Dans les technologies d'aujourd'hui, l’épitaxie est une technique indispensable pour la fabrication des composants. Avec la diminution continue de la taille des transistors les objets epitaxiés rétrécissent aussi. Par conséquence, des effets morphologiques qui sont négligeables à grande échelle, doivent être considéré dans les petits motifs, et de plus des anisotropies doivent être prises en compte. C'est pour cela que cette thèse est consacrée à l'étude de la morphologie en fonction de la taille et de l'orientation des motifs. La caractérisation de la morphologie du SiGe comme déposé sur des motifs orientés selon <100> et <110> nous conduit à introduire de nouveaux effets de charge, pas encore reportés dans la littérature. Après avoir étudié en profondeur la morphologie après croissance, les épitaxies sont soumises à des températures légèrement supérieures à celle de dépôt, et les changements sont discutés en fonction de l'orientation et de la largeur des lignes. Des recuits sous H2 à des températures plus élevées sont réalisés sur des motifs différents ce qui permet l'observation des effets morphologiques en bord et en coin de motif. Ces effets dominent la morphologie globale des couches epitaxiées quand la taille des motifs diminue. En particulier, la stabilité des lignes de Si et SiGe lors des recuits est étudiée, ce qui permet de déterminer les facteurs importants pour la stabilité des lignes. Dans des expériences supplémentaires un procédé est développé pour augmenter la stabilité thermique des couches SiGe. En outre, l'épitaxie cyclique - nécessaire pour réaliser les sources/drains des CMOS avancés - est discutée. L'influence des changements dans l'étape de gravure d'un procédé cyclique de Si, en gardant l'étape de dépôt inchangée, est étudiée pour des motifs orientés selon <100>. Nous avons trouvé des conditions dans lesquelles la couche n'est plus continue. Des expériences pour étudier la gravure séparément permettent d'expliquer les phénomènes observés. / In current technology nodes, epitaxy is an indispensable technique in device fabrication. With the continuous decrease of the transistor size, the epitaxial objects shrink as well. As a consequence, morphology effects which can be neglected at the large scale, have to be considered in small patterns and in addition, anisotropies have to be taken into account. Therefore, this thesis is dedicated to morphology studies as a function of pattern size and orientation. The characterization of the SiGe morphology in the as-deposited state on <100> and <110> oriented patterns leads to the introduction of new loading effects, which have not been reported elsewhere so far. After having studied thoroughly the as-deposited morphology, the epitaxial layers are exposed to a temperature slightly higher than the deposition temperature and the changes are discussed as a function of line width and orientation. H2 annealing at higher temperatures are performed with various Si and SiGe patterns leading to the observation of morphology effects at the pattern edges and corners. These effects dominate the global layer appearance with decreasing pattern size. In particular, the stability of annealed Si and SiGe lines is studied which allows to determine the crucial factors for line stability. In additional experiments, a process is developed which can increase the thermal stability of epitaxial SiGe. Moreover, cyclic epitaxy - required for sources/drains of advanced CMOS devices - is discussed. The influence of changes in the etch step of a cyclic Si process, by keeping the deposition step unchanged, is studied for <100> oriented patterns. Conditions are found, where cyclic epitaxy results in a discontinuous layer. Experiments, which consider the etching separately can explain the observed phenomena.

