Rôle de Paa dans la pathogénicité des Escherichia coli attachants et effaçants (AEEC)

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Interaction d'Escherichia coli entérohémorragique (EHEC) avec Acanthamoeba castellanii et rôle du régulon Pho chez les EHEC

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine).

Influence du microbiote intestinal sur le métabolisme et la virulence des Escherichia coli entérohémorragiques

Les Escherichia coli entérohémorragiques (EHEC) représentent un problème majeur de santé publique dans les pays développés. Les EHEC sont régulièrement responsables de toxi-infections alimentaires graves chez l’humain et causent des colites hémorragiques et le symptôme hémolytique et urémique, mortel chez les enfants en bas âge. Les EHEC les plus virulents appartiennent au sérotype O157:H7 et le bovin constitue leur réservoir naturel. À ce jour il n’existe aucun traitement pour éviter l’apparition des symptômes liés à une infection à EHEC. Par conséquent, il est important d’augmenter nos connaissances sur les mécanismes employés par le pathogène pour réguler sa virulence et coloniser efficacement la niche intestinale. Dans un premier temps, l’adaptation de la souche EHEC O157:H7 EDL933 à l’activité métabolique du microbiote intestinal a été étudiée au niveau transcriptionnel. Pour se faire, EDL933 a été cultivée dans les contenus caecaux de rats axéniques (milieu GFC) et dans ceux provenant de rats colonisés par le microbiote intestinal humain (milieu HMC). Le HMC est un milieu cécal conditionné in vivo par le microbiote. Dans le HMC par rapport au GFC, EDL933 change drastiquement de profile métabolique en réponse à l’activité du microbiote et cela se traduit par une diminution de l’expression des voies de la glycolyse et une activation des voies de l’anaplérose (voies métaboliques dont le rôle est d’approvisionner le cycle TCA en intermédiaires métaboliques). Ces résultats, couplés avec une analyse métabolomique ciblée sur plusieurs composés, ont révélé la carence en nutriments rencontrée par le pathogène dans le HMC et les stratégies métaboliques utilisées pour s’adapter au microbiote intestinal. De plus, l’expression des gènes de virulence incluant les gènes du locus d’effacement des entérocytes (LEE) codant pour le système de sécrétion de type III sont réprimés dans le HMC par rapport au GFC indiquant la capacité du microbiote intestinal à réprimer la virulence des EHEC. L’influence de plusieurs composés intestinaux présents dans les contenus caecaux de rats sur l’expression des gènes de virulence d’EDL933 a ensuite été étudiée. Ces résultats ont démontré que deux composés, l’acide N-acétylneuraminique (Neu5Ac) et le N-acétylglucosamine (GlcNAc) répriment l’expression des gènes du LEE. La répression induite par ces composés s’effectue via NagC, le senseur du GlcNAc-6-P intracellulaire et le régulateur du catabolisme du GlcNAc et du galactose chez E. coli. NagC est un régulateur transcriptionnel inactivé en présence de GlcNAc-6-P qui dérive du catabolisme du Neu5Ac et du transport GlcNAc. Ce travail nous a permis d’identifier NagC comme un activateur des gènes du LEE et de mettre à jour un nouveau mécanisme qui permet la synchronisation de la virulence avec le métabolisme chez les EHEC O157:H7. La concentration du Neu5Ac et du GlcNAc est augmentée in vivo chez le rat par le symbiote humain Bacteroides thetaiotaomicron, indiquant la capacité de certaines espèces du microbiote intestinal à relâcher les composés répresseurs de la virulence des pathogènes. Ce travail a permis l’identification des adaptations métaboliques des EHEC O157:H7 en réponse au microbiote intestinal ainsi que la découverte d’un nouveau mécanisme de régulation de la virulence en réponse au métabolisme. Ces données peuvent contribuer à l’élaboration de nouvelles approches visant à limiter les infections à EHEC.

