Xanthomonas axonopodis pv. citri (Xac) é o causador do cancro de plantas cítricas. Entre os potenciais fatores de virulência codificados por Xac, está o Sistema de Secreção do Tipo IV (T4SS), um grande complexo multiprotéico que atravessa o periplasma e as membranas interna e externa de bactérias Gram-negativas. O T4SS está envolvido com secreção de proteínas e/ou DNA para o meio extracelular ou diretamente no interior da célula do hospedeiro. Este Sistema requer tipicamente 12 proteínas para realizar suas funções: VirB1-VirB11 e VirD4. O T4SS codificado pelo cromossomo de Xac está aparentemente incompleto, devido a não codificar nenhuma proteína com similaridade de seqüência a VirB7. Os objetivos deste trabalho são estudar a estrutura, função e interações das proteínas do T4SS de Xanthomonas. Foram clonados 23 genes que codificam proteínas ou domínios relacionados ao T4SS, e os polipeptídeos foram produzidos de forma recombinante em E. coli. Treze deles foram purificados e submetidos a estudos estruturais, espectroscópicos e de interações proteína-proteína. A estrutura em solução de Xac262224-139 foi resolvida, apresentando uma região N-terminal desenovelada de aproximadamente 30 resíduos e um domínio globular. Este polipeptídeo oligomeriza em troca química rápida na escala de tempo de RMN e o seu N-terminal desenovelado reconhece o domínio C-terminal de VirB9 (VirB9154-255) em troca lenta. Análise de RMN demonstrou que VirB9154-255 possui uma estrutura flexível em solução, sofrendo uma marcante mudança conformacional na presença de Xac262224-139. Ambas proteínas se tornam rígidas após a interação. Xac2622 é o equivalente a VirB7 em Xanthomonas, baseado na localização do seu gene no lócus do T4SS, localização subcelular predita do polipeptídeo codificado e sua interação com VirB9. Porém, diferente de outras proteínas da família VirB7, Xac2622 possui um domínio globular adicional, com topologia e estrutura similares a domínios presentes apenas em proteínas associadas à membrana externa de bactérias Gram-negativas. Nocaute do gene xac2622, contudo, não afetou a virulência de Xac na infecção de plantas de laranja pêra. O domínio enovelado de Xac2622 foi cristalizado, e os cristais obtidos difrataram até uma resolução de 1,0 Å, pertencendo ao grupo espacial C2221. O modelo preliminar possui Rfactor de 0,121 e Rfree de 0,147. Foram obtidos cristais de outras 3 proteínas relacionadas ao T4SS de Xac, porém somente um deles difratou em alta resolução (2,0 Å, pertencendo ao grupo espacial C2). O potencial sinal de secreção pelo T4SS de Xanthomonas é um domínio C-terminal conservado de aproximadamente 115 resíduos, encontrado nos substratos putativos do T4SS. Caracterizamos um destes domínios, presente na proteína Xac2609, e ele é intrinsicamente desestruturado. Essa observação pode ter implicações funcionais, visto que os substratos são desenovelados antes de sua passagem pelo canal de secreção do T4SS / Xanthomonas axonopodis pv. citri (Xac) is a gram-negative bacterial phytopathogen that infects citrus. One possible virulence determinant is a chromosomally encoded Type IV Secretion System (T4SS), a multiprotein complex that spans the bacterial periplasm and both inner and outer membranes. The T4SS is used by some bacteria to secrete proteins and/or DNA to the extracellular milieu or the host interior. The model T4SS from Agrobacterium tumefaciens is made up of twelve structural proteins: VirB1-VirB11 and VirD4. The Xanthomonas T4SS is apparently incomplete because of the lack of a polypeptide with sequence similarity to VirB7. The aim of this project is the study of structure-function relationships in the Xanthomonas T4SS. Twenty-three T4SS protein-coding genes, including full-length proteins or domains, were cloned and the proteins were produced in different E. coli strains. Thirteen polypeptides were purified and some of them were submitted to structural, spectroscopic and protein-protein interaction studies. We used NMR to solve the solution structure of Xac262224-139 which consists of an unfolded N-terminal segment of ~30 residues followed by a globular domain. Xac262224-139 oligomerizes in fast exchange at the NMR time scale and interacts via its unfolded N-terminus with the VirB9 C-terminus (VirB9154-255) in slow exchange. NMR analysis showed that VirB9154-255 has a flexible structure in solution. However, this polypeptide undergoes a significant conformational