Cette thèse est dédiée à la pré-calibration des nouveaux systèmes d’aides à la conduite (ADAS). Le développement de ces systèmes est devenu aujourd’hui un axe de recherche stratégique pour les constructeurs automobiles dans le but de proposer des véhicules plus sûrs et moins énergivores. Cette thèse contribue à une vision méthodologique multi-critère, multi-modèle et multi-scénario. Elle en propose une instanciation particulière pour la pré-calibration spécifique au Lane Centering Assistance (LCA). Elle s’appuie sur des modèles dynamiques de complexité juste nécessaire du véhicule et de son environnement pour, dans le cadre du formalisme H2/H∞, formaliser et arbitrer les compromis entre performance de suivi de voie, confort des passagers et robustesse. Les critères élaborés sont définis de manière à être d’interprétation aisée, car directement liés à la physique, et facilement calculables. Ils s’appuient sur des modèles de perturbations exogènes (e.g. courbure de la route ou rafale de vent) et de véhicules multiples mais représentatifs, de manière à réduire autant que possible le pessimisme tout en embrassant l’ensemble des situations réalistes. Des simulations et des essais sur véhicules démontrent l’intérêt de l’approche. / This thesis deals with the tuning of the new Advanced Driving Assistance Systems (ADAS). The development of these systems has become nowadays a strategic line of research for the automotive industry towards the conception of safer and fuel-efficient vehicles.This thesis contributes to a multi-criterion, multi-modeland multi-scenario methodological vision of the tuning process. It is presented through a specific application of the tuning of the Lane Centering Assistance (LCA). It relies on vehicle and environment’s dynamical models of adequate complexity in the aim of formalizing and managing, in a H2/H∞ framework, the trade-off between performance, comfort and robustness. The formulated criteria are easy to compute and defined in a way to be understandable, closely linked to practical specifications. The whole methodology is driven by the research of a pertinent trade-off between realism (being as closest as possible to reality) and complexity (quick evaluation of the criterion). The efficiency and the robustness of the approach is demonstrated through high-fidelity simulations and numerous tests on real vehicles.

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.

À ses débuts, la conquête spatiale a pu bénéficier des rivalités politiques de la Guerre Froide pour se développer rapidement sans réellement se soucier des efforts économiques à fournir. Aujourd’hui, de nombreux pays subissent le revers de la médaille de cette course effrénée : pour maintenir une flotte de lanceurs viable économiquement, les différentes agences spatiales doivent faire face à un dilemme opposant la minimisation des coûts de lancement à la maximisation de leur fiabilité. Dans cette logique d’optimisation, les industriels présents dans ce processus de réflexion se tournent vers la simulation numérique pour tenter d’améliorer leurs connaissances des technologies existantes, en particulier sur les zones d’ombres inaccessibles aux mesures expérimentales. Dans la lignée de plusieurs études théoriques et expérimentales, ces travaux visent à apporter un éclairage nouveau sur les phénomènes se produisant lors de l’allumage d’un moteur fusée cryotechnique. Ces recherches se tournent dans un premier temps vers l’amélioration de la modélisation de la flamme H2/O2. La validation d’une cinétique chimique réduite initialement destinée à la combustion H2/Air permet de justifier son utilisation lors de l’allumage. Puis, le développement d’un modèle de combustion turbulente pour le régime de flamme de diffusion est mené dans le but de palier aux limitations du modèle de flamme épaissie. Enfin, une analyse du cas où les régimes prémélangés et non-prémélangés sont présents tous les deux permet d’étudier un moyen simple de les distinguer même dans le cas où ils sont très proches. Dans un second temps, ces travaux se tournent vers l’étude de l’allumage dans un moteur fusée cryotechnique. Après avoir analysé de manière globale le calcul d’une séquence simplifiée, deux études plus approfondies sont menées pour investiguer, d’une part, les différents régimes de combustion, et d’autre part, les différents modes de propagation de la flamme propres à cette configuration. / The beginning of the conquest of space received benefits from the political competition of the Cold War and consequently grow quickly without considering the cost of these advances. The end of this unrestrained technological race brings to light the other side of the coin. In order to keep a fleet of launch vehicles up-to-date with the market, spatial agencies must answer a question : how can the cost of a launch be reduced without decreasing its efficiency. Through the use of numerical simulation, industrial partners may investigate this logic of optimisation. This solution might provide improvement in the knowledge of existing technologies, especially when experimental measurements are impossible. Following the path of theoretical and experimental results, this study aims to present a new view about the different processes occurring during the ignition of a space rocket engine. First, this research will present an improvement of the modelling of H2/O2 flame. The validation of a reduced chemical scheme basically developed for H2/Air will justify its use during the ignition sequence. Then, a turbulent combustion model for non-premixed flames will be developed in order to compensate the limits of the thickened flame model implemented in AVBP. Additionally, a study of both premixed and non-premixed regimes in a closed position will bring a simple method to distinguish them for a further active use. Secondly, this research will study the ignition process of a representative cryogenic space rocket chamber. The calculation of a simplified ignition sequence will be globally investigated. Finally, two-detailed analysis will lead to different combustion regimes and flame spreading processes

