Ce travail de thèse s’inscrit dans le contexte de la sûreté nucléaire, et plus précisément, de l’intégrité des circuits secondaires des Réacteurs à eau pressurisée (REP). L’étude porte donc sur le comportement à rupture de structures minces soudées dans le domaine haut de la transition fragile/ductile. Elle a pour objectif de développer le modèle en contrainte seuil initialement développé par Chapuliot, qui permet de prédire la non-rupture par clivage de cette structure soudée. Le modèle est identifié pour la soudure de l’acier au C-Mn de construction nucléaire, en s’intéressant plus particulièrement à la limite supérieure du domaine de transition.Une contrainte seuil, en-dessous de laquelle le clivage ne peut avoir lieu, est identifiée à partir d’essais de traction à basses températures sur éprouvettes axisymétriques entaillées prélevées dans le joint soudé. Cette contrainte seuil permet de définir le volume seuil, ou volume dans lequel les contraintes principales maximales dépassent la contrainte seuil au cours de l’essai.L’analyse au MEB des faciès des éprouvettes rompues montre que la zone fondue brute de solidification dans la ZAT est la zone la plus susceptible de cliver. La relation entre la probabilité de rupture fragile et le volume seuil dans cette zone est établie via une fonction de sensibilité, grâce à des essais sur éprouvettes CT et à leur simulation multi-matériaux. Le modèle ainsi identifié est testé pour prévoir la non rupture par clivage d’éprouvettes SENT prélevées dans le joint soudé et sollicitées en traction. Les résultats obtenus sont encourageants relativement à la transférabilité du modèle à la structure réelle / This work concerns the nuclear safety, specifically the secondary circuit integrity of pressurized water reactors (PWR). Therefore, the present study focuses on the fracture behaviour of welded thin structures in brittle/ductile transition. It aims at developing the threshold stress model initially proposed by Chapuliot, to predict the non-brittle-fracture of this welded structure. The model is identified for the welded joint in C-Mn steel for nuclear construction, specifically in the upper part of the transition.A threshold stress, below which the cleavage cannot take place, is identified using tensile tests at low temperature on axis-symmetrical notched specimens taken in welded joint. This threshold stress is used to define the threshold volume where the maximum principal stress exceeds the threshold stress during the test.The analysis by SEM of specimen fracture surfaces shows that the gross solidification molten zone in the weld is the most likely to cleave. The relation between the brittle fracture probability and the threshold volume in the gross solidification molten zone is established via a sensitivity function, using multi-materials simulations. The model thus identified is tested for the prediction of non-brittle-fracture of SENT specimens taken in the welded joint and tested in tension. The results obtained are encouraging with regards to the transferability of the model to the actual structure

En prenant le soin de dépeindre un tableau de l'immigration étrangère en France telle qu'on la retrouve de nos jours, nous associons à ce travail descriptif une définition de l'intégration dans la société française contemporaine. Il importe après de présenter le contexte général du passage à l'acte délictueux, puis de décrire des mécanismes psychiques sous-jacents à celui-ci. Ceci nous met alors en mesure de développer des réflexions au sujet de dimensions importantes dans cette recherche clinique, comme par exemple celle du double clivage et celle du stress acculturatif. Au sujet des mécanismes psychiques, nous donnons une large place aux rites d'institution de soi, à la négativité, à l'agressivité, à la violence, au lien groupal et bien sûr au rapport entre la migration et la délinquance. Dans la réflexion sur le double clivage, nous nous arrêtons sur l'appartenance culturelle et sur l'identité, et nous mesurons aussi le lien entre l'histoire (ou l'Histoire) et le clivage dedans-dehors, un lien autour duquel prennent toute leur importance les notions de la filiation, de la transmission, du faux self ou encore de l'entre deux. Ensuite, nous rencontrons des notions telles que la vulnérabilité de l'estime de soi, la distorsion cognitive, la violence individuelle, les préjugés de groupe ou encore les processus d'acculturation. Cela nous amène à considérer les liens entre l'assimilation et l'immigration, en prenant le temps de saisir dès lors l'importance prise par la