Plants are submitted to repeated and fluctuating wind loads during their growth. In case of extreme winds, lodging or breaking of forest trees or crop plants occur with major economic and ecological consequences. Moreover, scenarii of global climatic changes forecast clear increases in the frequency of high wind occurrence in Europe, raising questions about the way plants may or not acclimate to these changes. A key biological process for such acclimation involves mechanical strain sensing (during wind-induced bending and torsion) and a syndrome of mechano-controlled growth responses called thigmomorphogenesis. However very little is known about the underlying mechanical and biological mechanisms. In particular, the mechanical and biological consequences of wind-induced vibrations have been disregarded up to now. In vertebrates, mechanosensitive responses to vibration frequency and strain rate have been shown to be central, for example for bone growth. Moreover a slower tuning of mechanosensitivity over successive mechanical stimulations was found to be a key process called accommodation. Many clues suggest that frequency dependence and accommodation may also exist in plants. Nevertheless, at the cellular and molecular level, the responses to vibration of MechanoSensors (MS) have not been well documented, whereas the physiological and molecular bases of accommodation remain elusive. Wind-induced vibrations in plants can broadly be separated into two ranges of frequencies: i) 'high' frequencies from 0.5 to 10 Hz, and very low frequencies related to diurnal and weather-induced wind changes (10-5-10-6 Hz). The high frequencies should have major influence on the mechanical dynamics of the plant and hence on their strains and strain rates. The very low frequencies are likely to be involved in the accommodation process. The Senzo project aims at understanding the sensing by plants of wind 'induced vibrations, and the accommodation process over time, as both are central to the thigmomorphogenetic acclimation of plants to wind. . The ambition of Senzo is to make a shifting-up in plant biomechanics and mechanobiology through broad and intensive interdisciplinary collaboration, joining vibration physicists, plant biomechanicists, cytologists, cellular electrophysiologists, molecular physiologists and molecular biophysicists in a focussed project. The plant mechanical structure clearly applies a mechanical filter/amplifier between external fluctuating loads and the mechanical strain regimes stimulating mechanosensitive tissues. By the same token the cellular structure is likely to mechanically influence cell responses. At the other end of the scale, specific genes and proteins may be involved. Two candidates have been selected from the bibliography and from our previous results: i) mechanosensitive channels (MSLs) recently cloned in plants and which bacterial homologs display relaxation time consistent with the 0.5-10 Hz range, ii) a mechanosensitive transcription factor (ZFP2) which expression seems to change over very low frequencies, thus being probably an entry to the genes-transcription-network involved in the accommodation process. However no phenotype has ever been described in the mutants or transgenic plants obtained for these two candidate-genes families, probably due to the lack of relevant stimulations and measurements. Three tasks have been designed to i) investigate the mechanical and biological features influencing the range of vibration that a plant can sense, ii) analyse the cellular and subcellular responses of MSLs to vibrations, and ii) analyse the genes-network involved in accommodation. All these tasks are interdisciplinary and involve a mix of experiments and modelling. The information gathered through these tasks will then be used in the fourth task in an attempt to reveal and assess the influence of the level of expression of the candidate genes (and proteins) on the quantitative phenotype of mutants and transgenic plants. Two contrasted plant systems will be studied, Arabidopsis and poplar (both being model species for genomics). In the field of plant biomechanics and mechanobiology, all this is relevant and completely novel. Even broadening to mechanobiology in Life Sciences, the systematic analysis of frequency-amplitude response of i) mechanosensors and ii) the genes involved in the accommodation of mechanosensitivity has not been conducted yet. Although fundamental in essence, this research may open avenues for more applied uses of mechanosensitive growth control. For example it may ground i) the design of ideotypes and markers of wind acclimation and hardening for plant breeding or ii) innovating chemical-free controls of plant growth and aspect in greenhouse. The project is also likely to foster a totally novel approach for high-rate plant phenotyping (mass, size'), beyond the field of plant biomechanics.

