La nécessité de diminuer la consommation à la fois des systèmes autonomes communicants à haute fréquence et des circuits CMOS implique l’utilisation de transistors fonctionnant sous faible tension d’alimentation. Les performances des composants à base de silicium se dégradant rapidement dans ce régime de fonctionnement, les semiconducteurs III-V à faible bande interdite sont aujourd’hui envisagés comme une alternative. Parmi ceux-ci, l’InAs paraît le plus prometteur. Dans ce contexte, ce travail a pour but d’ouvrir la voie à l'utilisation d’un canal à base d'InAs pour les systèmes analogiques et numériques. Plus précisément, nous étudions la croissance par épitaxie par jets moléculaires des hétérostructures InAs/AlSb sur des substrats (001) GaAs et GaP par l’intermédiaire d'une couche tampon Ga(Al)Sb. La microscopie à force atomique, la microscopie électronique en transmission et la diffraction d’électrons de haute énergie sont utilisées afin de mettre en évidence l’influence critique des conditions de croissance sur la nucléation des antimoniures. Cette étude sert de base à l’optimisation de canaux InAs à haute mobilité sur ces deux substrats fortement désadaptés en maille. Les résultats obtenus dans le cas de GaP sont ensuite étendus au cas de pseudo-substrats commerciaux GaP/Si de haute qualité cristalline pour l’intégration de matériaux à base d’InAs sur des substrats Si (001) exactement orientés. Des mobilités électroniques atteignant 28 000 cm-2.V-1.s-1 à 300K et supérieures à 100 000 cm-2.V-1.s-1 à 77K sont démontrées. / Low power consumption transistors operating at low supply voltage are highly required for both high frequency autonomous communicating systems and CMOS technology. Since the performances of silicon-based devices are strongly degraded upon low voltage operation, low bandgap III-V semiconductors are now considered as alternative active materials. Among them, one of the best candidates is InAs. Therefore, the present work aims on paving the way to the use of InAs in transistor channels for both high-speed analog and digital applications. We particularly investigate the molecular beam epitaxy growth of InAs/AlSb heterostructures on both (001) GaAs and GaP via an antimonide metamorphic buffer layer. Using atomic force microscopy, transmission electron microscopy and reflection high energy electron diffraction, we first show the critical influence of the growth conditions on the III-Sb nucleation. From this study, we then achieve optimized high mobility InAs layers on these two highly mismatched substrates. The results obtained in the GaP case are extended to commercially available high quality GaP/Si platforms for the integration of InAs-based materials on an exactly oriented (001) Si substrate. State of the art mobility of 28 000 cm-2.V-1.s-1 at 300K and higher than 100 000 cm-2.V-1.s-1 at 77K are demonstrated.

Le développement de produits électroniques flexibles futurs nécessite la combinaison de hautes performances électroniques (ondes millimétriques et sub-millimétriques) avec une bonne flexibilité mécanique et stabilité. Cependant, l’inconvénient majeur des matériaux utilisés pour concevoir ces transistors flexibles est leurs faibles mobilités des porteurs, limitant les performances fréquentielles. Transistors à haute mobilité d’électrons (HEMT) à base de matériaux III -V ont été utilisés dans le domaine des applications hyperfréquence depuis longtemps. Ce travail développe une méthode possible pour transférer des HEMT classiques sur le substrat flexible. Par un procédé de collage adhésif, des HEMT InAlAs/InGaAs de longueur de grille 100nm ont été transférés sur un film flexible et caractérisés électriquement en régime statique et dynamique. En optimisant la structure épitaxiale, d'excellentes fréquences de coupure ont été obtenues avec un ft=140GHz et un fmax=290GHz sans l’effet Kink. Ces performances sont comparables aux résultats obtenus sur des transistors HEMT sur substrat rigide de même dimension et de filière identique. Par ailleurs, les mesures électriques pour différents niveaux de déformation et différent directions de flexion démontrent très peu de dégradation électrique (inférieure à 15 %). / The development of future flexible electronics requires combining high electrical performance devices (i.e. millimeter and sub-millimeter wave electronic devices) with mechanical flexiblility and stability. However, a variety of existing technologies such as organic thin film transistors, amorphous silicon, and polycrystalline-silicon are limited by their poor transport properties. High electron mobility transistors (HEMTs) based on III-V materials have been used in the field of ultra-high frequency microwave applications for a long time. This work develops a feasible method for transferring conventional HEMTs onto the flexible substrate. By the means of adhesive bonding technique, 100nm-gate InAlAs/InGaAs HEMTs have been transferred onto polyimide film (Kapton) and electrically characterized in static and dynamic regime. Through the epitaxial layers optimization, finally, the fabricated devices are able to suppress Kink effect and provide high cut-off frequencies (ft=160GHz and fmax=290GHz) in unbent condition. These microwave characteristics are comparable to those obtained on 100nm-gate HEMTs on rigid substrate. Moreover, measured devices for various bending radius and bending directions show no obvious electrical degradation (lower than 15%).

