Our proposal integrates the scientific method with 21st century automation technology, with the goal of making scientific discovery more efficient (cheaper, faster, better). A Robot Scientist is a physically implemented laboratory automation system that exploits techniques from the field of artificial intelligence to execute cycles of scientific experimentation. Our vision is that within 10 years many scientific discoveries will be made by teams of human and robot scientists, and that such collaborations between human and robot scientists will produce scientific knowledge more efficiently than either could alone. In this way the productivity of science will be increased, leading to societal benefits: better food security, better medicines, etc. The Physics Nobel Laureate Frank Wilczek has predicted that the best scientist in one hundred years time will be a machine. The proposed project aims to take that prediction several steps closer. We will develop the AdaLab (an Adaptive Automated Scientific Laboratory) framework for semi-automated and automated knowledge discovery by teams of human and robot scientists. This framework will integrate and advance a number of ICT methodologies: knowledge representation, ontology engineering, semantic technologies, machine learning, bioinformatics, and automated experimentation (robot scientists). We will evaluate the AdaLab framework on an important real-world application in cell biology with biomedical relevance to cancer and ageing. The core of AdaLab will be generic. The expected project outputs include: - An AdaLab demonstrated to be greater than 20% more efficient at discovering scientific knowledge (within a limited scientific domain) than human scientists alone. - A novel ontology for modelling uncertain knowledge that supports all aspects of the proposed AdaLab framework. - The first ever communication mechanism between human and robot scientists that standardises modes of communication, information exchange protocols, and the content of typical messages. - New machine learning methods for the generation and efficient testing of complex scientific hypotheses that are twice as efficient at selecting experiments as the best current methods. - A significant advance in the state-of-the-art in automating scientific discovery that demonstrates its scalability to problems an order of magnitude more complex than currently possible. - Novel biomedical knowledge about cell biology relevant to cancer and ageing. - A strengthened interdisciplinary research community that crosses the boundaries between multiple ICT disciplines, laboratory automation, and biology.

In agriculture, abiotic stresses are major causes of yield loss. In the context of global warming, it is predicted that heat stresses will decrease crop yields by 3 to 7.4% for each degree Celsius increase. One of the urgent challenges for the seed industry is to improve the resilience of crop species to heat stress. Farmers are waiting for resilient varieties to guarantee the level of production in quantity and quality under a wider range of temperatures. Tomato is one of the main agricultural products of the European Union. Cultivated throughout Europe in the open field, under cover and in greenhouses, it is a model crop from a biological and agronomic point of view. Abiotic stresses are major problems for tomato producers and solutions are needed to stabilize yields. Despite its ability to grow in variable climates, tomato production is highly impacted by heat stress, among other abiotic stresses. In particular, heat stress affects the reproductive system, root development and seedling growth. Heat shock proteins represent a widely conserved class of proteins involved in stress response and plant growth and development. Although they were first discovered in the context of the heat shock response, most biotic and abiotic stress responses require the concerted action of heat shock proteins to regulate stress response and acclimation. Elucidation of the molecular mechanisms responsible for the regulation of heat shock proteins is essential to improve the tolerance of crop plants to biotic and abiotic stresses. The FloCad team and Gautier Semences have conducted several studies that have identified regulators of heat shock protein expression as targets for improving crops under abiotic stresses. The association of the two entities proposes the creation of the joint laboratory BioAdapt which aims to use these regulatory genes as breeding targets to improve the resilience of tomato to heat stress.