Recently, we discovered that a universal protein annotated as 'O-syaloglycoprotein-endopeptidase' in all sequenced genomes, is in fact a DNA binding protein that exhibits an atypical apurinic endonuclease (AP-endo) activity (Hecker et al., NAR, 2007). We also obtained in vivo evidence suggesting that this protein, known under various names in the literature (YgjD/Ggc, Kae1, OSGEP or else Qri7/OSGEPL) is involved in genome maintenance. We propose here to call these proteins UGM for Universal Genome Maintenance. In S. cerevisiae, the UGM protein Kae1 interacts with a conserved protein-kinase, Bud32, and three other small proteins to form a complex that associated with chromatin. This complex, called EKC or KEOPS, is involved in the transcription and in telomere maintenance. Four of the five proteins of the EK/KEOPS complex have homologues in Eukarya and Archaea. This conservation indicates that this complex is an ancient molecular machine of fundamental importance. Although UGM proteins and the EKC/KEOPS complex are certainly as important as other universal protein such as RecA/Rad51, they are still studied by a very limited number of laboratories, and their biochemical function(s) and biological role(s) are still largely unknown. The objective of the present project is thus to determine the biochemical function(s) and biological role(s) of UGM proteins in the three domains of life and of the EKC/KEOPS complex in Archaea and Eukarya. For that purpose, we will combine informatics, structural, biochemical, genetics and genomic approaches in E. coli, P. abyssi, M. jannashii, S. cerevisiae, and human cells. Our consortium involves four partners with complementary expertises. Patrick Forterre (P1), who is an expert in archaeal molecular biology and hyperthermophilic proteins involved in DNA metabolism, has already collaborated with Herman van Tilbeurgh (P2), who is an expert in protein structure analyses. In the framework of a previous ANR project, they solved the structure of the archaeal Kae1 protein and of a KEOPS/EKC subcomplex (Hecker et al., NAR, 2007, EMBO J. 2008, Biochemical Transaction, 2009). Domenico Libri (P3) and Carl Mann (P4), who discovered the EKC complex (EMBO J, 2008), are expert in yeast genetics and in the analysis of chromatin associated proteins in human cells. P2 and P3 have already collaborated in the exploitation of structural data to demonstrate that the physical interaction between the UGM protein Kae1 and the kinase Bud32 is essential in vivo (Hecker et al., EMBO J. 2008). In the present project, we will complete the biochemical and structural analyses of UGM proteins and the EKC/KEOPS complex in the three domains of life. One of our main objectives will be to solve the structure of the complete yeast and archaeal KEOPS/EKC complex (P2) and to reconstitute these complexes from individual components (the archael complex probably includes a fifth subunit that is missing in yeast but is present in humans) (P1-4). For enzymatic analyses, we will focus on the role of an atypical ATP-iron binding site that P2 discovered in the UGM protein Kae1, and on the interaction of UGM proteins and the KEOPS/EKC complex with nucleic acids. Preliminary experiments by P3 show that the EKC/KEOPS complex binds specifically a telomeric-like sequence (P1-4). A major goal is now to determine how these proteins bind to DNA, if they also interact with RNA, and if they have other activities than the AP endonuclease activity previously detected. Phenotypic analyses of mutants of UGM proteins and subunits of the EKC/KEOPS complex will be performed in E. coli and S. cerevisiae. A major aspect of this project will be to focus on the identification of proteins that interact with UGM proteins in the three domains of life and/or the EKC/KEOPS complex in Archaea and Eukarya (P1, P3, P4). These protein partners will be sought using in parallel in silico (genome context analysis), biochemical (pull down assays, TAP-tag screening) and genetic/genomic approaches (transcriptome profiling, genome-wide interaction analysis) both in E. coli, Archaea, S. cerevisiae, and human cells. The rational is that identification of proteins partners will foster predictions on the biological role of the UGM proteins and/or the EKC/KEOPS complex. These predictions will then be tested by biochemical and genetic analyses. A major novelty of this project will be to initiate the study of the EKC/KEOPS complex in humans (P4). This will be a very important task since one protein of this complex is already known to phosphorylate p53 in humans and another (triplicated in humans) is known as a cancer testis antigen. In general, since the proteins studied in this project are either universal (UGM) or at least conserved in two of the three domains (the EKC/KEOPS complex), the deciphering of their biological roles will have extremely important consequences for biologists working in very different research fields.