notion de l'honneur chez les jeunes résidant dans des quartiers dits sensibles et issus de populations migrantes. Cela nous permet de passer à une revue des liens potentiels entre le passage à l'acte délinquant et l'estime de soi, ainsi qu'entre le passage à l'acte délinquant et l'anxiété. La première revue nous amène à traiter notamment de la carence d'élaboration psychique et de son déterminisme dans le passage à l'acte délinquant. La deuxième revue nous donne l'occasion de prendre du temps sur l'analyse du modèle cognitif de traitement de l'information spécifique à l'anxiété, ce qui amène à considérer les notions de l'attention sélective et de l'hyper vigilance, ainsi que celle de la phobie sociale. Une dernière revue nous permet d'évoquer la place influente que prend l'exil dans l'histoire d'un sujet et dans l'histoire de sa famille. Cela n'est pas sans lien sur certaines formes de violence ou encore certaines manifestations délictueuses qui surgissent dans l'espace public comme dans l'espace privé. / Taking care to describe a board of the stranger immigration in FRANCE as it is existing nowadays, we connect to this descriptive work a definition of the integration in the contemporary french society. It is important after this to present the general context of the punishable acting out, and then to describe the subjacent psychic mecanisms of these. It authorizes us in this way to spread out reflexions about some importants dimensions in this clinical research, like for example, the "double cleavage". About psychics mecanisms, we accord a big place at rites, agressivity, violence, and of course at the ties between migration and juvenile delinquency. In the reflexion about the "double cleavage", we take a reflexion moment on the cultural appartenance and on the identity, and we also measure the ties between family 's history and the cleavage between "the inside" and "the outside", a bond on which take a big importance the notions of relationship, dependance and transmission, and the fact to navigate between two cultures. Then, we meet notions like the vulnerability of self esteem, the cognitive distorsion, individual violence, prejudiced people, etc. This leads us to consider the ties between immigration and assimilation, taking care to grasp the importance of the notion of honour for youg mens who are living in the slums, and who are coming from migrant people. This work leads us to be interested by the ties between delinquency and self esteem, and by the ties between delinquency and anxiety. The first review leads us to take care about the notion of the default of psychic elaboration and about its acting out so well-defined. The second review gives us the occasion to be interested by the analysis of the cognitive pattern of the treatment of the specific information of anxiety, so, we consider in this way the notions of vigilance and social phobia. A last review authorize us to evoke the influent place taken by the exile in the family story. This should have a tie with some violence forms or some punishable manifestations which are rising in the private space or in the public space.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The Binder of Sperm Protein 1 (BSP1) is a protein produced by seminal vesicles of animals. It helps into the fertilization process of oocytes, promoting sperm capacitation, mediating the ligation from itself with the oviduct epithelium, and increasing the motility in the oviduct. When spermatozoa with purified BSP1 were incubated, acrosomal reaction rates were determined with and without the presence of heparin, as controls. The percentage of acrosomal reaction was higher (p <0.05) in control 1 (44.5%) compared with the other treatments (BSP1:10 Âg/mL: 24.5%; 20 Âg/mL: 25.5%; 40 Âg/mL: 26.0 %), and lower the rate (p> 0.05) when sperm were incubated only in Fert-TALP medium without heparin (14.5%). Cleavage and blastocyst rates were evaluated on days two and seven respectively, after fertilize oocytes in a medium containing BSP1. 1274 cumulus- oocyte complexes were recovered and incubated for 18 hours with frozen-thawed ejaculated semen in a Fert-TALP medium, containing: only heparin, 10, 20 or 40 &#956;g/mL of BSP1. Cleavage and blastocyst rates (34.1  4.4 vs 40.8  5.0%) were similar (p> 0.05) when incubation was made with 10 Âg/mL of BSP1 and heparin, respectively. However, the concentrations of 20 and 40 Âg/mL decreased the formation of blastocysts (22.4  2.9 and 19.3  4.1%) compared with heparin (40.8  5.1%, p <0.02). Subsequently, cumulus- oocyte complexes (n = 2239) were incubated with frozen-thawed ejaculated semen. Spermatozoa were selected by a gradient of 45% -90% Percoll containing: only heparin, 10, 20, 40 &#956;g/mL of BSP1 and there was a control 2 of Percoll without heparin or BSP1. Cleavage rate was similar (p> 0.05) for the incubation with concentrations of 10, 20 and 40 &#956;g/mL of BPS1 (67.7  3.0, 68.7  3.5, 75.2  3.8, respectively). However, these rates were similar when it was used just heparin and when oocytes were incubated with 40Âg/mL of BSP1 (78.9  1.7 vs 75.2  3.8%). For blastocyst rates was found that they were similar (p> 0.05) when the selection was made with heparin (44.1  4.3%) and 40 Âg/mL of BSP1 (30.0  3.3%). Finally, 1213 cumulus-oocyte complexes were recovered and incubated with freeze-thawed epididymal semen in Fert-TALP medium, which contained: only heparin, with heparin, and 10, 20 or 40 Âg/mL of BSP1. Cleavage and blastocyst rates were similar after incubation with and without heparin, but the concentration of 40Âg/mL of BSP1 produced higher cleavages rates (79.0  1.1%) than control 1 (68.5  1.3%, p <0.05). About blastocyst rates, there were rates significantly higher when concentrations of 20 and 40 Âg/mL of BSP1 were used (35.6  2.5 and 41.1  2.0%) than with (24.7  3.2; p <0.05) and without heparin (27.3  1.6%; p <0.0003). In conclusion, BSP1 allowed proper cleavage and development, when the protein was added to the fertilization medium and the Percoll gradient using ejaculated semen, and to the fertilization medium when used epididymal sperm, without requiring the use of heparin. / A âBinder of Sperm Protein 1â (BSP1) à uma proteÃna produzida pelas vesÃculas seminais dos animais, que ajuda no processo de fertilizaÃÃo do oÃcito promovendo a capacitaÃÃo espermÃtica, mediando a sua ligaÃÃo com o epitÃlio do oviduto e aumentando sua motilidade no oviduto. As taxas de reaÃÃo de acrossoma dos espermatozoides foram determinadas ao serem incubados com BSP1 purificado, e com e sem a presenÃa de heparina, como controles. A porcentagem de reaÃÃo de acrossoma foi maior (p<0.05) no controle 1 (44.5%) comparado com os tratamentos restantes (BSP1: 10 Âg/mL: 24.5%; 20 Âg/mL: 25.5%; 40 Âg/mL: 26.0%), sendo menor esta taxa (p>0.05) quando os espermatozoides sà foram incubados no meio Fert-TALP sem heparina (14.5%). As taxas de clivagem e de blastocistos foram avaliadas nos dias dois e sete respectivamente, depois de fertilizar oÃcitos em um meio que continha BSP1. Foram recuperados 1274 COCs e incubados durante 18 horas com sÃmen ejaculado congelado- descongelado em meio Fert-TALP, que continha: sà heparina, 10, 20 ou 40 Âg/mL de BSP1. As taxas de clivagem e de blastocistos (34.1  4.4 vs 40.8  5.0%) foram similares (p>0.05) quando se fez a incubaÃÃo com 10 Âg/mL de BSP1 e heparina, respectivamente. PorÃm, as concentraÃÃes de 20 e 40 Âg/mL diminuÃram a formaÃÃo de blastocistos (22.4  2.9 e 19.3  4.1%) em relaÃÃo com a heparina (40.8  5.1%; p < 0.02). Posteriormente foram incubados COCs (n= 2239), com sÃmen ejaculado congelado-descongelado que foi selecionado por um gradiente de Percoll 45%-90% que continha: sà heparina, 10, 20 ou 40 Âg/mL de BPS1 e foi realizado um controle 2 de Percoll sem heparina nem BSP1. A taxa de clivagem foi similar (p > 0.05) para a incubaÃÃo com as concentraÃÃes de 10, 20 e 40 Âg/mL de BSP1 (67.7Â3.0, 68.7Â3.5, 75.2Â3.8). PorÃm, estas taxas foram similares quando sà foi usada heparina e quando se incubou com 40 Âg/mL de BSP1 (78.9Â1.7 vs 75.2Â3.8%). Para as taxas de blastocistos encontrou-se que foram similares (p > 0.05) quando se fez a seleÃÃo com heparina (44.1Â4.3%) e com 40 Âg/mL de BSP1 (30.0Â3.3%). Finalmente, foram recuperados 1213 complexos cumulus-oÃcitos e incubados com espermatozoides epididimÃrios congelados-descongelados em meio Fert-TALP, que continham: sà heparina, sem heparina, e 10, 20 ou 40 Âg/mL de BSP1. As taxas de clivagem e de blastocistos foram similares apÃs as incubaÃÃes com e sem heparina, mas a concentraÃÃo de 40 Âg/mL de BSP1 produziu maiores taxas de clivagem (79.0  1.1%) que o controle 1 (68.5  1.3%; p < 0.05). Quanto Ãs taxas de blastocistos, encontraram-se maiores taxas quando foram usadas as concentraÃÃes de 20 e 40 Âg/mL de BSP1 (35.6  2.5 e 41.1  2.0%) que com (24.7  3.2; p < 0.05) e sem heparina (27.3  1.6%; p < 0.0003). Em conclusÃo, a BSP1 adicionada ao meio de fertilizaÃÃo e ao gradiente de Percoll quando usado o sÃmen ejaculado, e ao meio de fertilizaÃÃo quando usados espermatozoides epididimÃrios, permitiu clivagem e desenvolvimento prÃprio, sem a necessidade do uso de heparina.