modification in the presence of Xac2622,24-139 and both proteins become rigid upon interaction. Xac2622 is the Xanthomonas VirB7, based on the chromosomal localization of its gene, predicted subcellular localization and protein interaction analysis. But surprisingly, unlike other VirB7 proteins, Xac2622 has an extra C-terminal folded domain whose topology and structure are strikingly similar to that of periplasmic domains found in outer membrane proteins of many bacterial Secretion Systems. Knockout of the xac2622 gene, however, does not affect the Xac virulence in orange leaf infection assays. The Xac2622 folded domain was also crystallized, and these crystals diffracted up to 1.0 Å resolution and belong to the space group C2221. The preliminary refined model has Rfactor of 0.121 and Rfree of 0.147. Crystals of three other T4SS proteins have been obtained, but only one of them diffracted to high resolution (2.0 Å; space group C2). Xac2610 is a hypothetical protein whose gene is located in the T4SS locus, and its interactions were studied with VirB9, VirB11 and Xac2609, a putative T4SS substrate. The potential T4SS secretion signal is a conserved, approximately 115 residues, C-terminal domain found in the putative substrates of the Xanthomonas T4SS. This sequence mediates interactions with VirD4. We have characterized this domain from one substrate and it is mainly unfolded. This observation may have functional implications, as the substrates are unfolded before their secretion through the T4SS channel

Des liens de caténation sont progressivement crées lors de la réplication de l’ADN et sont responsables de la cohésion des chromatides sœurs. La topoisomérase IV est une topoisomérase de type II impliquée dans la résolution de ces liens de caténation accumulés derrière la fourche de réplication, et lors de la dernière étape de séparation des chromatides sœurs à la fin de la réplication. Nous avons étudié la liaison de la topoIV à l’ADN ainsi que son activité catalytique à l’aide de méthodes de biologie moléculaire et de génomique. Une expérience de ChIPseq a révélé que l’interaction de la topoIV de chez E.coli avec l’ADN est contrôlée par la réplication. Durant la réplication, la topoIV a accès à des centaines de sites sur l’ADN mais ne se lie qu’à quelques sites où elle exerce son activité catalytique. La conformation locale de la chromatine et l’expression des gènes influencent la sélection de certains sites. De plus, une forte liaison et une activité catalytique renforcée a été trouvée au site de résolution des dimers, dif. Le site dif est situé à l’opposé de l’origine de réplication dans le macrodomaine ter. Nous avons montré qu’il existe une interaction physique et fonctionnelle entre la topoIV et la recombinase XerCD, qui agit au site dif. Cette interaction est médiée par MatP, une protéine essentielle dans l’organisation du macrodomaine ter. L’ensemble de ces résultats montre que la topoIV, XerCD/dif et MatP œuvrent ensemble pour permettre l’étape finale de ségrégation des chromosomes lors du cycle cellulaire. / Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle.

The master ‘s thesis deals with the studying behavior of nuclear reactors cooled by liquid salts and with application of liquid salts for IV. Generation of nuclear reactors. For the use of liquid salts, it’s necessary to change the concept of the structure of nuclear reactors. There are new ways to use new fuel in a liquid state. Each salt compound or a set thereof is original in its nature. Therefore, it is necessary to consider a suitable compound for the purposes employed. These points are discussed in the theoretical part. The thesis examines particularly chloride salts, with which there is not much experience. The practical part monitors and verifies the nuclear properties of chloride salts, in our case it is a solid-state chloride sodium chloride’s compound. In addition to the experimental measurement, the MCNP simulation program is used also.

Tato práce se zabývá zpracováním snímků Slunce pořízených kosmickou sondou SDO na různých vlnových délkách a vizualizací výskytu třikrát ionizovaného uhlíku C IV jejich vhodným složením. V práci jsou uvedeny základní informace o Slunci a jeho atmosféře, dále je shrnuta potřebná teorie a možné postupy vizualizace, které jsou následně vyhodnoceny a porovnány. Součástí je i vytvořený program pro snadnou tvorbu snímků vizualizujících uhlík C IV.

