Thanks to the development of native chemical ligation (NCL) in early 90's by Kent and co-workers, chemical protein synthesis has shifted into the realms of the achievable. The basis of this methodology is the chemo- and regioselective reaction of a peptide thioester and a N-terminal cysteinyl peptide, and produces a 'natural' peptide bond. This allowed the production of site-specific modified proteins (glycoproteins, phosphorylation) as well as cytotoxic or hydrophobic proteins previously unattainable by molecular biology. Contrasting with the major scientific breakthrough, the applicability of NCL is restricted to proteins containing a cysteine residue which participates in the reaction as nucleophilic reaction partner in inducing a preliminary dynamic thioester exchange step : this dramatically reduces the usefulness of this reaction to a handful of proteins, as cysteine residues are seldom conveniently distributed throughout a peptide sequence so as to facilitate NCL. To remove this restriction, a lot of strategies are currently being actively explored by a few world-leading groups, mostly based in the USA but also in UK and Germany (French teams being almost virtually absent from this emerging discipline). The goal of my project resides in overtaking one the main drawback of classic NCL, the first step based on a slow, reversible, thioester exchange. To explore innovative native ligation procedures where the initial reaction that link the two unprotected peptide fragment would be a fast and irreversible process, two dramatically different concepts will be investigated: the triazole-capture ligation (TCL) and a more universal 'DMAP-handle Native chemical Ligation' (DMAP-NL). If the former implies the exceptional chemo- and regioselectivities of the recently-discovered Copper(I)-catalyzed Alkyne-Azide Cycloaddition to assist the native ligation process, the latter is based on a totally new concept of DMAP-organocatalyzed native ligation. The breakthrough provided by the exploration of the TCL and DMAP-NL will be extended to the multi-ligation strategies which are required for the synthesis of proteins bigger than 60 to 100 amino acids. The final biological application of this chemical project concerns the production of MccJ25, a new anti-microbial peptide whose production is extremely challenging due to its original [1]rotaxane peptidic topology. In a long-term course, this project will output on the synthesis of modified microcins J25 lasso-peptides, crucial chemical tools to clarify the molecular bases of the biochemical pathway to this naturally-occurring, mechanically-interlocked peptide.

Symbiotic associations between species are of major importance as drivers of ecological function and evolutionary processes. The symbiotic association of termites with their intestinal microorganisms provides interesting symbiotic systems from both functional and evolutionary point of views. Contrasting with many symbioses in insects, each termite individual harbors a complex symbiotic community composed by an astonishing diversity of microorganisms including eukaryotes (flagellated protozoa) and prokaryotes (bacteria and archea). Wood-feeding termites depend on their microorganisms to degrade lignocelluloses, and the symbionts cannot live outside their hosts. Although functional aspects of nutritional symbioses in termites have made progresses these last years, there has been relatively little work done on the evolution of these symbiotic systems. The main goal of this project is to initiate a multi-disciplinary research program focusing on the evolutionary dynamics of symbiotic communities in field populations of the European wood-feeding pest termites of the genus Reticulitermes (Rhinotermitidae). Symbiotic associations in termites seem to be highly unstable and generally do not persist for long periods of co-evolution. This is mainly the case of flagellate protozoa living in termite hindgut. The number and identity of flagellate species vary significantly between termite families as well as between and within genus of the same family. Even closely related termite species often harbor different flagellate species. Our principal research interest is to understand how and why flagellate communities of the hindgut of termites vary among host species. Our central hypothesis is that species composition of flagellate communities is in perpetual dynamics within termite species, and that these dynamics result from the occurrence of two events: (1) flagellate species can be occasionally lost by termite species, and (2) new flagellate species can be occasionally gained by host species. The objectives of our project are to infer the occurrence and the mechanisms of flagellate loss and gain events in termite species, and to study the consequences of such events on the diversity and structure of the entire microbial community. Using our well-developed termite model system, our research investigations will combine both empirical and experimental studies. More precisely, we will conduct five specific aims. 1. Using microscopy and molecular methods, we aim to infer the co-evolutionary history between the Reticulitermes species and their flagellates. The obtained data will inform us on the diversity, prevalence, host specificity and evolutionary transmission (vertical versus horizontal) of the flagellates within the Reticulitermes lineage. 2. Similarly, we aim to infer the co-evolutionary history between the flagellates and their ecto- and endo-symbiotic prokaryotes. Using Fluorescent In Situ Hybridization, we will assign the physical association and the degree of specificity between flagellates and the symbiotic prokaryotes. Molecular results will inform us of the diversity, prevalence, host specificity and evolutionary transmission of the symbiotic prokaryotes associated with flagellates. 3. We aim to infer the evolutionary dynamics of flagellates within host species. Using an original method for screening flagellate species (diagnostic PCR-RFLP), we will determine the colony-level variation of the flagellate composition within termite populations, and test the effect of several factors on this variation. Comparison between the population genetic structures of termites and the variation of the flagellate composition will inform us on the transmission modes of flagellates within host populations. 4. As a potential mechanism of flagellate loss by termite lineage, we aim to measure the efficiency of vertical transmission of flagellates from one colony generation to another. Using swarming reproductives collected in field populations, our experiments will consist of founding colonies under laboratory conditions, determine the flagellate composition of their offspring and compare it to the flagellate composition of the parental colonies. 5. As a potential mechanism of flagellate gain by termite lineage, we aim to test whether flagellates can be transferred from one termite species to another. In particular, we will perform experiments that will demonstrate whether termite individuals that kill and consume individuals of another termite species gain new flagellate species. The expected results of this project will improve our fundamental knowledge on the diversity, structure, function and evolution of the microbial communities of termites, and can also improve our methods for controlling pest termite populations, either by identifying new targets for termiticides, or by improving our current termite management strategies.

