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It is recognized that a large variety of the organic compounds contaminating the environment can form inclusion complexes with cyclodextrins resulting in enhanced solubility as mentioned in a recent review on cyclodextrins for environmental biotechnologies [1]. Micelle-clay composites have been used for environmental remediation and for the removal of organic pollutants from water [2-5]. In this research we explore the use of cyclodextrins, known to for inclusion complexes and layers onto negatively charged surfaces when combined with cationic surfactants [6-7], to form a composite with clays using similar protocol to the described for micelle-clay composite preparation by Gonzalez-Perez and Macia [8] for the removal of bacteria from water.

In bacteria, DNA is not separated in the cell by a membrane, converse to eukaryotes, which have a nucleus. Therefore, DNA must be protected, while remaining accessible to allow for gene expression. The DNA structuring is achieved via proteins called NAPs (standing for "Nucleoid Associated Protein"). Among these NAPs, the Hfq protein is found. Hfq is a pleiotropic factor involved in bacterial physiology. Its best-known function is to regulate RNA-dependent processes. However, its ability to form an amyloid structure allows it to interact with DNA, to form bridges between distant parts of DNA and thus to compact it strongly. If this property was demonstrated in vitro, analyses were lacking in cellulo. This PhD work highlights the direct and indirect effects of Hfq on the compaction of the nucleoid. The complexes or the cells were observed using cryo-(scanning) transmission electron microscopy (cryo-(S)TEM) and cryo-soft X-ray tomography (cryo-SXT) techniques. The latter is a three-dimensional imaging technique that permits to quantify the volume and density of the bacterial nucleoid. This PhD project made possible to characterize the impact of Hfq on the structure of DNA and bacterial morphology, which will make it possible to test in cellulo the effects of new antibiotics, which could be developed in connection with this property in the future., Chez les bactéries, l'ADN n'est pas séparé du reste de la cellule par une membrane, contrairement aux eucaryotes qui possèdent un noyau. Cet ADN a donc besoin d'être à la fois protégé tout en restant accessible pour permettre l'expression génétique. Cette structuration de l'ADN se fait via des protéines appelées NAPs (pour "Nucleoid Associated Protein"). Parmi ces NAPs, on trouve la protéine Hfq. Hfq est un facteur pléiotrope impliqué dans la physiologie bactérienne. Sa fonction la plus connue est de réguler des processus ARNs dépendants. Cependant, sa capacité à former une structure amyloïde lui permet d'interagir avec l'ADN, de former des ponts entre des parties éloignées de l'ADN et ainsi de le compacter fortement. Si cette propriété a pu être démontrée in vitro, les analyses in cellulo manquaient. Ce travail de thèse met en lumière les effets directs et indirects d'Hfq sur la l'état de compaction du nucléoïde. Ainsi les complexes ou les cellules ont pu être observés à l'aide de techniques de cryo-microscopie électronique en transmission (à balayage) (cryo-(S)TEM) et de cryo-tomographie à rayons-X mous (cryo-SXT). Cette dernière, est une technique d'imagerie tri-dimensionnelle qui permet de quantifier le volume et la densité du nucléoïde bactérien. Ce projet de thèse a permis de caractériser l'impact d'Hfq sur la structure de l'ADN et la morphologie bactérienne, ce qui permettra de tester in cellulo l'effet de nouveaux antibiotiques qui pourront être développés en lien avec cette propriété dans le futur.

International audience; It was recently shown that radiation, conduction and convection can be combined within a single Monte Carlo algorithm and that such an algorithm immediately benefits from state-of-the-art computer-graphics advances when dealing with complex geometries. The theoretical foundations that make this coupling possible are fully exposed for the first time, supporting the intuitive pictures of continuous thermal paths that run through the different physics at work. First, the theoretical frameworks of propagators and Green’s functions are used to demonstrate that a coupled model involving different physical phenomena can be probabilized. Second, they are extended and made operational using the Feynman-Kac theory and stochastic processes. Finally, the theoretical framework is supported by a new proposal for an approximation of coupled Brownian trajectories compatible with the algorithmic design required by ray-tracing acceleration techniques in highly refined geometry.

International audience; endothelial or epithelial tissues form semipermeable barriers that restrict and regulate the transport of solutes to their surrounding tissues. Microphysiological systems aim to recapitulate this hallmark function and provide models for e.g. drug screening. Here, we develop, and validate by extensive simulations, two assays dedicated to the characterization of diffusion- and pressure-driven transport across epithelial and endothelial ductal tissues fabricated in collagen matrices. We first prove that, upon pressure actuation, barrier crossing is enhanced, although transport in paracellular space remains dominated by diffusion. Using the monopore model, we show that diffusion-driven measurements are consistent with paracellular pore radii of 2.1 and 24 nm for epithelial and endothelial tissues, respectively, but also demonstrate that these estimates fail to account for pressure-driven transport data. We solve this contradiction by introducing the deformable monopore model, which assumes the same pore sizes of 2.1 and 24 nm, and includes the deformation, essentially elongational thinning of paracellular cleft, induced by intraluminal stress to fit pressure-driven transport. Hence, this study suggests that transport through barrier tissues can be regulated by mechanical strain

International audience; Collective motions of epithelial cells in vivo are essential for morphogenesis in developmental biology. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear, with various behaviors depending on the tissues and species. Moreover, investigations on in vitro tissue behavior usually focus on only one type of cell dynamics and use diverse theoretical approaches, making systematic comparisons between studies challenging. Here, we show that a single epithelial monolayer of Madin Darby Canine Kidney (MDCK) cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows. We analyzed these distinct motions by using quantitative live imaging. We also report that these motions can be controlled with internal and external cues such as specific inhibitors, and friction modulation of the substrate by microcontact printing method. We further demonstrate with a unified vertex model that both behaviors depend on the competition between velocity alignment and random diffusion of cell polarization. When alignment and diffusion are comparable, a pulsatile flow emerges, whereas the tissue undergoes long-range flows when velocity alignment dominates. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating the dynamics of tissue morphogenesis.

The brain is a highly complex system. Most of such complexity stems from the intermingled connections between its parts, which give rise to rich dynamics and to the emergence of high-level cognitive functions. Disentangling the underlying network structure is crucial to understand the brain functioning under both healthy and pathological conditions. Yet, analyzing brain networks is challenging, in part because their structure represents only one possible realization of a generative stochastic process which is in general unknown. Having a formal way to cope with such intrinsic variability is therefore central for the characterization of brain network properties. Addressing this issue entails the development of appropriate tools mostly adapted from network science and statistics. Here, we focus on the recent advances of exponential random graph models (ERGMs), as a powerful means to identify the local connection mechanisms behind observed global network structure. Efforts are reviewed on the quest for basic organizational properties of human brain networks, as well as on the identification of predictive biomarkers of neurological diseases such as stroke. We conclude with a discussion on how emerging results and tools from statistical graph modeling, associated with forthcoming improvements in experimental data acquisition, could lead to a finer probabilistic description of complex systems in network neuroscience.

Gene expression consists in the synthesis of proteins from the information encoded on DNA. One of the two main steps of gene expression is the translation of messenger RNA (mRNA) into polypeptide sequences of amino acids. Here, by taking into account mRNA degradation, we model the motion of ribosomes along mRNA with a ballistic model where particles advance along a filament without excluded volume interactions. Unidirectional models of transport have previously been used to fit the average density of ribosomes obtained by the experimental ribo-sequencing (Ribo-seq) technique. In this case an inverse fit gives access to the kinetic rates: the position-dependent speeds and the entry rate of ribosomes onto mRNA. The degradation rate is not, however, accounted for and experimental data from different experiments are needed to have enough parameters for the fit. Here, we propose an entirely novel experimental setup and theoretical framework consisting in splitting the mRNAs into categories depending on the number of ribosomes from one to four. We solve analytically the ballistic model for a fixed number of ribosomes per mRNA, study the different regimes of degradation, and propose a criteria for the quality of the inverse fit. The proposed method provides a high sensitivity to the mRNA degradation rate. The additional equations coming from using the monosome (single ribosome) and polysome (arbitrary number) ribo-seq profiles enable us to determine all the kinetic rates in terms of the experimentally accessible mRNA degradation rate., 10 pages, 7 figures, 2 tables

International audience; Frost hardiness is the main factor affecting plant species distribution at high latitudes and altitudes. The main effects of freeze-thaw cycles on trees are damages to living cells, as well as the formation of gas embolism in xylem vessels. Frost effect on trees is also quantified through changes in branch diameter.In order to resorb embolism, some species (walnut, maple, birch, etc.) exhibit an increase in xylem sap pressure during successive freeze-thaw cycles. The modelling of such phenomenon is very challenging due to its multi-physics and multi-scale nature. In this work, we present a numerical model coupling heat transfer, phase change, water and osmotic fluxes, taking into consideration different cell types within walnut branch tissues. We show how diameter and pressure variations are inter-related, and we validate the model against experimental results from the literature. We eventually show how this work can be adapted in order to explore inter-species differences.

Flavins are derivatives of vitamin B2 that form a highly versatile group of chromophores found in a large variety of enzymes and photoreceptor proteins. They can adopt three different redox states with various protonation states, leading to at least five physiologically relevant forms, with distinct electronic absorption spectra. Despite the diverse photophysical properties of the flavin cofactors, there are only very few natural photo-responsive flavoproteins. The vast majority of flavoproteins perform non-light-driven physiological functions (“non-photoactive”), although ultrafast, reversible photoinduced redox reactions between flavins and surrounding residues still widely occur in these systems, which can be viewed as photo-protective “self-quenching”. The past few decades have seen a blooming in the study of flavoproteins for their photocatalytic and photo-biotechnological applications. Moreover, a newly emerging approach in the development of novel photocatalysts from canonical “non-photoactive” flavoenzymes is making use of the photochemistry of reduced flavins instead of the oxidized resting state. Furthermore, practical implications of photochemistry of yet different flavin species are envisaged, but a basic understanding of their mechanisms is still required. In this thesis, applying ultrafast absorption and fluorescence spectroscopy combined with molecular simulations and quantum chemistry approaches, a variety of fundamental photochemical processes in flavoproteins is investigated. First the photoreduction of oxidized flavins was revisited in a flavoprotein, ferredoxin-NADP+ oxidoreductase (FNR), with closely packed reactant configurations, allowing ultrafast formation of intermediate radical pairs. Combining experimental and modeling techniques allowed a detailed assessment of the influence of the environment on the spectral properties of both the anionic flavin and the cationic amino acid (tyrosine or tryptophan) radicals. We further investigated the photochemistry of protein-bound flavin species in different chemical states that are largely unexplored in the literature. It is demonstrated that photooxidation of anionic flavin radicals, which act as reaction intermediates in many biochemical reactions, efficiently occurs within ~100 fs in several flavoprotein oxidases. This process, effectively the reverse of the well-known photoreduction of oxidized flavin may constitute a universal decay pathway. The excited-state properties of fully reduced flavins were studied in several FNR systems where they are involved as functional intermediates, and compared with those in solution. Valuable information concerning their electronic structures and the flavin flexibility was obtained and compared with atomic simulations, with important catalytic implications. Finally, an unprecedented photo-dissociation phenomenon was revealed for a non-covalent charge transfer complex of flavin and a inhibitor in the flavoenzyme monomeric sarcosine oxidase. This process occurs on the timescale of a few hundred femtoseconds and can be attributed to a well-defined photoinduced isomerization of the inhibitor. Altogether, the described findings, which include the discovery of two hitherto undocumented photochemical processes in flavoproteins, expand the repertoire of photochemistry involving flavin cofactors. This work may open new avenues for the exploration of flavin photochemistry with ultimately possible practical implications as novel photocatalysts and optogenetic tools., Les flavines sont des dérivés de la vitamine B2 qui forment un groupe de chromophores très polyvalents présents dans une grande variété d'enzymes et de protéines photoréceptrices. Elles peuvent adopter trois états redox différents avec divers états de protonation, menant à au moins cinq formes physiologiquement pertinentes, avec des spectres d'absorption électronique distincts. Malgré les propriétés photophysiques diverses des cofacteurs flavine, il n'y a que très peu de flavoprotéines naturellement photoréactives. La grande majorité des flavoprotéines remplissent des fonctions physiologiques non générées par la lumière ("non-photoactives"), bien que des réactions d'oxydoréduction photo-induites ultra-rapides et réversibles entre les flavines et les résidus proches s’y produisent largement, ce qui peut être considéré comme "auto-quenching" photo-protectrice. Ces dernières décennies, l'étude des flavoprotéines a connu un essor pour leurs applications photocatalytiques et photo-biotechnologiques. De plus, une nouvelle approche émergeant dans le développement de nouveaux photocatalyseurs à partir de flavoenzymes canoniques "non-photoactives" utilise la photochimie de flavines réduites au lieu de l'état de repos oxydé. En outre, des implications pratiques de la photochimie d'espèces de flavine encore différentes sont envisagées, mais une compréhension fondamentale de leurs mécanismes est requise. Dans cette thèse, par l'application de spectroscopie d'absorption et de fluorescence ultrarapide combinée avec des simulations moléculaires et d’approches de chimie quantique, une variété de processus photochimiques fondamentaux dans les flavoprotéines est étudiée. Tout d'abord, la photoréduction des flavines oxydées a été revisitée dans une flavoprotéine, la ferrédoxine-NADP+ oxydoréductase (FNR), ayant des configurations des réactifs très compactes, permettant la formation ultrarapide de paires de radicaux intermédiaires. La combinaison de techniques expérimentales et de modélisation a permis une évaluation détaillée de l'influence de l'environnement sur les propriétés spectrales des radicaux anioniques de flavine et cationiques d'acide aminé (tyrosine ou tryptophane). Par ailleurs, nous avons étudié la photochimie de flavines liées aux protéines dans différents états chimiques largement inexplorés dans la littérature. Il est démontré que la photooxydation des radicaux anioniques de flavine, qui agissent comme des intermédiaires de réaction dans de nombreuses réactions biochimiques, se produit efficacement en ~100 fs dans plusieurs flavoprotéines oxydases. Ce processus, qui est effectivement l'inverse de la photoréduction bien connue de la flavine oxydée, pourrait constituer une voie de désactivation universelle. Les propriétés de l'état excité des flavines complétement réduites ont été étudiées dans plusieurs systèmes FNR où elles sont impliquées comme intermédiaires fonctionnels, et comparées à celles en solution. Des informations précieuses concernant leurs structures électroniques et la flexibilité des flavines ont été obtenues et comparées avec des simulations atomiques, avec des implications catalytiques importantes. Enfin, un phénomène de photo-dissociation sans précédent a été révélé pour un complexe de transfert de charge non covalent de la flavine et d'un inhibiteur dans la flavoenzyme sarcosine oxydase monomérique. Ce processus se produit sur une échelle de temps de quelques centaines de femtosecondes et peut être attribué à une isomérisation photoinduite bien définie de l'inhibiteur. Dans l'ensemble, les résultats décrits, qui incluent la découverte de deux processus photochimiques jusqu'alors non documentés dans les flavoprotéines, élargissent le répertoire de la photochimie impliquant des cofacteurs de flavine. Ce travail peut ouvrir de nouvelles voies pour l'exploration de la photochimie des flavines avec, à terme, des implications pratiques possibles comme nouveaux photocatalyseurs et outils optogénétiques.