Diese Arbeit umfasst die Präparation sowie Charakterisierung von neuartigen Uran(IV)-Kolloiden, die in nahneutralen pH-Bereichen und unter umweltrelevanten Bedingungen stabilisiert vorliegen. Rückschlüsse auf Stabilitätsverhalten und Partikelgrößenverteilungen wurden durch dynamische Lichtstreuung, Zetapotentialmessungen sowie Ultrafiltration und Ultrazentrifugation in Kombination mit Elementanalysen getroffen. UV-Vis- und Laserfluoreszenzspektroskopie bestätigten den tetravalenten Zustand des Urans bei den Experimenten. Anders als bisherige Untersuchungen vermuten lassen, ist es möglich langzeitstabile Uran(IV)-Kolloide in höheren Konzentrationen zu erzeugen. Durch Zusatz von geochemischen Komponenten, wie Carbonat und Silikat sind diese sedimentationsstabil und im nahneutralen bis basischen pH-Bereich über längere Zeiträume beständig. Dabei zeigte sich, dass gelöstes Silikat bei der Herstellung der Kolloide eine wesentliche Rolle spielt und Uran(IV) bis zu einer Konzentration von 10-3 mol/L, entsprechend 0,238 g/L in Lösungen stabilisieren kann. Diese Urankonzentration ist dabei ca. drei Potenzen höher als für bisher bekannte silikatfreie, wässrige Uran(IV)-Kolloide. Durch die Verwendung unterschiedlicher analytischer Methoden konnte gezeigt werden, dass die Durchmesser der entstandenen Uran(IV)-Kolloide im nanoskaligen Bereich von teilweise unter 20 nm liegen. Durch diesen kolloidalen Zustand kann eine hohe Mobilität in aquatischen Systemen unterstellt werden. Zusätzlich weisen Langzeituntersuchungen darauf hin, dass diese Kolloide in einem abgeschlossenen System über Jahre stabilisiert werden. Je höher dabei das Verhältnis zwischen Silikat- und Uran(IV)-Gehalt und je höher der pH-Wert der Lösung ist, desto kleiner und stabiler sind diese Partikel. Es ist anzumerken, dass sich keine Kolloide in Abwesenheit von Uran(IV) bilden. Silikat ist in der Lage, die negative Oberflächenladung der Uran(IV)-Kolloide im nahneutralen pH-Bereich zu erhöhen. Dies führt zu einer stärkeren elektrostatischen Abstoßung bzw. repulsiven Wechselwirkungen, womit eine bessere Stabilisierung gewährleistet wird. Der isoelektrische Punkt der erzeugten Partikel wird zu niedrigeren pH-Werten verschoben. Extended-X-ray-absorption-fine-structure-Untersuchungen zeigen, dass die innere Struktur der Kolloide durch den steigenden Silikatgehalt bei deren Bildung von U-O-U-Bindungen (entsprechend Uran(IV)oxyhydroxiden) zu U-O-Si-Bindungen verändert wird. Die Koordination in der benachbarten Region von U(IV) in den U(IV)-Silikat-Kolloiden ist vergleichbar mit der des Coffinits, USiO4. Dieses, für tetravalentes Uran noch nicht beschriebene Phänomen, wurde bereits bei silikatstabilisierten Eisen(III)- oder Mangan(III,IV)-Kolloiden beobachtet und als „Sequestrierung“ bezeichnet. Die silikatstabilisierten U(IV)-Kolloide sind in Laborexperimenten unter kontrollierten Bedingungen erzeugt worden, d.h. es ist noch nicht bekannt, ob diese Phasen in der Natur frei auftreten können. Die qualitative Zusammensetzung der Matrix der experimentellen Lösungen (H+, OH-, Na+, HCO3-/CO32-, Silikat) wurde ähnlich der geochemischen Natur von Grund- bzw. Porenwässern gewählt. Dadurch kann prinzipiell von einem Vorhandensein solcher Kolloide in Wässern natürlichen Ursprungs ausgegangen werden. Die Existenz solcher Partikel würde eine Erklärung für das beobachtete Auftreten von Uran(IV)-Kolloiden in anoxischen Porenwässern oder anoxischen Grundwässern liefern. Es ist jedoch zu beachten, dass experimentell die Reduktion von Uran(VI)-Phasen vorausgesetzt wurde und eine anschließende Verdünnung in Anwesenheit von Silikat erfolgt. Umweltbezogene Untersuchungen zur Mobilität und Stabilität in aquatischen Systemen dieser Kolloide waren nicht Gegenstand der Arbeit und so kann eine umweltrelevante Beurteilung dieser neuartigen Uran(IV)-Kolloide in Bezug auf den Eintrag in die Biossphäre noch nicht getroffen werden. Die hier präsentierten Ergebnisse bieten aber die Grundlage für weitere, intensive Untersuchungen zu Möglichkeiten der Mobilisierung und Stabilisierung verwandter Actinide und Schwermetalle und sollten Bestandteil der Sicherheitsanalyse bei der Lagerung radioaktiven Abfälle in tiefen geologischen Formationen