Nowadays, subsurface landfill is the main final destination of household and industrial wastes and residues obtained after incineration (bottom and fly ashes). According to the European legislation, engineered clay barriers are put on the sides and bottom of each pit, in order to prevent any pollution of soils and water tables by waste leachates. For this reason, clay materials must have a low hydraulic conductivity (less than 10-9 m.s-1) and a high cation exchange capacity for the retention of pollutants. However, even if clay barriers are initially in accordance with legislation, no information is known concerning their long term behaviour. The METALCLAY project aims to determine the evolution of macroscopic clay properties, when submitting to metallic solutions. Some experiments were already performed at ISTO on natural compacted clay materials, percolated with copper or lead solution. These first studies have shown that clays rapidly and significantly lose their swelling and cohesive properties, and also became more permeable. The METALCLAY project intends to better understand mechanisms responsible for these dramatic evolutions. Besides, smectites, one of the most abundant phyllosilicates in soils and sediments, have specific characteristics (high specific area, high cation exchange capacity, good hydration properties), which give to this clay mineral family a high industrial interest for waste confinement. For this reason, the METALCLAY team has chosen to deal with smectites. Two important aspects of METALCLAY project have to be stressed. Firstly, in order to better apprehend mechanisms of interaction between clays and metal cations, it is vital to use pure clays. For this reason, the METALCLAY team plans to work, not only with reference natural smectites, but also with synthetic smectites. Indeed, natural smectites also contain minor phases, such as calcite for the hectorite of Hector, quartz and feldspar for the montmorillonite Swy-2 from Wyoming, which can have an influence on the interactions with metals. In contrast, synthetic smectites are pure, and their local tetrahedral or octahedral charge deficit can be fixed. Hydrothermal synthesis requires three autoclaves (type 'Tuttle'), in which pressure (up to 3000 bars) and temperature (up to 750°C) are controlled separately. This specific equipment will be set up, tested and used by METALCLAY team, using the experimental facilities and expertise already at work at ISTO. Secondly, one of main technical skill of the METALCLAY team is its ability to reproduce at the laboratory scale the subsurface landfill conditions. Previous work done at the University of Orleans (Jullien and Lecomte, 2000), has lead to the development of an œdometric cell equipped with a separated injection system characterized by a controlled mechanical pressure. Moreover, experimental parameters have been defined and optimized, to reproduce the hydromechanical conditions of subsurface landfill as well (Pothier, 2001; Pothier et al., 2003). Natural and synthetic smectite samples will be compacted then percolated under constant pressure, with pure water or with solutions having a fixed metal concentration. Copper, nickel, zinc, and chromium will be investigated; these metals are all present in landfill leachates. Hydraulic conductivity will be calculated from measured parameters using Darcy's law. Moreover, chemical analyses (cations and anions) and pH values of leachates will give us information about the evolution of clay-metal exchanges. Samples obtained at the end of each œdometer experiment will be characterized in detail, by several methods: ICP-ES and ICP-MS for chemical analyses, cryo-scanning electron microscopy and transmission electron microscopy for structural pictures, X-ray diffraction for mineralogy, X-ray absorption spectrocopy (EXAFS) for the determination of metal speciation. X-ray diffractograms will be simulated, to have information about the evolution of interfoliar space and about possible modifications in the perpendicular plan ab. In addition, the transport/chemical modelling aspect will be investigated, in terms of hydrological aspects, major species equilibrium in clayey media, and sorption and cation exchange processes. This geochemical modelling at the interface between clays and solution will be performed with the PHREEQC2 software and PHAST coupled code, and will be enhanced thanks to the obtained experimental results. The METALCLAY project aims to combine an experimental approach with a fine characterization and a geochemical modelling, in order to understand the main mechanisms of metal-smectite interactions (sorption either on surface or in interlayer smectites) and to better define the evolution of clay macroscopic properties. The main objective is to contribute to the elaboration of a tool for predicting the long term behaviour of clay barriers.