Microbial fuel cells (MFC) can transform directly into electrical energy the chemical energy contained in various organic compounds. A MFC uses microorganisms, which adhere spontaneously on the surface of the anode and form a biofilm that oxidizes organic compounds by directly transferring the electrons to the electrode. With this new type of electrocatalysis, discovered in the early 2000s, MFCs produce electrical energy by oxidizing various organic compounds (acetate, volatile fatty acids, alcohols, (poly)saccharides ...) contained in natural environments or can be obtained from biomass. The majority of the MFCs consists of a microbial bioanode associated with an abiotic air cathode. This configuration produces electricity by oxidizing generally acetate and using the reduction of oxygen at the cathode. The performance of these MFCs grew rapidly in the beginning, but has leveled off at a few Watt per square meter of electrode surface area from 2008. Unfortunately, no serious tracks are now available for the engineer to exceed this threshold around a few W/m2. However, some groups, including partners gathered in the previous AgriElec (ANR-08-BIOE 001) project, have developed microbial bioanodes that produced current densities beyond 50 A/m2. For comparison, the current density provided by photovoltaic panels is of the order of 100 to 200 A/m2. The partners have also produced abiotic air cathodes and microbial biocathodes at the best level of the state of the art, but they have only managed to increase the power density limit of a few W/m2. The experience gained during the previous project brings several conclusions: bioanodes and biotic or abiotic cathodes that are designed separately in optimal conditions do not work in harmony when they met in a MFC module; engineering-oriented analyses of MFCs are rare and generally focus one or the other elements of the stack but do not embrace the overrall process; finally, cathodes represent a major bottleneck, the abiotic air cathodes have limited performance, the microbial biocathodes look promising but their potential has been little investigated. The Bioelec project will launch an engineering approach "in the right direction." The overall MFC process will first be analyzed in terms of thermodynamic, mass transfer and electrochemical kinetics to extract the optimal operating conditions for each element. These conditions extracted from the analysis of the whole process, will then be imposed on the design of the electrodes. A first prototype will be built with an abiotic air cathode associated with a separator (or membrane) and a bioanode. This separator-electrode assembly will ensure a power of 1 Watt. A second prototype will aim to overcome the cathode bottleneck by developing an air microbial biocathode. Here, the objective is to link the concepts of gas diffusion cathode and microbial biocathode to overcome the low solubility of oxygen in solution. This is an exploratory, more ambitious goal. The consortium is composed by five partners: an industrial company that manufactures hydrogen-air fuel cells, three laboratories that provide expertise in engineering, microbiology and physical-chemistry of surfaces and a company devoted to technology transfer.

Personalized medicine is based on medical biology, whose purpose is to define the metabolic profile of patients to adapt their treatment and minimize secondary effects. These metabolic profiles consist of an ensemble of biomarkers, which allow the early diagnosis of diseases like cancer and establish vital prognoses. This proposal aims to develop a biosensor platform based on new hybrid nanopores and electrical detection at the single-molecule level to identify and quantify peptide biomarkers involved in dangerous human diseases like cancer and coagulopathies. By using an engineered protein nanopore inserted into a solid-state nanopore, this project proposes to detect specifically peptides differing by a single amino acid or post-translational modification from patients’ serum at very low concentrations. The channel will be engineered to increase the peptide detection specificity. The hybrid nanopore platform will ensure stable measurements for a longer time than the traditional suspended bilayer system, increasing the detection sensitivity limit.

Heparan sulfate (HS) are complex polysaccharides abundantly found in extracellular matrices and cell surfaces. These polysaccharides participate to major cellular processes through their ability to bind and modulate a wide array of signalling proteins. HS/ligands interactions occur through saccharide domains (termed S-domains) of specific sulfation pattern, present within the polysaccharide. Assembly of such functional domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes, including extracellular sulfatases of the Sulf family. Sulfs specifically target HS S-domains and catalyze the selective removal of 6-O-sulfate groups, which are required for the recognition of many proteins. Although structurally subtle, these modifications have great functional consequences, and Sulfs have emerged as critical regulators of HS activity, in physiological processes such as embryogenesis and tissue regeneration, and in diseases such as cancer. There are two identified isoforms of Sulfs, Sulf-1 and Sulf-2, which share a very similar molecular organization. They are composed of two regions that are essential for enzyme activity: the catalytic domain (CAT-D), which includes the enzyme active site and is well conserved amongst sulfatases, and a highly basic, hydrophilic domain (Hyd-D), which is responsible for recognition and binding to HS substrates and is a unique feature of the Sulfs. However, despite increasing interest, Sulfs still remain poorly understood. During our recent studies of these enzymes, we have shed light on an original processive desulfation mechanism and on remarkable structural features. Based on these data, the SULF@AS project proposes to deliver an integrated study of the human isoforms HSulf-1 and HSulf-2, combining biochemical and biophysical approaches to characterize their structure and post-translational modifications ; in vitro, in cellulo and in vivo functional analysis to determine their substrate specificities and respective role during tumour progression ; and the development of specific inhibitors based on HS mimetics. This project should provide major insights into the regulatory role played by these enzymes in many biological processes and deliver the structural basis for the development of therapeutic strategies targeting HSulfs.

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

The HomeoGAG project aims to improve our understanding of brain plasticity and provide therapeutic strategies through the design and synthesis of innovative active molecules based on peptide mimics of glycosaminoglycans (GAGs). These GAG mimics will be capable of modulating cerebral cortex critical periods (CPs). CPs are windows of heightened brain plasticity that open during postnatal development and are closed in part by the formation of extracellular matrix perineuronal nets (PNNs) composed of GAGs. Mistiming in CPs has been attributed to brain disorders and inducing ectopic plasticity holds the promise of brain repair. The homeoprotein Otx2 is a transcription factor that binds to PNNs and plays a key role in regulating CPs by acting as a switch on both the opening and closing of plasticity windows. In the adult brain, Otx2 actively dampens brain plasticity. One of our aims is to control this Otx2 switch through the design of GAG peptide mimics capable of blocking Otx2-PNN interactions. Natural GAGs are very complex and structurally heterogeneous polysaccharides that are impractical to purify and very time consuming to prepare chemically. Our GAG mimics will provide access to structurally defined molecules that are easily modifiable through automated peptide synthesis. Preliminary results with pioneer GAG mimics show significant Otx2 binding and in vivo activity, thus providing strong proof-of-concept and a solid argument for the feasibility of our novel approach. We will build several libraries of GAG mimics of varying size, sequence and structural organization. Their affinity and specificity to Otx2 will first be evaluated in vitro by screening methods that couple online thermodynamic analysis of interactions (capillary electrophoresis of affinity and surface plasmon resonance) to structural analysis by mass spectrometry. Validated GAG mimics will then be tested on mouse model to determine their ability to modify postnatal CPs and to induced plasticity in adult mice for therapeutic purposes. Another aim is to use GAG mimic libraries designed not only for Otx2 but also other GAG-dependent signaling proteins; subsequent in vivo experiments will impact our understanding of CP mechanisms. A final aim will be to develop non-invasive approaches to disrupt Otx2 in the choroid plexus and induce adult cortical plasticity. This project has strong pharmacological potential aimed in particular at restoring the plasticity of the visual cortex, and it will have a significant impact on research in neurobiology of development and in molecular neuroscience.