sein. / This work includes the preparation and characterization of new uranium(IV) colloids which are formed and stabilized in the near neutral pH range and under environmentally relevant conditions. Conclusions on stability behavior and particle size distributions were drawn based on results obtained by dynamic light scattering, zeta potential measurements, as well as ultrafiltration and ultracentrifugation in combination with element analyzes. Spectroscopic methodes confirmed the tetravalent state of uranium in the experiments. Unlike empirical data, it is possible to generate long-term stable uranium(IV) colloids at higher concentrations. By addition of geochemical components such as carbonate and silicate, they are stable and resistant in the near neutral pH range over a long period. It was found that dissolved silica plays an essential role in the preparation of colloids. Colloid-borne uranium(IV) up to a concentration of 10-3 mol/L, corresponding to 0,238 g/L, is stabilized in solutions. This concentration is about three orders of magnitude higher than so far known silicate-free aqueous uranium(IV) colloids. Through the use of different analytical methods (invasive and non-invasive) it could be shown that the resulting uranium(IV) colloids are in the nanoscalar range. A high mobility can be assumed in aquatic systems. Evidence is provided by photon correlation spectroscopy, ultrafiltration, and ultracentrifugation that uranium(IV) can form silicate-containing colloids of a size lower than 20 nm. The particles are generated in near neutral to slightly alkaline solutions containing geochemical relevant components (carbonate, silicate, sodium ions). They remain stable in aqueous suspension over years. Electrostatic repulsion due to a negative zeta potential in the near-neutral to alkaline pH range caused by the silicate stabilizes the uranium(IV) colloids. The isoelectric point of the nanoparticles is shifted towards lower pH values by the silicate. The higher the silicate to uranium(IV) content ratio and the higher the pH of the solution are, the smaller and more stable (in terms of pH-changes) are the particles. It should be noted that no colloids were formed in absence of uranium(IV). The mechanism of the colloidal stabilization can be regarded as “sequestration” by silicate, a phenomenon well known from heavy metal ions of high ion potential such as iron(III) or manganese(III,IV), but never reported for uranium(IV) so far. Extended X-ray absorption fine structure (EXAFS) spectroscopy showed that U–O–Si bonds, which increasingly replace the U–O–U bonds of the amorphous uranium(IV) oxyhydroxide with increasing silicate concentration, make up the internal structure of the colloids. The next-neighbor coordination of uranium(IV) in the uranium(IV)-silica colloids is comparable with that of coffinite, USiO4. The assessment of uranium behavior in the aquatic environment should take the possible existence of uranium(IV)-silica colloids into consideration. Their occurrence might influence uranium migration in anoxic waters. The silicate-stabilized colloids have been generated in laboratory experiments under controlled conditions; i.e., it is not known yet whether these phases can occur in natural water. The qualitative composition of the matrix of the experimental solutions (H+, OH-, Na+, HCO3-/CO32-, silicate) was chosen similar to the geochemical nature of groundwater. Thereby, it can be assumed that such colloids are present in natural waters. The existence of such particles would provide an ex-planation for the occurrence of uranium(IV) colloids in anoxic pore waters or groundwaters. However, it should be noted that these results were observed by the reduction of uranium(VI) carbonate and dilution in the presence of silicate. Studies on the mobility and stability of these colloids in aquatic systems were not subject of this work and an environmental assessment of these novel uranium(IV) colloids with respect to the entry in the biosphere cannot be taken into account. But the possibilities of mobilization and stabilization can be applied to surrogate actinides and heavy metals, and point to the need for more intensive research in this area.

