Tous les organismes, les animaux, les plantes et les microbes, sont composés de cellules polarisées, en affichant un positionnement asymétrique des organites sub-cellulaires ou des structures. Le contrôle de polarité a été étudié chez les eucaryotes pendant une longue période, et a été montré pour être impliqués dans de nombreux processus physiologiques, tels que l'embryogenèse, le cancer métastatique et les maladies dégénératives des neurones. Chez les procaryotes, des études de polarité ne sont apparues récemment avec le développement de la microscopie à fluorescence sensibles. Ces études ont révélé que les cellules procaryotes sont en fait très organisé et une masse croissante de la littérature a montré que les cellules bactériennes également utiliser des radeaux lipidiques, courbure membranaire, la paroi cellulaire et un cytosquelette complexe pour diriger le positionnement spécifique de structures subcellulaires.Petites GTPases de la superfamille Ras sont des éléments réglementaires polarisation répandue chez les eucaryotes. Malgré l'existence depuis longtemps de ces petites GTPases dans les génomes procaryotes, leur fonction a jamais été étudiée. Pendant ce travail de thèse, nous avons trouvé, pour la première fois, qu'une petite GTPase, MglA et de sa protéine apparentée Activation GTPase (GAP) MglB, directe une dynamique axe antéro-postérieur à la motilité directe en forme de tige deltaproteobacterium Myxococcus xanthus. Dans ce processus, MglA s'accumule dans son état lié au GTP au niveau du pôle leader de cellules, en activant les machineries motilité. Ce schéma de localisation est maintenue par MglB, qui localise le pôle opposé, le blocage de l'accumulation MglA à ce pôle à travers son activité GAP. Remarquablement, les deux protéines passer leur localisation synchrone, ce qui correspond à un changement dramatique dans la direction du mouvement cellulaire (inversion). Ce commutateur est réglementé par un système chimiosensoriels-like, Frz. Dans une deuxième partie de ce travail, nous avons identifié un régulateur de protéine de réponse, RomR qui est essentiel pour le regroupement polaire de MglA. Interdépendances complexes entre la localisation RomR, MglA et MglB indiquent que ces protéines pourraient constituer un complexe de polarité dynamique de trois protéines qui reçoit Frz de signalisation pour passer l'axe de polarité. En conclusion, les résultats de ce travail de thèse suggère que M. xanthus intégré un module de polarité eucaryotes-like (MglAB) dans un procaryote spécifique (Frz) réseau de signalisation pour réguler sa motilité. Une telle réglementation est distincte sous forme de petites protéines G des règlements, qui sont généralement couplés à la protéine G récepteurs couplés (GPCR) chez les eucaryotes. Enfin, ce travail ouvre la voie pour comprendre comment la réglementation seule la motilité cellulaire sont intégrés pour générer des comportements commandés multicellulaires donnant naissance à des structures primitives de développement, par exemple, la morphogenèse du corps fructifères. D'autre part, ce travail fournit également un exemple d'analyser les étapes évolutives donnant lieu à des réseaux de signalisation. / All organisms, animals, plants and microbes, are composed of polarized cells, displaying asymmetric positioning of sub-cellular organelles or structures. Polarity control has been studied in eukaryotes for a long time, and has been shown to be involved in many physiological processes, such as embryogenesis, cancer metastasis and neuron degenerative diseases. In prokaryotes, polarity studies only emerged recently with the development of sensitive fluorescent microscopy. These studies revealed that prokaryotic cells are in fact highly organized and a growing body of literature has shown that bacterial cells also use lipid rafts, membrane curvature, the cell wall and a complex cytoskeleton to direct the specific positioning of subcellular structures.Small GTPases of the Ras superfamily are widespread polarization regulatory elements in eukaryotes. Despite the long known existence of such small GTPases in prokaryotic genomes, their function has never been studied. During this thesis work, we found, for the first time, that a small GTPase, MglA and its cognate GTPase Activating Protein (GAP) MglB, direct a dynamic anterior- posterior axis to direct motility of the rod-shaped deltaproteobacterium Myxococcus xanthus. In this process, MglA accumulates in its GTP-bound state at the leading cell pole, activating the motility machineries. This localization pattern is maintained by MglB, which localizes at the opposite pole, blocking MglA accumulation at this pole through its GAP activity. Remarkably, both proteins switch their localization synchronously, which correlates with a dramatic change in the direction of cell movement (reversal). This switch is regulated by a chemosensory-like system, Frz. In a second part of this work, we identified a response regulator protein, RomR which is essential for the polar clustering of MglA. Intricate localization interdependencies between Romr, MglA and MglB indicate that these proteins might constitute a dynamic three-protein polarity complex that receives Frz-signaling to switch the polarity axis. In conclusion, the results from this thesis work suggest that M. xanthus integrated a eukaryotic-like polarity module (MglAB) into a prokaryotic- specific (Frz) signaling network to regulate its motility. Such regulation is distinct form small G- protein regulations, which are generally coupled to G-protein coupled receptors (GPCRs) in eukaryotes. Finally, this work paves the way to understand how single cell motility regulations are integrated to generate ordered multicellular behaviors giving rise to primitive developmental structures, for example fruiting body morphogenesis. On the other hand, this work also provides an example to analyze the evolutionary steps giving rise to signaling networks.