L’électronique flexible est une thématique en plein essor, et impacte de nombreux secteurs applicatifs. L’objectif de cette thèse est de développer des composants sur substrats flexibles, pour des applications dans le domaine des radiofréquences. Elle est constituée de deux grandes parties : (i) la fabrication de composants passifs RF en utilisant la technologie d’impression par jet d’encre ; (ii) la fabrication de transistors graphène sur substrats flexibles. Ces travaux sont partiellement intégrés au projet Européen flagship GRAPHENE, et au projet ANR GRACY. La technique d’impression jet d’encre est particulièrement adaptée à la fabrication de composants sur substrats flexibles. L’un des challenges de cette approche technologique est de pouvoir atteindre une définition et une résolution adaptée au fonctionnement en régime radiofréquence. Le travail mené dans cette thèse a permis de réaliser des lignes homogènes de largeur minimale de 50 µm, et une résolution (distance entre 2 lignes de l’ordre de 15 µm. Différents composants passifs ont été fabriqués et caractérisés avec succès, et ce même en appliquant des contraintes en flexion aux dispositifs.Nous avons également développé et optimiser un procédé technologique, adapté à la fabrication de transistors à effet de champ à base de graphène (GFET), sur substrat flexible. Ce procédé présente un bilan thermique faible, et est basé sur l’utilisation d’une grille arrière à base d’aluminium dont l’oxyde naturel sert d’oxyde de grille. De nombreux transistors ont été fabriqués sur substrat kapton, et avec un bon rendement. Les meilleures performances en termes de fréquence de coupure du gain en courant (ft=39 GHz) et la fréquence maximale d’oscillation (fmax=13GHz) ont été mesurées sur un transistor de longueurs de grille Lg=100 nm et un développement de 12µm. Cette performance est à l’état de l’art de GFET flexibles. Ces performances sont conservées pour des contraintes atteignant 0,5%. / Flexible electronic has drawn growing attentions for past several years due to its largely potential applications. The objective of my PhD work is to develop devices based on flexible substrate, for RF applications. There are mainly two parts involved: (i) fabrication of passive devices (transmission lines, antenna, etc) using inkjet printing technology; (ii) fabrication of graphene field effect transistors on flexible substrate using graphene growth by CVD technique. This work is partially involved in the European Flagship program GRAPHENE, and the ANR program GRACY. Inkjet printing is a promising fabrication technology for flexible electronics. The challenge of this technology is the quality and reliability of printed patterns in terms of geometry. Based on optimized printing parameters, the structures of coplanar wave guide (CPW) transmission lines with nice printing quality were realized (definition of 50 µm, resolution down to 20 µm). The RF characterization of these transmission lines combining the considerations of geometric dimensions, sintering temperature, and substrate bending are presented. The outstanding electrical and mechanical properties make graphene suitable for flexible transistors. In this thesis, we have developed and optimized a new low temperature process based on back-gated structure either on rigid substrate than on flexible substrate (here kapton). From flexible transistors, we report as measured current gain cut-off frequency ( ft-DUT ,without any de-embedding) of 39 GHz and maximum oscillation frequency (fmax) of 13 GHz in devices with 100 nm gate length and 12 µm gate width. This result is at the level of the state of art for flexible GFETs.

Ce travail a été réalisé dans le cadre du projet GRACY regroupant l’IEMN et d’autres laboratoires de recherche : CALISTO et IMS Bordeaux. Ce manuscrit fait état d’une synthèse exhaustive des études et avancées menées dans le cadre de ce travail de thèse au sein de l’Institut d’Électronique, de Microélectronique et de Nanotechnologie (IEMN) dans le groupe CARBON. Le principal axe de réflexion de ce travail repose sur la conception, la modélisation et la caractérisation des dispositifs actifs et passifs sur substrat souples et rigides en vue du développement de nouveaux composants et de circuits électroniques avec des critères de performances de plus en plus importants. Au cours de ce travail, l’accent a été principalement portée sur les étapes essentielles à la réalisation de circuit intégré en ondes millimétriques utilisant la technologie coplanaire en impression jet d’encre et les transistors à effet de champ à base de graphène (GFETs). Ce mémoire montre en particulier l’intérêt et les potentialités du graphène pour son intégration au sein des circuits électroniques. De plus, une attention particulière a été portée sur la modélisation et les techniques de caractérisations relatives aux dispositifs passifs sur substrat souple. Par conséquent, un banc de caractérisation de ces éléments sur substrat flexibles a été développé au cours de cette thèse afin de vérifier et consolider expérimentalement leurs comportements. / This work was carried out under the project involving GRACY IEMN and other research laboratories: CALISTO and IMS Bordeaux. This manuscript reports a comprehensive overview of studies and advanced conducted as part of this thesis in the Institute of Electronics, Microelectronics and Nanotechnology (IEMN) in CARBON group. The main reflection axis of this work is based on the design, modeling and characterization of active and passive devices on flexible and rigid substrates for the development of new components and electronic circuits with increasingly important performance criteria. During this work, the focus was mainly focused on the essential steps to achieving integrated circuit millimeter wave using coplanar technology by inkjet printing and field effect transistors based on graphene (GFETs). This memory in particular shows the importance and potential of graphene for integration into electronic circuits. In addition, special attention was paid on modeling and characterization techniques related to passive devices on flexible substrates. Therefore, a characterization bench of these elements on flexible substrate has been developed during this thesis to verify and consolidate their behavior experimentally.