Cytokines are small size proteins that, by diffusing in the extracellular space carry distinct signals (activation, inhibition, proliferation, differentiation), and provide cells a system to communicate. Current research increasingly shows that most cytokines, in addition to their specific receptors, bind to a class of molecules, collectively known as glycosaminoglycans, and in particular to one of them called heparan sulfate (HS). These long, unbranched polysaccharides are near ubiquitous constituents of cell surfaces, and are carried by a specialized family of glycoprotein: the proteoglycans. Cytokine-HS interactions importantly regulate cytokine-receptor binding and activation, signaling, storage and stabilization in the extracellular space, and/or induce structural changes that modify cytokines activity. Interactions with HS are thus crucial and impact the dynamic of the cytokines at the cell surface or in the extracellular matrix and thus, their proper tissue localization and many of their biological functions. Given the importance of HS-cytokine interactions both in normal and pathologic states, understanding the mechanism and the structural bases that drive these interactions and their functions represent an important issue. However, progresses in that field have been hampered by the extraordinary complexity of HS. In particular, the characterization of the protein-HS interface, the isolation of the corresponding binding domains, and the elucidation of the mechanism by which HS regulate cytokine activity and dynamic remain particularly challenging. Based on a preliminary determination of the structure that a cytokine: interferon-gamma (IFNg), recognizes along the HS chain, a set of glycoconjugate mimetics has been synthesized, and evaluated for their ability to interact with the protein. One of these molecules, composed of two N-sulfated octasaccharides (dp8), linked to each other through a 5 nm long spacer displays high affinity for the cytokine. This molecule, termed 2O10 inhibits the binding of IFNg to HS with an IC50 of 35-40 nM, and most interestingly also blocks the binding of the cytokine to its cell surface receptor. This achievement represented the first synthetic HS-like molecule that targets a cytokine, and provides a potentially powerful strategy to inhibit IFNg. Although this molecule enables us to define the structural organization of the binding site (i.e. two binding octasaccharides spaced apart by an internal domain of appropriate length), the sulfation profile of each of the dp8 (currently homogeneously sulfated) has not been address yet. Our working hypothesis, supported by preliminary results recently obtained, is that only a selected (and specific) number of sulfate groups are actually engaged in the complex with IFNg. By combining structural, chemical, analytical and biological approaches, the goal of our proposal is to characterize the molecular determinants involved in the interaction of IFNg with HS, and to investigate, at the cellular level, the biological relevance of this interaction. In particular, this project should significantly contribute to outline the importance of the sulfation profile within the octasaccharides that interact with IFNg. This important point has never been addressed before, due to the structural heterogeneity of natural HS molecules, but is now possible in the context of the 2O10 template that can be obtained in pure form and large amount by chemical synthesis. Based on these results, we want to enhance the affinity and specificity of such glycoconjugates towards IFNg. An ultimate goal of this work will be the development of an inhibitor of the cytokine, which could be then evaluated in a number of pathology in which the cytokine has been identified as a target. The new knowledge generated in this complex field of biology will be the basis of the development of new therapeutic strategies based on the use of 'small glycanic drugs' to control a fundamental immunologic process. This project results from a long-term and sustained collaboration carried out by partners 1 (glycobiologist) and 2 (organic chemist) in the investigation of the IFNg/HS interaction, bringing together structure/function analysis in the field of protein-HS interaction and HS carbohydrate chemistry. Coupled to partner 3 (mass spectrometry specialist), it will lead to a further step in the characterization of the IFNg/HS complex. Such knowledge and collaboration are shared and mastered by only a very small number of groups over the world. In this regard, the collaboration between the 3 partners, on which this proposal is based is rather unique and has already allowed addressing important issues in the field.

A decade ago, it was thought that the global star formation history of galaxies at cosmological scales was relatively well constrained by the optical and UV data. Soon after, 15 microns observations with ISOCAM, onboard the Infrared Space Observatory (ISO), and sub-millimeter SCUBA results from the ground, brought a revolution in the field by revealing that Luminous and Ultra-luminous infrared galaxies were much more common in the past than they are now. These galaxies are forming stars at a high pace, and most of the energy radiated by the forming stars and also by active galactic nuclei, when present, is absorbed by dust and re-radiated in the IR. It became possible to re-establish the global history of star formation at z'2, and to identify the sources from which the bulk of the Cosmic Infrared Background originates. The next IR Observatory, Spitzer, combined with multiwavelength observations on specific areas of the sky, and with the UV satellite Galex, brought a second revolution in the field: the abundance of active galactic nuclei in distant massive galaxies had been largely underestimated, even when the deepest X-ray surveys were used; galaxies behave in what appears as anti-hierarchical with massive galaxies formed first and then stopping abruptly to form stars, but environment effects and AGNs thought to quench star formation, either accelerate or are coeval with star formation in galaxies. At each of these stages of understanding of galaxy evolution, members of the team in this ANR proposal played a leading role at the world level. But mostly it was shown by us and others that the far infrared luminosity, hence the star formation rate (SFR), of distant galaxies (z>1) cannot be safely