Motions, order and consistency : a story based on the study of the dynamics of the class A beta-Lactamase PSE-4 by NMR

Partie I L’analyse de données de relaxation des spins à l’aide de l’approche model-free est très répandue pour obtenir des informations sur la dynamique des protéines aux échelles de temps ps-ns et μs-ms. Afin d’extraire des informations de qualité, les données sont enregistrées à plusieurs champs magnétiques. Combiner de telles données est cependant sujet aux erreurs expérimentales. Ainsi, la consistence des données de relaxation à plusieurs champs doit être vérifiée. Malheureusement, cela s’effectue rarement, i.e. on assume simplement que les données sont correctes. Nous proposons donc une approche simple pour la vérification de la consistence de données de relaxation enregistrées à plusieurs champs. L’utilisation des test proposés améliore l’analyse et réduit la présence artéfactuelle d’échange conformationnel. Ainsi, comme les données d’échange conformationnel sont souvent discutées en terme de liaison de substrat ou de catalyse, s’assurer de leur validité améliore la compréhension biologique du système étudié. Partie II Les b-lactamases de classe A sont impliquées dans la résistance aux antibiotiques. Elles y participent en hydrolysant les b-lactamines. Ces enzymes ont été étudiées par différentes approches : études mutationnelles, simulations de dynamique moléculaire, cristallographie des rayons X et RMN. L’enzyme modèle de cette classe, TEM-1, a précédemment été étudiée par RMN dans notre laboratoire. TEM-1 est très rigide sur l’échelle de temps des ps-ns, mais subit des mouvements lents μs-ms au niveau du site actif. Afin de mieux caractériser la dynamique des b-lactamases de classe A, l’homologue PSE-4 a aussi été étudié par RMN avec des données de relaxation des spins, de dispersion de relaxation par CPMG et d’échange d’amides. Les mêmes conclusions que pour TEM-1 ont été obtenues : rigidité générale élevée et présence de mouvements lents près du site actif. Ces mouvements pourraient être conservés chez les b-lactamases de classe A et ainsi avoir un lien avec la catalyse enzymatique. Cette hypothèse est renforcée par les données RMN pour cTEM-17m, une chimère TEM-1/PSE-4, pour laquelle plusieurs résonances près du site actif sont non observées dû à un élargissement causé par ces mouvements lents.Part I The analysis of spin relaxation data using the model-free formalism is a widely used approach to get insights into protein dynamics on the ps-ns and μs-ms timescales. In order to extract high quality data, multiple magnetic field datasets are required. Combining datasets recorded using different NMR magnets is prone to experimental errors. Hence, the consistency of multiple field spin relaxation data must be carefully verified. Analysis of multiple field spin relaxation data generally proceeds without verification of consistency, i.e. with only the assumption that data is fine. We propose a simple approach to verify the consistency of multiple field relaxation data. Using the proposed tests improves the analytical approach by reducing the presence of artifactual conformational exchange terms. Since these terms are often rationalised in relation with ligand binding or catalysis, improving their confidence yields a better understanding in terms of biology. Part II Class A b-lactamases are involved in antibiotics resistance. They do so by hydrolysing the b-lactam antibiotics. These enzymes have been widely studied by different approaches including mutational studies, MD simulations, X-ray crystallography and NMR. The model enzyme for this class of proteins, TEM-1, has previously been studied by NMR in the laboratory. It was observed that TEM-1 is a highly ordered protein on the ps-ns timescale, with slower μs-ms motions clustered around the active site. In order to characterize further the backbone dynamics of class A b-lactamases, the homologous enzyme PSE-4 was studied by NMR using different approaches such as spin relaxation, CPMG relaxation dispersion, and amide exchange experiments. The same conclusions as for TEM-1 were obtained with a high rigidity along the sequence balanced by slower motions in the vicinity of the active site. These motions might be conserved in class A b-lactamases and potentially be important for catalysis. This hypothesis is further enforced by the backbone resonance assignments for cTEM-17m, a TEM-1/PSE-4 chimera, where many resonances are unobservable around the active site, potentially suffering from line broadening caused by slow motions