A produ&ccedil;&atilde;o de energia &eacute; um dos grandes desafios deste s&eacute;culo, principalmente com a necessidade no desenvolvimento de processos que utilizem preferencialmente fontes renov&aacute;veis. Nesse contexto, &eacute; claro o interesse por pesquisas baseada no hidrog&ecirc;nio. Por&eacute;m, os ganhos ambientais efetivos est&atilde;o associados tamb&eacute;m &agrave; mat&eacute;ria-prima usada no processo de gera&ccedil;&atilde;o do hidrog&ecirc;nio, senso assim mais significativo quando do uso de fontes renov&aacute;veis. No presente trabalho foi estudado o efeito da adi&ccedil;&atilde;o de diferentes teores de CeO2 em catalisadores de NiZnO, preparados pelo m&eacute;todo da co-precipita&ccedil;&atilde;o. Os catalisadores foram estudados frente &agrave; rea&ccedil;&atilde;o de reforma a vapor do etanol (RVE) para produ&ccedil;&atilde;o de H2, e no decorrer do trabalho foi pertinente incluir o estudo da reforma a vapor da acetona (RVA), como complemento dos estudos da RVE. Al&eacute;m disso, esta &eacute; uma mol&eacute;cula modelo para reforma a vapor do bio-&oacute;leo. Nos sistemas catal&iacute;ticos sintetizados houve a forma&ccedil;&atilde;o da liga NiZn3, o que aparenta proporcionar um efeito sin&eacute;rgico entre esses elementos. Observou-se um efeito altamente promotor do CeO2 com rela&ccedil;&atilde;o a diminui&ccedil;&atilde;o do coque formado, devido a uma menor da forma&ccedil;&atilde;o da acetona, durante as etapas de reforma do etanol. O que consequentemente interferiu na produ&ccedil;&atilde;o de H2. O catalisador contendo 20% de CeO2 denominado NiZn20Ce apresentou um desempenho altamente promissor, pois de acordo com as an&aacute;lises de DRIFT, a presen&ccedil;a do CeO2 levou a forma&ccedil;&atilde;o de esp&eacute;cies de formiato, o que consequentemente interferiu em uma menor forma&ccedil;&atilde;o de coque e maior produ&ccedil;&atilde;o de H2. Foi comprovado tamb&eacute;m que a natureza dos dep&oacute;sitos carbon&aacute;ceos depende tanto do substrato utilizado quanto das etapas precursoras inicias que levar&atilde;o a forma&ccedil;&atilde;o desse coque, sendo o ponto chave para um melhor desempenho do catalisador. / Energy production is one of the great challenges of this century, especially with the need to develop processes that use preferentially renewable sources. In this context, it&acute;s evident the interest in research based on hydrogen. However, the environmental benefits are also associated with the raw material used on the hydrogen generation process, being more significant when used renewable sources. In the present work, it was studied the effect of CeO2 addition in NiZnO catalysts, prepared by coprecipitation method. The catalysts were studied front of the ethanol steam reforming reaction (ESR) to produce H2, and in the course of this study was relevant include study of acetone steam reforming (ASR), as a complement of ESR studies. Moreover, acetone is a molecule model for steam reforming of bio-oil. In the catalyst systems there has been the NiZn3 alloy formation, which provides a synergistic effect between these elements. It was observed a high CeO2 promoter effect regarding reduction of the formed coke, due to lower formation of acetone in the ethanol reforming steps, and consequently interfere in the H2 production. The catalyst denominated NiZn20Ce showed a high performance, according to DRIFT analyzes, the presence of the CeO2 led to the formation of species formate, which in turn interfere through a lower coke formation and higher H2 production. It was also established that the nature of the carbonaceous deposits depends as the substrate used as the precursors initial steps that led the formation of this coke, and it is the key to a better performance of the catalyst.