1-Scientific background and objectives : Control and understanding of the ubiquitous functions of lipid membranes forming domains is among the more fundamental and practical challenge in membrane biophysics. Assemblies and lateral heterogeneities provide remarkable functions, including the regulation of channels and permeability (e.g. pore formation by antimicrobial peptides pipes are developed in pharmaceutics). Recent investigations of amphiphilic copolymers binding onto lipid vesicles show similar control of membrane properties (domains&well-define pores) by much simpler macromolecules devoid of even a secondary structure. Of practical importance, similar macromolecules have been patented for drug delivery and release of liposomes content in cytosol after cellular uptake. Little is known to date on the physics of their anchoring and effect on membranes, but their hydrophobicity appears a critical parameter. We recently showed that such polymers impart bilayers with the ability to transiently form nano-channels with no disruption of membrane. We obtained similarly the permeabilisation of plasmamembranes of mammalian cells (HEK, COS). The milder impact of polymers as compared to detergents, absence of membrane solubilisation, and the remarkable responsiveness of amphipilic polymers open a route toward the development of new tools for handling membranes and cells, and would significantly help investigation on non-specific stabilisation of channels, provided that the properties of polymer can be controlled. We propose to develop macromolecules, whose hydrophobicity has been made light-sensitive. We seek at triggered permeabilisation of lipid membranes for applications to cell biology and liposome-carriers (e.g. ophthalmology). Reversible adjustment and rapid switch of the properties of membranes with exposure to light, would provide an unprecedented degree of control that bring a novel tool to characterise the dynamics and stability of the nano-channels formed by anchored macromolecules. 2-Description of the project, methodology : Amphiphilic polymers known to affect membranes and cells will be modified by grafting photochrome groups (e.g. hydrophobic azobenzene) to obtain light-responsive macromolecules. The modification procedure was developed by the coordinator and is published. The project will address in this context the following points: (i) Obtaining high magnitude of membrane response to light-exposure, both in liposomes and planar bilayers ; and characterizing the efficient structures. (ii) Permeabilising mammalian cells, with the aim of minimal perturbation of intra-cytosol functions. Using giant vesicles (GUV), thethered bilayers and black films, we would consider the physics of pore opening&closure (conductivity isolated pores, lipid translocation), and physical-chemistry of anchoring and permeability (dynamics and surface density of polymers by fluorescence correlation, kinetics of leakage of isolated GUV). Our approach gather together chemists (polymer synthesis and adjustment of structure), physicists (study of single pore dynamics), physical-chemists (colloids and liposomes), and biologists (permeabilisation of cells and preservation of cytosolic functions, proof of concept using the example of G-protein coupled receptors). Stimulation that is both specific and sharply focused would made possible totally new investigation on pore formation, including their dynamics, by the use of light beams to trigger the onset of phenomena, and eventually cycles. Polymer tools for mild and targeted (light-triggered) 'poration' of cells, and liposomes delivery of drugs could readily be made available at large scale for interested users. 3-Expected results : (i) Rapidly switch the release of internal compartment of lipid vesicles upon exposure to UV-visible light (possible development of liposomes for drug release in ophthalmology) (ii) Reversal of permeability to the impermeable state of the bilayer by exposure to another wavelength (reversibility of light-triggered hydrophobicity is achieved) (iii) Taking advantage of the degree of control so achieved to study pore opening/closure on black lipid membranes and GUVs. The investigation would benefit of the unprecedented possibility to swing (even cycles) specifically the properties of polymer, in absence of additives and with no other perturbation of lipids. (iv) Permeation of mammalian cells with substrates introduced in their cytosol at controlled time (and eventually targeting a few cells in a tissue), to follow the expression of functions in the internal leaflet (here G-protein coupled receptors). Proof of concept include toxicity assays and low perturbation of membrane proteins in presence of polymer, at condition of weak or no permeabilisation (UV exposure).

Numerous chemical or biological processes involve the transport of macromolecules through tiny channels of nanometric size. We have been the firsts in France to study these processes (as early as 2003) using natural channels and artificial channels obtained by drilling nanopores in ultra-thin SiC and Si3N4 solid-state membranes with a Focused Ions Beam Apparatus (FIB). The molecules passing through a pore are detected by a simple electrical method. We would like to pursue this research by developing their different aspects : fabrication, detection and applications. We first propose to drill nanopores in single sheets of graphene by using an optimized system of focused Gallium or Helium ions beam, and then to study its use as an ultra-fast DNA and proteins sequencing tool. This domain, which we explore since two years is growing explosively. For what concerns detection, we wish to develop the optical and mechanical detection of the translocation of a macromolecule through a nanopore. The optical detection requires the use of fluorescent or luminescent macromolecules. Spurious light created while illuminating a pore is eliminated by absorting it or by hindering its propagation (condition of zero mode waveguide). This is obtained by coating the surface of the pore and of the silicon nitride membrane by silicon or gold.The mechanical detection of the forces exerted on a macromolecule confined in a nanopore is obtained when the molecule is attached to the tip of an atomic force microscope or to a bead trapped in optical tweezers. We propose to measure the work exerted on a translocating (out of equilibrium) macromolecule and to use the recent Jarzynski’s relation for studying the energetic lanscape explored by the molecule. Our experience in drilling nanopores by focused ions beam enables us to make nanopores in various materials, controlling their size, their position, their organization We are also able to produce a large amount of nanopore which may serve the needs of research laboratories and future applications. We have constructed our project in order to propose valuable applications of nanopores in the fields of Biology and Biotechnoly, avoiding the well-know application to DNA sequencing, which is outside our scope. We have made a association with a small spin-off company created by a partner laboratory of this consortium in order to study the production of DNA vectors for gene tranfer and gene therapy by molecular extrusion through a nanopore. An electric field or a pressure force the passage of a DNA plasmid through a Silicon Nitride Nanopore and put the molecule in contact with a solution of cationic polyelectrolyte at the exit of the pore. An electrostatic complex is formed with a controlled size and composition, with a single DNA molecule per nanoparticle. We propose to use the same principle of molecular extrusion for studying the synthesis of polymers through a nanopore coated with a suitable catalyst and for controlling the folding and unfolding of proteins buy nanopores. We will use a new experimental prtein model, the Luciferase protein, which allows an optical detection of its transport and functionnal foldind after translocation through a nanopore. We thus hope to create new biomimetic objects enabling the analysis and manipulation of macromolecules with a never achieved spatial and temporal resolution