Effet du magnésium, des traitements thermiques et de la porosité sur les propriétés mécaniques de traction et de fatigue de l'alliage sous pression A380.1

L'alliage A38O.1, un alliage d'aluminium-silicium-cuivre-magnésium (série 300), est produit à partir de rebuts d'aluminium et mis en forme sous pression. Cet alliage qui est particulièrement utilisé dans le domaine automobile peut subir des traitements thermiques pour obtenir une combinaison optimale de résistance et de ductilité. La composition chimique, la procédure observée lors de la fusion du métal, la technique de mise en forme, les défauts et le type de traitement thermique déterminent les propriétés mécaniques de cet alliage. Comme l'alliage A380.1 est obtenu à partir de métal recyclé, il contient une quantité variable de plusieurs éléments. Afin d'obtenir la composition souhaitable pour chacun de ces éléments, différents procédés coûteux et souvent dommageables pour l'environnement, tel le nettoyage au chlore, sont utilisés. Pour contrer ce problème de pollution, un moyen efficace pour éviter tous ces procédés serait certainement le bienvenu. Le magnésium est un des éléments dont la proportion moyenne doit être inférieure ou égale à 0,10 % pour répondre aux normes de l'industrie nord-américaine. Toutefois certaines études ont démontré qu'une quantité supérieure de magnésium influençait peu les caractéristiques mécaniques de l'alliage sauf peut-être celle de la ductilité. Donc, en justifiant l'augmentation du titre en magnésium dans cet alliage, on pourrait éviter l'application de traitements malsains tant sur le plan économique qu'environnemental. L'objectif de ce travail est de déterminer l'impact sur les propriétés mécaniques de posséder une proportion en magnésium supérieure au taux standard nord-américain de 0,10 % pour l'alliage A380.1. Des taux respectifs de 0,10 %, 0,30 % et de 0,50 % en magnésium sont utilisés pour constater l'effet sur les principales propriétés mécaniques à savoir: la limite élastique, la limite ultime et le pourcentage d'allongement à la rupture. Une grande partie de la recherche est également axée sur l'effet de cet élément (magnésium) sur la résistance en fatigue dudit alliage. Cette section comprend aussi l'analyse du rôle des défauts de surface (joint du moule) et des défauts internes (porosité) sur la résistance. Les propriétés mécaniques de traction sont analysées dans le but d'optimiser les traitements thermiques T6 (mise en solution, trempe et vieillissement artificiel) et T7 (mise en solution, trempe et survieillissement). Par la suite, l'influence de plusieurs paramètres est évaluée au moyen d'essais de traction et de la micrographie optique. La résistance en fatigue est observée en faisant des essais en flexion rotative. Une fois de plus, cette étude est réalisée dans le but d'optimiser les traitements T6 et T7 de même que pour vérifier l'effet des défauts de surface et des défauts internes. Pour examiner la contribution de ces défauts de même que pour les quantifier, des fractographies prises à l'aide d'un microscope électronique à balayage sont utilisées. Autant pour la traction que pour la fatigue, les paramètres qui nous intéressent plus spécialement sont: l'addition de magnésium, la température et le temps de vieillissement. De plus, pour la fatigue uniquement, la porosité est ciblée. En ce qui concerne les résultats, on peut affirmer notamment qu'un taux de 0,3 % de magnésium ne les altère pas, ni en traction ni en fatigue, et qu'un taux de 0,5 % de magnésium entraîne la formation de phases complexes sujettes à la fusion instantanée. De plus, on note que les propriétés de traction fluctuent selon les zones de Guinier-Preston apparues lors des traitements thermiques tandis que celles de fatigue diminuent au fur et à mesure que le taux de magnésium augmente

L'impact de l'utilisation du protocole semi-structuré du NICHD et de l'âge des enfants sur les scores du CBCA : grilles standard et révisée

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Tar conversion over olivine and sand in a fluidized bed reactor using toluene as model compound