The cytokine Interleukin-4 (IL-4) plays a crucial role in the pathophysiology and progression of asthma and other atopic diseases. Its activities are signaled into the cells upon binding to and signaling through a shared receptor complex composed of the subunits IL-4Rα and common γc. Another cytokine, Interleukin-13 shares many functions with IL-4. This can be explained by the fact that both, IL-4 and IL-13, can signal via a shared receptor complex comprising the IL-4R and the IL-13R1 subunit. Therefore, the IL-4Rα receptor subunit has become a highly promising drug target, since it mediates IL-4 and IL-13 responses and blocking IL-4Rα will abrogate IL-4 as well as IL-13 effector functions. Currently, an IL-4 based mutein (Pitrakinra), acting as a dual IL-4/IL-13 receptor antagonist is in clinical development. This work describes the generation and production of biologically active IL-4 muteins, which contain a single additional engineered cysteine. The introduction of a free thiol group allows site-specific chemical modification. The muteins were expressed in E. coli in insoluble form, refolded and purified. The thiol group of the mutein was protected as mixed disulfide with the tripeptide glutathione. A first attempt to chemically reduce the engineered cysteine residue failed, because the three native disulfide bonds of IL-4 exhibit a similar reactivity and chemical reduction of the native disulfide resulted in full deactivation and precipitation of the IL-4 protein. Therefore, an enzymatic approach was developed which specifically reduces the mixed disulfide bonds with an attached glutathion moiety and thus leaves the native structurally essential disulfide bonds unaltered. For optimization, four different IL-4 cysteine muteins with four cysteine residues introduced at positions close to the IL-4Rα binding site were tested and their reduction rates by glutaredoxin was determined. The enzymatic reduction occured at different rates for all four muteins indicating that accessibility is an important influence and must be determined individually for each mutant protein. After optimization of the pH value and particularly the reaction time, all muteins could be prepared with the engineered thiol group being released in reasonable yield. The proteins exhibiting the free thiol group were then modified by N-ethylmaleimide (NEM) or maleimido-PEG. The effects of these modifications at different positions on binding to IL-4R were measured employing SPR biosensor technology. In the second project of this study, foldamers, which represent a new class of stable, compactly folded biomolecules and can specifically interact with proteins and nucleic acids, were examined to identify their potential as new drugs to interfere with IL-4 activities. Fragment-based drug discovery offers great promise for providing new starting points for drug discovery and facilitates the lead optimization. As foldamers equipped with a thiol-group for tethering could not to be produced; only the effect of foldamers present in a synthesized foldamer library on the binding to IL-4R could be tested. Two libraries containing different foldamers based on aromatic amide were synthesized by Michael Grotz and Dr. Michael Deligny and tested in our lab for their capability to disrupt the ligand-receptor interaction of IL-4 and its receptor IL-4Rα [ECD] using surface plasmon resonance technology. None of the studied foldamers could specifically inhibit the IL-4/IL-4Rα interaction. Some foldamers showed non-specific binding. The study presented here shows the design and production of a potentially new type of IL-4 antagonists, which employ site-specific chemical modification to exert their antagonistic function. / Das Zytokin Interleukin-4 (IL-4) spielt eine entscheidende Rolle in der Entstehung und Pathophysiologie von Asthma und anderen atopischen Krankheiten. Seine Aktivitäten können in die Zelle durch die Bindung an einen Rezeptorkomplex übertragen werden, welcher aus den Untereinheiten IL-4Rα und γc besteht. Interleukin-13 (IL-13), ein verwandtes Zytokin, und IL-4 besitzen viele gemeinsame Funktionen. Das kann dadurch erklärt werden, dass IL-4 wie auch