Un connecteur électrique automobile est le siège de points de contact électriques soumis à des échauffements supplémentaires par effet Joule provoqués par leurs résistances de contact. La dégradation d’une résistance de contact peut provoquer des échauffements critiques au niveau des points de contact. La présente étude propose deux approches pour détecter les variations anormales des résistances de contact. La première, appliquée à un connecteur de boîtier électronique automobile, repose sur un diagnostic à base de modèle afin de détecter les variations anormales de la résistance de contact par rapport à sa valeur nominale. La seconde approche,appliquée à un connecteur de recharge de véhicule électrique, consiste d’abord à estimer un flux de chaleur lié à l’effet Joule provoqué par la résistance de contact. La valeur de cette dernière est ensuite estimée grâce à la méthode des moindres carrés. Ces deux approches reposent sur des modèles électrothermiques des connecteurs considérés et de leurs environnements. Cette étude présente également le développement de ces modèles, et notamment l’optimisation du maillage d’un fil électrique basée sur la minimisation de la norme H2 de l’erreur entre un modèle maillé d’ordre entier et un modèle analytique de référence d’ordre non entier. / An automotive electrical connector contains electrical contact points subject to additional temperature rises by Joule effect caused by their contact resistances. The deterioration of a contact resistance can cause critical overheating at the contact points. This study proposes two approaches to detect abnormal changes in contact resistance. The first one, applied to an automotive electric case connector, relies on a model-based diagnosis to detect an abnormal variation in the contact resistance with respect to its nominal value. The second one, applied toan electric vehicle charging connector, consists in estimating a heat flow related to the Joule effect caused by the contact resistance. The value of the latter is then estimated using the least squares.These approaches rely on electrothermal models considered connectors and their environments.This study also presents the development of these models, including the optimization of the mesh of an electrical wire based on the minimization of the H2-norm of the error between ameshed integer order model and a reference analytical fractional order model.

Les systèmes implicites (dits aussi « descripteurs ») peuvent décrire des processus régis à la fois par des équations dynamiques et statiques et permettent de préserver la structure des systèmes physiques. Ils comportent trois types de modes : dynamiques finis, infinis (réponse temporelle impulsive (en cas continu) ou acausale (en cas discret)) et statiques. Dans le cadre du formalisme descripteur, les contributions de cette thèse sont triples : i) revisiter des résultats existants pour les systèmes d’état, ii) étendre certains résultats classiques au cas des systèmes implicites, iii) résoudre rigoureusement des problèmes de commande non standard. Ainsi, le présent mémoire commence par revisiter les résultats concernant la caractérisation LMI stricte de la dissipativité, les caractérisations de l’admissibilité et des performances H2 ou H∞ par LMI étendues et les équations de Sylvester et de Riccati généralisées. Il aborde dans un deuxième temps, le problème de stabilisation simultanée, avec ou sans critère H∞, à travers l’extension de certains résultats récents au cas des systèmes implicites. La solution proposée s’appuie sur la résolution combinée d’une équation algébrique de Riccati généralisée (GARE) et d’un problème de faisabilité sous contrainte LMI stricte. Il traite enfin des problèmes H2 et H∞ non standards : i) en présence de pondérations instables voire impropres, ii) sous contraintes de régulation; dans le cas des systèmes implicites. Ces dernières contributions permettent désormais de traiter rigoureusement, sans approximations ou transformations, de nombreux problèmes H2 ou H∞ formalisant des problèmes pratiques de commande, dont ceux faisant intervenir une pénalisation haute fréquence de la commande ou un modèle interne instable des signaux exogènes. / The descriptor systems have been attracting the attention of many researchers over recent decades due to their capacity to preserve the structure of physical systems and to describe static constraints and impulsive behaviors. Within the descriptor framework, the contributions of this dissertation are threefold: i) review of existing results for state-space systems, ii) generalization of classical results to descriptor systems, iii) exact and analytical solutions to non standard control problems. A realization independent Kalman-Yakubovich-Popov (KYP) lemma and dilated LMI characterizations are deduced for descriptor systems. The solvability and corresponding numerical algorithms of generalized Sylvester equations and generalized algebraic Riccati equations (GARE) associated with descriptor systems are provided. In addition, the simultaneous H∞ control problem is considered through extending recently reported results. A sufficient condition is proposed through a combination of a generalized algebraic Riccati equation and a set of LMIs. Moreover, the nonstandard H2 and H∞ control problems with unstable and/or nonproper weighting functions or subject to regulation constraints are addressed. These contributions allow, without approximation or transformation, dealing with many practical problems defined within H2 or H∞ control methodologies, where the control signals are penalized at high frequency or unstable internal models specified by external signals is involved.