The aim of this work is to study the tars conversion in conditions representative of biomass gasification in a fluidized bed reactor. Experiments are conducted at 850 °C and atmospheric pressure in a fluidized bed reactor with toluene as tar model. Influences of the nature of the media (sand and olivine) and of the reactive atmosphere (steam and hydrogen partial pressures) on toluene conversion are particularly studied. The steam and hydrogen partial pressures were varied in the range of 0.05 to 0.4 bars and 0 to 0.2 bars, respectively. Results showed a strong influence of these parameters on toluene conversion. Olivine was found to have a catalytic activity towards steam reforming reactions which depends on the ratio in the reactor. Both thermodynamic equilibrium and surface analyses (EDX and XRD) of olivine particles suggested that this ratio controls the oxidation/reduction of iron at the olivine surface. Besides, iron is more active towards tars removal when its oxidation state is low. At 850 °C and , the iron is reduced to form native iron (Fe0) on the olivine surface which favors the steam reforming of toluene

Kinetic study and modelling of char combustion in TGA in isothermal conditions

The purpose of this work is the kinetic study of biomass char combustion in isothermal conditions in TGA. This char was obtained from fast pyrolysis of beech bark pellet in a fluidized bed reactor at 850 °C and atmospheric pressure. Kinetic study of isothermal char combustion was performed for temperatures up to 400 °C, oxygen partial pressures ranging from 5065 to 21,273 Pa and a char particles size of 25 μm. Mass transfer effects around and within the crucible were thoroughly characterized by naphthalene vaporization. Oxygen diffusion was found to have no effect on char combustion for temperatures below 400 °C. A novel method including the transfer function of the TGA which describes the variation of oxygen partial pressure just after switching the gas from inert to reactive in the TGA was taken into consideration in the kinetic modelling. Two kinetic models (the Grain Model and the Random Pore Model) were used to determine kinetic parameters. The Grain Model was found to be in very good agreement with experimental data. Values of activation energy and reaction order with respect to oxygen are respectively equal to 124 kJ/mol and 0.74. Besides, the maximum combustion rate commonly observed in the literature during char combustion was found to be the result of the non-uniform oxygen partial pressure in the TGA at the initial stage of the char combustion

Synergistic Applications of MD and NMR for the Study of Biological Systems

Modern biological sciences are becoming more and more multidisciplinary. At the same time, theoretical and computational approaches gain in reliability and their field of application widens. In this short paper, we discuss recent advances in the areas of solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations that were made possible by the combination of both methods, that is, through their synergistic use. We present the main NMR observables and parameters that can be computed from simulations, and how they are used in a variety of complementary applications, including dynamics studies, model-free analysis, force field validation, and structural studies

Biomass gasification in a fluidized bed reactor: effect of temperature on properties and oxidative reactivity of chars

Introduction: Biomass gasification is a promising alternative to fossil fuels for the synthesis of highly energetic products via Fischer-Tropsch or methanation processes. It is a thermochemical conversion occurring at high temperatures with many simultaneous reactions. For temperatures above 350°C, biomass undergoes a thermal decomposition called pyrolysis which leads to the formation of volatile products either condensable (steam and tars) or incondensable (H2, CO, CO2, CH4 and C2Hx) and a solid residue called char [1]. Then, the char reacts with steam and carbon dioxide at temperatures greater than 700°C to produce syngas. These transformations are endothermic. Therefore, a contribution of energy is required to maintain the temperature and the different reactions. One of the most encouraging and advanced technology is dual fluidized beds [2]. Its principle relies on the circulation of a media (sand, olivine or catalyst particles) which acts as a heat carrier between an endothermic reactor, where biomass gasification produces syngas, and an exothermic reactor where combustion of a part of the char from the gasification of biomass produces heat. Therefore, it is of importance to carefully understand the effect of operating conditions on char structure and composition which are directly related to its reactivity in combustion and steam gasification. During pyrolysis of biomass, many changes occur in the solid structure including (1) the loss of functional groups on the carbon surface, (2) ordering of the carbon microstructure to tend to a graphitic crystalline form, (3) the formation of pores which directly influence the surface area, (4) the modification in ash content and its distribution that affect the catalytic activity of chars. Together, these changes are responsible for the steam gasification and combustion reactivity of the chars. Please click Additional Files below to see the full abstract

