La maladie de Parkinson (PD) est considérée comme la deuxième maladie neurodégénérative la plus fréquente. L'examen post-mortem de patients parkinsoniens et des modèles physiologiques d’études de la maladie de Parkinson suggèrent la participation de la mort cellulaire programmée, l'inflammation et l'autophagie dues au stress oxydatif, à des mutations ou l’agrégation de protéines au sein des neurones DA. Les aptamères peptidiques sont de petites protéines combinatoires, consistitués d’une plateforme (dans notre cas, la thiorédoxine humaine, hTRX) et une boucle variable insérée dans le domaine actif de hTRX. Deux aptamères peptidiques ont été identifiés par la sélection fonctionnelle. L’aptamère peptide 32 (Apta-32) ,est spécifique liant deux paralogues T32 impliqués dans le processus d'endocytose. L’aptamère peptidique 34(Apta-34) lie à une cible "T34", une protéine pro-apoptotique ayant un rôle dans la voie apoptotique provenant du noyau. Le travail de cette thèse visait à étudier la fonction anti-apoptotique de nos deux aptamères peptidiques dans deux modèles d’étude de la maladie de Parkinson: un modèle cellulaire (in vitro) et un modèle transgénique D. melanogaster (in vivo). Deux toxines majeures ont été appliquées dans ce travail, 6-hydroxindopamine (6-OHDA) et le paraquat, un pesticide couramment utilisé. Nos observations montrent que la drosophile exprimant Apta-32 dans tous les neurones ont montré une meilleure résistance après 48h de traitement avec le paraquat comparé à deux autre aptamères peptidiques, Apta-34 et Apta-TRX (sans boucle de contrôle variable). Une autre étude a révélé un défaut dans la phagocytose des corps apoptotiques au cours du développement embryonnaire de la drosophile exprimant Apta-32 dans les macrophages, ce qui suggère qu’Apta-32 pourrait participer à et peut-être interférer avec le processus de l’autophygie, et que Apta-32 pourrait protéger contre l'autophagie induite par paraquat dans les neurones. / Parkinson’s disease is considered as the second most common neurodegenerative disease. Although the cause of the progressive cell loss of PD remains unclear to date, programmed cell death, inflammation and autophagy due to oxidative stress, gene mutations or protein aggregations within DA neuron have been suggested as potential causes. Peptide aptamers are small combinatorial proteins, with a variable loop inserted into a scaffold protein, human thioredoxin, hTRX. They are used to facilitate dissection of signaling networks by modulating specific protein interactions and functions. Two peptide aptamers were identified by functional selection which inhibit Bax-dependent cell death in mammalian models. One peptide aptamer (Apta-32) is binding two paralogues involved in endocytotic trafficking T32. The second peptide aptamer (Apta-34) is binding to a target "T34", a pro-apoptotic protein mediating apoptosis emanating from the nucleus. The work of my PhD thesis aimed to investigate the anti-apoptotic function of our two peptide aptamers in different PD models including cell model (in vitro), brain tissue slice and D. melanogaster (in vivo) ; in particular their impact on neuron survival after exposure to specific toxins. Two major toxins were applied in this work, 6-hydroxindopamine (6-OHDA) and Paraquat, a commonly used pesticide. Our observations indicated that Drosophila expressing Apta-32 in all neurons showed more resistance 48h after treatment with Paraquat, compared to drosophila expressing Apta-34 or TRX. Another study revealed a defect in phagocytosis of apoptotic bodies in drosophila embryo’s expressing Apta-32 in macrophage, suggesting Apta-32 could be involved in, and perhaps interfere with, the process of autophagy. This suggests that Apta-32 could protect against paraquat induced autophagy in neurons.