Virulence Potential And Antibiotic Susceptibility Pattern Of Motile Aeromonads Associated With Freshwater Ornamental Fish Culture Systems: A Possible Threat To public Health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF–ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

Elektrochemische und spektroelektrochemische Untersuchungen symmetrischer und unsymmetrischer Spiroverbindungen

In dieser Arbeit wurden elektronische Eigenschaften der sogenannten Spiroverbin-dungen untersucht, die aus zwei durch ein gemeinsames Spiro-Kohlenstoffatom miteinander verbundenen π-Systemen bestehen. Solche Untersuchungen sind notwendig, um die gezielte Synthese organischer Materialien mit bestimmten optischen, elektrischen, photoelektrischen oder magnetischen Eigenschaften zu ermöglichen. Im einzelnen wurden mit Hilfe der Cyclovoltammetrie, Square-Wave-Voltammetrie und Spektroelektrochemie eine Reihe homologer Spiro-p-oligophenyle, sowie symmetrisch und unsymmetrisch substituierte Spiroverbindungen und Spirocyclopentadithiophene unter-sucht. Dabei ergaben sich folgende Einflussfaktoren: Kettenlänge, verschiedene Substituenten (Trimethylsilyl, tert-Butyl, Fluor, Pyridyl, perfluoriertes Pyridyl, Dimethylamino-Gruppe), verschiedene Positionen der Substitution, Lage der Spiroverknüpfung und Art des π-Systems im Spirokern. Die elektronischen Eigenschaften der untersuchten Verbindungen variieren systema-tisch mit der Kettenlänge. So vermindert sich der Betrag der Redoxpotentiale der Spiroverbin-dungen mit Zunahme der Kettenlänge, während die Anzahl der übertragenen Elektronen mit zunehmender Kettenlänge wächst. Die Absorption der neutralen und geladenen Spezies ver-schiebt sich mit steigender Kettenlänge bathochrom. Der Substituenteneinfluss auf die Poten-tiallage hängt davon ab, welcher der Effekte +I, -I, +M, -M überwiegt; dabei spielt auch die Position der Substitution eine Rolle. Weiter lässt sich der Einfluss der Lage der Spiroverknüpfung auf die Redoxpotentiale mit der verschiedenen Coulomb-Abstoßung innerhalb oder/und zwischen Phenylketten bei symmetrisch und unsymmetrisch verknüpften Spiroverbindungen begründen. Schließlich wurden die Redoxmechanismen der untersuchten Spiroverbindungen er-mittelt. Die meisten Verbindungen werden zum Bis(radikalion) reduziert bzw. oxidiert (Me-chanismus A). Nur wenige Verbindungen werden nach Mechanismus B reduziert, in dem das Elektron unter Bildung eines Dianions in die schon einfach reduzierte Molekülhälfte über-geht. Die Unterschiede der Redoxpotentiale, der Lage der Absorption, des Reduktionsme-chanismus der Verbindungen mit unterschiedlichen Spirokernen (Spirobifluoren und Spiro-cyclopentadithiophen) konnten mit den unterschiedlichen elektronischen Strukturen von Phe-nyl- und Thiophenring (aromatisches und heteroaromatisches π System) erklärt werden.

Alley cropping of willows and grassland for bioenergy provision: productivity, tree-crop interactions and energy balance