The aim of this work is to design an experiment, where client-server systems can be compared. They belong to the group of systems that are called component systems. Client-server systems are difficult to compare, because they are complex. The client-server systems are documented in different ways. Notations in the implementation of them are in different ways. There is a large difference in the communication between the client and server. The architectures between the client-server systems differ also, but they are not totally different. Therefore it is possible to construct an experiment for comparing them. Client-server systems that will be compared are Java RMI, Web Services, CORBA, and Enterprise JavaBeans. We are going to use Java as the programming language. Some of these systems, for example CORBA, can be implemented in other languages. The designed experiment is among other things going to answer how long time is needed to implement a specific application, how fast a specific client-server system is, and how long time is spent for learning about a specific system.

In this thesis project we have successfully added an MPI extension layer to the LOIS framework. The framework defines an infrastructure for executing and connecting continuous stream processing applications. The MPI extension provides the same amount of stream based data as the framework’s original transport. We assert that an MPI-2 compatible implementation can be a candidate to extend the given framework with an adaptive and flexible communication sub-system. Adaptability is required since the communication subsystem has to be resilient to changes, either due to optimizations or system requirements.

of Doctoral Thesis Title: Components and Services in Resource-Constrained Envi- ronments Author: Tomáš Pop tomas.pop@d3s.mff.cuni.cz Department: Department of Distributed and Dependable Systems Faculty of Mathematics and Physics Charles University in Prague Advisor: Prof. František Plášil plasil@d3s.mff.cuni.cz Abstract Appliances of every day use such as consumer electronics, automotive and telecommunication devices as well as various kinds of control systems have be- come a common and important part of our everyday lives. In comparison to general-purpose desktop systems, they are limited in terms of various resources, for example memory, CPU power, and battery capacity. Component-based soft- ware engineering is a well-established development technique, which has found its way to industry and has been successfully used for a long time in multiple do- mains. However, the industrially adopted component frameworks were designed for general-purpose systems and they can be hardly used in resource-constrained environments. In the thesis, we explore the potential of component-based system engineer- ing in resource-constrained environments. Stemming from a survey of existing component frameworks targeted to this domain, we identify the most important approaches to overcome resource constraints in...