Les aciers ODS (Oxide Dispersion Strengthened) sont envisagés comme matériau de gainage du combustible pour les futurs réacteurs nucléaires au sodium de Génération IV. Ces matériaux présentent une excellente résistance au fluage à haute température et au gonflement sous irradiation, mais des interrogations subsistent quant à leur propriété de résilience. L'objectif de ce travail de thèse est alors de comprendre l'effet de différents paramètres (composition chimique, texture, vieillissement thermique…) sur le comportement à l'impact des aciers ODS et sur l'apparition de la rupture de type fragile. L'objectif à terme est d'appréhender l'apparition de ce type de rupture afin d'assurer l'intégrité des composants en acier ODS en conditions normales ou incidentelles.Dans un premier temps, cette étude a permis d'évaluer la stabilité de deux nuances d'acier ODS contenant 14%Cr et 18%Cr dans des conditions de vieillissement thermique. La nuance à 18%Cr a été écartée à cause de la formation d'une phase intermétallique fragilisante de type sigma à 600°C. Par contre, la nuance à 14%Cr est plus prometteuse puisque son comportement est resté stable entre 400°C et 600°C pour une durée de vieillissement maximale de 10000 heures, et ce même si des précipités de phases alpha', de Laves Fe2W et des carbures de chrome ont été observés. D'autre part, la texture morphologique, caractérisée par des grains allongés selon la direction de filage, est propice à la propagation de fissures intergranulaires selon cette direction. La texture cristallographique régit, quant-à elle, les micromécanismes de clivage. En effet, les entités microstructurales qui contrôlent la propagation du clivage sont des groupes de grains faiblement désorientés les uns des autres d'un point de vue cristallographique, et qui sont associés à la notion de grains effectifs. Enfin, le comportement en traction et en flexion d'une nuance d'acier ODS à 14%Cr a été modélisé, et un critère de rupture fragile basé sur une valeur de contrainte critique a été proposé. Ce modèle nous a alors permis de simuler des essais sur différentes géométries et de prédire l'apparition de la rupture fragile sur cette nuance. / The Oxide Dispersion Strengthened (ODS) steels have been identified as potential materials for fuel cladding in Generation IV nuclear reactors. They are characterized by a very good resistance to swelling under irradiation and to high temperature creep, but questions still remain about the impact toughness of these materials. The first aim of this work is to understand the effects of different parameters (chemical composition, texture, thermal ageing...) on the impact behaviour of ODS steels. The final objective is to predict the occurrence of the brittle failure on the ODS steel components in normal or incidental conditions.Firstly, this study assesses the stability of two ODS steel grades containing either 14%C or 18%Cr during thermal ageing. The 18%Cr steel grade has been discarded due to the growing of the brittle intermetallic sigma phase at 600°C. However, the 14%Cr steel grade showed a promising behaviour given the stability of its mechanical properties after thermal ageing between 400°C and 600°C, for a maximum duration of 10000 hours, and regardless of the presence of chromium carbides, alpha' and Laves phases. Then, the morphological texture characterized by elongated grains along the extrusion direction enhances the propagation of intergranular cracks along this direction. Meanwhile, the crystallographic texture controls the cleavage micromechanisms. Indeed, the microstructural entities that control the propagation of the cleavage crack are groups of grains which are characterized by low crystallographic misorientations and referred to as effective grains. At last, the tensile and bending behaviour of a 14%Cr ODS steel grade has been modelled. A brittle failure criterion based on a critical stress value had also been suggested. This model allowed us to simulate mechanical tests on different geometries and to predict the occurrence of the brittle failure.