The demand for biomass for bioenergy has increased rapidly in industrialized countries in the recent years. Biogenic energy carriers are known to reduce CO2 emissions. However, the resource-inefficient production of biomass often caused negative impacts on the environment, e.g. biodiversity losses, nitrate leaching, and erosion. The detrimental effects evolved mainly from annual crops. Therefore, the aim of modern bioenergy cropping systems is to combine yield stability and environmental benefits by the establishment of mixed-cropping systems. A particular emphasis is on perennial crops which are perceived as environmentally superior to annual crops. Agroforestry systems represent such mixed perennial cropping systems and consist of a mix of trees and arable crops or grassland within the same area of land. Agroforestry practices vary across the globe and alley cropping is a type of agroforestry system which is well adapted to the temperate zone, with a high degree of mechanization. Trees are planted in rows and crops are planted in the alleyways, which facilitates their management by machinery. This study was conducted to examine a young alley cropping system of willows and two grassland mixtures for bioenergy provision under temperate climate conditions. The first part of the thesis identified possible competition effects between willows and the two grassland mixtures. Since light seemed to be the factor most affecting the yield performance of the understory in temperate agroforestry systems, a biennial in situ artificial shade experiment was established over a separate clover-grass stand to quantify the effects of shade. Data to possible below- and aboveground interactions among willows and the two grassland mixtures and their effects on productivity, sward composition, and quality were monitored along a tree-grassland interface within the alleys. In the second part, productivity of the alley cropping system was examined on a triennial time frame and compared to separate grassland and willow stands as controls. Three different conversion technologies (combustion of hay, integrated generation of solid fuel and biogas from biomass, whole crop digestion) were applied to grassland biomass as feedstock and analyzed for its energetic potential. The energetic potential of willow wood chips was calculated by applying combustion as conversion technique. Net energy balances of separate grassland stands, agroforestry and pure willow stands evaluated their energy efficiency. Results of the biennial artificial shade experiment showed that severe shade (80 % light reduction) halved grassland productivity on average compared to a non-shaded control. White clover as heliophilous plant responded sensitively to limited radiation and its dry matter contribution in the sward decreased with increasing shade, whereas non-leguminous forbs (mainly segetal species) benefited. Changes in nutritive quality could not be confirmed by this experiment. Through the study on interactions within the alleys of the young agroforestry system it was possible to outline changes of incident light, soil temperature and sward composition of clover-grass along the tree-grassland interface. Nearly no effects of trees on precipitation, soil moisture and understory productivity occurred along the interface during the biennial experiment. Considering the results of the productivity and the net energy yield alley cropping system had lower than pure grassland stands, irrespective of the grassland seed mixture or fertilization, but was higher than that for pure willow stands. The comparison of three different energetic conversion techniques for the grassland biomass showed highest net energy yields for hay combustion, whereas the integrated generation of solid fuel and biogas from biomass (IFBB) and whole crop digestion performed similarly. However, due to the low fuel quality of hay, its direct combustion cannot be recommended as a viable conversion technique, whereas IFBB fuels were of a similar quality to wood chip from willow.

FastAero – A Precorrected FFT – Fast Multipole Tree Steady and Unsteady Potential Flow Solver

In this paper a precorrected FFT-Fast Multipole Tree (pFFT-FMT) method for solving the potential flow around arbitrary three dimensional bodies is presented. The method takes advantage of the efficiency of the pFFT and FMT algorithms to facilitate more demanding computations such as automatic wake generation and hands-off steady and unsteady aerodynamic simulations. The velocity potential on the body surfaces and in the domain is determined using a pFFT Boundary Element Method (BEM) approach based on the Green’s Theorem Boundary Integral Equation. The vorticity trailing all lifting surfaces in the domain is represented using a Fast Multipole Tree, time advected, vortex participle method. Some simple steady state flow solutions are performed to demonstrate the basic capabilities of the solver. Although this paper focuses primarily on steady state solutions, it should be noted that this approach is designed to be a robust and efficient unsteady potential flow simulation tool, useful for rapid computational prototyping.

Simulation of monsoon disturbances in the LMD GCM

The monsoon depressions that form over India during the summer are analyzed using simulations from the Laboratoire de Meteorologie Dynamique general circulation model. This type of synoptic system often occurs with a frequency of one to two per month and can produce a strong Indian rainfall. Two kinds of analyses are conducted in this study. The first one is a subjective analysis based on the evolution of the precipitation rate and the pattern of the sea level pressure. The second one is an objective analysis performed using the TRACK program, which identifies and tracks the minima in the sea level pressure anomaly held and computes the statistics for the distribution of systems. The analysis of a 9-yr control run, which simulates strong precipitation rates over the foothills of the Himalayas and over southern India but weak rates over central India, shows that the number of disturbances is coo low and that they almost never occur during August, when break conditions prevail. The generated disturbances more often move north, toward the foothills of the Himalayas. Another analysis is performed to study the effect of the Tibetan Plateau elevation on these disturbances with a 9-yr run carried out with a Tibetan Plateau at 50% of its current height. It is shown that this later integration simulates more frequent monsoon disturbances, which move rather northwestward, in agreement with the current observations. The comparison between the two runs shows that the June-July-August rainfall difference is in large part due to changes in the occurrence of the monsoon disturbances.