Nowadays, earthquakes are one of the most catastrophic natural events that have a significant human, socio-economic and environmental impact. Besides, based on both observations of damage following recent major/moderate seismic events and numerical/experimental studies, it clearly emerges that critical non-structural components (NSCs) that are ubiquitous to most industrial facilities are particularly and even disproportionately vulnerable to those events. Nonetheless and despite their great importance, seismic provisions for industrial facilities and their process equipment are still based on the classical load-and-resistance factor design (LRFD) approach; a performance-based earthquake engineering (PBEE) approach should, instead, be preferred. Along this vein, in recent years, much research has been devoted to setting computational fragility frameworks for special-risk industrial components and structures. However, within a PBEE perspective, studies have clearly remarked: i) a lack of definition of performance objectives for NSCs; ii) the need for fully comprehensive testing campaigns data on coupling effects between main structures and NSCs. In this respect, this doctorate thesis introduces a computational framework for an efficient and accurate seismic state-dependent fragility analysis; it is based on a combination of data acquired from an extensive experimental shake table test campaign on a full-scale prototype industrial steel frame structure and the most recent surrogate-based UQ forward analysis advancements. Specifically, the framework is applied to a real-world application consisting of seismic shake table tests of a representative industrial multi-storey frame structure equipped with complex process components, carried out at the EUCENTRE facility in Italy, within the European SPIF project: Seismic Performance of Multi-Component Systems in Special Risk Industrial Facilities. The results of this experimental research campaign also aspire to improve the understanding of these complex systems and improve the knowledge of FE modelling techniques. The main goals aim to reduce the huge computational burden and to assess, as well, when the importance of coupling effects between NSCs and the main structure comes into play. Insights provided by innovative monitoring systems were then deployed to develop and validate numerical and analytical models. At the same time, the adoption of Der Kiureghian's stochastic site-based ground motion model (GMM) was deemed necessary to severely excite the process equipment and supplement the scarcity of real records with a specific frequency content capable of enhancing coupling effects. Finally, to assess the seismic risk of NSCs of those special facilities, this thesis introduces state-dependent fragility curves that consider the accumulation of damage effects due to sequential seismic events. To this end, the computational burden was alleviated by adopting polynomial chaos expansion (PCE) surrogate models. More precisely, the dimensionality of a seismic input random vector has been reduced by performing the principal component analysis (PCA) on the experimental realizations. Successively, by bootstrapping on the experimental design, separate PCE coefficients have been determined, yielding a full response sample at each point. Eventually, empirical state-dependent fragility curves were derived.

Made available in DSpace on 2014-12-17T14:08:38Z (GMT). No. of bitstreams: 1 Ernesto Vargas Bautista_DISSERT_01_59.pdf: 4396872 bytes, checksum: 70a69eb1e9973135d541fd7ce76f4ed9 (MD5) Previous issue date: 2010-02-26 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / In Brazil and around the world, oil companies are looking for, and expected development of new technologies and processes that can increase the oil recovery factor in mature reservoirs, in a simple and inexpensive way. So, the latest research has developed a new process called Gas Assisted Gravity Drainage (GAGD) which was classified as a gas injection IOR. The process, which is undergoing pilot testing in the field, is being extensively studied through physical scale models and core-floods laboratory, due to high oil recoveries in relation to other gas injection IOR. This process consists of injecting gas at the top of a reservoir through horizontal or vertical injector wells and displacing the oil, taking advantage of natural gravity segregation of fluids, to a horizontal producer well placed at the bottom of the reservoir. To study this process it was modeled a homogeneous reservoir and a model of multi-component fluid with characteristics similar to light oil Brazilian fields through a compositional simulator, to optimize the operational parameters. The model of the process was simulated in GEM (CMG, 2009.10). The operational parameters studied were the gas injection rate, the type of gas injection, the location of the injector and production well. We also studied the presence of water drive in the process. The results showed that the maximum vertical spacing between the two wells, caused the maximum recovery of oil in GAGD. Also, it was found that the largest flow injection, it obtained the largest recovery factors. This parameter controls the speed of the front of the gas injected and determined if the gravitational force dominates or not the process in the recovery of oil. Natural gas had better performance than CO2 and that the presence of aquifer in the reservoir was less influential in the process. In economic analysis found that by injecting natural gas is obtained more economically beneficial than CO2 / No Brasil e no mundo, as empresas de petr?leo est?o ? procura, desenvolvimento e ? espera de novas tecnologias e processos que possam elevar o fator de recupera??o de ?leo em seus reservat?rios, com um equilibrado custo-benef?cio. Na procura de novas tecnologias foi desenvolvido o processo de drenagem gravitacional assistida por g?s (GAGD Gas Assisted Gravity Drainage) que foi classificado dentro dos m?todos de recupera??o avan?ada de ?leo. O processo GAGD, que se encontra em fase de teste piloto