This diploma thesis aims to compare the results in the field of cognitive functions obtained by static and dynamic assessment of children with attachment disorder at preschool age. The ACFS-cz assessment battery was used for the dynamic assessment, specifically four subtests: Categorisation, Short-term auditory memory (Story Retelling), Short-term visual memory, Sequential Pattern Completion. The method of static assessment was Woodcock- Johnson IV COG, specifically four subtests: Visualization, Verbal Attention, Picture Recognition, Analysis-Synthesis. The research sample consisted of 16 preschool children (3 to 6 respectively 8 years) placed in institutional care. The results showed that there were no differences between the of the ACFS-cz pretest scores and the Woodcock-Johnson IV COG scores. While the comparison of the results of the ACFS-cz posttest with Woodcock- Johnson IV COG showed significant differences in the three examined areas (Categorisation, Short-term auditory memory, Short-term visual memory). The outcomes of the dynamic assessment lead to suitable interventions for the cognitive development of children in institutional care. Keywords ACFS, Woodcock-Johnson IV COG, cognitive functions, attachment disorder, preschool age.

The system code TRACE is the latest development of the U.S. Nuclear Regulatory Commission (US NRC). TRACE, developed for the analysis of operational conditions, transients and accidents of light water reactors (LWR), is a best-estimate code with two fluid, six equation models for mass, energy, and momentum conservation, and related closure models. Since TRACE is mainly applied to LWR specific issues, the validation process related to innovative nuclear systems (liquid metal cooled systems, systems operated with supercritical water, etc.) is very limited, almost not existing. In this work, essential contribution to the validation of TRACE related to lead and lead alloy cooled systems as well as systems operated with supercritical water is provided in a consistent and corporate way. In a first step, model discrepancies of the TRACE source code were removed. This inconsistencies caused the wrong prediction of the thermo physical properties of supercritical water and lead bismuth eutectic, and hence the incorrect prediction of heat transfer relevant characteristic numbers like Reynolds or Prandtl number. In addition to the correction of the models to predict these quantities, models describing the thermo physical properties of lead and Diphyl THT (synthetic heat transfer medium) were implemented. Several experiments and numerical benchmarks were used to validate the modified TRACE version. These experiments, mainly focused on wall-to-fluid heat transfer, revealed that not only the thermo physical properties are afflicted with inconsistencies but also the heat transfer models. The models for the heat transfer to liquid metals were enhanced in a way that the code can now distinguish between pipe and bundle flow by using the right correlation. The heat transfer to supercritical water was not existing in TRACE up to now. Completely new routines were implemented to overcome that issue. The comparison of the calculations to the experiments showed, on one hand, the necessity of these changes and, on the other hand, the success of the new implemented routines and functions. The predictions using the modified TRACE version were close to the experimental data. After validating the modified TRACE version, two design studies related to the Generation IV International Forum (GIF) were investigated. In the first one, a core of a lead-cooled fast reactor (LFR) was analyzed. To include the interaction between the thermal hydraulic and the neutron kinetic due to temperature and density changes, the TRACE code was coupled to the program system ERANOS2.1. The results gained with that coupled system are in accordance with theory and helped to identify sub-assemblies with the highest loads concerning fuel and cladding temperature. The second design which was investigated was the High Performance Light Water Reactor (HPLWR). Since the design of the HPLWR is not finalized, optimization of vital parameters (power, mass flow rate, etc.) are still ongoing. Since most of the parameters are affecting each other, an uncertainty and sensitivity analysis was performed. The uncertainty analysis showed the upper and lower boundaries of selected parameters, which are of importance from the safety point of view (e.g., fuel and cladding temperature, moderator temperature). The sensitivity study identified the most relevant parameters and their influence on the whole system.