L’objectif de cette thèse était de développer et d’optimiser de nouvelles méthodologies et approches analytiques afin d’améliorer le potentiel de l’analyse protéomique pour les études biologiques.La première partie de ce travail est consacrée à la détermination massive et exacte de la position N-Terminale des protéines (N-Terminome). Pour cela, nous avons utilisé et développé une approche basée sur une dérivation N-Terminale au TMPP. Cette méthodologie de marquage de la position N-Terminale a permis d’aborder l’étude des clivages protéolytiques des protéines exportées par le parasite P. falciparum (pathogène de la malaria) dans le globule rouge.Afin de permettre une exploitation automatique à haut débit des données de MS/MS, nous avons élaboré une nouvelle méthodologie (dénommée dN-TOP). Celle-Ci repose sur l’utilisation de TMPP portant des isotopes stables et permet ainsi d’accéder à la détermination des positions N-Terminales pour des études de N-Terminome à large échelle.La seconde partie est dédiée aux développements de différentes stratégies analytiques de quantification, aussi bien au niveau peptidique qu’au niveau protéique, appliquées à une série de problématiques biologiques. Ces optimisations ont été réalisées dans le contexte de l’étude des complexes protéiques, du dosage de prion par SRM, de quantification des glycations d’anticorps monoclonaux thérapeutiques et de l’hémoglobine HbA2 pour la standardisation des méthodes de référence. / The objective of this Ph.D. thesis was to develop and optimize new methodologies and analytical approaches to improve the potential of the mass spectrometry based proteomics.The first part of this work focused on the development of the N-Termini proteomics. This topic was addressed with a specific N-Termini chemical derivatization based on TMPP. We have shown that our method allowed both specific N-Terminomics and classical proteomics studies in the same experiment.This N-Terminus methodology was applied to study the proteolytic cleavages of the exported proteins in P. falciparum, a parasite responsible for the malaria.In order to automatize the complex and tedious informatics processsing of the MS/SM data of ourTMPP based N-Terminomics method, we have introduced a new approach (named dN-TOP), based on the use of a stable isotope labeled TMPP which made now N-Terminome proteomics compatible with high throughput studies.The second part addresses quantitative aspects of proteomics. It describes the optimization of quantitative methods at the peptide level or at the protein level for five different proteomic studies in the context of protein complex subunits, targeted SRM based prion, quantification of monoclonal antibodies glycation and hemoglobin HbA2 for reference measurement methods standardization.

No description available.