em campo, est? sendo muito estudado atrav?s de modelos f?sicos em escala de laborat?rio e em testemunhos, isto devido ?s altas recupera??es de ?leo em rela??o aos outros processos de inje??o de g?s. Este m?todo consiste em injetar g?s no topo de um reservat?rio atrav?s de po?os injetores verticais ou horizontais e deslocar o ?leo, aproveitando a segrega??o gravitacional natural dos fluidos, para um po?o produtor horizontal colocado na base do reservat?rio. Para estudar este processo foi idealizado um reservat?rio homog?neo e um modelo de fluido multicomponente com caracter?sticas parecidas aos campos de ?leo leve do Brasil para que, atrav?s de um simulador composicional, se analisem os par?metros operacionais. O processo foi simulado no programa comercial GEM da CMG (Computer Modelling Group). Os par?metros operacionais estudados foram a vaz?o de inje??o de g?s, o tipo de g?s injetado (g?s natural e CO2), a localiza??o do po?o injetor e do po?o produtor. Tamb?m foi estudada a presen?a de aq??fero no processo. Os resultados mostraram que o m?ximo espa?amento vertical entre os dois po?os, provocou a m?xima recupera??o de ?leo no GAGD. Tamb?m, verificou-se que a maiores vaz?es de inje??o, se obtiveram os maiores fatores de recupera??o. Este par?metro controlou a velocidade da frente do g?s injetado e determinou se a for?a gravitacional domina ou n?o sobre o processo na recupera??o de ?leo. O g?s natural teve melhor desempenho que o CO2 e que, a presen?a de aq??fero no reservat?rio influenciou pouco no processo. Na an?lise econ?mica verificou-se que a inje??o de g?s natural obteve o melhor beneficio econ?mico que com CO2

Face au défi des entreprises pour le maintien de leurs équipements au maximum de leur fiabilité, de leur disponibilité, de leur rentabilité et de leur sécurité au coût de maintenance minimum, des stratégies de maintenance telles que le CBM et le PHM ont été développées. Pour mettre en œuvre ces stratégies, comme pour la planification des activités de production il est nécessaire de connaître l’aptitude des systèmes à réaliser les futures tâches afin de réaliser le séquencement des opérations de production et de maintenance. Cette thèse présente les éléments d'une fonction générique qui évalue la capacité des systèmes techniques multi-composants à exécuter les tâches de production de biens ou de services assignées. Ce manuscrit présente une proposition de modélisation de systèmes techniques multi-composants représentant les différentes entités qui les composent, leurs états et leurs relations. Plusieurs types d’entités ont été identifiés. Pour chacun d’eux, des inférences sont proposées pour définir à l’intérieur du système l’aptitude de l’entité à accomplir les futures tâches de production à partir des évaluations de son état présent et futur et des évaluations des états présents et futurs des autres entités avec lesquelles elle est en relation. Ces évaluations des états présents et futurs sont basées sur l’exploitation de pronostics locaux des composants. Ces pronostics sont des prévisions qui intrinsèquement comportent des incertitudes pouvant être aléatoires ou épistémiques. La fonction proposée et les inférences prennent en compte ces deux formes d’incertitudes. Pour cela, les traitements et la fonction proposée exploite des éléments de la théorie de Dempster-Shafer. La modélisation des systèmes multi-composants pouvant être représentée sous la forme de graphes dont les états des nœuds dépendent de données comportant des incertitudes, des éléments des réseaux bayésiens sont également utilisés. Cette fonction fournit des indicateurs, sur l’aptitude de chaque entité du système à accomplir les futures tâches de production mais aussi indique les composants nécessitant une intervention afin d’améliorer cette aptitude. Ainsi, ces indicateurs constituent les éléments d'aide à la décision pour la planification des opérations de maintenance de façon conditionnelle et préventive, mais aussi pour la planification des opérations de production. / One major challenge of companies consists in maintaining their technical production resources at the maximum level of reliability, availability, profitability and safety for a minimum maintenance cost, maintenance strategies such as CBM and PHM have been developed. To implement these strategies, as with production activity planning, it is necessary to know the ability of systems to perform future tasks to order production and maintenance operations. This thesis presents the generic function that evaluates the ability of multi-component technical systems to perform the production tasks of goods or services. This manuscript presents a proposal for the modelling of multi-component technical systems representing the different entities that compose them, their states and their relationships. Several types of entities have been identified. For each of them, inferences are proposed to define within the system the entity's ability to perform future production tasks based on its own assessment of its present and future state and the assessments of the present and future states of the other entities with which it is involved. These assessments of present and future states are based on the use of local prognoses of components. These prognoses are predictions that inherently involve uncertainties that may be aleatory or epistemic. The proposed function and inferences take into account these two kinds of uncertainty. To do this, the inferences and the proposed function uses elements of the Dempster-Shafer theory. Since the modelling of multi-component systems can be represented in the form of graphs whose node states depend on data with uncertainties, elements of Bayesian networks are also used. This function provides indicators on the ability of each system entity to perform future production tasks but also indicates the components that require maintenance to improve this ability. Thus, these indicators constitute the elements of decision support for the planning of maintenance operations in a conditional and preventive way, but also for the planning of production tasks.