A Galerkin boundary element method for high frequency scattering by convex polygons

In this paper we consider the problem of time-harmonic acoustic scattering in two dimensions by convex polygons. Standard boundary or finite element methods for acoustic scattering problems have a computational cost that grows at least linearly as a function of the frequency of the incident wave. Here we present a novel Galerkin boundary element method, which uses an approximation space consisting of the products of plane waves with piecewise polynomials supported on a graded mesh, with smaller elements closer to the corners of the polygon. We prove that the best approximation from the approximation space requires a number of degrees of freedom to achieve a prescribed level of accuracy that grows only logarithmically as a function of the frequency. Numerical results demonstrate the same logarithmic dependence on the frequency for the Galerkin method solution. Our boundary element method is a discretization of a well-known second kind combined-layer-potential integral equation. We provide a proof that this equation and its adjoint are well-posed and equivalent to the boundary value problem in a Sobolev space setting for general Lipschitz domains.

A multiwave range test for obstacle reconstructions with unknown physical properties

We develop a new multiwave version of the range test for shape reconstruction in inverse scattering theory. The range test [R. Potthast, et al., A ‘range test’ for determining scatterers with unknown physical properties, Inverse Problems 19(3) (2003) 533–547] has originally been proposed to obtain knowledge about an unknown scatterer when the far field pattern for only one plane wave is given. Here, we extend the method to the case of multiple waves and show that the full shape of the unknown scatterer can be reconstructed. We further will clarify the relation between the range test methods, the potential method [A. Kirsch, R. Kress, On an integral equation of the first kind in inverse acoustic scattering, in: Inverse Problems (Oberwolfach, 1986), Internationale Schriftenreihe zur Numerischen Mathematik, vol. 77, Birkhäuser, Basel, 1986, pp. 93–102] and the singular sources method [R. Potthast, Point sources and multipoles in inverse scattering theory, Habilitation Thesis, Göttingen, 1999]. In particular, we propose a new version of the Kirsch–Kress method using the range test and a new approach to the singular sources method based on the range test and potential method. Numerical examples of reconstructions for all four methods are provided.

A combined BIE-FE method for the Stokes equations

A numerical algorithm for the biharmonic equation in domains with piecewise smooth boundaries is presented. It is intended for problems describing the Stokes flow in the situations where one has corners or cusps formed by parts of the domain boundary and, due to the nature of the boundary conditions on these parts of the boundary, these regions have a global effect on the shape of the whole domain and hence have to be resolved with sufficient accuracy. The algorithm combines the boundary integral equation method for the main part of the flow domain and the finite-element method which is used to resolve the corner/cusp regions. Two parts of the solution are matched along a numerical ‘internal interface’ or, as a variant, two interfaces, and they are determined simultaneously by inverting a combined matrix in the course of iterations. The algorithm is illustrated by considering the flow configuration of ‘curtain coating’, a flow where a sheet of liquid impinges onto a moving solid substrate, which is particularly sensitive to what happens in the corner region formed, physically, by the free surface and the solid boundary. The ‘moving contact line problem’ is addressed in the framework of an earlier developed interface formation model which treats the dynamic contact angle as part of the solution, as opposed to it being a prescribed function of the contact line speed, as in the so-called ‘slip models’. Keywords: Dynamic contact angle; finite elements; free surface flows; hybrid numerical technique; Stokes equations.