Fur (Ferric Uptake Regulator) est un régulateur transcriptionnel spécifique des bactéries qui intervient dans le contrôle de l'homéostasie du fer, ce qui en fait une cible antibactérienne intéressante. Avant mon arrivée au laboratoire, quatre inhibiteurs interagissant spécifiquement avec Fur avaient été isolés. La partie active de ces inhibiteurs consiste en des peptides de 13 acides aminés. Au cours de cette thèse, j'ai utilisé une double-approche : théorique et expérimentale pour étudier l'interaction de ces peptides avec Fur afin de comprendre le mécanisme d'inhibition. J'ai synthétisé plusieurs séquences peptidiques, montré par des tests biochimiques que certaines inhibaient Fur et déterminé les interactions importantes à l'activité inhibitrice. J'ai obtenu des modèles théoriques des complexes Fur/peptides par amarrage moléculaire, cohérents avec les résultats expérimentaux, qui ont mis en évidence une zone d'inhibition de Fur. Des criblages in silico dans cette zone ont permis de sélectionner de petites molécules, inhibitrices potentielles de Fur et donc intéressantes pour des applications thérapeutiques. / Fur (Ferric Uptake Regulator) is a transcriptional regulator involved in the control of iron homeostasis. Specific to bacteria, Fur is an attractive antibacterial target. Before my arrival in the laboratory, four inhibitors interacting specifically with Fur had been isolated. The active part of these inhibitors consists of peptides of 13 amino acids. In this thesis I have used both theoretical and experimental approaches to study interactions of these peptides with Fur in order to understand the inhibition mechanism. I have synthesized several peptide sequences, shown through biochemical assays that some of them could inhibit Fur and I have identified residues important to the inhibitory activity. I‘ve obtained theoretical models of Fur/peptide complexes consistent with experimental results, which reveal an inhibition pocket in Fur. Small molecules have then been selected though In silico screening of this pocket, that could potentially inhibit Fur, and thus be interesting for therapeutic applications.

Today, development of accurate early diagnosis of cancers thus became the number one challenge of medicine during the 21st century as the current techniques relies on imaging methods that suffer from low sensitivity and misdiagnosis.For these reasons, in this work, we aimed at developing a no-wash colorimetric biosensor for the detection of the oncoprotein Mdm2. Indeed, abnormal levels of Mdm2 could be related to the early formation of tumors.This thesis is devoted to the conception of a no-wash colorimetric biosensor for the detection of the oncoprotein Mdm2. This work can be divided in four parts:(i) The detection strategy and the design of the recognition elements (Chapter I).(ii) The conjugation of gold nanoparticles with the recognition elements (Chapter II, III, IV, V and VI).(iii) The modification of the metallic core of the nanoparticles (Chapter VII).(iv) The use of the optimized biosensor for the detection of Mdm2 (Chapter VIII).In the first part, we investigated the sensing strategy. An aggregation-based assay with plasmonic nanoparticles was selected, as the detection signal is a change of color of the suspension that can be observed to the naked eye or by UV-Vis spectroscopy. We designed the recognition elements, two peptide aptamers coming from endogenous proteins p53 and p14, and we grafted them separately on two batches of gold nanoparticles (AuNPs) via thiol end-groups. We used these latter for the detection of various concentrations of Mdm2 in buffer using our dual-trapping strategy with these two batches of functionalized AuNPs. We demonstrated that both peptides are able to interact with Mdm2 even after grafting onto the particles and that this detection strategy is highly specific. However, this first sensor presented some drawbacks, such as a poor colloidal stability of the AuNPs and a limited dynamic range.With the aim to encompass these issues we investigated, in the second part of this thesis, alternative strategies to conjugate the peptides to the particles. We investigated the functionalization of the particles with stabilizing ligands such as thiolated poly(ethyleneglycol) (HS-PEG). We first studied their simultaneous grafting with the peptides on the AuNPs. We observed that grafting HS-PEGs and peptides side-by-side allowed to control the density of peptides conjugated to the AuNPS and increased drastically the stability of the particles. However, the detection of Mdm2 was strongly hindered by the presence of PEG on the particles carrying the p14 peptide. In a second step, we