Mycobacterium tuberculosis, the causative organism of the disease tuberculosis (TB) in humans, leads to nearly two million deaths each year. This versatile pathogen can exist in highly distinct physiological states such as asymptomatic latent TB infection where bacilli lie dormant or as active TB disease in which the bacilli replicate in macrophages. The pathogenic lifestyle requires the tubercle bacillus to sense and respond to multiple environmental cues to ensure its survival. Such stimuli include hypoxia, nutrient limitation, presence of reactive oxygen and reactive nitrogen intermediates, pH alterations, and cell wall/ membrane stress. Two component systems (TCSs) form the primary apparatus for sensing and responding to environmental cues in bacteria. A prototypical TCS is composed of a sensory protein called sensor kinase (SK) and a response generating protein called response regulator (RR). M. tuberculosis encodes 11 genetically paired TCSs, 2 orphan sensor kinases and six orphan response regulator proteins. Studies of the TB bacilli using transcriptional profiling and gene knockouts have revealed that TCSs play an important role in facilitating successful adaptation to diverse environmental conditions encountered within the host. The mtrAB and prrAB genes encoding corresponding TCSs have been shown to be essential for survival, mprAB for persistence and devRS for hypoxic adaptation. Further, inactivation of the TCSs regX3-senX3, tcrXY, trcRS, phoPR or kdpDE was shown to affect the growth and/or virulence of M. tuberculosis in animal infection models. The SK and RR proteins of TCSs are modular and contain variable input and output domains coupled to conserved ‘transmitter’ and ‘receiver’ domains. Despite the modular nature and extensive homology of SK and RR proteins across TCSs, which may allow non-cognate interactions, it is believed that crosstalk across different TCSs is not favored and that individual pathways are generally well insulated. The existing profiling studies have been performed on the TCSs of bacterial species containing a relatively large number of TCSs. In those studies, specificity and insulation have been the norm and thus become the prevalent paradigm of TCS signaling. In vitro genome wide phosphotransfer profiling has revealed only a few cross- communication nodes in the TCSs of Escherichia coli (~3%), while none in Caulobacter crescentus (in 352 interactions tested, in short time duration) and Myxococcus xanthus (in 250 interactions tested). Yet, many instances of cross talk have been reported in literature. For example, E. coli TCSs PmrAB and EnvZ-OmpR show cross-communication with QseBC and ArcBA, and many more. In M. tuberculosis, indirect evidence of the existence of such cross regulation has originated from studies where mutations in phoPR have been shown to affect the expression of the TCS devRS and its regulon. It is thus interesting to examine the extent of crosstalk in the TCSs of M. tuberculosis, which has an exceptionally small number of TCS proteins compared to E. coli. As mentioned earlier, M. tuberculosis H37Rv has 11 cognate pairs of TCSs, 2 orphan sensor kinases and 6 orphan response regulators. To study the entire landscape, we aimed to study all 221 connections between SK and RR proteins including 12 cognate interactions. While 10 of the cognate TCS interactions were established in the literature, two putative systems KdpDE and NarSL and 5 orphan response regulators were still uncharacterized, therefore we initiated our work with the characterization of these TCSs. At the biochemical level, the KdpDE two component system of M. tuberculosis is not well studied, though one report showed interaction of the C-terminal domain of KdpD SK and KdpE RR using yeast two hybrid assay and another reported the interaction of the SK with LRP protein. Besides these associations, there is no evidence for the functionality of KdpDE system. Similarly, NarSL system also has not been characterized and it not known whether these putative two component proteins are functional. The initial part of the study includes the characterization of these two TCSs, NarS-NarL and KdpD-KdpE, at biochemical and physiological levels. In our studies we demonstrated that KdpDE system is a bonafide two component system of M. tuberculosis, and KdpD SK undergoes autophosphorylation at His642 residue in presence of Mg+2 ions and then it transfers phosphoryl group to a conserved Asp52 residue on the KdpE RR protein. The acid-base stability analysis revealed the nature of chemical bonds present in the KdpD and KdpE proteins, and further confirmed that KdpD and KdpE are typical SK and RR respectively. SPR analysis demonstrated that KdpD and KdpE proteins interact under basal non-phosphorylated conditions and the interaction affinity reduced when SK was phosphorylated. The reduction in the interaction affinity indicated towards a possible dissociation of SK and RR protein during phosphotransfer, which allows RRs to exert their regulatory effect. On the similar line, the phosphorylation defective SK (KdpDH642Q) had least affinity with KdpE suggesting that perhaps this mutant SK, fails to interact with the RR. We have also shown that both the kdpD and kdpE genes are in the same operon and are up regulated in potassium ions limitation and osmotic stress conditions. Overall, using the biochemical approaches, we have established that Rv1027c–Rv1028c operon of M. tuberculosis encodes a functional and a typical KdpDE two component signal transduction system. Using the similar biochemical and biophysical approaches, we have demonstrated that NarS-NarL proteins constitute a functional TCS and His241 and Asp61 are the phosphorylatable residues. In contrast to KdpDE which shows typical behaviour of TCS, NarSL TCS showed atypical behaviour. Malhotra and group’s work on NarSL suggested that there is cross-regulation between NarS/NarL and DevS/DosT/DevR systems. We addressed this possibility on three separate levels, by examining (i) the cross-phosphorylation of DevR and NarL RRs by non-cognate sensor kinases NarS and DevS/DosT respectively, (ii) the interaction between DevR and NarL RR proteins, and (iii) examining the effect of DevR-NarL interactions on their DNA binding properties. Our studies ruled out the presence of any physiologically relevant phosphorylation mediated cross-talk between NarS/NarL and DevS/DosT/DevR. We identified that the cross talk between these TCSs could be explained on the basis of interaction between NarL and DevR RRs and their subsequent binding to the target gene promoter regions for concerted regulation of gene expression. We also identified that DevR activation is critical for cooperative action with NarL. This process comes out as a novel mechanism of gene regulation via heteromerization of RRs. We hypothesized that formation of NarL-DevR heteromers may arise because of high sequence similarities. Conclusively, our study provides insights into the functionality of M. tuberculosis NarL/NarS TCS and regulatory function of NarL protein which acts in concert with another RR, DevR. Overall, NarS-NarL system showed an atypical, novel mode of gene regulation involving RR heteromerization. Subsequent to the basic biochemical characterization of NarSL and KdpDE system, the genome wide phosphotransfer profiling was done to identify the cross-connections between TCSs. Remarkably, we found that specificity was the exception rather than the rule. While only three of the TCS pairs were completely specific, all the other nine TCS pairs exhibited crosstalk, including a few that were highly promiscuous. We classified the interactions as specific, one-to-many, and many-to-one signaling circuits. We also profiled all the RRs including the orphans for their ability to accept phosphoryl group from a low molecular weight donor, acetyl phosphate, and interestingly found that only two RRs DevR and NarL were capable of accepting phosphoryl group from such a donor. Interestingly, none of the orphan RRs accepted phosphoryl group from any donor, neither SKs nor low molecular weight phospho donors, warranting further analysis of their roles and presence in the M. tuberculosis genome. Our exhaustive map of the crosstalk between the TCSs of M. tuberculosis sets the stage for a renewed view of TCS signaling and proposes a dispersive-integrative landscape for TCS signaling rather than one of insulation. As an extension of our basic characterization work of NarSL TCS, we also attempted to understand the localization pattern of NarS sensor kinase in M. smegmatis cells using fluorescence approaches. It is known that many bacterial receptors including sensor kinases form clusters or show specific localization patterns inside the cell. We found that NarS shows distinct cellular localization pattern. However, the functional significance of this localization pattern is not obvious yet and warrants further investigations. We also developed a few non-radioactive methods to study interaction between two component systems to overcome the limitations associated with radioactive experiments in studying TCSs. We developed fluorescence resonance energy transfer (FRET) to study in vitro interaction between two component proteins which was sensitive to the phosphorylation status of the proteins. Using fluorescently tagged SKs and RRs, we determined a change in FRET for KdpDE and NarSL TCS pairs in vitro. Our study thus also provides an alternative approach to study TCS signaling, using an easier, non-radioactive and high throughput approach. In summary, our study presents the evidence of an alternative paradigm of bacterial signaling, where significant crosstalk between the underlying TCSs prevails. The new paradigm is expected to have important implications in our understanding of the virulence and pathogenesis of bacterial infections. Overall, our studies (i) allowed the establishment of functionality of all paired TCSs encoded in the genome of M. tuberculosis including NarSL and KdpDE TCSs, (ii) identified the novel mechanism of gene regulation by NarL RR and DevR, (iii) demonstrated the existence of TCS signaling which is contrary to the existing notion of specificity (iv) showed the distinct localization pattern of NarS and (v) developed non-radioactive approaches to study two component interactions.