Sensitivity of radar backscatter to desert surface roughness

Synthetic aperture radar (SAR) data have proved useful in remote sensing studies of deserts, enabling different surfaces to be discriminated by differences in roughness properties. Roughness is characterized in SAR backscatter models using the standard deviation of surface heights (sigma), correlation length (L) and autocorrelation function (rho(xi)). Previous research has suggested that these parameters are of limited use for characterizing surface roughness, and are often unreliable due to the collection of too few roughness profiles, or under-sampling in terms of resolution or profile length (L-p). This paper reports on work aimed at establishing the effects of L-p and sampling resolution on SAR backscatter estimations and site discrimination. Results indicate significant relationships between the average roughness parameters and L-p, but large variability in roughness parameters prevents any clear understanding of these relationships. Integral equation model simulations demonstrate limited change with L-p and under-estimate backscatter relative to SAR observations. However, modelled and observed backscatter conform in pattern and magnitude for C-band systems but not for L-band data. Variation in surface roughness alone does not explain variability in site discrimination. Other factors (possibly sub-surface scattering) appear to play a significant role in controlling backscatter characteristics at lower frequencies.

The acoustic signature of fluid flow in complex porous media

Effective medium approximations for the frequency-dependent and complex-valued effective stiffness tensors of cracked/ porous rocks with multiple solid constituents are developed on the basis of the T-matrix approach (based on integral equation methods for quasi-static composites), the elastic - viscoelastic correspondence principle, and a unified treatment of the local and global flow mechanisms, which is consistent with the principle of fluid mass conservation. The main advantage of using the T-matrix approach, rather than the first-order approach of Eshelby or the second-order approach of Hudson, is that it produces physically plausible results even when the volume concentrations of inclusions or cavities are no longer small. The new formulae, which operates with an arbitrary homogeneous (anisotropic) reference medium and contains terms of all order in the volume concentrations of solid particles and communicating cavities, take explicitly account of inclusion shape and spatial distribution independently. We show analytically that an expansion of the T-matrix formulae to first order in the volume concentration of cavities (in agreement with the dilute estimate of Eshelby) has the correct dependence on the properties of the saturating fluid, in the sense that it is consistent with the Brown-Korringa relation, when the frequency is sufficiently low. We present numerical results for the (anisotropic) effective viscoelastic properties of a cracked permeable medium with finite storage porosity, indicating that the complete T-matrix formulae (including the higher-order terms) are generally consistent with the Brown-Korringa relation, at least if we assume the spatial distribution of cavities to be the same for all cavity pairs. We have found an efficient way to treat statistical correlations in the shapes and orientations of the communicating cavities, and also obtained a reasonable match between theoretical predictions (based on a dual porosity model for quartz-clay mixtures, involving relatively flat clay-related pores and more rounded quartz-related pores) and laboratory results for the ultrasonic velocity and attenuation spectra of a suite of typical reservoir rocks. (C) 2003 Elsevier B.V. All rights reserved.

Acoustic scattering by mildly rough unbounded surfaces in three dimensions

For a nonlocally perturbed half- space we consider the scattering of time-harmonic acoustic waves. A second kind boundary integral equation formulation is proposed for the sound-soft case, based on a standard ansatz as a combined single-and double-layer potential but replacing the usual fundamental solution of the Helmholtz equation with an appropriate half- space Green's function. Due to the unboundedness of the surface, the integral operators are noncompact. In contrast to the two-dimensional case, the integral operators are also strongly singular, due to the slow decay at infinity of the fundamental solution of the three-dimensional Helmholtz equation. In the case when the surface is sufficiently smooth ( Lyapunov) we show that the integral operators are nevertheless bounded as operators on L-2(Gamma) and on L-2(Gamma G) boolean AND BC(Gamma) and that the operators depend continuously in norm on the wave number and on G. We further show that for mild roughness, i.e., a surface G which does not differ too much from a plane, the boundary integral equation is uniquely solvable in the space L-2(Gamma) boolean AND BC(Gamma) and the scattering problem has a unique solution which satisfies a limiting absorption principle in the case of real wave number.