investigated the conjugation of peptides on the top of a PEG layer carrying functional groups (HS-PEG-X where X is a carboxylate or an alkyne). AuNPs were first functionalized with mixtures of HS-PEG and HS-PEG-X, and the peptides were conjugated to the functional groups via amide bond formation or CuAAC coupling in a second step. However, we noticed that it was not possible to control the composition of the mixed layer of PEGs and thus the peptide grafting density.Due to the lack of recognized protocols in the literature for (i) the determination of the chemical and colloidal stabilities of AuNPs and (ii) the determination of the proportion of different ligands in the organic coating of the particles, we developed two interesting tools. The first one was a convenient method allowing to evaluate by UV-Vis spectroscopy the efficiency of the citrate exchange process using thiol-, alkyne- or diazonium-ligands from gold nanoparticles synthesized via a Turkevich method. The second protocol was a method allowing to quantify the proportion of two HS-PEGs ligands grafted in mixtures onto gold nanoparticles via 1H NMR spectroscopy.As we couldn’t find conditions in which the proportion of multiple thiolated ligands can be controlled on AuNPs, we decided to investigate another functionalization strategy based on the use of calix4arene-diazonium salts.We first studied the grafting on AuNPs of calixarenes bearing four PEG chains at the level of their small rim, one ended by a carboxylic acid and three by a methoxy group. The calixarene layer allowed to obtain AuNPs covered by a very dense PEG shell (with more PEG chains/nm2 that what was obtained previously with thiol anchoring). In addition to that, this PEG shell was strongly anchored to the AuNPs, conferring them a very high colloidal and chemical robustness. We then combined the grafting of this calixarene with the grafting of another non-functional calixarene, bearing four PEG chains ended by a methoxy group, and we quantified the conjugation capacity of such particles by amide bond formation. We demonstrated that this strategy allows to (i) increase drastically the stability of the AuNPs and (ii) control the proportion of peptide conjugated at their surface. Finally, we showed that calixarene-coated AuNPs to which to the p53 and p14 peptides have been conjugated could be used to detect Mdm2.With the evidence that the peptide conjugation density could be controlled using calixarene-coated AuNPs, we investigated the simultaneous grafting of two functional calixarenes on particles: one bearing four carboxylic acids groups and one bearing four PEG chains ended by alkyne groups. We optimized the grafting of these calixarenes in mixed layers on the AuNPs as well as their conjugation. We demonstrated that the grafting of two functional calixarenes led to the production of bi-functional AuNPs, capable of conjugation with two molecules via two distinct chemistries.In the third part, we optimized the composition of the metallic core of the biosensor. As it is well known that silver nanoparticles express better optical properties than gold nanoparticles of the same size, we aimed to incorporate silver nanoparticles (AgNPs) in the biosensor. This was a true challenge due to the intrinsic low chemical stability of silver nanoparticles that greatly limits their use in IVD. For this purpose, we developed an innovative in situ synthesis of silver nanoparticles in the presence of the calixarene-diazonium salts. After optimization of the synthesis, we observed that calixarenes bearing four carboxylic acids groups at the level of their small rim allowed the production of ultra-stable silver nanoparticles to which biomolecules can easily be conjugated. This in situ synthesis procedure even allowed us to produce alloy nanoparticles, with metallic cores whose composition could easily be tuned from pure silver to silver/gold alloys or pure gold. With this synthesis, the composition of the organic layer could also be easily tuned by using mixtures of calixarenes-diazonium salts.Finally, in the last part, we investigated the detection of Mdm2 with the optimized version of the biosensor, i.e. silver nanoparticles coated by a calixarene layer to which the p53 and p14 peptides were conjugated. With this novel class of nanoparticles, we could encompass the two initial drawbacks of the initial sensor. First, we were able to detect Mdm2 with a wider detection range and a lower limit. Secondly, the particles were sufficiently stable and robust to be dispersed in physiological fluids and we could detect Mdm2 in human serum without interference. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished