Large assemblies of living or synthetic self-propelled particles make up Active Matter. They operate far from equilibrium without necessarily leading to macroscopic currents, hence a superficial resemblance to their equilibrium counterparts. Our project focuses on the theoretical challenges posed by the emergent local and global order observed in such systems. It builds on the counterintuitive idea, supported by encouraging attempts, that thermodynamics-based ideas will help rationalize and predict the wealth of phase behaviors observed in active systems. Our threefold approach is based on exploring statistical concepts like entropy (without its thermal meaning, understood as a means of counting states), exploring mechanical or chemical concepts like pressure, chemical potential, surface tension (without their free energy interpretation), and connecting local structure to effective interactions, by means of energetics or dynamic approaches (without invoking the Boltzmann measure).

Over the last decade, Extracellular Vesicles (EVs), including Exosomes, emerged as an important vector of intercellular communication. EVs have been proposed to transfer cargoes such as lipids, nucleic acids and proteins from donor to acceptor tissues or cells. EVs have been associated with several physiological functions and diseases. Today, EV biology is an intense and high impact research topic, which promises to revolutionize translational research through the development of EV-mimetics designed for targeted delivery of therapeutics. Tremendous progresses have been made to better understand the mechanisms that regulate EV secretion. However, our knowledge of EV uptake and content delivery within the acceptor cells is still very limited. In this project, we propose a combination of cell biology experiments coupled with in vitro studies and structural replicate electron microscopy to study how EVs fuse with their target membranes. In a first aim, we will capitalize on our previously published cell-based assays and imaging methods (optical-and electron-microscopy) to 1) further characterize EV uptake and content delivery 2) identify new proteins involved in the fusion process through a candidate approach, 3) determine basic parameters such as cargo size/ type that condition EV delivery through fusion. We will test several proteins candidates that are suspected to control EV uptake and delivery. We will focus our work on a family of proteins name hEnvs, derived from ancestral viral envelop proteins that have been integrated in human genome, and also IFITM1 and 3 proteins. We suspect that hEnv might be involved in pH-dependent EV delivery whereas IFITM1 and 3 would inhibit EV-content delivery. The major actors of endocytosis pathways (clathrin, dynamin, caveolin) will also be tested. Our bulk assays will be complemented with morphological analysis (fluorescent microcopy and classical electron microscopy), to analyze the distribution of EV markers on cells In a second aim, we plan to develop novel fluorescent-based assays to image EV membrane fusion in real time using fluorescent microscopy and correlative EM to capture all the intermediates. Our main goal is to formally establish that membrane fusion is the mechanism responsible for EV content delivery. This mechanism has never been proved so far. Our work will provide an important breakthrough in understanding and proving this delivery content. We will adapt a cell-free assay that used acceptor Plasma Membrane (PM) sheets in suspension coupled with single particle tracking diffusion. Using fluorescent microscopy, we will follow in real-time and on single EV the diffusion of EV, the docking and the fusion reactions. In addition, the same in vitro assay will be used to directly visualize the fusion process at the ultrastructural level. We propose to image the samples through platinum-replica electron microscopy (PREM). EVs will be loaded on the top of the deposited PM sheets and fixed. Initially we expect to dissect the fusion reaction and capture all the fusion-intermediates (including the fusion pore itself) by rapidly fixing the samples at different time points under different conditions Note that all candidates mentioned in Aim1 can be quickly tested within this cell-free imaging assay to directly demonstrate if cell phenotypes observed in Aim 1 correspond to perturbation of the fusion reaction. In other term we will establish causality and not just correlation.

Epithelium folding transforms simple tissue sheets in complex three-dimensional structures and underlies processes such as gastrulation, neurulation and eye formation. To advance the understanding of the mechanisms of tissue folding, our project aims to investigate (i) the link between homeotic gene domains and tissue folding, (ii) the concerted action of the apical and basal actomyosin cell networks in force production and cell shape changes; and (iii) how collective cell migration promotes folding of tissues. Using the consortium expertise in biochemical and mechanical regulations of tissue morphogenesis in Drosophila epithelia tissues, these aims will be achieved by combining advanced light microscopy approaches, genetics, optogenetics and biophysical modeling. By focusing on both genetics and mechanical regulations controlling tissue folding from the subcellular scale to animal scale, our work should shed light on the conserved mechanisms sculpting epithelial sheets into complex shapes.

Understanding sediment transport in rivers, lakes and along the ocean floor is key to sustainable management of open water bodies and aquatic ecosystems. Prominent processes are river morphodynamics, turbidity currents, and tsunamis running up a beach. Predicting and managing these processes requires in-depth knowledge of the rheology to describe macroscopic properties of the fluid-sediment mixture. However, the constitutive laws to describe these processes have so far mostly been based on studies of dense suspensions of neutrally buoyant particles in either highly viscous shearing flows or at much larger flow rates where inertial effects play the dominant role. The transition between the two regimes, however, has not been investigated in a systematic manner yet, and, hence, remains only poorly understood. This may be problematic for the predictive modeling of situations that are more relevant for engineering practices and natural flows involving sediment transport. This transitional regime will be the focus of the present study and our objectives are twofold: First, the French and German partners aim to conduct a joint complementary campaign of state-of-the art sediment transport experiments and numerical simulations, respectively. The campaign will yield highly-resolved data of laminar pressure-driven shearing flows across an idealized sediment bed for a wide range of Stokes numbers as the ratio of competing inertial and viscous effects. In a second step, these data will be used to improve existing two-phase modeling approaches that have become popular for macroscopic sediment transport models.

The release of metal nanoparticles (NPs) in ecosystems is constantly growing. NPs are massively manufactured for their useful properties such as UV-filter, anti-bacterial agents, remediation agent, fertilizers, or pesticides. Sampling from lakes, rivers, seas and even tap water indicated the presence of NPs, as well as in soils and in the air of densely urbanized and industrial areas. Whether animals living in these ecosystems are contaminated by NPs remains unknown.With iron oxide (Fe2O3) NPs, titanium dioxide (TiO2)-NPs is one of the most manufactured nanomaterial. Very emergent literature suggests an actual contamination in humans by TiO2-NPs in placental tissue, newborn stools, blood and colon tumor tissues. Whether animals, especially those raised for human consumption may be contaminated is unknown. Scarce studies in lactating rodents exposed to some NPs suggested the passage of NPs in milk and in offsprings whose development and survival were affected. In this context, NANOMILK proposes to evaluate the existence of an actual contamination by NPs in milk (WP1). Using combination of cutting-edge biophysical approaches, we will analyse the presence of NPs in milk from animals raised in urban farms with a known metal pollution in soils and vegetables. This will be compared to milk from animals raised in unpolluted arctic farms in Norway. To understand the mechanisms by which NPs may be released in milk, we will run a comprehensive analysis to characterize the molecular mechanisms underlying the secretion and intercellular transfer of NPs from mammary cells (WP2). To that end, we will investigate NP secretion in extracellular vesicles (EVs) which are an ensemble of membrane-limited carriers playing key roles in cell-to-cell communication and in physio-pathological processes such as immune response or cancer progression. Very recently, EVs have been detected in milk (milk-EVs). Secreted by mammary cells, they may transport information to offspring and influence their immunity and development. Whether milk-EVs secreted by mammary cells may also transport NPs is unknown but partners of NANOMILK have previously shown that Fe2O3-NPs and TiO2-NPs were transferred between non-mammary cells in a process involving EVs. Thorough analysis of the metal and protein content of EVs deriving from NPs-exposed mammary cells will be combined to transfer assays, indirect or direct in co-culture, coupled to silencing of genes involved in cell-cell communication. Furthermore the impact of NPs transferred from mammary cells to recipient cells will be evaluated on genome-wide expression profiles by transcriptomics in recipient cells. Finally, we propose to investigate in vivo the route of NPs within the entire mother-to-offpsring continuum (WP3). After a dose escalation study, we will analyse the behavior of NPs administrated by drinking water in lactating female rabbits. Biodistribution of NPs will be analysed in mother and offspring tissues and in milk-EVs collected at different lactation time. In addition to monitoring the effect of NPs on offspring growth and survival, we will also analyse their effect on milk-EV proteome to uncover potential effect on EVs biogenesis, origin and abundance. From on-field, to in vitro and to in vivo, NANOMILK investigates the spreading of contaminant NPs from mother-to-progeny. NANOMILK may reveal an actual contamination in milk samples from urban farms, influencing this agricultural trend of producing locally when location is polluted. Expected results will generate knowledge on mother-to-offspring communication by milk, and how this can be highjacked by pollutants. NANOMILK may provide new grounds for the use of NPs, for dairy industry, for nanoagriculture and breastfeeding in polluted areas.