International audience; Membrane fusion is not a spontaneous process. Physiologically, the formation of coiled-coil protein complexes, the SNAREpins, bridges the membrane of a vesicle and a target membrane, brings them in close contact and provides the energy necessary for their fusion. In this review, we utilize results from in vitro experiments and simple physics and chemistry models to dissect the kinetics and energetics of the fusion process from the encounter of the two membranes to the full expansion of a fusion pore. We find three main energy barriers that oppose the fusion process: SNAREpin initiation, fusion pore opening and expansion. SNAREpin initiation is inherent to the proteins and makes in vitro fusion kinetics experiments rather slow. The kinetics are physiologically accelerated by effectors. The energy barriers that precede pore opening and pore expansion can be overcome by several SNAREpin acting in concert.

Nanopores combined with optical approaches can be used to detect viral particles. In this work, we demonstrate the ability of hydrodynamical driving and optical sensing to identify and quantify viral particles in a biological sample. We have developed a simple and rapid method which requires only fluorescent labeling of the particles and can therefore be applied to a wide range of virus type. The system operates in real time and at the single particle level while providing a low error on concentration (4%) and a low limit of detection of 10

Ion transport across the cell membrane is an essential process for living cells. Active transport of ions is carried out by various ion transporters, including microbial rhodopsins (MRs).Microbial rhodopsins are light-driven membrane proteins that can perform various functions such as proton or ion pumping, photo-sensing, ion channeling, and others. Ion pumps MRs are known to be able to pump various monovalent ions such as Na+, K+, Cl-, I-. However, until recently, no microbial rhodopsin was known capable of pumping divalent ions.In 2017 first microbial rhodopsin able to pump divalent ions was described. It is rhodopsin from cyanobacteria Synechocystis sp. PCC 7509 called Synechocystis halorhodopsin or SyHR. This protein belongs to the family of cyanobacterial anion pumps. It is a chloride pump with a unique ability to pump sulfate in the absence of chloride.In this thesis, the author obtained high-resolution three-dimensional structures of different states of this protein for the first time. There are chloride-bound form, sulfate-bound form, and two structures of active states: O and presumably K. All four structures have resolution better than 2.0 Å resolution. Obtained results give insights into the unique ability of this protein to pump divalent ions. In addition, the structures of active states explain the molecular mechanism of chloride transport in cyanobacterial anion pumps. This mechanism is incredibly similar to archaeal halorhodopsins, despite the fact that these two groups of MRs are not the closest relatives.The results obtained make it possible to understand better the molecular principles of ion transport by cyanobacterial anion pumps and assume that proteins from this family can become promising tools for optogenetics., Le transport des ions à travers la membrane cellulaire est un processus essentiel pour les cellules vivantes. Le transport actif des ions est assuré par divers transporteurs d'ions, dont les rhodopsines microbiennes (MR). Les rhodopsines microbiennes sont des protéines membranaires lumineuses qui peuvent remplir diverses fonctions telles que le pompage de protons ou d'ions, la photodétection, la canalisation des ions et autres. Les pompes à ions Les MR sont connues pour être capables de pomper divers ions monovalents tels que Na+, K+, Cl-, I-. Cependant, jusqu'à récemment, aucune rhodopsine microbienne n'était connue capable de pomper des ions divalents.En 2017, la première rhodopsine microbienne capable de pomper des ions divalents a été décrite. Il s'agit de la rhodopsine de la cyanobactérie Synechocystis sp. PCC 7509 appelé Synechocystis halorhodopsin ou SyHR. Cette protéine appartient à la famille des pompes à anions cyanobactériennes. C'est une pompe à chlorure avec une capacité unique à pomper le sulfate en l'absence de chlorure.Dans cette thèse, l'auteur a obtenu pour la première fois des structures tridimensionnelles à haute résolution de différents états de cette protéine. Il existe une forme liée au chlorure, une forme liée au sulfate et deux structures d'états actifs : O et vraisemblablement K. Les quatre structures ont une résolution meilleure que la résolution 2,0 Å. Les résultats obtenus donnent un aperçu de la capacité unique de cette protéine à pomper des ions divalents. De plus, les structures des états actifs expliquent le mécanisme moléculaire du transport du chlorure dans les pompes à anions cyanobactériennes. Ce mécanisme est incroyablement similaire aux halorhodopsines archéennes, malgré le fait que ces deux groupes de MR ne sont pas les plus proches parents. Les résultats obtenus permettent de mieux comprendre les principes moléculaires du transport d'ions par les pompes à anions cyanobactériennes et supposent que les protéines de cette famille peuvent devenir des outils prometteurs pour l'optogénétique.

International audience; In this work, we compared the dynamics of motion in a linear shear flow of individual red blood cells (RBCs) from healthy and pathological donors (Sickle Cell Disease (SCD) or Sickle Cell-β-thalassemia) and of low and high densities, in a suspending medium of higher viscosity. In these conditions, at lower shear rates, biconcave discocyte-shaped RBCs present an unsteady flip-flopping motion, where the cell axis of symmetry rotates in the shear plane, rocking to and fro between an orbital angle ±ϕ observed when the cell is on its edge. We show that the evolution of ϕ depends solely on RBC density for healthy RBCs, with denser RBCs displaying lower ϕ values than the lighter ones. Typically, at a shear stress of 0.08 Pa, ϕ has values of 82 and 72° for RBCs with average densities of 1.097 and 1.115, respectively. Surprisingly, we show that SCD RBCs display the same ϕ-evolution as healthy RBCs of same density, showing that the flip-flopping behavior is unaffected by the SCD pathology. When the shear stress is increased further (above 0.1 Pa), healthy RBCs start going through a transition to a fluid-like motion, called tank-treading, where the RBC has a quasi-constant orientation relatively to the flow and the membrane rotates around the center of mass of the cell. This transition occurs at higher shear stresses (above 0.2 Pa) for denser cells. This shift toward higher stresses is even more remarkable in the case of SCD RBCs, showing that the transition to the tank-treading regime is highly dependent on the SCD pathology. Indeed, at a shear stress of 0.2 Pa, for RBCs with a density of 1.097, 100% of healthy RBCs have transited to the tank-treading regime vs. less than 50% SCD RBCs. We correlate the observed differences in dynamics to the alterations of RBC mechanical properties with regard to density and SCD pathology reported in the literature. Our results suggest that it might be possible to develop simple non-invasive assays for diagnosis purpose based on the RBC motion in shear flow and relying on this millifluidic approach.

Both classical and quantum electrodynamics predict the existence of dipole-dipole long-range electrodynamic intermolecular forces; however, these have never been hitherto experimentally observed. The discovery of completely new and unanticipated forces acting between biomolecules could have considerable impact on our understanding of the dynamics and functioning of the molecular machines at work in living organisms. Here, using two independent experiments, on the basis of different physical effects detected by fluorescence correlation spectroscopy and terahertz spectroscopy, respectively, we demonstrate experimentally the activation of resonant electrodynamic intermolecular forces. This is an unprecedented experimental proof of principle of a physical phenomenon that, having been observed for biomacromolecules and with long-range action (up to 1000 Angstroms), could be of importance for biology. In addition to thermal fluctuations that drive molecular motion randomly, these resonant (and thus selective) electrodynamic forces may contribute to molecular encounters in the crowded cellular space., 40 pages, 8 figures, Supplementary material for this article (additional data, theory and 4 movies) is available at https://www.science.org/doi/10.1126/sciadv.abl5855#con1

Protein adsorption on surfaces is used in analytical tools as an immobilization mean to trap the analyte to be detected. However, protein adsorption can lead to a conformational change in the protein structure, resulting in a loss of bioactivity. Here, we study adsorption of a streptavidin−biotin complex on amorphous SiO2 surfaces functionalized with five different silane self-assembled monolayers by all-atom molecular dynamics simulations. We find that the streptavidin global conformational change increases linearly with the adsorption energy, which depends, as well as the nature of residues with high mobility, on the alkyl chain length and head-group charge of silane molecules. Effects on interactions with biotin are further investigated by steered molecular dynamics (SMD) simulations, which mimics atomic force microscopy (AFM) with the biotin attachedon the tip. We show the combined effects of adsorption-induced global conformational changes and of the position of residues with high mobility on the force of biotin detachment. By comparing our results to experimental and SMD detachment forces obtained in water, without any surface, we conclude that silane with uncharged and short alkyl chains allows streptavidin immobilization, with high adsorption>energy, while keeping biotin interactions better than silanes with>long alkyl chains or charged head groups.

We prepared new organoclay and micelle-clay composites using bentonite and hexadecylpyridinium bromide (C16PyBr) for bacterial removal from water. Using different surfactant concentrations, we ensure that monomeric, spherical micelles and rod-like micelles are present for the clay modification. The modification process was monitored for several days to ensure the saturation and the modified clay was extracted via centrifugation and posterior drying. Preliminary tests for bacterial removal are in agreement with the results of Benitez et al 2022 and indicate that bacteria can be removed from a stock solution opening the door for further systematic experiments on bacterial removal and filter recovery. The method can be used for small-scale drinking water production units and have potential uses in general for small to medium scale decentralized systems.

Frédérique Bordignon, Y J Erden, Raphaël Lévy. ERC Scientific Seminar - How, when and why science fails to correct itself?. ERC Scientific Seminar, Jul 2022, Paris, France; International audience

International audience

In this work, three different cold atmospheric-pressure plasma reactors were studied as sources of reactive oxygen and nitrogen species for biomedical applications. The reactors used were a single-plasma jet, a multi-plasma jet and a micro-cathode sustained discharge. This work is divided in three parts. The first part contains the study of the gaseous phase of the plasmas and its main purpose is to provide information on the physical and chemical of the produced plasmas. We found that the electric field in the front of the ionization wave increases when the plasma approaches the treated liquid. As a result, the gas temperature as well as the electron density of the plasma are also greater closer to the liquid. The second part of this work is a study of the chemical composition of plasma-treated PBS (pPBS). Thus, the production of H₂O₂, NO₂⁻, NO₃⁻, •OH, O₂(a1Δg), O₃, HCl⁻ and ONOO⁻ was investigated in the pPBS. Alongside, the pH and conductivity of the pPBS were also measured. On top of that, the ability to conserve the long-lived reactive species (H₂O₂, NO₂⁻ and NO₃⁻) over a long period was investigated as a function of the time and temperature of storage of the pPBS. It is shown in this work that if the pPBS is stored at pertinent conditions, the concentration of these long-lived reactive species, and, thus, the anti-cancer capacity of the pPBS remains stable for more than 75 days. Knowing that these long-lived reactive oxygen and nitrogen species are the main drivers of the anti-cancer capacity of plasma treatment in oncology, the third part of this work, performed in close collaboration with biologists, was the plasma application on biological targets and the assessment of these reactive species’ contribution to its cytotoxicity. Thus, the anti-tumor capacity of direct and indirect plasma treatments, using two models of head & neck cancer cells and three normal cell lines, was assessed in terms of metabolic cell activity, cell viability, lipid peroxidation, intracellular ROS production and caspase 3/7 induction. We found that the selectivity of plasma treatment towards killing cancer cells instead of normal cells is highly dependent on the short-, intermediate- and long-lived reactive species that are present in the pPBS. More specifically, when the cells are treated directly with the plasma, we observed a strong effect on the normal cells, while the cancer cells were moderately affected. On the other hand, when indirect plasma treatment was used, the effect on the normal cells was minor, while the effect on the cancer cells was significant (slightly less efficient than direct plasma treatment). Our hypothesis is that short- and intermediate-lived reactive species that are present only during the direct plasma treatment damage the cellular membrane of the normal cells allowing the long-lived reactive species to penetrate into the cells. To support this hypothesis and to better understand the interaction of the cellular membrane with these reactive species, the effect of the plasma treatment on bovine serum albumin proteins and different types of phospholipids was investigated. Followingly, we investigated the potential capabilities of the combined application of indirect plasma treatment and microsecond pulsed electric fields (μsPEFs) to outperform in vitro cell electropermeabilisation, the basis of electrochemotherapy (ECT). We showed that the combined treatment opens the possibility to reduce the amplitude of the electric pulses used in ECT, allowing an ECT treatment with reduced side-effects. Finally, the in vivo application of the pPBS on inbred female immunocompetent C57Bl/6J mice was also studied. It is shown that the combination of indirect plasma treatment and μsPEFs delays the tumour growth of the mice., Dans ce travail, trois différents réacteurs à plasma froid à pression atmosphérique ont été étudiés comme sources d'espèces réactives d'oxygène et d'azote pour des applications biomédicales. Les réacteurs utilisés étaient un jet de plasma, un multi-jet de plasma et une micro-cathode sustained discharge. Ce travail est divisé en trois parties. La première partie concerne l'étude de la phase gazeuse des plasmas et son objectif principal est de fournir des informations sur les propriétés physiques et chimiques des plasmas produits. Nous avons constaté que le champ électrique sur le front de l'onde d'ionisation augmente lorsque le plasma s'approche du liquide traité. En conséquence, la température du gaz ainsi que la densité électronique du plasma sont également plus importantes plus près du liquide. La deuxième partie de ce travail est une étude de la composition chimique du PBS traité au plasma (pPBS). Ainsi, la production de H₂O₂, NO₂⁻, NO₃⁻, •OH, O₂(a1Δg), O₃, HCl⁻ et ONOO⁻ a été étudiée dans le pPBS. Parallèlement, le pH et la conductivité du pPBS ont également été mesurés. De plus, la capacité à conserver les espèces réactives à longue durée de vie (H₂O₂, NO₂⁻ et NO₃⁻) sur une longue période a été étudiée en fonction du temps et de la température de stockage du pPBS. Il est montré dans ce travail que si le pPBS est stocké dans des conditions pertinentes, la concentration de ces espèces réactives à longue durée de vie, et donc la capacité anticancéreuse du pPBS, reste stable pendant plus de 75 jours. Sachant que ces espèces réactives de l'oxygène et de l'azote sont les principaux moteurs de la capacité anticancéreuse du traitement par plasma en oncologie, la troisième partie de ce travail, réalisée en étroite collaboration avec des biologistes, concerne l'application du plasma sur des cibles biologiques et l’évaluation de la contribution de ces espèces réactives à sa cytotoxicité. Ainsi, en utilisant deux modèles de cellules cancéreuses de la tête et du cou et trois lignées cellulaires normales, la capacité anti-tumorale des traitements directs et indirects au plasma a été évaluée selon l’activité métabolique cellulaire, la viabilité cellulaire, la peroxydation lipidique, la production de ROS intracellulaires et l’induction de la caspase 3/7. Nous avons constaté que la sélectivité du traitement au plasma pour tuer les cellules cancéreuses au lieu des cellules normales dépend fortement des espèces réactives à courte, moyenne et longue durée de vie présentes dans le pPBS. Plus précisément, lorsque les cellules sont traitées directement avec le plasma, nous avons observé un fort effet sur les cellules normales, tandis que les cellules cancéreuses étaient modérément affectées. En revanche, lorsque le traitement indirect au plasma était utilisé, l'effet sur les cellules normales était mineur, tandis que l'effet sur les cellules cancéreuses était significatif (légèrement moins efficace que le traitement direct au plasma). Notre hypothèse est que les espèces réactives à courte et moyenne durée de vie présentes uniquement lors du traitement direct au plasma endommagent la membrane cellulaire des cellules normales, permettant aux espèces réactives à longue durée de vie de pénétrer dans les cellules. Par la suite, nous avons étudié les capacités potentielles de l'application combinée du traitement indirect au plasma et des champs électriques pulsés microsecondes (μsPEF), pour surpasser l'électroperméabilisation cellulaire in vitro, la base de l'électrochimiothérapie (ECT). Nous avons montré que le traitement combiné ouvre la possibilité de réduire l'amplitude des impulsions électriques utilisées en ECT, permettant un traitement ECT avec des effets secondaires réduits. Enfin, l'application in vivo du pPBS sur des souris femelles consanguines immunocompétentes C57Bl/6J a également été étudiée. Il est démontré que la combinaison du traitement indirect au plasma et des μsPEF retarde la croissance tumorale des souris.

International audience; Gene expression consists in the synthesis of proteins from the information encoded on DNA. One of the two main steps of gene expression is the translation of messenger RNA (mRNA) into polypeptide sequences of amino acids. Here, by taking into account mRNA degradation, we model the motion of ribosomes along mRNA with a ballistic model where particles advance along a filament without excluded volume interactions. Unidirectional models of transport have previously been used to fit the average density of ribosomes obtained by the experimental ribosome sequencing (Ribo-Seq) technique. In this case an inverse fit gives access to the kinetic rates: the position-dependent speeds and the entry rate of ribosomes into mRNA. The degradation rate is not, however, accounted for and experimental data from differentexperiments are needed to have enough parameters for the fit. Here, we propose an entirely novel experimental setup and theoretical framework consisting in splitting the mRNAs into categories depending on the number of ribosomes from one to four. We analytically solve the ballistic model for a fixed number of ribosomes per mRNA, study the different regimes of degradation, and propose a criterion for the quality of the inverse fit. The proposed method provides a high sensitivity to the mRNA degradation rate. The additional equations coming from using the monosome (single ribosome) and polysome (arbitrary number) Ribo-Seq profiles enables us to determine all the kinetic rates in terms of the experimentally accessible mRNA degradation rate.

Cell generated forces play a major role in coordinating large-scale behavior of cells, in particular during development, wound healing and cancer. Mechanical signals based on cellular force generation propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissue with tight cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing due to the lack of appropriate model systems. Here we show that such a setup can be realized by combining optogenetics and micropatterning. Our minimal system are two epithelial cells on an H-pattern ("cell doublet"). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In contrast to single cells on the same pattern ("cell singlet"), whose force generation after half-activation suffers from internal flows, in the cell doublet the cell boundary suppresses global flows and leads to a stable contractile situation. Force propagation and response of the receiver cell strongly depends on the actin organization in the sender cell, which we control by the aspect ratio of the H-pattern. Thus both cell-cell boundary and organization of the sender cell are essential for stimulation of the receiver cell. We quantify the active response of the receiver cell by comparing it with the passive response calculated with a mathematical model. We find that the response essentially matches the signal strength of the sender cell, and that it is the stronger the more organized the actin cytoskeleton is perpendicular to the direction of the cell-cell boundary, reminiscent of the Poisson effect in passive material. We finally show that the same effects are at work in small t issues. Our work demonstrates that cellular organization and active mechanical response of tissue is key to avoid global actin flows and to generate an elastic response that can lead to long-range mechanical signaling across the tissue., Les forces générées par les cellules jouent un rôle majeur dans la coordination de leur comportement à grande échelle, notamment au cours du développement, de la cicatrisation et du cancer. Les signaux mécaniques se propagent plus rapidement que les signaux biochimiques, mais leurs effets peuvent être similaires, notamment au sein des tissus épithéliaux avec une forte adhésion cellule-cellule. Cependant, une description quantitative de la chaîne de transmission (de la génération de forces dans une cellule émettrice se propageant via les jonctions entre cellules, à la réponse induites des cellules réceptrices) manque en raison de l'absence d'outils permettant un bon contrôle spatio-temporel de la force. Nous proposons ici un modèle minimal de deux cellules épithéliales sur un motif en H ("doublet cellulaire"). Après l'activation par optogénétique de RhoA, un régulateur majeur de la contractilité cellulaire, la cellule émettrice se contracte. Nous mesurons ensuite la réponse mécanique de la cellule réceptrice par microscopie des forces de traction et contrainte de monocouche. On observe que la propagation de la force ainsi que la réponse de la cellule réceptrice dépendent fortement de la polarisation mécano-structurelle de la cellule émettrice, que nous contrôlons par le rapport d'aspect du motif en H. Nous constatons que la réponse de la cellule réceptrice est plus forte lorsque l'axe de polarisation mécano-structurelle est orienté perpendiculairement à la direction de propagation de la force, ce qui rappelle l'effet de Poisson dans les matériaux passifs. Nous montrons finalement que les mêmes effets sont à l'œuvre dans les petits tissus. Ainsi, nos travaux démontrent que l'organisation cellulaire et la réponse mécanique active d'un tissu sont essentiels pour maintenir la force du signal et conduisent à l'émergence de l'élasticité, ce qui signifie que les signaux ne sont pas dissipés comme dans un système visqueux, mais peuvent se propager sur de grandes distances, comme dans un système élastique.

Locomotion is essential for motile animal to adapt to their environment. This ability to navigate is modulated by the sensory information animals are continuously integrating from their environment in order to find food or mates and avoid predators. Both locomotion and information integration are part of the sensorimotor program that is controlling animal exploratory behavior. In fish, these operations are already functional at early development stage and evolve with animal development. In this thesis, we explored how stereotyped stimuli are integrated and influence locomotion, and we also investigate how the underlying neuronal circuit of locomotion change through animal development. We used larvae zebrafish to answer the first question. Our involvement in developing a new vertebrate model Danionella cerebrum (DC), allowed us to initiate the exploration of the second question. Sensorimotor program requires the integration of multisensory stimulation that necessitates processing time. To study this, we combined two reflexes already described independently in larvae zebrafish, the optomotor response and the acoustic startle response. We confirmed that larvae are delaying their first movement when exposed to a visual stimulus, and we showed that this visual information did not bias the escape direction triggered by an acoustic signal. Our laboratory was involved in developing a new vertebrate model for neuroscience, the miniature fish DC. With its small size, around 15 mm at adulthood, its optical transparency from larval to adult stage, its wide behavior repertoire and its close phylogenetic relation with zebrafish, this fish is suitable for whole-brain functional imaging with a single cell resolution at different developmental stages. We created a specific fish facility in our university and we established raising and feeding protocols based on already existing knowledge. During a collaborative study, we took benefits of the phylogenetic proximity between zebrafish and DC to propose a comparative study between those two larvae. We especially focused on the spontaneous locomotion and long-term exploration. We showed that despite their different swimming patterns, both species have similar long-term exploration. To explore in more details this, we decided to model both trajectories with simple models. We are also interested in understanding how light affects the locomotor program of DC. In parallel, neuronal calcium activity was recorded with light-sheet microscope in larvae to determine neuron populations that are correlated with swimming activity. It has been published that both species have in common brain region that are recruited during swimming behaviors. Thanks to its different locomotion, we highlighted neuronal populations in DC that are firing when the fish is not swimming. We also demonstrated that it is possible to record whole-brain calcium activity in juveniles DC with the same protocol and setup used for larvae, which will allow following the maturation of the locomotor neuronal circuit and to identify mechanisms involved in brain evolution through the development. This thesis work is part of a global effort to establish DC as an interesting model for neuroscience. Further studies in our laboratory will aim at comparing the simple exploration models defined for zebrafish and DC larvae to have an insight on the evolution strategies which create divergent behaviors in close species. We will also follow modification on the simple model developed for larvae DC and on the locomotor neuronal circuit, in juvenile fish to reveal some mechanisms involved in maturation through development., Se déplacer est essentiel pour que les animaux puissent s'adapter à leur environnement. Cette capacité est modulée par les informations sensorielles environnementales intégrées en permanence par les animaux. Chez les poissons, ce programme sensorimoteur est déjà fonctionnel au stade larvaire et évolue avec le développement de l'animal. Dans cette thèse, nous avons exploré comment les stimuli stéréotypés sont intégrés et influencent la locomotion. Nous avons également étudié comment le circuit neuronal sous-jacent de la locomotion évolue au cours du développement animal. Nous avons utilisé des larves de poisson zèbre pour répondre à la première question. Notre implication dans le développement d'un nouveau modèle vertébré, Danionella cerebrum (DC), nous a permis de commencer l’étude de la deuxième question. L’intégration de stimulations multisensorielles est un processus qui nécessite un temps de traitement des informations. Pour l’étudier, nous avons combiné deux réflexes déjà décrits indépendamment chez la larve de poisson zèbre : la réponse optomotrice et la réponse de fuite à une stimulation acoustique. Nous avons confirmé que les larves retardent leur premier mouvement lorsqu'elles sont exposées à un stimulus visuel, et nous avons montré que cette information visuelle n’influence pas la direction de fuite déclenchée par un signal acoustique. Notre laboratoire participe au développement d'un nouveau modèle vertébré, le poisson DC. Par sa petite taille, sa transparence optique de la larve à l’adulte, son large répertoire de comportements et sa relation phylogénétique étroite avec le poisson zèbre, cet organisme est adapté pour l'imagerie fonctionnelle du cerveau en entier avec une résolution cellulaire et à différents stades du développement. Nous avons créé une installation spécifique pour cette espèce et nous avons établi des protocoles d'élevage et d'alimentation basés sur des connaissances déjà existantes. En tant que collaborateurs pour une étude, nous avons profité de la proximité phylogénétique entre le poisson zèbre et le DC pour mener une étude comparative. Nous nous sommes particulièrement intéressés à la locomotion spontanée et à l'exploration à long terme chez la larve. Nous avons montré que malgré leurs différents modes de nage, les deux espèces ont une exploration à long terme similaire. Pour explorer plus en détail ce phénomène, nous avons décidé de modéliser les trajectoires avec des modèles simples. Nous souhaitons également comprendre comment la lumière affecte le programme locomoteur de DC. En parallèle, l'activité calcique neuronale a été enregistrée avec un microscope à nappe laser afin de déterminer les populations de neurones corrélées à la nage, chez la larve. Il a été publié que les deux espèces ont en commun des régions du cerveau recrutées pendant les phases de mouvements. Grâce à son comportement de nage différent, nous avons mis en évidence des populations neuronales dans le cerveau de DC qui sont activées lorsque le poisson ne nage pas. Nous avons aussi démontré qu'il est possible d'enregistrer l'activité calcique du cerveau entier chez des DC juvéniles avec le même protocole et la même configuration que pour les larves. Cela permettra de suivre la maturation du circuit neuronal locomoteur et d'identifier les mécanismes impliqués dans l'évolution du cerveau au cours du développement. Ce travail de thèse fait partie d'un effort global pour établir DC comme modèle animal en neurosciences. Les futures études dans notre laboratoire viseront à comparer les modèles d'exploration simple définis pour les larves de poisson zèbre et de DC afin d'avoir un aperçu des stratégies d'évolution créant des divergences de comportement. Nous comparerons aussi les modifications du modèle des trajectoires développé pour les larves de DC et celles du circuit neuronal locomoteur, chez les poissons juvéniles, pour révéler certains mécanismes impliqués dans la maturation au cours du développement.

Establishing accurate as well as interpretable models of networks activity is an open challenge in systems neuroscience. Here we infer an energy-based model of the ARTR, a circuit that controls zebrafish swimming statistics, using functional recordings of the spontaneous activity of hundreds of neurons. Although our model is trained to reproduce the low-order statistics of the network activity at short time-scales, its simulated dynamics quantitatively captures the slowly alternating activity of the ARTR. It further reproduces the modulation of this persistent dynamics by the bath temperature and visual stimulation. Mathematical analysis of the model unveils a low-dimensional landscape-based representation of the ARTR activity, where the slow network dynamics reflects Arrhenius-like barriers crossings between metastable states. Our work thus shows how data-driven models built from large neural populations recordings can be reduced to low-dimensional functional models in order to reveal the fundamental mechanisms controlling the collective neuronal dynamics.

International audience; • Stimulation of a dissected retina with a sequence of periodic dark flashes. • Extracellular spike recordings. 2. Identification of necessary components via pharmacological inhibition of synaptic transmission onto : • ON bipolar cells via mGluR6 receptor antagonist LAP-4. • OFF bipolar cells via AMPA receptor antagonist ACET. Results ON BC inhibition cancels the OSR.

We summarize here the recent advances in UV-LED technology with focus on their use for drinking water disinfection on small scale drinking water production plants and with a main target on decentralized systems as well as point-of-use applications. The state of the art on the UV-LED technology and the challenges for their use for drinking water production are highlighted together with regulation and standardization issues. Technological/operational differences compared with the existing UV technology on mercury-based UV lamps are also shown. The possibility to use flashing modes and combinations of different UV wavelengths to enhance disinfection are two of the most advantageous options available for devices using LED technology for disinfection. The UV-LED disinfection units are already implemented into small and medium-sized water treatment units characteristic of water decentralized systems.

All living organisms are surrounded by fluids, either air or water, which create unique sensory landscapes. For example, chemical signals disperse in the flow by diffusion and advection and when the flow is turbulent odor concentration breaks up in filaments and discrete patches of varying intensity. In my thesis I focused on olfactory navigation in turbulent environments and I aimed at understanding how organisms overcome uncertainties to make decisions. I developed three-dimensional direct numerical simulation of a turbulent channel flow to recreate a realistic environment for olfactory search. I realized these state of the art simulations by customizing an open software called Nek5000, which solves the Navier-Stokes equations for the velocity field and the advection-diffusion equation, which regulates the evolution of the odor (passive scalar) in a fluid. After generating large fluid dynamics datasets of odorant evolution in a channel, I analyzed which features of the olfactory signal are more relevant to locate the odor source. Surprisingly, not only the signal, but also its absence can be informative to infer the distance from the odor source. Using supervised learning algorithms I showed that the intensity of odor concentration is an informative measure, but that the temporal dynamics of the signal allow robust predictions in different conditions and at different ranges from the source. These theoretical results suggest that it is computationally advantageous to measure both odor intensity and timing. I analyzed a set of neural recording from awake mice, demonstrating that they are indeed able to store both quantities, and that the neural representation depends on the underlying flow. I then considered the problem of navigating to the source of the turbulent odor. Although animals (for example moths and crustaceans) robustly perform this task, the algorithms they use are not understood. I modeled olfactory navigation using the framework of Partially Observable Markov Decision Processes (POMDP) and I proposed a normative theory to explain the alternation between sniffing in the air and sniffing the ground, typical of mammals like rodents and dogs. Alternation stems from the physics of fluids, prescribing that odor near the ground is more continuous than up in the air, but remains relatively close to the source. In contrast, at nose level the odor is transported quickly away from the source, but is more noisy and intermittent. An agent searching for the odor source should thus sniff in the air when it is far from the source to increase its chances of detecting the odor. Once the agent localizes the odor plume, it should continue the search sniffing the ground where the trail is less intermittent. The exact timing for alternation stems from marginal value theory. Finally, the commonly observed behavior of searchers proceeding in casts and surges emerges from this computational framework, and alternation naturally complements this dynamics to ensure optimal exploration., Tous les organismes vivants sont entourés de fluides, air ou eau, qui créent des paysages sensoriels uniques. Par exemple, les signaux chimiques se dispersent dans le fluide par diffusion et advection et lorsque l'écoulement est turbulent, la concentration d'odeurs se décompose en filaments et en taches discrètes d'intensité variable. Dans ma thèse, je me suis concentré sur la navigation olfactive dans des environnements turbulents et j'ai cherché de comprendre comment les organismes surmontent les incertitudes pour prendre des décisions. J'ai développé des simulations numériques (DNS) tridimensionnelles d'un écoulement turbulent dans un canal afin de recréer un environnement réaliste pour la recherche olfactive. J'ai réalisé ces simulations en personnalisant un open-source software appelé Nek5000, qui résout les équations de Navier-Stokes pour le champ de vitesse et l'équation d'advection-diffusion, qui régule l'évolution de l'odeur (scalaire passif) dans un fluide. Après avoir généré de grands ensembles de données de dynamique des fluides sur l'évolution des odeurs dans un canal, j'ai analysé quelles caractéristiques du signal olfactif sont les plus pertinentes pour localiser la source de l'odeur. Étonnamment, non seulement le signal, mais aussi son absence peut être informative pour déduire la distance de la source de l'odeur. En utilisant des algorithmes de Reinforcment Learning, j'ai montré que l'intensité de la concentration d'odeur est une mesure informative, mais que la dynamique temporelle du signal permet des prédictions robustes dans différentes conditions et à différentes distances de la source. Ces résultats théoriques suggèrent qu'il est avantageux sur le plan informatique de mesurer à la fois l'intensité et la dynamique de l'odeur. J'ai analysé un ensemble d'enregistrements neuronaux de souris éveillées, démontrant qu'elles sont bien capables de stocker les deux quantités, et que la représentation neuronale dépend du flux sous-jacent. J'ai alors considéré le problème de la navigation jusqu'à la source de l'odeur turbulente. Bien que les animaux (par exemple les papillons de nuit et les crustacés) effectuent cette tâche de manière robuste, les algorithmes qu'ils utilisent ne sont pas compris. J'ai modélisé la navigation olfactive en utilisant le cadre des Partially Observable Markov Decision Processes (POMDP) et j'ai proposé une théorie normative pour expliquer l'alternance entre renifler dans l'air et renifler le sol, typique des mammifères comme les rongeurs et les chiens. L'alternance découle de la physique des fluides, prescrivant que l'odeur près du sol est plus continue que dans l'air, mais reste relativement proche de la source. En revanche, au niveau du nez, l'odeur est transportée rapidement loin de la source, mais est plus bruyante et intermittente. Un agent recherchant la source de l'odeur doit donc renifler l'air lorsqu'il est loin de la source pour augmenter ses chances de détecter l'odeur. Une fois que l'agent a localisé le panache d'odeur, il doit continuer la recherche en reniflant le sol où la piste est moins intermittente. Le moment exact de l'alternance découle de la Marginal Value Theory. Enfin, le comportement communément observé chez les organismes recherchant une source d'odeur, procédant en cast et surge émerge spontanément de ce cadre computationnel.

Hydrogels connected by multivalent reversible crosslinkers are a versatile design platform for biocompatible viscoelastic materials. Their linear response to a step strain displays a fast, exponential relaxation when using low valence crosslinkers, while larger supramolecular crosslinkers bring about much slower dynamics involving a wide distribution of time scales whose physical origin is still debated. Here, we are proposing a model where the gels' relaxation originate from elementary events in which the bonds connecting two neighboring crosslinkers all disconnect. Larger crosslinkers allow for a greater average number of bonds connecting them, but also generate more heterogeneity. We characterize the resulting distribution of relaxation time scales analytically, and accurately reproduce rheological measurement on metal-coordinate hydrogels with a variety of crosslinker sizes including ions, metal-organic cages, and nanoparticles. Our approach is simple enough to be extended to any crosslinker size, and could thus be harnessed for the rational design of complex viscoelastic materials.

Flavins are derivatives of vitamin B2 that form a highly versatile group of chromophores found in a large variety of enzymes and photoreceptor proteins. They can adopt three different redox states with various protonation states, leading to at least five physiologically relevant forms, with distinct electronic absorption spectra. Despite the diverse photophysical properties of the flavin cofactors, there are only very few natural photo-responsive flavoproteins. The vast majority of flavoproteins perform non-light-driven physiological functions (“non-photoactive”), although ultrafast, reversible photoinduced redox reactions between flavins and surrounding residues still widely occur in these systems, which can be viewed as photo-protective “self-quenching”. The past few decades have seen a blooming in the study of flavoproteins for their photocatalytic and photo-biotechnological applications. Moreover, a newly emerging approach in the development of novel photocatalysts from canonical “non-photoactive” flavoenzymes is making use of the photochemistry of reduced flavins instead of the oxidized resting state. Furthermore, practical implications of photochemistry of yet different flavin species are envisaged, but a basic understanding of their mechanisms is still required. In this thesis, applying ultrafast absorption and fluorescence spectroscopy combined with molecular simulations and quantum chemistry approaches, a variety of fundamental photochemical processes in flavoproteins is investigated. First the photoreduction of oxidized flavins was revisited in a flavoprotein, ferredoxin-NADP+ oxidoreductase (FNR), with closely packed reactant configurations, allowing ultrafast formation of intermediate radical pairs. Combining experimental and modeling techniques allowed a detailed assessment of the influence of the environment on the spectral properties of both the anionic flavin and the cationic amino acid (tyrosine or tryptophan) radicals. We further investigated the photochemistry of protein-bound flavin species in different chemical states that are largely unexplored in the literature. It is demonstrated that photooxidation of anionic flavin radicals, which act as reaction intermediates in many biochemical reactions, efficiently occurs within ~100 fs in several flavoprotein oxidases. This process, effectively the reverse of the well-known photoreduction of oxidized flavin may constitute a universal decay pathway. The excited-state properties of fully reduced flavins were studied in several FNR systems where they are involved as functional intermediates, and compared with those in solution. Valuable information concerning their electronic structures and the flavin flexibility was obtained and compared with atomic simulations, with important catalytic implications. Finally, an unprecedented photo-dissociation phenomenon was revealed for a non-covalent charge transfer complex of flavin and a inhibitor in the flavoenzyme monomeric sarcosine oxidase. This process occurs on the timescale of a few hundred femtoseconds and can be attributed to a well-defined photoinduced isomerization of the inhibitor. Altogether, the described findings, which include the discovery of two hitherto undocumented photochemical processes in flavoproteins, expand the repertoire of photochemistry involving flavin cofactors. This work may open new avenues for the exploration of flavin photochemistry with ultimately possible practical implications as novel photocatalysts and optogenetic tools., Les flavines sont des dérivés de la vitamine B2 qui forment un groupe de chromophores très polyvalents présents dans une grande variété d'enzymes et de protéines photoréceptrices. Elles peuvent adopter trois états redox différents avec divers états de protonation, menant à au moins cinq formes physiologiquement pertinentes, avec des spectres d'absorption électronique distincts. Malgré les propriétés photophysiques diverses des cofacteurs flavine, il n'y a que très peu de flavoprotéines naturellement photoréactives. La grande majorité des flavoprotéines remplissent des fonctions physiologiques non générées par la lumière ("non-photoactives"), bien que des réactions d'oxydoréduction photo-induites ultra-rapides et réversibles entre les flavines et les résidus proches s’y produisent largement, ce qui peut être considéré comme "auto-quenching" photo-protectrice. Ces dernières décennies, l'étude des flavoprotéines a connu un essor pour leurs applications photocatalytiques et photo-biotechnologiques. De plus, une nouvelle approche émergeant dans le développement de nouveaux photocatalyseurs à partir de flavoenzymes canoniques "non-photoactives" utilise la photochimie de flavines réduites au lieu de l'état de repos oxydé. En outre, des implications pratiques de la photochimie d'espèces de flavine encore différentes sont envisagées, mais une compréhension fondamentale de leurs mécanismes est requise. Dans cette thèse, par l'application de spectroscopie d'absorption et de fluorescence ultrarapide combinée avec des simulations moléculaires et d’approches de chimie quantique, une variété de processus photochimiques fondamentaux dans les flavoprotéines est étudiée. Tout d'abord, la photoréduction des flavines oxydées a été revisitée dans une flavoprotéine, la ferrédoxine-NADP+ oxydoréductase (FNR), ayant des configurations des réactifs très compactes, permettant la formation ultrarapide de paires de radicaux intermédiaires. La combinaison de techniques expérimentales et de modélisation a permis une évaluation détaillée de l'influence de l'environnement sur les propriétés spectrales des radicaux anioniques de flavine et cationiques d'acide aminé (tyrosine ou tryptophane). Par ailleurs, nous avons étudié la photochimie de flavines liées aux protéines dans différents états chimiques largement inexplorés dans la littérature. Il est démontré que la photooxydation des radicaux anioniques de flavine, qui agissent comme des intermédiaires de réaction dans de nombreuses réactions biochimiques, se produit efficacement en ~100 fs dans plusieurs flavoprotéines oxydases. Ce processus, qui est effectivement l'inverse de la photoréduction bien connue de la flavine oxydée, pourrait constituer une voie de désactivation universelle. Les propriétés de l'état excité des flavines complétement réduites ont été étudiées dans plusieurs systèmes FNR où elles sont impliquées comme intermédiaires fonctionnels, et comparées à celles en solution. Des informations précieuses concernant leurs structures électroniques et la flexibilité des flavines ont été obtenues et comparées avec des simulations atomiques, avec des implications catalytiques importantes. Enfin, un phénomène de photo-dissociation sans précédent a été révélé pour un complexe de transfert de charge non covalent de la flavine et d'un inhibiteur dans la flavoenzyme sarcosine oxydase monomérique. Ce processus se produit sur une échelle de temps de quelques centaines de femtosecondes et peut être attribué à une isomérisation photoinduite bien définie de l'inhibiteur. Dans l'ensemble, les résultats décrits, qui incluent la découverte de deux processus photochimiques jusqu'alors non documentés dans les flavoprotéines, élargissent le répertoire de la photochimie impliquant des cofacteurs de flavine. Ce travail peut ouvrir de nouvelles voies pour l'exploration de la photochimie des flavines avec, à terme, des implications pratiques possibles comme nouveaux photocatalyseurs et outils optogénétiques.

In this thesis we report new extensions and applications of next generation neural mass models. Montbrió, Pazó, and Roxin (MPR) have shown that the collective behavior of an ensemble of quadratic integrate and fire (QIF) neurons can be described in terms of the mean membrane potential and firing rate in an exact manner, reducing the dimension of the problem from an infinitely large microscopic network to a low dimensional macroscopic description. Since the neural mass gives access to the mean membrane potentials, it can be used as a proxy for local field potential and EEG signals.One contribution of this thesis is the implementation of short-term synaptic plasticity (STP) into the MPR model. Based on a synaptic theory of working memory (WM), we reproduce WM mechanisms using a QIF network and its exact mean-field limit in a multi-population setup. In particular, we report agreement between the behavior of the network and the extended neural mass model and we make a comparison between our model and experimental results on cortical oscillations associated to WM tasks. The neural mass model exhibits oscillations in the β-γ range during memory loading and maintenance, as also observed in experiments, while we encounter empty β-γ bands using a heuristic model. Furthermore we report how these power bands are formed by resonances among fundamental frequencies, which are linked to the number of items retained in memory. We also provide an analytical estimate for the maximal WM capacity of around five items.The second contribution is the application of a multi-population model in order to test clinical hypothesis of seizure propagation. We use structural connectomes obtained from dMRI scans of healthy subjects and epileptic patients. We describe how seizure-like events can be modeled as a recruitment from low activity to high activity states. External inputs can trigger such an event and lead to a cascade of recruitments, hence mimicking the spatio-temporal propagation of seizures. The numerical results suggest that epileptic patients are more susceptible to extensive recruitment events than healthy subjects. We also find good agreement between the first brain areas to be recruited in our model and the pre-surgical assessment of recruited secondary networks.As a third contribution we study the network and neural mass in presence of STP using slow-fast dynamics. Depending on the amplitude of a slow periodic current applied to the population, the collective behavior can be either in a regime of subthreshold oscillations or bursting, i.e., alternating between a quasi-static drift and fast large amplitude oscillations. The two regimes are separated by a narrow parameter interval, resembling a canard explosion. In this region we report jump-on canards, which approach otherwise repelling invariant sets. We study their impact on the emergence of bursting. For intermediate timescale separations bursting emerges in a continuous manner via a spike-adding mechanisms mediated by mixed-type-like torus canards, trajectories which evolve nearby families of repelling equilibria and limit cycles. For stronger timescale separation the continuous transition is blocked by jump-on canards. The mechanisms observed in the neural mass are also responsible for the emergence of bursting in the network. To summarize, this thesis puts next generation neural mass models into a broader context of modeling in neuroscience and provides new perspectives for future work. This includes approaches to take better models of STP into account and ideas for exact neural masses based on biologically plausible neuron models., Dans cette thèse, nous présentons de nouvelles extensions et applications des modèles de masse neurale de nouvelle génération. Montbrió, Pazó et Roxin (MPR) ont montré que le comportement collectif d’un ensemble de neurones intègre-et-tir quadratiques (QIF) peut être décrit en termes de potentiel de membrane moyen et de taux de décharge de façon exacte, ramenant la dimension du problème d’un réseau microscopique infiniment grand à une description macroscopique de petite dimension. Vu que la masse neurale donne accès aux potentiels de membrane moyens, elle peut être utilisée comme un indicateur pour le potentiel de champ local et les signaux EEG.Une des contributions de cette thèse est l’implémentation de la plasticité synaptique à court terme (STP) dans le modèle MPR. Sur la base d’une théorie synaptique de la mémoire de travail (WM), nous reproduisons les mécanismes de la WM avec un réseau QIF et de sa limite champ moyen exacte dans une configuration multi-population. Le modèle de masse neurale présente des oscillations dans la bande β-γ durant le chargement et le maintien de la mémoire, comme observé dans les expériences, alors que nous rencontrons des bandes β-γ vides en avec un modèle heuristique. De plus, nous indiquons comment ces bandes de puissance sont formées par des résonances entre les fréquences fondamentales, liées au nombre d’éléments retenus en mémoire. Nous fournissons aussi une estimation analytique de la capacité maximale de la WM d’environ cinq éléments.La deuxième contribution est l’application d’un modèle multi-population pour tester l’hypothèse clinique de la propagation des crises épileptiques. Nous utilisons des connectomes structurels obtenus à partir de scans de MRI de diffusion de sujets sains et de patients épileptiques. Nous décrivons comment les événements de type crise épileptique peuvent être modélisés comme un recrutement d’un état de faible activité à un état de forte activité. Des entrées externes peuvent déclencher un tel événement et conduire à une cascade de recrutements, imitant ainsi la propagation spatio-temporelle des crises. Les résultats numériques suggèrent que les patients épileptiques sont plus sensibles aux événements de recrutement étendus que les sujets sains. Nous trouvons aussi un bon accord entre les premières aires cérébrales à être recrutées dans notre modèle et l’évaluation pré-chirurgicale des réseaux secondaires recrutés.Comme troisième contribution, nous étudions le réseau et la masse neurale en présence de STP en utilisant des dynamiques lentes-rapides. En fonction de l’amplitude d’un courant périodique lent appliqué à la population, le comportement collectif peut être soit dans un régime d’oscillations sous-seuil, soit dans un régime de bursting, c’est-à-dire alternant entre une dérive quasi-statique et des oscillations rapides de grande amplitude. Les deux régimes sont séparés par un étroit intervalle de paramètres, ressemblant à une explosion de canards. Dans cette région, nous signalons des jump-on canards, qui s’approchent d’ensembles invariants normalement répulsifs. Pour des séparations d’échelles de temps intermédiaires, le bursting émerge d’une manière continue via des mécanismes d’addition de spike organisés par des mixed-type torus canards, des trajectoires qui évoluent à proximité de familles d’équilibres et de cycles limites répulsifs. Pour une séparation d’échelles de temps plus forte, la transition continue est bloquée par des jump-on canards. Les mécanismes observés dans la masse neurale sont également responsables de l’émergence du bursting dans le réseau. Pour résumer, cette thèse place les modèles de masse neurale de nouvelle génération dans un contexte plus large de modélisation en neurosciences et fournit de nouvelles perspectives pour des travaux futurs. Cela inclut des approches visant à prendre en compte de meilleurs modèles de STP et des idées pour des masses neurales exactes basées sur des modèles de neurones biologiquement plausibles.

Viruses are right at the interface of inanimate matter and life. However, recent experiments [T. Sakai, et al., J.~Virol.~{\bf 92}, e01522-17 (2018)] have shown that some influenza strains can actively roll on glycan-covered surfaces. In a previous letter [F. Ziebert and I. M. Kuli\'{c}, Phys. Rev. Lett. {\bf 126}, 218101 (2021)] we suggested this to be a form of viral surface metabolism: a collection of spike proteins that attach to and cut the glycans act as a self-organized mechano-chemical motor. Here we study in more depth the physics of the emergent self-rolling states. We give scaling arguments how the motion arises, substantiated by a detailed analytical theory that yields the full torque-angular velocity relation of the self-organized motor. Stochastic Gillespie simulations are used to validate the theory and to quantify stochastic effects like virus detachment and reversals of its direction. Finally, we also cross-check several approximations made previously and show that the proposed mechanism is very robust. All these results point together to the statistical inevitability of viral rolling in presence of enzymatic activity., Comment: 17 pages, 10 Figures

International audience; Motility is a crucial activity of immune cells allowing them to patrol tissues as they differentiate, sample or exchange information, and execute their effector functions. Although all immune cells are highly migratory, each subset is endowed with very distinct motility patterns in accordance with functional specification. Furthermore individual immune cell subsets adapt their motility behaviour to the surrounding tissue environment. This review focuses on how the generation and adaptation of diversified motility patterns in immune cells is sustained by actin cytoskeleton dynamics. In particular, we review the knowledge gained through the study of inborn errors of immunity (IEI) related to actin defects. Such pathologies are unique models that help us to uncover the contribution of individual actin regulators to the migration of immune cells in the context of their development and function.

Cell mechano-sensing is based on biomolecule deformations and reorganizations, yet the molecular mechanisms are still unclear. Super-resolution microscopy (SRM) and single protein tracking (SPT) techniques reveal the dynamic organization of proteins at the nanoscale. In parallel, stretchable substrates are used to investigate cellular responses to mechanical forces. However, simultaneous combination of SRM/SPT and cell stretching has never been achieved. Here, we present a cell stretching device compatible with SRM and SPT, composed of an ultra-thin Polydimethylsiloxane (PDMS) layer. The PDMS sheet is gliding on a glycerol-lubricated glass cover-slip to ensure flatness during uniaxial stretching, generated with a 3D-printed micromechanical device by a mobile arm connected to a piezoelectric translator. This method enables to obtain super-resolved images of protein reorganization after live stretching, and to monitor single protein deformation and recruitment inside mechanosensitive structures upon stretching. This protocol is related to the publication ‘Cell stretching is amplified by active actin remodeling to deform and recruit proteins in mechanosensitive structures’, in Nature Cell Biology.

International audience; Artificial intelligence is nowadays used for cell detection and classification in optical microscopy during post-acquisition analysis. The microscopes are now fully automated and next expected to be smart by making acquisition decisions based on the images. It calls for analysing them on the fly. Biology further imposes training on a reduced data set due to cost and time to prepare the samples and have the data sets annotated by experts. We propose a real-time image processing compliant with these specifications by balancing accurate detection and execution performance. We characterised the images using a generic, high-dimensional feature extractor. We then classified the images using machine learning to understand the contribution of each feature in decision and execution time. We found that the non-linear-classifier random forests outperformed Fisher's linear discriminant. More importantly, the most discriminant and time-consuming features could be excluded without significant accuracy loss, offering a substantial gain in execution time. It suggests a feature-group redundancy likely related to the biology of the observed cells. We offer a method to select fast and discriminant features. In our assay, a 79.6 ± 2.4% accurate classification of a cell took 68.7 ± 3.5 ms (mean ± SD, 5-fold cross-validation nested in 10 bootstrap repeats), corresponding to 14 cells per second, dispatched into eight phases of the cell cycle, using 12 feature groups and operating a consumer market ARM-based embedded system. A simple neural network offered similar performances paving the way to faster training and classification, using parallel execution on a general-purpose graphic processing unit. Finally, this strategy is also usable for deep neural networks paving the way to optimizing these algorithms for smart microscopy.

The thesis describes the intricacy of local dynamics of unfolded proteins on ps-ns timescales probed by 15N NMR relaxation in complex macromolecular environments across a range of conditions. Application of temperature-dependent Model-Free analysis to 15N backbone relaxation rates, reveals existence of three dominant dynamic modes, which represent the stochastic processes leading to loss of memory function of the NH-backbone vector. The distinct dynamic profile of the unfolded protein under investigation to the one previously studied (JACS 2016), featuring ultra-flexible tail dominated by single ns dynamics, reaffirms previous assignment of the dynamic modes to librational motions in a flat potential well, local dihedral sampling of the backbone and correlated motions of several peptide units (segmental motions). Systematic modification of the composition of the medium by addition of viscogen allows to assess the influence of crowding on local backbone dynamics of three unfolded proteins, in turn revealing tight coupling of local backbone motions to the solvent dynamics on different length- and timescales. The proportionality constants associated with relative deceleration of correlated backbone fluctuations to the rotational perturbation of the solvent (friction coefficients) arising due to crowding reveal marked sequence-dependence, which to a good extent are influenced by the molecular architecture of the unfolded protein. The analysis is fully consistent with the previously developed temperature-dependent Model-Free analysis and valid within the bounds of adiabatic approximation. The orthogonality of the two analyses (temperature- and viscosity-dependent) allows to combine the expression relating the timescales of motion as a function of temperature and solvent nanoviscosity to a single Arrhenius-Einstein equation, allowing its facile application to the Model-Free analysis of 15N relaxation rates across a range of conditions and macromolecular settings. Relating the relative changes in viscosity to the relative perturbation of the solvent dynamics, rather than to the concentration of the viscogen, allows to predict the local dynamics of unfolded proteins in complex macromolecular environments of different physicochcemical nature, beyond the range of conditions studied, with application in prediction of NH-backbone fluctuations in cell and in condensed phases (JACS 2019). The presented work sheds light on the complex dynamics exhibited by intrinsically disordered proteins in complex environmental conditions on pico- to nanosecond timescales., La thèse décrit la complexité de la dynamique locale des protéines dépliées sur des échelles de temps ps-ns sondées par relaxation RMN 15N dans des environnements macromoléculaires complexes dans une gamme de conditions. L'application de l'analyse sans modèle dépendant de la température aux taux de relaxation du squelette 15N révèle l'existence de trois modes dynamiques dominants, qui représentent les processus stochastiques conduisant à la perte de la fonction de mémoire du vecteur du squelette NH. Le profil dynamique distinct de la protéine dépliée à l'étude par rapport à celui précédemment étudié (JACS 2016), avec une queue ultra-flexible dominée par une dynamique simple ns, réaffirme l'affectation précédente des modes dynamiques aux mouvements de libration dans un puits de potentiel plat, échantillonnage dièdre local du squelette et des mouvements corrélés de plusieurs unités peptidiques (mouvements segmentaires). La modification systématique de la composition du milieu par addition de viscogène permet d'évaluer l'influence de l'encombrement sur la dynamique du squelette local de trois protéines dépliées, révélant à son tour un couplage étroit des mouvements du squelette local avec la dynamique du solvant à différentes échelles de temps et de longueur. Les constantes de proportionnalité associées à la décélération relative des fluctuations du squelette corrélées à la perturbation rotationnelle du solvant (coefficients de frottement) résultant de l'encombrement révèlent une dépendance séquentielle marquée, qui dans une bonne mesure est influencée par l'architecture moléculaire de la protéine dépliée. L'analyse est entièrement cohérente avec l'analyse sans modèle dépendant de la température développée précédemment et valide dans les limites de l'approximation adiabatique. L'orthogonalité des deux analyses (dépendant de la température et de la viscosité) permet de combiner l'expression reliant les échelles de temps du mouvement comme une fonction de la température et de la nanoviscosité du solvant à une seule équation d'Arrhenius-Einstein, permettant son application facile à l'analyse sans modèle des taux de relaxation 15N dans une gamme de conditions et de paramètres macromoléculaires. Relier les changements relatifs de viscosité à la perturbation relative de la dynamique du solvant, plutôt qu'à la concentration du viscogène, permet de prédire la dynamique locale des protéines dépliées dans des environnements macromoléculaires complexes de nature physico-chimique différente, au-delà de la gamme des conditions étudiées, avec application à la prédiction des fluctuations du squelette NH dans les phases cellulaires et condensées (JACS 2019). Le travail présenté met en lumière la dynamique complexe présentée par les protéines intrinsèquement désordonnées dans des conditions environnementales complexes sur des échelles de temps de la pico à la nanoseconde.

Self organization is crucial for the wealth of living systems, both at the molecular and cellular level. To correctly achieve their role, a tight control over the shape and structure of the protein, for instance, is required. Any mistake can lead to various diseases like Alzheimer, where proteins aggregate into fibrous structures. On a larger scale, cells need to probe the mechanical response of their environment -the extra cellular matrix- and to adapt their own rigidity to collectively orientate. In this thesis, we are looking at two different models. In the first part, we study protein aggregation beyond the microscopical details. By considering elastic assembling particles, we are able to derive generic laws and understand the persistent formation of fibers. In the second part, we model the dynamical response of a fashionable class of hydrogels for their biocompatibility. Material engineers are now able to synthesize materials with more and more subtle behavior, although understanding certain emergent properties -such as non-exponential relaxation- can be a major challenge. We design a simple model for the dynamical response of hydrogels connected by large multivalent crosslinkers. We are able to account for experimental results, and rationalize their origin., Les systèmes biologiques sont entièrement auto-assemblés, à la fois à l’échelle moléculaire et cellulaire. Pour réussir la forme et la structure de chaque protéine par exemple, doit être précisément contrôlée. La moindre erreur conduit à des maladies telles qu’Alzheimer où des protéines s’agrègent entre elles pour former des structures fibreuses. À une échelle plus large les cellules ont besoin de sonder la réponse mécanique de leur environnement -la matrice extra cellulaire- et d’adapter leur propre rigidité pour être capable de s’orienter collectivement. Dans cette thèse nous étudions deux modèles différents. Dans la première partie, nous étudions l’agrégation de protéines au-delà des détails microscopiques. En considérant l’assemblage de particules élastiques, nous sommes capables de comprendre la formation générique de structures fibreuses. Dans la seconde partie, nous modélisons la réponse dynamique d’une catégorie d’hydrogel qui suscite beaucoup d’intérêt en raison de leur compatibilité biologique. Les ingénieurs en matériaux sont aujourd’hui capables de synthétiser des matériaux aux propriétés surprenantes, néanmoins comprendre l’origine de ces propriétés émergentes peut s’avérer compliqué. Nous modélisons la réponse dynamique des hydrogels connectés par de gros agents réticulants multivalent. Ceci nous permet de reproduire les résultats expérimentaux et d’en comprendre l’origine.

X-ray crystallography is the predominant method for investigating the three-dimensional structures of biomacromolecules. X-rays interact with electrons in the crystalline sample with limited visibility of hydrogen atoms at sub-atomic resolution with only one electron. In neutron macromolecular crystallography (NMX) neutrons interact with atomic nuclei, thus opening up the possibility to locate hydrogen atoms even at a medium resolution. These hydrogen position allow to understand the protonation states of amino acid chains involved in enzymatic mechanisms or even help in investigating the orientation of critical water molecules. Practical reasons such as requirement of large crystals (~1 mm3) is the major limitation, which also implies a requirement for large amounts of protein material. This becomes a major issue while working with membrane proteins as they are known for low protein yields in general. Thus, no membrane protein neutron structure has so far been deposited in PDB. Hydrogen atoms also give a significant background due to their large incoherent cross-section. These problems of large crystal volume and high background can be alleviated by isotope labelling the sample with deuterium (D).The most common strategy for isotope labelling is to produce the recombinant protein in microbial hosts such as Escherichia coli fed on deuterated media allowing the protein to be fully deuterated (‘perdeuterated’). However, D2O can be toxic due to its slightly different physicochemical properties and restricts the growth of bacteria. In this work, a methodology is optimized for production of perdeuterated membrane protein using different carbon sources such as deuterated algae, labelled and unlabeled glycerol. Outer membrane protein F (OmpF) was chosen as the model system to conduct this study as it is known for its high stability and well-characterized with a high-resolution X-ray structure deposited in PDB. It forms a water-filled channel across the outer membrane of E. coli serving as a passage of nutrients and as a barrier for toxic agents. An optimum yield sufficient for growing neutron-sized crystals was achieved by this protocol. Deuteration level and thermal stability of the sample was characterized by mass spectrometry and differential scanning fluorimetry. Perdeuterated crystals diffracted X-rays to a resolution of 1.9 Å and deuteration had no significant effect on structural properties of protein. This work lays the foundation for membrane protein neutron diffraction studies in future.In parallel, we also tested this methodology for a cytoplasmic protein, Triose phosphotase isomerase (TIM) and later used the perdeuterated protein to understand the reaction mechanism by neutron crystallography. TIM is a glycolytic enzyme that catalyzes the inter-conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate in glycolysis cycle. In this reaction Glu167 is proposed as the catalytic base in the rate limiting step and deprotonates the substrate to generate an enediolate intermediate. These intermediates are further stabilized by other catalytic residues including Glu97, the role of which in enzymatic mechanism is not yet well characterized. Here, neutron structure of TIM E97Q and X-ray structure of TIM E167Q variant have been determined using an inhibitor 2-phosphoglycolate as a mimic of enediolate intermediate. Glu97 was shown to have a significant role in reaction mechanism to allow easy relaying of protons. The E617Q structure highlighted the importance of the hydrogen bond geometry along with electrostatic behavior for the formation of the enediolate intermediate. Perdeuteration is therefore critical in obtaining good neutron diffraction data that can reveal the structural details that are indispensable for a mechanistic understanding of protein function., La cristallographie aux rayons X est la méthode prédominante pour étudier les structures tridimensionnelles des biomacromolécules. Les rayons X interagissent avec les électrons dans l'échantillon cristallin avec une visibilité limitée des atomes d'hydrogène à une résolution subatomique avec un seul électron. Dans la cristallographie macromoléculaire neutronique (NMX), les neutrons interagissent avec les noyaux atomiques, ouvrant ainsi la possibilité de localiser les atomes d'hydrogène. La taille des cristaux est la principale limite, ce qui implique la nécessité de disposer de grandes quantités de protéines. Cela devient un problème majeur lorsqu'on travaille avec des protéines membranaires, car elles sont connues pour leur faible rendement protéique en général. Ainsi, aucune structure neutronique de protéine membranaire n'a été déposée jusqu'à présent dans la base de données PDB. Ces problèmes de grand volume cristallin et de bruit de fond élevé peuvent être atténués par le marquage isotopique de l'échantillon avec du deutérium (D).La stratégie la plus courante pour le marquage isotopique consiste à produire la protéine recombinante dans des hôtes microbiens tels que Escherichia coli, nourris avec des milieux deutérés permettant à la protéine d'être entièrement deutérée ("perdeutérée"). Dans ce travail, une méthodologie est optimisée pour la production des protéines membranaires perdeutérées en utilisant différentes sources de carbone telles que des algues deutérées, du glycérol marqué et non marqué. La protéine de la membrane externe F (OmpF) a été choisie comme système modèle pour mener cette étude car elle est connue pour sa grande stabilité et bien caractérisée par une structure aux rayons X à haute résolution déposée dans la PDB. Elle forme un canal rempli d'eau à travers la membrane externe d'E.coli servant de passage aux nutriments et de barrière aux agents toxiques. Ce protocole a permis d'obtenir un rendement optimal suffisant pour la croissance des cristaux de taille neutronique. Le niveau de deutération et la stabilité thermique de l'échantillon ont été caractérisés par spectrométrie de masse et fluorimétrie à balayage différentiel. Les cristaux perdeutérés ont diffracté les rayons X avec une résolution de 1,9 Å et la deutération n'a eu aucun effet significatif sur les propriétés structurales de la protéine. Ce travail pose les bases d'études futures de la diffraction neutronique des protéines membranaires.En parallèle, nous avons également testé cette méthodologie pour une protéine cytoplasmique, la Triose phosphotase isomérase (TIM) et avons ensuite utilisé la protéine perdeutérée pour comprendre le mécanisme de réaction par cristallographie neutronique. La TIM est une enzyme glycolytique qui catalyse l'interconversion du dihydroxyacétone phosphate en glycéraldéhyde-3-phosphate dans le cycle de la glycolyse. Dans cette réaction, Glu167 est proposé comme base catalytique dans l'étape limitant la vitesse et déprotonise le substrat pour générer un intermédiaire enediolate. Ces intermédiaires sont ensuite stabilisés par d'autres résidus catalytiques dont Glu97, dont le rôle dans le mécanisme enzymatique n'est pas encore bien caractérisé. Ici, la structure neutronique de TIM E97Q et la structure aux rayons X de la variante TIM E167Q ont été déterminées en utilisant un inhibiteur, le 2-phosphoglycolate, comme mimétique de l'intermédiaire enediolate. Il a été démontré que le Glu97 a un rôle important dans le mécanisme de réaction pour permettre un relais facile des protons. La structure E617Q a mis en évidence l'importance de la géométrie de la liaison hydrogène ainsi que le comportement électrostatique pour la formation de l'intermédiaire enediolate. La perdeutération est donc essentielle pour obtenir des bonnes données de diffraction neutronique qui peuvent révéler les détails structuraux indispensables à la compréhension mécanistique de la fonction des protéines.

International audience; Introduction:The flux-force relationship between free energy dissipated during growth (∆Gmet) and microbial growth rate (µ) is often postulated. Temperature has also a strong influence on growth rate, which exhibits a non-monotonic behavior with a temperature at which the growth rate is maximum. We therefore questioned if changes in microbial growth rate associated to different temperature is sustained by a change in free energy dissipation. For that, we grew aerobically Escherichia coli K12 NCM3722 strain (∆motA), in minimal medium (Modified MOPS culture with 6 amino acids) containing glucose as the sole energy source in an isothermal calorimeter. Indeed, for aerobic metabolism, ∆Gmet mainly results from heat (∆Hmet) and isothermal calorimetry is a useful tool to monitor jointly microbial growth rate and total heat evolution. Materials and methods:Calorimetric experiments were performed with isothermal titration calorimeter connected to a specific software for controlling, acquiring, and processing data. The setup enabled real-time monitoring of sample temperature and heat flow with thermograms. Overnight grown precultures of E. coli were mixed with growth media. The initial quantity of inoculum to be transferred into calorimetric cells was precisely determined to allow a sufficiently long lag phase to allow stable thermal equilibrium before the onset of the growth process. The temperature range of calorimetric experiments varied between 28-44°C with a 1°C step and at least three replicated cultures for each temperature. Thermal profiles were normalized to calculate power vs time curves at each temperature. Heat profiles were integrated to determine heat evolution at each temperature. The exponential parts of heat evolution curves were used to estimate corresponding µmax values. Results and discussion:Evaluation of power curves showed that the µmax vs T curve exhibits the characteristic hump shaped behavior with a temperature optimum of 42°C. The heat exchanged during microbial growth ranged between 420 to 568 mJ with standard deviations from 10 to 23%. Statistical analysis of variance indicated that there was no significant difference between heat measurements at different temperatures. Therefore, our results open the debate on the precise thermodynamic driving force for microbial growth and call for further detailed investigations.

Fluid-filled biological cavities are ubiquitous, but their collective dynamics has remained largely unexplored from a physical perspective. Based on experimental observations in early embryos, we propose a model where a cavity forms through the coarsening of myriad of pressurized micrometric lumens, that interact by ion and fluid exchanges through the intercellular space. Performing extensive numerical simulations, we find that hydraulic fluxes lead to a self-similar coarsening of lumens in time, characterized by a robust dynamic scaling exponent. The collective dynamics is primarily controlled by hydraulic fluxes, which stem from lumen pressures differences and are dampened by water permeation through the membrane. Passive osmotic heterogeneities play, on the contrary, a minor role on cavity formation but active ion pumping can largely modify the coarsening dynamics: it prevents the lumen network from a collective collapse and gives rise to a novel coalescence-dominated regime exhibiting a distinct scaling law. Interestingly, we prove numerically that spatially biasing ion pumping may be sufficient to position the cavity, suggesting a novel mode of symmetry breaking to control tissue patterning. Providing generic testable predictions, our model forms a comprehensive theoretical basis for hydro-osmotic interaction between biological cavities, that shall find wide applications in embryo and tissue morphogenesis., Author summary The formation of a single biological cavity, or lumen, in tissues and embryos has been widely studied experimentally but the collective dynamics of multiple lumens has received much less attention. Here, we focus on a particular type of lumens, which are located at the adhesive side of cells and can therefore interact directly through the intercellular space, as recently observed in the very first stages of embryogenesis. We propose a generic model to describe the hydraulic and osmotic exchanges between lumens themselves, and with the surrounding cellular medium. Lumens are pressurized by a surface tension, which leads naturally to their coarsening into a single final cavity through hydraulic exchanges. With extensive numerical simulations and mean-field theory we predict that such coarsening dynamics follows a robust scaling law, that barely depends on concentration heterogeneities between lumens. On the contrary, active osmotic pumping largely influences the collective dynamics by favoring lumen coalescence and by biasing the position of the final cavity. Our theoretical work highlights the essential role of hydraulic and osmotic flows in morphogenesis.

International audience; Reported transcranial current stimulation (tCS) effects on cerebral activity leave numerous questions open regarding the involved mechanisms of action. A number of studies have identified clear physiological effects, however it remains to be understood how to take into account interindividual variability to tCS: For example, to what extent does individual brain geometry, such as cerebral tissues morphology, impact this prediction? Here, we aimed at evaluating how the geometry of cortical tissue surfaces impacts the charge distribution and therefore the electric fields induced by tCS.

International audience; We describe a highly sensitive and stable quantum-cascade laser-based attenuated total reflection (ATR) terahertz sensor for the detection of very low concentration solutions, using a dual-modulation differential approach and ATR geometry. This sensor offers a very high dynamic range and along-term stability of 40 dB, which extends the potential of terahertz radiation for the analysis of liquid and biological samples. The performance is illustrated by measurements on standard solutions of ions, sugars, and proteins, for concentrations down to 1 M.

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets., Currently, bidirectional control of activity in the same neurons in the same experiment is difficult. Here the authors report a Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES, which they use in a range of organisms including worms, fruit flies, mice and ferrets.

G-quadruplexes play important roles in cellular regulatory functions, but despite significant experimental and theoretical efforts, their folding mechanisms remain poorly understood. In this context, we developed a T-jump experiment to access the thermal denaturation and renaturation dynamics of short intramolecular G-quadruplexes in vitro, on the time scale of a few hundred milliseconds. With this new setup, we compared the thermal denaturation and renaturation kinetics of three antiparallel topologies made of the human telomeric sequences d[(5'-GGG(TTAGGG)3-3']/Na+ and d[5'-AGGG(TTAGGG)3-3']/Na+ and the thrombin-binding aptamer sequence d[5'-GGTTGGTGTGGTTGG-3']/K+, with those of the parallel topology made of the human CEB25 minisatellite d[5'-AAGGGTGGGTGTAAGTGTGGGTGGGT-3']/Na+. In all cases, exponential kinetics of the order of several hundred milliseconds were observed. Measurements performed for different initial temperatures revealed distinct denaturation and renaturation dynamics, ruling out a simple two-state mechanism. The parallel topology, in which all guanines adopt an anti conformation, displays much slower dynamics than antiparallel topologies associated with very low activation barriers. This behavior can be explained by the constrained conformational space due to the presence of the single-base propeller loops that likely hinders the movement of the coiled DNA strand and reduces the contribution of the entropy during the renaturation process at high temperatures.

Membrane proteins are potential targets for drug design due to their vital biological functions. Investigating the 3D structure of proteins is essential to understand their function. Macromolecular X-ray crystallography is the most widely used technique to determine the structure of proteins. However, obtaining high diffraction quality crystals of membrane proteins, whilst maintaining their native conformation during the production, purification and crystallization steps, is challenging. Screening and optimization of crystallization conditions as well as finding optimal methods for growing high-quality crystals with appropriate size distribution is another bottleneck in modern structural studies of membrane proteins. Neutron protein crystallography requires crystals with a volume larger than 0.1 mm3, whereas micro- or nano-sized crystals are sufficient for Microcrystal Electron Diffraction (MicroED).Knowledge of the protein crystallization phase diagram allows the quality of the crystals to be controlled and subsequently optimized. Thanks to these tools, it is possible to adjust crucial physico-chemical parameters such as the temperature or the chemical composition of the crystallization solutions to optimize the crystallization process. Moreover, the automated control of these parameters has been achieved using a recently designed crystallization bench developed in our laboratory. Automated dialysis-based crystallization with variation of the temperature and composition of the crystallization solution by this instrument allows to screen different crystallization conditions using a single sample as well as to optimize the crystal growth in a reversible manner. Therefore, crystallization optimization and phase diagram exploration are possible using a small amount of protein. The crystallization of model soluble and membrane proteins has been demonstrated using this instrument to obtain crystals suitable for downstream structural studies by crystallography.This rational approach to crystallization was applied to the membrane proteins Acriflavine resistance protein B (AcrB) from Escherichia coli and TonB-dependent heme outer membrane transporter (ShuA) from Shigella dysenteriae. The purification of these proteins for crystallization was optimized. The purified proteins were then crystallized using different crystallization techniques, identifying the dialysis method as the most successful and consistent although comparable crystals were obtained with almost all techniques, but not always reproducible. AcrB crystals co-crystallized with rifampicin were used for systematic X-ray diffraction studies and the best statistics for the AcrB-rifampicin crystal from each method were compared. By co-crystallization of ShuA with europium (III) chloride, the 3D structure of this protein was solved at 2.49 Å. The results also showed that the purity of the protein sample and well-chosen additives improve the crystal quality.MicroED is an ideal method for studying membrane proteins and protein complexes that do not readily form crystals large enough for other diffraction techniques. The Coulomb potential maps determined by MicroED also provide information on the electrostatic environment and charge states of amino acids, which can complement the structural information obtained by other techniques. Preliminary studies applying this approach have been performed for our two model membrane proteins co-crystallized with additives.For further characterization of the crystallization process using the dialysis method, the mass transport of detergents across semi-permeable membranes was studied. This information, combined with previously determined knowledge of the diffusion of salts and polymeric precipitants across semi-permeable membranes, can be used to establish effective strategies for optimizing the crystallization of membrane proteins using this technique with automated control., Les protéines membranaires sont des cibles potentielles pour la conception des médicaments en raison de leurs fonctions biologiques vitales. L'étude de la structure 3D des protéines est essentielle pour comprendre leur fonction. La cristallographie macromoléculaire aux rayons X est la technique la plus largement utilisée pour déterminer la structure des protéines. Cependant, obtenir des cristaux de protéines membranaires de haute qualité de diffraction, tout en maintenant leur conformation native pendant les étapes de production, de purification et de cristallisation, est un défi. Le criblage et l'optimisation des conditions de cristallisation ainsi que la recherche de méthodes optimales pour la croissance des cristaux de haute qualité, avec une distribution de taille appropriée, constituent un autre goulot d'étranglement dans les études structurales modernes des protéines membranaires.La connaissance du diagramme de phase de la cristallisation des protéines permet de contrôler la qualité des cristaux, puis de les optimiser. Grâce à ces outils, il est possible d'ajuster des paramètres physico-chimiques cruciaux tels que la température ou la composition chimique des solutions de cristallisation pour optimiser le processus de cristallisation. De plus, le contrôle automatisé de ces paramètres a été réalisé grâce à un banc de cristallisation récemment conçu dans notre laboratoire. La cristallisation automatisée par dialyse avec variation de la température et de la composition de la solution de cristallisation par cet instrument permet de cribler différentes conditions de cristallisation en utilisant un seul échantillon ainsi que d'optimiser la croissance des cristaux de manière réversible. Par conséquent, l'optimisation de la cristallisation et l'exploration du diagramme de phase sont possibles en utilisant une petite quantité de protéine. La cristallisation d'un modèle de protéine soluble et membranaire a été démontrée à l'aide de cet instrument afin d'obtenir des cristaux adaptés aux études structurales avancées par cristallographie.Cette approche rationnelle de la cristallisation a été appliquée aux protéines membranaires, AcrB et ShuA. La purification de ces protéines pour la cristallisation a été optimisée. Les protéines purifiées ont ensuite été cristallisées à l'aide des différentes techniques de cristallisation. La méthode de dialyse a été identifiée comme la plus réussie et la plus cohérente, bien que des cristaux comparables aient été obtenus avec presque toutes les techniques, mais pas toujours reproductibles. Des cristaux d'AcrB co-cristallisés avec de la rifampicine ont été utilisés pour des études systématiques de diffraction des rayons X et les meilleures statistiques pour le cristal d'AcrB-rifampicine de chaque méthode ont été comparées. Par la co-cristallisation de ShuA avec du chlorure d'europium (III), la structure 3D de cette protéine a été résolue à 2,49 Å. Les résultats ont également montré que la pureté de l'échantillon de protéine et des additifs bien choisis améliorent la qualité des cristaux.La MicroED est une méthode idéale pour étudier les protéines membranaires et les complexes protéiques qui ne forment pas facilement des cristaux suffisamment grands pour les autres techniques de diffraction. Des études préliminaires appliquant cette approche ont été réalisées pour nos deux protéines membranaires modèles co-cristallisées avec des additifs.Pour caractériser davantage le processus de cristallisation par la méthode de dialyse, le transport de masse des détergents à travers les membranes semi-perméables a été étudié. Ces informations, combinées aux connaissances précédemment déterminées sur la diffusion des sels et des polymères à travers les membranes semi-perméables, peuvent être utilisées pour établir des stratégies efficaces pour optimiser la cristallisation des protéines membranaires en utilisant cette technique avec un contrôle automatisé.

The present work follows a series of investigations of ours aimed at understanding whether longrange intermolecular electrodynamic forces could play a role in accelerating the encounter of the cognate partners of biochemical reactions in living matter and by which experimental methods and available techniques these forces could be detected. After the experimental detection of collective intramolecular oscillations of protein molecules [that we reported in Phys. Rev.X8, 031061 (2018)] the possibility of observing the mentioned electrodynamic interactions appears more realistic and thus deserving a deeper theoretical investigation of possible experimental detection of these forces with new data at hand.

A microfluidic chip has been developed coupling the advantages of the microfluidic technology and the microdialysis protein crystallization method allowing for accurate, fine-tuned and reversible control over the experimental parameters of the on chip crystallization. Microchips with 0.1 µL and 0.3 µL maximum volume of the protein reservoir have been developed for the on chip crystallization of proteins via microdialysis. Dialysis is based on the diffusion and equilibration of the precipitant molecules through a semi-permeable membrane. A small piece of regenerated cellulose dialysis membrane is embedded within two layers of the chip. The molecular weight cut off of the membrane can be chosen according to the molecular weight of the protein sample and the precipitant solution. Dialysis offers a kinetic trajectory that allows an extensive exploration of the phase diagram and it can be used in combination with temperature control to decouple nucleation from crystal growth, while a multitude of crystallization conditions can be screened with the same protein sample.Two model soluble proteins (Hen Egg White Lysozyme (HEWL) and Thaumatin from Thaumatococcus danielli) and four membrane proteins (AcrB from Escherichia coli, ShuA from Shigella dysenteriae, SERCA from Oryctolagus cuniculus and TmPPase from Thermotoga maritima) were crystallized on chip with the microdialysis method and used for room temperature in situ X-ray diffraction experiments. The background noise generated by the interaction of X-rays with the materials comprising the dialysis chip was evaluated, rendering the chips compatible with in situ X-ray data collection. Detailed electron density maps were produced for HEWL crystals at a resolution higher than 2 Å. Moreover, partial diffraction data sets were collected from numerous, small Thaumatin crystals and HEWL crystals grown on chip with the lipidic cubic phase (LCP) crystallization method, verifying the compatibility of the chip for in situ synchrotron serial X-ray crystallography (SSX). As expected, in the case of membrane protein targets, on chip crystallization was more challenging. However, the crystals of all four membrane proteins tested were produced on the dialysis chips. SERCA crystals diffracted close to 7 Å, which gives the best resolution so far acquired with membrane protein crystals on our chip.A platform (MicroCrys) has been developed to automate the on chip crystallization via microdialysis for investigating phase diagrams through chemical composition and temperature control. The platform contributes to the overall goal of optimizing and rationalizing protein crystallization. All the separate components of the platform are controlled by a custom-built LabVIEW software displaying a user-friendly graphical interface. Moreover, a commercial fluidic system (Elveflow) for mixing and circulating the crystallization solution has been incorporated in the platform. This fluidic system contributes to the automation of the on chip crystallization via microdialysis allowing the exchange of crystallization solutions in the fluidic channel of the chip. A qualitative study of the HEWL phase diagram was performed by nucleating, growing, dissolving and re-nucleating HEWL crystals on chip in different precipitant concentrations using MicroCrys and the pressure-driven fluidic system. Finally, a prototype for thermal regulation using the Peltier effect has been developed to couple the on chip microdialysis with temperature control. A LabVIEW program has been written for real-time acquisition, displaying and recording of the temperature value at the protein reservoir of the chip. Once, the thermal regulation system and the pressure-driven fluidic system will be integrated into the main software of the platform, MicroCrys will be a fully-automated, user-friendly platform for on chip crystallization experiments., Une puce microfluidique a été développée, combinant les avantages de la technologie microfluidique et de la méthode de cristallisation par microdialyse, permettant un contrôle précis et réversible des paramètres expérimentaux de la cristallisation sur puce. Des micropuces avec un volume maximal de réservoir de protéines de 0.1 µL et 0.3 µL ont été développées. Un petit morceau de membrane de dialyse en cellulose régénérée est incorporé entre deux couches de la puce. La coupure du poids moléculaire de la membrane peut être choisie en fonction du poids moléculaire de l'échantillon de protéine et de la solution de précipitant. La dialyse offre une trajectoire cinétique qui permet une exploration étendue du diagramme de phase et elle peut être utilisée en combinaison avec le contrôle de la température pour découpler la nucléation de la croissance cristalline, tandis qu'une multitude de conditions de cristallisation peut être criblée avec le même échantillon de protéine.Deux protéines solubles modèles (le lysozyme du blanc d'œuf de poule (HEWL) et la thaumatine de Thaumatococcus danielli) et quatre protéines membranaires (AcrB d'Escherichia coli, ShuA de Shigella dysenteriae, SERCA d'Oryctolagus cuniculus et TmPPase de Thermotoga maritima) ont été cristallisées sur puce et utilisées pour des expériences de diffraction in situ des rayons X à température ambiante. Le bruit de fond généré par l'interaction des rayons X avec les matériaux composant la puce a été évalué, rendant les puces compatibles avec la collecte des données de diffraction in situ. Des cartes de densité électronique détaillées ont été produites pour les cristaux de HEWL à une résolution supérieure à 2 Å. De plus, des ensembles de données de diffraction partiels ont été collectés à partir de nombreux petits cristaux de thaumatine et de cristaux de HEWL cultivés sur la puce avec la méthode de cristallisation en phase cubique lipidique (LCP), vérifiant la compatibilité de la puce pour la cristallographie sérielle aux rayons X (SSX) in situ au synchrotron. Comme attendu, dans le cas des protéines membranaires, la cristallisation sur puce était plus difficile. Cependant, les cristaux de toutes les quatre protéines membranaires testées ont été produits sur les puces. Les cristaux de SERCA ont diffracté avec une résolution proche de 7 Å, ce qui donne pour l’instant la meilleure résolution acquise sur notre puce avec des cristaux de protéines membranaires.Une plateforme (MicroCrys) a été développée pour automatiser la cristallisation sur puce par microdialyse afin d'étudier les diagrammes de phase par le biais de la composition chimique et du contrôle de la température. La plate-forme contribue à l'objectif global d'optimisation et de rationalisation de la cristallisation des protéines. Tous les composants séparés de la plateforme sont contrôlés par un logiciel LabVIEW personnalisé. De plus, un système fluidique commercial (Elveflow), pour mélanger et faire circuler la solution de cristallisation, a été incorporé dans la plateforme. Ce système fluidique permet l'échange des solutions de cristallisation dans le canal fluidique de la puce. Une étude qualitative du diagramme de phase de HEWL a été réalisée en faisant germer, croître, dissoudre et ré-germer des cristaux de HEWL sur la puce dans de différentes concentrations de précipitant. Enfin, un prototype de régulation thermique utilisant l'effet Peltier a été développé. Un programme LabVIEW a été écrit pour l'acquisition et l'affichage en temps réel de la valeur de la température au niveau du réservoir de protéine de la puce. Une fois que le système de régulation thermique et le système fluidique sous pression seront intégrés dans le logiciel principal de la plateforme, MicroCrys sera une plateforme entièrement automatisée et conviviale pour les expériences de cristallisation sur puce.

Animal cells are active, contractile objects. While bioassays address the molecular characterization of cell contractility, the mechanical characterization of the active forces in cells remains challenging. Here by confronting theoretical analysis and experiments, we calculated both the resistive and the active components of the intracellular stresses that build up following cell adhesion. We obtained a linear relationship between the divergence of the resistive stress and the traction forces, which we show is the consequence of the cell adhering and applying forces on the surface only through very localized adhesion points (whose size is inferior to our best resolution, of 400 nm). This entails that there is no measurable forces outside of these active point sources, and also that the resistive and active stresses inside cells are proportional., 5 pages, 4 figures

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.