Un nouveau système d'expression en levure Saccharomyces cerevisiae basé sur la répression catabolique azotée

SUMMARYGap1, the general amino acid permease of Saccharomyces cerevisiae, is a plasma membrane protein which is synthesized and most active under conditions of poor nitrogen supply. Under these conditions, the role of Gap1 is to scavenge external amino acids to be used as nitrogen sources or directly as building blocks for protein synthesis. Gap1 is a member of the Yeast Amino Acid Transporter (YAT) family, a family of amino acid transporters highly conserved in bacteria and fungi. The intracellular trafficking of Gap1 has been the subject of intense investigation as well as the role of lipids (in particular sphingolipids) in its activity and folding. These studies have all been carried out in the cellular context using versatile yeast genetics as exploratory tools. While such in vivo investigations allow to identify physiologically relevant features, they do not provide the details to understand the molecular basis of these phenomena. In order to decipher the molecular features responsible for biological functions, physiological analysis must be combined with biochemical, biophysical and structural studies which typically will be performed on isolated and purified proteins.Our work during this study fits in this trans-disciplinary approach that aims at understanding different properties of the protein: (I) how sphingolipids modulate the activity of Gap1, (II) what part of the protein, and more specifically, what residues, are implicated in its regulation and (III) what are the molecular determinants of the multi-specificity of Gap1.At the beginning of this work, Gap1 had not been previously produced and purified. During this thesis we have identified suitable expression and purification strategies for Gap1 and initiated first characterization studies. In the process, we have developed a new expression system in S. cerevisiae based on the nitrogen catabolite repression. The capacity of this system to express other proteins was successfully tested for two other yeast transporters (Mep2 and Uga4) and for two human proteins: MD-2, a soluble protein and Vglut1, a vesicular transporter.Therefore, we propose our expression design as a viable alternative to existing systems of production in yeast and as a valuable tool to be tested when starting the expression of a new target.Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Unsupervised regularized inverse method for 3D reconstruction in tomographic diffractive microscopy

Regularized inverse approaches are now widely used for the three dimensional reconstructions in Tomographic Diffractive Microscopy. This family of methods consists in the minimization, under some constraints, of a cost function often composed with a data fidelity term and one or several regularization terms. On one hand, the data fidelity term is based on the likelihood of the data. On the other hand, the regularization terms are based on physical a priori (morphology, values,…). It is important to keep a good trade off between data fidelity and regularization. The contribution of these last ones is weighted through hyperparameters which require a precise tuning. The minimization of the Generalized Stein’s Unbiased Risk Estimator (GSURE) is efficient to select a set of optimal hyperparameters however it requires an accurate approximation of the data formation. In this work, we compare the efficiency of an unsupervised regularized approach using GSURE depending of the data model approximation. We compare the optimal reconstructions obtained using the first Born approximation on one side and the Beam Propagation Method on the other side. We chose the regularization of the Total Variation, which favor piece-wise constant structures. For the reconstruction, we used the Primal-Dual Condat-Vũ algorithm with backtracking. We apply both reconstruction methods on experimental data. Our results show that our unsupervised regularized method manages in both cases to find an optimal reconstruction

Reconstruction d’image régularisée non supervisée en microscopie tomographique diffractive

Ce travail présente une approche inverse régularisée non supervisée pour la reconstruction de la carte 3D d’indice de réfraction d’un échantillon en microscopie tomographique diffractive (MTD), récemment publiée dans le Journal of the Optical Society of America A (JOSAA) [1]. La technique MTD permet l’imagerie d’échantillons transparents en 3D sans requérir de marquage. La MTD apporte une information morphologique complémentaire aux phénomènes métaboliques observés via les techniques de nanoscopie fluorescente (STED, PALM/STORM). Bien que moins résolue et moins spécifique que ces dernières, la MTD permet tout de même d’imager un champ de vue plus étendu à haute résolution (2 fois la limite de diffraction). Notre méthode de reconstruction se base sur la minimisation de l’estimateur généralisé non biaisé du risque de Stein – GSURE pour Generalized Stein’s Unbiased Risk Estimator – afin d’estimer automatiquement des valeurs optimales des hyperparamètres de régularisation (parcimonie, préservation de bords, Variation Totale). Nous avons évalué cette méthode sur des reconstructions 3D à partir de données simulées et expérimentales, en utilisant différents modèles de formation d’image (approximation de Rytov à l’ordre 1, modèle de propagation de proche en proche – Beam Propagation Method), et en les comparant avec des méthodes de reconstruction usuelles (inversion de Rytov, algorithme de Gerchberg-Papoulis). Nos résultats montrent qu’un réglage approprié du poids des régularisations dans la résolution du problème de reconstruction permet d’exploiter au mieux l’information disponible dans les données, ce qui peut s’avérer particulièrement critique si cette dernière est limitée (nombre d’angles de vues, couverture angulaire limitée). Disposer d’une méthode de réglage non supervisée de ces hyperparamètres constitue alors un avantage certain, et dans ce contexte nous montrons que le critère GSURE est un bon candidat

Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae.

Decades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe