieve phase information of the nonlinear electric susceptibility in SHG and CARS imaging while also enhancing the resolution and correcting for distortions in the input beams. I can also use much larger laser spot sizes than in conventional experiments without compromising the obtained resolution, thus fewer measurements are required. The technique is not limited to SHG and CARS either; it will work for other nonlinear optical processes as well. Experimental verification of nonlinear ptychography will be done soon. This technique has to potential to significantly improve current imaging techniques since access to the phase information allows one to observe additional information about the sample as we showed with the I-SHG microscope.

Ce travail de thèse propose de réaliser un nanofil électrique auto-assemblé constitué de protéines. L'unité de base de ce nanofil est une protéine chimère comprenant un domaine capable de former des fibres amyloïdes (Het-s 218-289) et un domaine capable d'effectuer des transferts d'électrons (une rubrédoxine). Le premier domaine permet la réalisation d'une fibre par auto-assemblage tandis que le deuxième est exposé à la surface de cette structure. Les caractéristiques redox du domaine exposé permettent aux électrons de se déplacer d'un bout à l'autre de la fibre par sauts successifs. Un tel nanofil a été créé et caractérisé par différentes techniques biophysiques. Ensuite, la preuve de la conduction des nanofils a été apportée sur des ensembles d'objets, de manière indirecte par électrochimie, et de manière directe par des mesures tension/courant. Ces travaux ouvrent la voie à la réalisation d'objets biocompatibles, biodégradables, possédant des propriétés électroniques exploitables dans des dispositifs technologiques. / The research described in this thesis aims at creating a self-assembled nanowire only made of proteins. The building block of this wire is a chimeric protein that comprises an amyloid fibril forming domain (Het-s 218-289) and an electron transfer domain (rubredoxin). The first one self-assembles in amyloid fibrils which display the second at their surface. Redox characteristics of the exposed domain allow electrons to move from one extremity of the fibril to the other by successive jumps. Such a nanowire has been created and characterized by various biophysical experiments. Then, the conductivity of the nanowires has been demonstrated on sets of wires by electrochemistry and by direct current measurements. These experiments pave the way for future design of biocompatible and biodegradable objects that possess usable electronic properties.

Nanomaterials such as graphene oxide and carbon nanotubes, have demonstrated excellent properties for membrane desalination, including decrease of maintenance, increase of flux rate, simple solution casting, and impressive chemical inertness. Here, two projects are studied to investigate nanocarbon based membrane desalination. The first project is to prepare hybrid membranes with amyloid fibrils intercalated with graphene oxide sheets. The addition of protein amyloid fibrils expands the interlayer spacing between graphene oxide nanosheets and introduces additional functional groups in the diffusion pathways, resulting in increase of flux rate and rejection rate for the organic dyes. Amyloid fibrils also provide structural assistance to the hybrid membrane, which supresses cracking and instability of graphene oxide sheets. The second project is to fabricate polymer nanocomposite membranes with carbon nanotubes encapsulated by polymerized surfactants. The designed polymerizable surfactant forms lyotropic liquid crystalline mesophases in an aqueous medium with hexagonal packing of cylindrical micelles. The adsorption of surfactants on the surface of carbon nanotubes allows a stable dispersion of carbon nanotubes encapsulated in the cylindrical micelles, resulting in the ordered structure. After photo-polymerization, the composite membranes display enhanced dye rejection. Both projects have shown promising ways to improve membrane filtration by using nanomaterials.

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L'α-Synuclein (αSyn) fibrillaire, impliqué dans la maladie de Parkinson et d’autres synucleinopathies, peut se propager entre cellules de manière « prion-like » et cette propagation est liée à la progression de la maladie. Durant cette étude, nous nous sommes tournés vers les chaperons moléculaires impliqués dans l’agrégation de l’αSyn ou bien dans sa toxicité afin de trouver des candidats capables d’interférer avec la propagation. Nous avons ensuite testé l’effet des chaperons capables de se lier aux fibres d’αSyn sur l’internalisation des fibres d’αSyn par les cellules Neuro-2a. Nous démontrons que l’interaction avec l’αSyn agrégeant avec αB-crystallin (αBc) ou Carboxyl terminus of Hsc70-interacting protein (CHIP) a mené à la formation de fibres qui sont moins internalisées par les cellules. Enfin, en passant par une stratégie de pontage chimique optimisé couplé à la spectrométrie de masse, nous avons identifié les zones d’interaction entre l’αSyn fibrillaire et soit αBc, soit CHIP. Ces résidus issus des chaperons, se trouvant à proximité des fibres d’αSyn dans les complexes, pourraient être développés dans des mini-chaperons peptidiques, capables d’enrober la surface des fibres et ainsi de bloquer la liaison à la membrane et l’internalisation des fibres. De surcroît, des polypeptides issus des partenaires précédemment identifiés d’αSyn ont été testé pour leur liaison aux fibres et leur effet sur la propagation des fibres. / Fibrillar α-Synuclein (αSyn) is the molecular hallmark of Parkinson’s Disease and other synucleinopathies. Its prion-like propagation between cells is linked to disease progression. In this study, we looked to molecular chaperones previously implicated in αSyn fibrillation and/or toxicity to identify proteins capable of binding αSyn fibrillar aggregates in order to target their propagation. We further assessed the effect of the fibril-binding chaperones on internalization of αSyn fibrils by Neuro-2a cells. We demonstrate that the interaction of aggregating αSyn with αB-crystallin (αBc) or Carboxyl terminus of Hsc70-interacting protein (CHIP) led to the formation of fibrils that are less internalized by cells. Finally, using an optimized chemical cross-linking and mass spectrometry strategy, we identified the interaction areas between fibrillar αSyn and either αBc or CHIP. These chaperone residues, located proximally to αSyn fibrils, could be subsequently developed into peptidic mini chaperones, capable of coating the fibril surface and thereby blocking fibrillar cell binding and internalization. Furthermore, polypeptides derived from previously identified αSyn binding partners were tested for their binding to αSyn fibrils and subsequent effect on fibril propagation.

Les dépôts intracellulaires de la protéine Tau agrégée sont la caractéristique commune des tauopathies, une famille de maladies neurodégénératives dont fait partie la maladie d’Alzheimer. Alors que les isoformes de Tau contenant trois (3R) ou quatre (4R) domaines de liaison aux microtubules sont retrouvées à des niveaux similaires dans le cerveau des individus sains, elles diffèrent au sein des inclusions intracellulaires en fonction des tauopathies. Notre étude repose sur l’identification de déterminants structuraux communs et distincts de fibres de Tau 3R et 4R. Pour cela deux approches de protéomique structurale complémentaires ont été mises au point à partir de fibres de Tau 1N3R et 1N4R produites in vitro. La première stratégie, reposant sur l’utilisation de protéolyses ménagées, nous a permis d’identifier les fragments protéolytiques qui composent un « code-barre » moléculaire propre à chaque assemblage. La seconde stratégie a utilisé un marquage chimique covalent des lysines accessibles suivi de l’analyse qualitative et quantitative des acides aminés marqués par spectrométrie de masse. Nous avons ainsi pu montrer que la partie N-terminale de la protéine était accessible au sein des fibres 1N3R et 1N4R tandis que la région C-terminale de la protéine est protégée pour Tau 1N3R et accessible au solvant pour Tau 1N4R. Nos résultats ouvrent la voie à de nouveaux outils moléculaires pour discriminer les tauopathies. / Intracellular deposits of Tau protein aggregates are the common hallmark of tauopathies, a range of neurodegenerative diseases including Alzheimer's disease. Levels of tau with three (3R) or four (4R) microtubule binding repeats are found similar in the normal adult brain, whereas they differ in neuropathological intracellular Tau inclusions, according to the type of tauopathy. Our study consists of the identification of common and different structural molecular determinants of 3R and 4R Tau fibrils. To this end, two proteomic approaches were optimized using 1N3R and 1N4R recombinant fibrils. The first strategy, using limited proteolysis, allowed us to identify the proteolytic fragments composing the molecular “bar-code” for each type of fibril. The second strategy we optimized used chemical covalent surface labelling of accessible lysines, and qualitative and quantitative analysis of the biotinylated residues using mass spectrometry. We show that, while the N-terminal part of the protein remains accessible within 1N3R and 1N4R fibrils, the C-terminal region is protected within 1N3R yet solvent accessible for 1N4R assemblies. Our results pave the way to new molecular tools to discriminate tauopathies.