extrapolated from data at other wavelengths: the correction for extinction of the optical-UV regime saturates at high SFR; z~2 SCUBA galaxies exhibit unique spectral energy distributions in the IR with e.g. colder dust temperatures than extrapolated from local galaxies; the total IR luminosity of z'1.5 galaxies extrapolated from the mid IR is overestimated by increasingly large factors with increasing luminosity, partly due to the presence of Compton Thick AGNs. Thus, many of the key questions on galaxy formation/evolution, such as the global star formation rate or the frequency of AGNs in galaxy cores, and their link with star formation/quenching, remain open. When the Herschel satellite, to be launched on April 12, 2009, will produce the deepest images of the sky in the far infrared, it will become possible, for the first time, to address these unsolved questions, by measuring the bolometric luminosity of distant galaxies, as produced not only by young and massive stars, but also by accreting super massive black holes. An international team (PI David Elbaz; this is the only Herschel Open Time Key Program with a French PI) has obtained considerable Herschel time to carry out crucial observations in a region of the sky where data at other wavelengths is already available. The French part of this team, author of this ANR proposal, will profit from the know how of the colleagues, but also suffer from the competition with the rest of the team, especially considering that the US part of the team will be well funded to carry out this project. To enable the French team to extract the key observational information from Herschel and to use/design the modeling tools necessary for the interpretation, it is mandatory that, in France too, young researchers (postdocs from the ANR and PhD students from other funding sources). The main scientific objectives of this ANR project rely on the GOODS-Herschel Open Time Key Program that we are leading: (A) To resolve most of the cosmic SFR density up to z~4, thanks to the ~2000 galaxies that will be detected in GOODS-Herschel in the unexplored regimes of normal galaxies up to z~1, luminous and ultra-luminous infrared galaxies up to z~2 and 4 respectively. This will be done by bridging IR and UV selected galaxies down to the level where both SFR agree up to z~1.5 and potentially up to z~4. Hence the need to use/design models dealing with UV-optical-IR emission of galaxies. (B) To identify the buried Compton Thick AGNs responsible for the still unresolved 30% fraction of the cosmic X-ray background (CXB), which peaks at 30 keV, hence above the 10 keV limit of XMM-Newton and Chandra, and study their link with star formation or its quenching in distant massive galaxies. This will be done by searching for mid over far infrared excess galaxies using and extending a technique that we previously validated on a statistical scale (stacking). (C) To identify the sources making the bulk of the extragalactic infrared background, second in intensity after the CMB and which contains the majority ofthe energy radiated by star formation over the Hubble time.

Secretins are a unique class of outer membrane proteins involved in protein secretion or the assembly of multi-protein complexes in this membrane. In contrast to the major class of beta-barrel outer membrane proteins such as porins, secretin PulD, the secretin studied here, is composed of more subunits (12) that form a plugged or gated channel n the outer membrane. PulD exhibits remarkable resistance to dissociation in ionic detergents at 100°C, can assemble and insert spontaneously in the presence of lipid bilayers, does not require the canonical outer membrane protein targeting and insertion machinery (Bam), does not possess readily identifiable amphipathic beta strands that could span the outer membrane (as in porins), and requires a dedicated chaperone for targeting and protease protection but not for assembly. These unique features are based on equally unique but hitherto poorly explored structural and physical properties of the PulD polypeptide and the dodecamer it forms. Technical problems associated with the production, extraction and purification of PulD dodecamers have hitherto limited structural analysis to low-resolution cryo-electron microscopy of single PulD particles. These technical problems have now been overcome, opening up new and exciting avenues for research into the high-resolution structure-function analysis that we propose here. Particular emphasis will be placed on two main features of the PulD polypeptide and its complex. First, the PulD protein complex will be produced in an in vitro system that we have developed, and will be purified in non-denaturing detergents using affinity chromatography or examined directly as 2D crystalline arrays in the liposome membranes. We will determine what parts of the polypeptide form the transmembrane spanning, subunit interaction and plug segments of dodecamer. Both 2D and 3D crystallography will be used for this purpose. The corresponding regions of the polypeptide will be mapped on the 3D model derived from electron microscopy. Specific selection and screening methods will be set up to obtain mutations in the pulD gene that affect channel opening (screening for constitutively open channel variants that cannot close the plug that normally seals the outer membrane) and dodecamer assembly. Variants affected in channel formation will be analyzed in vitro to determine the dimensions of the channel and the extent to which it has been opened (or locked in place). The in vitro synthesis system will also be used to examine the kinetics of dodecamer assembly and to identify potential assembly intermediates. Assembly-defective variants will be examined in this system to determine at which stage their assembly is blocked. Second, we will examine in greater detail the pathway by which PulD reaches the outer membrane. We will examine the structural basis for the interaction between PulD and its specific chaperone, PulS, to identify key amino acids in both partners. We will also examine further the preliminary evidence that we have that the two effects of PulS, protease protection and outer membrane targeting, are dissociable, determine the effects of PulS on the kinetics of PulD folding and assembly and examine the similarities and differences between PulS and two other well-characterized secretin chaperones, MxiM and PilW.