The Influence of William of Ockham teaching to the politics of Louis IV of Bavaria The aim of this work is to find out how far was the King of the Romans and Emperor Louis IV of Bavaria willing or able to use the idea fulfilled in work of the Franciscan monk William of Ockham while patterning his state policy. William of Ockham was living for many years at Louis's court and he reacted to the actual political situation in the Roman Empire by his work. Two elections took place after the death of the Roman Emperor Henry VII of Luxembourg. Louis of Bavaria and Friedrich of Habsburg became two Kings of the Romans and enemies to each other, too. This was followed by many years lasting fight for the throne which paralysed all Empire politics and was only finished when Friedrich was taken captive in the battle of Mühldorf. The new Pope John XXII was elected after two years lasting sede vacante in 1316. When Louis of Bavaria began exercise his right to the Italian part of the Empire after the battle of Mühldorf fully, he got into a disagreement with John XXII. It was ended up not only by a denial of king's right to the Northern Italy but also to Germany and Burgundy and to the start of the trial against the person of Louis IV. The king refused all Pope's demands through three appellations - of Nuremberg,...

Les oscillateurs de référence de haute pureté sont actuellement utilisés dans un grand nombre d’applications allant du contrôle de fréquence aux horloges pour les radars, les GPS et l’espace... Les tendances actuelles dans ce domaine requièrent des architectures miniaturisées avec la génération de signaux directement dans la gamme de fréquences d’intérêt, autour de quelques GHz. Récemment, de nouvelles architectures basées sur les principes de l’optomécanique ont vu le jour dans ce but. De tels oscillateurs optomécanique génèrent non seulement des signaux hyperfréquences directement dans la gamme de fréquences GHz avec éventuellement un faible bruit de phase, mais permettent également un degré élevé d'intégration sur puce. Ce travail de thèse s'inscrit dans cette démarche. L’oscillateur optomécanique étudié se compose de cavités à cristaux photoniques suspendues couplées à des guides d’ondes silicium sur isolant intégrés dans une architecture tridimensionnelle. Ces cavités abritent des modes optiques fortement confinés autour de 1550nm et des modes mécaniques dans le GHz. De plus, ces structures présentent un recouvrement spatial entre phonon et photon élevé. Il en résulte un couplage optomécanique amélioré. Cette force de couplage optomécanique améliorée est ici sondée optiquement sur des structures à cristaux photoniques de conception optimisée. Ces cavités sont réalisées dans des matériaux semi-conducteurs III-V dont la piézoélectricité nous permet d'intégrer des outils supplémentaires pour sonder et contrôler les vibrations mécaniques via un pilotage capacitif, piézoélectrique ou acoustique. Ce contrôle total des modes mécaniques et de l’interaction optomécanique ouvre la voie à la mise en œuvre de circuits intégrés pour le verrouillage par injection et des boucles de rétroaction permettant de réduire le bruit de phase de l’oscillateur. / High purity reference oscillators are currently used in a wide variety of frequency control and timing applications including radar, GPS, space... Current trends in such fields call for miniaturized architectures with direct signal generation in the frequency range of interest, around few GHz. Recently, novel optomechanically-enhanced architectures have emerged with this purpose. Such optomechanically-driven oscillators not only generate microwave signals directly in the GHz frequency range with possibly low phase noise but also are amenable to a high degree of integration on single chip settings. This PhD work falls within this scope. The optomechanically-driven oscillator under study consists of suspended photonic crystal cavities coupled to integrated silicon-on-insulator waveguides in a three-dimensional architecture. These cavities harbor highly-confined optical modes around 1,55 µm and mechanical modes in the GHz and most importantly, feature a high phonon-photon spatial overlap, all resulting in an enhanced optomechanical coupling. This enhanced optomechanical coupling strength is here probed optically on photonic crystal structures with optimized design. These cavities are hosted in III-V semiconductor materials whose piezoelectricity enable us to integrate additional tools for probing and controlling mechanical vibrations via capacitive, piezoelectric or acoustic driving. This full control over the mechanical modes and optomechanical interaction, paves the way towards the implementation of integrated injection locking circuits of feedback loops for reducing the phase noise of the oscillator.

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.