La Loire, espace d’une gouvernance environnementale ?

Nous sommes passés sur la Loire d’une politique principalement de l’État (énergétique, sécuritaire, économique) à une action publique multi-niveaux. Nous entendons par action publique Loire : un territoire de l’action publique à géométrie variable (la zone d’action du Plan Loire grandeur nature, le Val de Loire patrimoine mondial de l’Unesco) en fonction des politiques et des acteurs concernés. Cette action publique Loire, au contenu politique varié (sécurité, économie, environnement, patrimoine), met en scène une pluralité d’acteurs publics (État, collectivités locales) et privés (associations de protection de l’environnement).Il faut se rappeler que cet espace devait être aménagé (quatre barrages étaient programmés) et qu’à la faveur d’un mouvement écologiste, le réseau Loire Vivante, à la fin des années 1980, ce programme a été largement revu. L’intérêt ici se situe moins sur le conflit que sur ses effets : la réorientation de l’action publique Loire et les modalités de production de cette dernière.L’intervention de la société civile a modifié l’action publique dans ses objectifs mais aussi dans ses moyens. Nous nous accordons avec Vincent Simoulin pour penser la gouvernance comme un référentiel (au sens de Pierre Muller) de l’action publique (Simoulin, 2003).Le débat d’idées opéré par l’intrusion d’acteurs écologistes dans le programme d’aménagement de la Loire a-t-il conduit à l’ouverture du processus de décision ? Pouvons-nous pour autant parler de gouvernance ?Le processus de crises-conflits n’a pas donné lieu à une ouverture mais à une percolation, une transfusion, un passage des idées d’une sphère à une autre (des écologistes vers les aménageurs notamment ceux de l’Établissement public d’aménagement de la Loire et de ses affluents). Au final nous n’observons pas de renouvellement des modalités de la coopération entre acteurs. Ainsi une analyse en terme de régulation plutôt que de gouvernance se dégage.This work analyses the Loire river policy over a thirty year period. This policy was at first meant to be a politically-driven project to spatially and economically regenerate a particular area but became later a transversal public policy whereby the environment, and then the notion of heritage, became key factors. The Loire river was approached from both a spatial perspective as well as a temporal one. Within this thesis it was necessary to reconstruct the decision making process over time in order to understand where we started from and where we are today. The Loire river public policy is a mixture of diverse purposes : regeneration of a hydrosystem, ecology, urban regeneration, landscape, the notion of heritage and the notion of world heritage. The Loire river has been a platform upon which many projects have been developed, but rarely leading to concrete results. The few results achieved are truly complex. The Loire river has been a place around which many debates, conflicts of ideology, and action have occurred.The objective is to understand the Loire river related public policy as well as its evolution. This was done from the perspective of the changes introduced by the involvement of “territorial agents” i.e. some local public actors, as well as some ecology-driven ones. The latter took part in the gradual reshaping the public policy objectives of the area under scrutiny.We also wanted to address the hypothesis according to which territorial governance was to be put in place. This territorial governance was to symbolize the renewal of the public policy. We wish to see if, as is often said, the sort of actions and relations between different agents involved in the Loire projects have had an impact on public policies of other geographical sites and areas

Gestion des temps et régulations sociales : quelles incidences de la loi Aubry II sur l'organisation des entreprises et les conditions de travail des salariés ?

International audienc

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 °C in 0.1 M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge–dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds. A primary objective in this study was to investigate the influence of charge–charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the α-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge–dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge–charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pKSa−pK5K) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson–Boltzmann equation, which considers desolvation and charge–dipole interactions in addition to charge–charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge–charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.This work was supported by grants GM-37039 and GM-52483 from the National Institutes of Health (USA), grants BE-1060 and BE-1281 from the Robert A. Welch Foundation, and a grant PB-93-06777 to M.R. from the Dirección General de Investigación Cientı́fica y Técnica (Spain

OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU* and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.htm

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de-novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized

Peptide Conformer Acidity Analysis of Protein Flexibility Monitored by Hydrogen Exchange†

ABSTRACT: The amide hydrogens that are exposed to solvent in the high-resolution X-ray structures of ubiquitin, FK506-binding protein, chymotrypsin inhibitor 2, and rubredoxin span a billion-fold range in hydroxide-catalyzed exchange rates which are predictable by continuum dielectric methods. To facilitate analysis of transiently accessible amides, the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin were determined under near physiological conditions. With the previously reported NMR-restrained molecular dynamics ensembles of ubiquitin (PDB codes 2NR2 and 2K39) used as representations of the Boltzmann-weighted conformational distribution, nearly all of the exchange rates for the highly exposed amides were more accurately predicted than by use of the high-resolution X-ray structure. More strikingly, predictions for the amide hydrogens of the NMR relaxation-restrained ensemble that become exposed to solvent in more than one but less than half of the 144 protein conformations in this ensemble were almost as accurate. In marked contrast, the exchange rates for many of the analogous amides in the residual dipolar coupling-restrained ubiquitin ensemble are substantially overestimated, as was particularly evident for the Ile 44 to Lys 48 segment which constitutes the primary interaction site for the proteasome targeting enzymes involved in polyubiquitylation. For both ensembles, “excited state ” conformers in this active site region having markedly elevated peptide acidities are represented at a population level that is 102 to 103 abov