Designing Resilient Distributed embedded Systems is a challenging task. One of the design issues is to guarantee correct behavior of the system during the runtime. It demands verification that information propagated through the system is reliable. The goal of this thesis is a research and implementation of an analysis that should identify obsolescence of variables due to delays caused by scheduling and communication in real-time systems. Analysis will be designed for Ensemble-Based Component System (EBCS) semantics because it enables precise specification and analysis of important properties. The main problem is to find a suitable input model of the analysis and find its possible limits. Effort should be put in balancing between the level of abstraction given to a RDS developer and power of the analysis itself. The main benefit of the analysis will be detection of situations in which data processed in RDS are outdated and can cause incorrect behavior of particular components.

La résistance aux polymyxines chez P. aeruginosa résulte en partie de la modification du lipide A par addition de 4-amino-aL-arabinose (L-Ara4N), due à l'expression de l'opéron arnBCADTEF-ugD (arn), activée par au moins 6 systèmes de régulation à deux composants (S2C). Nous avons mis en évidence que P. aeruginosa était capable de s'adapter de manière transitoire à la présence de fortes concentrations de polymyxines (8 x CMI) aussi bien in vitro que in vivo dans un modèle murin d'infection pulmonaire aiguë. La délétion de l'opéron arn chez la souche sauvage n'a pas modifié la capacité d'adaptation de P. aeruginosa. Afin d'identifier les gènes impliqués dans le processus adaptatif, le transcriptome global du mutant PAOl/z«;-Aar«-ATCS délété des principaux S2C (parRS, pmrAB, cprRS eiphoPQ) et de l'opéron arn a été réalisé en présence de différentes concentrations de colistine par RNA-Seq. Deux nouveaux mécanismes ont ainsi été identifiés. L'un repose sur l'expression de l'opéron mmsAB codant des enzymes du catabolisme des acides gras et l'autre fait intervenir le facteur sigma alternatif AlgU. Seule la délétion conjointe du gène algW participant à l'activation de AlgU et des opérons arn, mmsAB, pmrAB, parRS, phoPQ et cprRS a permis d'abolir complètement la résistance adaptative à la colistine. Par ailleurs, nous avons mis en évidence le rôle des vésicules de membrane externe (OMVs) dans la séquestration de l'antibiotique, dont la production semble régulée au moins par AlgU et le PQS. Ces travaux offrent des perspectives intéressantes pour l'identification de nouvelles cibles antibactériennes et pour l'amélioration de l'effet bactéricide des polymyxines. / Resistance to polymyxins in Pseudomonas aeruginosa involves the addition of 4-amino-L-arabinose (Ara4N) to LPS phosphates, thanks to an enzymatic modification due to the operon named arnBCADTEF-ugD (arn) whose expression is activated by at least 6 two component regulatory Systems (TCS). We demonstrated that P. aeruginosa was able to resist in a transient way to high concentrations of polymyxins (8x MIC) in vitro and in vivo in a mice lung infection model. Arn operon deletion in the wild type strain did not modify the ability to adapt to polymyxins. In order to identify gènes involved in adaptive resistance, we performed RNA-seq transcriptomes of quintuple mutant PAO\lux-Aaw-ATCS exposed to different concentrations of colistin or non exposed. Two new mechanisms were identified. The first one is based upon mmsAB operon encoding fatty acid catabolism enzymes and the second one is due to the sigma factor AlgU. Only the deletions of algW%enz involved in AlgU activation and arn, mmsAB, pmrAB, parRS, phoPQ and cprRS operons completely abolished the adaptive process. We also demonstrated the role of outer membrane vesicles in the sequestration of colistin whose production is regulated by AlgU and PQS. This study provides knoweldge essential for the design of novel strategies aimed at tackling the adaptive resistance to polymyxins.