Restriction modification (R-M) systems are important components of the prokaryotic arsenal against invading genomes. R-M systems directly target the foreign DNA and are often considered as primitive immune systems in bacteria. The defense system comprises of two contrasting enzymatic activities – a restriction endonuclease (REase) and a methyltransferase (MTase). Functionally, REases cleave a specific DNA sequence endonucleolytically at the phosphodiester bonds generating 5' or 3' overhangs or blunt ends. MTases catalyze the transfer of a methyl group from S-adenosyl-Lmethionine to adenine or cytosine. Four types of R–M systems are found in bacteria, viz., Types I, II, III and IV. Type II R-M systems, comprising of a separate REase and MTase, are the most abundant and well-studied enzymes. Type II REases recognize and cleave DNA within or near their recognition sequences. Surprisingly, these enzymes share little or no sequence homology amongst them. All the enzymes identified so far can be grouped into conventional PD-(D/E)XK, ββα-Me, GIY-YIG, phospholipase-derived and half-pipe endonucleases according to their folds and active site structures. Owing to their high specificity and defined cleavage pattern, they have become indispensable tools in molecular biology and have been widely exploited for studying protein–DNA interactions. The work presented in this thesis deals with R.KpnI, which belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. The REase isolated from Klebsiella pneumoniae recognizes the palindromic DNA sequence GGTAC/C and cleaves DNA as indicated. The enzyme is unique in exhibiting promiscuous DNA cleavage in the presence of Mg2+, a natural co-factor for a vast majority of REases. Surprisingly, Ca2+ and Zn2+ completely suppress the Mg2+ mediated promiscuous activity and induce high fidelity cleavage. These unusual features of R.KpnI led to the functional characterization of the ββα-Me finger active site motif. In addition, the studies were aimed at understanding the mechanism and the biological significance of substrate and co-factor promiscuity exhibited by the enzyme. The salient aspects of the thesis are summarized below. A general introduction and overview of the literature on structure-function studies, mechanism of recognition and catalysis by REases with special emphasis on Type II enzymes is presented in the Chapter 1. An account of co-factor specificity in REases, role of metal ions in DNA binding as well as in phosphodiester bond hydrolysis is provided. The various aspects of R-M systems that target the invading DNA elements and counter strategies employed by the foreign genomes to evade the restriction are also covered. The new developments that provide insights in understanding the diversity of R-M systems and additional biological roles that could increase the fitness of the host organism harboring them are described. The features of substrate and metal ion specificity in REases and the efforts undertaken to alter the specificity have been dealt at the end of the chapter. From the structures of the several ββα-Me finger nucleases, the α-helix has been implicated in providing a structural scaffold for the correct juxtapositioning of the catalytic residues. However, no mutagenesis data exists to delineate its role. Homology modeling studies of R.KpnI suggested a crossover structure for the α-helix of the ββαMe finger active site motif, which could possibly form dimeric interface and/or structural scaffold for the active site. Chapter 2 describes the computational modeling and mutational analysis performed to understand the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. Mutation of the residues present in the α-helix lead to the loss of the enzyme activity, but not dimerization ability. Subsequent biophysical experiments showed that the α-helix of the ββα-Me finger of R.KpnI plays an important role for the stability of the protein–DNA complex needed for its function. In Chapter 3, unusual co-factor flexibility for R.KpnI is shown by using a battery of divalent metal co-factors differing in ionic radii and coordination geometries. A number of alkaline earth and transition group metal ions function as co-factors for DNA cleavage. The metal ions replaced each other readily from the enzyme’s active site revealing the active site plasticity. Mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all the co-factors indicating that the enzyme follows single metal ion mechanism for DNA cleavage. The indispensability of the invariant His in nucleophile activation together with the broad co-factor tolerance of the enzyme indicated the role of metal ions in electrostatic stabilization during catalysis. At higher concentrations, Mg2+, Mn2+ or Co2+ stimulate promiscuous cleavage while Cd2+, Ni2+ or Zn2+ inhibit phosphodiester bond hydrolysis. The underlying molecular mechanisms for the modulation of the enzyme activity by the metal ion binding to the second site are presented. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The identification of additional metal ion binding residues would help in engineering REase variants with enhanced activity and/or specificity. Chapter 4 describes the generation of an R.KpnI variant with altered co-factor specificity by exploiting the active site plasticity of the enzyme. The mutant enzyme is a Mn2+ -dependent endonuclease defective in DNA cleavage with Mg2+ and other divalent metal ions. In the engineered mutant, only Mn2+ is selectively bound at the active site, imparting in vitro activity while being dormant in vivo. In addition to the Mn2+ selectivity, the mutant is impaired in concerted double-stranded DNA cleavage leading to the accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altering the reaction conditions. Thus, a single point mutation in the active site of R.KpnI generates a Mn2+ -dependent REase and a sequence specific nicking endonuclease. The potential applications of such enzymes engineered for selective metal ion dependent activities have been discussed. R.KpnI is peculiar in retaining robust promiscuous cleavage despite being a typical Type II REase in all other characteristics. Chapter 5 presents results of the growth properties and phage titer analysis carried out with R.KpnI and its high fidelity variant to understand the biological significance of promiscuous activity. The enzyme isolated from the K. pneumoniae exhibited biochemical properties similar to that of R.KpnI overexpressed in E.coli. It was observed that the wild type but not the high fidelity variant could effectively restrict bacteriophages methylated at GGTACC. These results show that the REase exhibits promiscuous activity in vivo, which would be advantageous for the organism to better target the incoming foreign DNA. The promiscuous behavior of the R.KpnI could be one of the counter strategies employed by the bacteria against the constantly evolving phages in the co-evolutionary arms race. In conclusion, the work described in this thesis provides new insights about structure, function and biology of REases in general and R.KpnI in particular. The co-factor and substrate promiscuity of R.KpnI may indicate its evolutionarily intermediate form that is yet to attain a high degree of specificity. Alternatively, it is possible that this unique feature is retained during the evolution of the HNH REases serving some unknown function(s) in the cell, in addition to having an edge in countering the phage infections.