[pt] Os materiais nanoestruturados têm sido estudados ao longo das últimas décadas, por apresentarem propriedades particulares, promissoras propriedades térmicas, mecânicas e catalíticas, que muitas vezes não estão presentes no material não manométrico. Alguns avanços recentes têm mostrado que estas propriedades podem ser reforçadas pela inclusão de materiais com propriedades diferentes na sua estrutura, formando assim nanocompósitos. Por exemplo, as ligas de CuNi são muito dúcteis, mas a presença de nanopartículas de Al2O3 depositadas na matriz pode melhorar consideravelmente a dureza do material. Tal nanocompósito pode ser obtido, por exemplo, através de decomposição térmica nitratos, seguido por redução seletiva com hidrogênio. Nesse contexto, o presente trabalho tem como foco a síntese de ligas de CuNi e CuNi com adição de nanoparticulas de Al2O3, baseados na redução seletiva de CuO e de NiO com H2, e óxidos coformados com o óxido de alumínio por meio da decomposição térmica de seus nitratos metálicos. Cálculos termodinâmicos mostraram que a redução de Cu e Ni pode ser realizada a temperaturas relativamente baixas (400 mais ou menos 5 Graus C), e também que o processo se desenvolve seletivamente (apenas os óxidos de Ni e de Cu reagem nas condições impostas), resultando em compósitos de CuNi/Al2O3, que consiste na formação de uma liga CuNi contendo 1 por cento de Al2O3 como finas nanopartículas distribuídas homogeneamente. Precursores e amostras reduzidas foram caracterizados por difração de raios x (DRX) para determinar a natureza das fases individuais presentes (óxidos e ligas), microscopia electrónica de varredura (MEV) como uma primeira aproximação da morfologia das partículas e microscopia eletrônica de transmissão (MET). Os resultados obtidos indicam que a via química proposta resultou satisfatória para a elaboração das ligas CuNi contendo nanopartículas de Al2O3 homogeneamente distribuídas. Os resultados obtidos também indicam que, para as condições experimentais impostas tanto a decomposição dos nitratos como as reações de redução alcançaram conversões de 100 por cento. / [en] Materials containing nanostructured particles have been studied over the last decades in order to take advantage of their promising thermal, mechanical and catalytic properties. Some recent progress has shown that these properties can be further enhanced by the inclusion of materials with different properties in their structure, thereby forming nanocomposites. For instance, Ni-Cu alloys are highly ductile, but the presence of Al2O3 nanoparticles deposited inside the alloy matrix can considerably improve the material s hardness. Such a nanocomposite can be obtained, for example, through nitrate solutions thermal decomposition followed by selective reduction with hydrogen. In this context, the present work focuses on the synthesis of CuNi alloys and CuNi/Al2O3 composites based on the selective reduction of copper and nickel oxides with pure H2, co-formed with aluminum oxide through thermal decomposition of aqueous solutions of their metal nitrates. Thermodynamic computations showed that the Cu and Ni reduction can be accomplished at relatively low temperatures (400 plus or minus 5 C degrees), and also that the process develops selectively (only the oxides of Ni and Cu react at the imposed conditions), resulting in Cu-Ni-Al2O3 composites, consisting in a Cu-Ni alloy crystals containing 1 percent of Al2O3 as fine homogeneously distributed nanoparticles. Both the original (co-formed) and reduced oxide samples were characterized using x ray diffraction (XRD) for determining the nature of the individual phases present (oxides and alloys) and scanning electron microscopy (SEM) as a first approach to the investigation of the morphology of the particles. The results indicate that the proposed chemical route resulted in composite materials containing CuNi alloy and Al2O3 particles of controllable composition and homogeneously distributed among the samples. The achieved results also suggest that for the imposed experimental conditions both the nitrate decomposition as well as the reduction reactions could be conducted to 100 percent conversion.

The laser tool is predestined for automated manufacturing processes and has already proven its suit-ability in many areas. In particular, joining technologies in the field of hydrogen production, its storage and for bi-polar plates have to meet the highest requirements in terms of seam quality, reproducibility and manufacturing efficiency. By the examples  700 bar car H2 pressure tank with laser welded connection (welding depth 25 mm)  Laser Remote Welding of HT Fuel Cell Stacks  Laser-welded aluminum tube-ground heat exchanger for gas liquefaction the article presents the possibilities of modern laser beam welding technologies. Furthermore, it gives an outlook on future challenges, especially with regard to the requirements of welding bi-polar plates for upcoming applications in the field of mobility

Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.