Evaluation of fixed point H2S gas detectors intended for use in industrial safety applications

International audienceH2S gas detectors are used by a wide variety of industrial companies for safety reasons, e.g: Oil&Gas, Chemistry, Water Treatment. The detectors are usually installée! where H2S can be found in ambient air in order to prevent toxic risk. The tests, requested by the French Ministry for Environment, were conducted by INERIS in partnership with Exera (an association of companies and organizations which invest in instruments, measurement, control and automation Systems). This évaluation was carried out to investigate if H2S gas detectors can operate efficiently under différent operating conditions on industrial sites (température, humidity, pressure, interfering species, etc.). The measurement range of the detectors that were evaluated is 0- 20 ppm

Hydrodynamic study of a circulating fluidized bed used for biomass gasification between 20 °c and 900 °c

This work, carried out in the GAYA project frame and subsidized by ADEME, concerns the hydrodynamic study of a 60 kWth circulating fluidized bed pilot plant constructed at the Laboratoire de Génie Chimique in Toulouse for biomass gasification. In that process, biomass gasification and heating of fluidisation material (sand, olivine, catalyst…) which also acts as heat transfer medium, are performed in two separate reactors: a dense fluidized bed and a transported bed. Thermal energy, required in order to heat the fluidisation material, is supplied by the partial combustion of the char produced in the gasification reactor. The hydrodynamic study carried out at atmospheric pressure between 20 °C and 900 °C leads to the determination of the influence of the temperature and media average particle size (olivine), the reactor geometric properties and the fluidization gas nature (air or steam) on the media hydrodynamic parameters such as the minimum fluidization velocity and porosity, the bed expansion and the solid transport velocity. Results have shown that the minimal fluidization and transport velocity decrease with the bed temperature increase. However, the bed expansion is not affected by this factor. Furthermore, the particles transport happens from the terminal falling velocity and increases with the gas velocity. The fluidization gas nature has a significant effect on the minimal fluidization velocity, but not on the bed expansion. Correlations are proposed in order to estimate the characteristic parameters of the olivine hydrodynamic behavior depending on the temperature, the fluidization gas nature and the solid particle size

New platinum(II) complexes conjugate at position 7α of 17β-acetyl-testosterone as new combi-molecules against prostate cancer : design, synthesis, structure-activity relationships and biological evaluation

Prostate cancer is a major public health problem worldwide and, more specifically, new treatments for hormone-refractory cancers are highly sought by several research groups. Although platinum(II)-based chemotherapy and other strategies grow in interest to treat castration-resistant prostate cancer (CRPC), they still exhibit modest activity on CRPC and overall patient survival. In this study, we designed and prepared new combi-molecules using 17β-acetyl-testosterone and amino acid platinum(II) complexes linked at the position 7α to target and to improve the antiproliferative activity of platinum(II)-based chemotherapy on prostate cancer cells. Twelve chemical intermediates and six new combi-molecules were prepared and characterized. Structure-activity relationships studies show that the platinum complex moiety is essential for an optimal cytocidal activity. Moreover, stereochemistry of the amino acid involved in the platinum complexes had only minor effects on the antiproliferative activity whereas pyridinyl (10a and b) and thiazolyl (10f) complexes exhibited the highest cytocidal activities that are significantly superior to that of cisplatin used as control on human prostate adenocarcinoma LNCaP (AR+), PC3 (AR-) and DU145 (AR-). Compounds 10a, b and f arrested the cell cycle progression in S-phase and induced double strand breaks as confirmed by the phosphorylation of histone H2AX into γH2AX. Compounds 10a and f showed 33 and 30% inhibition, respectively of the growth of HT-1080 tumors grafted onto chick chorioallantoic membranes. Finally, compounds 10a and 10f exhibited low toxicity on the chick embryos (18 and 21% of death, respectively), indicating that these new combi-molecules might be a promising new class of anticancer agents for prostate cancer