L'activation de petites molécules azotées telles que l'azote et l'ammoniac a été développé dans notre laboratoire via la chimie organométallique de surface (COMS). Les recherches effectuées durant cette thèse ont permis d'établir la réactivité de complexe de tantale imido amido supporté sur silice, [(SiO)2Ta(=NH)(NH2)] vis-à-vis de l'hydrogène et de l'ammoniac. Des étapes élémentaires de clivage hétérolytique de liaison H-H ou N-H ont été établies. En particulier, l'importance d'une molécule d'ammoniac dans la deuxième sphère de coordination (outer sphere assistance) du système s'est avérée cruciale pour la diminution des barrières d'énergie des états de transition pendant le transfert de protons. Les études ont été faites pour déterminer et expliquer le mécanisme de réduction de N2 par les complexes d'hydrures de tantale. La compréhension du mécanisme a été établie grâce aux études avec N2, N2H4 et N2H2 pour trouver les intermédiaires de cette réduction suivis par in-situ infrarouge, RMN et l'analyse élémentaire, et à l'aide de calcul DFT. Un mécanisme de clivage de N2 par des complexes dihydrogènes de Ta(V) est proposé. Enfin, la réactivité du complexe [(SiO)2Ta(=NH)(NH2)] vers l'activation de liaison C-H de C6H6, C6H5-CH3, t-Bu-Ethylène et CH4 a été étudié par la spectroscopie infrarouge / The activation of small molecules such as nitrogen and ammonia was already developed in our laboratory using the surface organometallic chemistry (SOMC) approach. This thesis focused on understanding the reactivity of tantalum imido amido complex [(SiO)2Ta(=NH)(NH2)], under hydrogen and/ or ammonia atmosphere. Heterolytic H-H and N-H cleavage across Ta-NH2 and Ta=NH bonds appeared crucial. The assistance of an additional ammonia molecule in the outer sphere of the d0 tantalum(v) imido amido ammonia model complex in order to reduce the energy barriers of the transition states during proton transfer was also shown. Studies were done to identify the mechanism of N2 reduction by tantalum hydride complexes. Studies with N2, N2H4 and N2H2 allowed identifying the intermediaries via in situ IR, NMR and elemental analysis. Combined with DFT calculations, these experiments led to the proposal of a novel mechanism for N2 cleavage based on the central role of Ta(H2) adducts. Finally, the reactivity of imido amido complex toward C-H bond activation was studied with C6H6, C6H5-CH3, t-Bu-Ethylene and CH4

京都大学 / 新制・課程博士 / 博士(情報学) / 甲第23397号 / 情博第766号 / 新制||情||131(附属図書館) / 京都大学大学院情報学研究科知能情報学専攻 / (主査)教授 阿久津 達也, 教授 山本 章博, 教授 鹿島 久嗣 / 学位規則第4条第1項該当 / Doctor of Informatics / Kyoto University / DFAM

This thesis concentrates on the methodology of semiconductor samples preparation for low voltage scanning electron microscopy. In the first part a detailed theory of sample imaging using electron beam and difference between classical scanning electron microscopy (SEM) and low voltage scanning electron microscopy (LVSEM) is described. It is given a description of a contrast formation in SEM and LVSEM and theories describing a contrast formation of differently doped semiconductors. The second part contains experimental data. The advantages and disadvantages of cleavage and focused ion beam (FIB) milling as sample preparation techniques are discussed. FIB was found as the best method for sample preparation for the analysis of precisely defined location on the sample. It is necessary to use the lowest possible FIB accelerating voltage for final polishing, ideally 1 kV.