IL-13 ihre Signale über einen gemeinsamen Rezeptorkomplex übertragen können, der aus der IL-4R und der IL-13R1 Untereinheit besteht. Die IL-4R Untereinheit ist ein vielversprechendes Zielmolekül für die Entwicklung von Pharmaka, da sie IL-4 und IL-13 Reaktionen vermittelt. Durch Blockieren von IL-4R werden die Aktivitäten von IL-4 sowie IL-13 unterdrückt. Ein IL-4 basiertes Doppelmutein (Pitrakinra), welches als Gegenspieler zu IL-4 und IL-13 Rezeptoren fungiert, befindet sich derzeit in der klinischen Entwicklung. In dieser Arbeit wird die Bildung und Produktion von biologisch aktiven IL-4 Muteinen mit einem einzelnen zusätzlich eingefügten Cysteinrest beschrieben. Die Einführung einer freien Thiol-Gruppe ermöglicht ortsspezifische chemische Modifizierungen. Ein „Tethering“ Ansatz sollte dann auch eine sehr schwach Bindung von thiol-reaktiven Verbindungen an IL-4 messbar machen. Die Muteine wurden in unlöslicher Form in E. coli exprimiert, zurückgefaltet und auf gereinigt. Dabei wurde die Thiolgruppe des Muteins als Disulfid mit dem Tripeptid Glutathion geschützt. Erste Versuche gezielt den eingeführten Cysteinrest selektiv chemisch zu reduzieren schlugen fehl, da die drei proteineigenen Disulfidbrücken von IL-4 eine ähnliche Reaktivität zeigten, und die Reduktion zur vollständigen Desaktivierung und Fällung des IL-4 Proteins führte. Daher wurde ein enzymatischer Ansatz entwickelt, der gezielt die Disulfidbrücke zum Glutathionrest reduziert und die proteineigenen strukturell essentiellen Disulfidbrücken unverändert lässt. Zur Optimierung wurden vier verschiedene IL-4 Cystein-Muteine mit Cysteinresten an verschiedenen Positionen nahe der IL-4Rα Bindungsstelle getestet und die Reduktionsgeschwindigkeit in Gegenwart von Glutaredoxin bestimmt. Die enzymatische Reduktion verlief für alle vier Muteine mit verschiedenen Geschwindigkeiten. Dies deutet darauf hin, dass die Zugänglichkeit der Disulfidgruppe einen wichtigen Einfluss besitzt. Die Reduktionsbedingungen mussten daher für jedes Mutein neu bestimmt werden. Nach Optimierung des pH Wertes und insbesondere der Reaktionszeit konnten alle Muteine mit einer freien Thiolgruppe in angemessener Ausbeute erhalten werden. Die Proteine mit jeweils einer freien Thiolgruppe wurden daraufhin mit N-Ethylmaleinimid (NEM) oder Maleimido-PEG modifiziert. Die Effekte der Modifizierung an verschiedenen Positionen des IL-4 auf die Bindung an IL-4R wurden mit Hilfe der SPR-Spektroskopie (Oberflächen Plasmon Resonanz Spektroskopie) gemessen. Im zweiten Teil dieser Arbeit wurde die Interaktion von Foldameren mit der IL-4Ra Rezeptorkette untersucht. Foldamere stellen eine neue Klasse von stabilen, kompakt gefalteten Biomolekülen dar, die möglicherweise spezifisch mit Proteinen und Nukleinsäuren wechselwirken können. Es sollten Vorversuche durchgeführt werden um zu sondieren, ob aus Foldameren Hemmstoffe für IL-4 und IL-13 entwickelt werden können. Da Foldamere mit einer Thiolgruppe zur Anbindung (Tethering) an IL-4 nicht hergestellt werden konnten, wurden zunächst nur nichtreaktive Foldamare aus einer synthetisierten Foldamer-Bibliothek getestet. Zwei Bibliotheken mit verschiedenen auf aromatischen Amiden basierenden Foldameren wurden von Michael Grotz und Dr. Michael Deligny synthetisiert und von mir mit Hilfe der SPR Spektroskopie auf ihre Fähigkeit getestet, die Ligand-Rezeptor Wechselwirkung von IL-4 und derIL-4Rα Rezeptoruntereinheit zu unterbinden. Keines der untersuchten Foldamere konnte die IL-4/IL-4Rα Wechselwirkung spezifisch hemmen. Einige Foldamere zeigten eine unspezifische Bindung. Die hier dargestellten Studien zeigen das Design und die Herstellung eines potentiell neuen Typs von Gegenspieler zu IL-4, welcher ortsspezifische chemische Modifikationen ausnutzt um seine antagonistische Funktion zu erfüllen.

Das Cytokin Interleukin-4 (IL-4) ist ein essentieller Faktor bei der Entstehung von Sofort-Typ Allergien. Die Bindung von IL-4 an seinen Rezeptor und die anschließende Phosphorylierung des IL-4 aktivierten Transkriptionsfaktors Stat6 ist ein Schlüsselereignis bei der allergischen Immunantwort. In der vorliegenden Arbeit werden Ergebnisse zur Hemmung der Stat6 vermittelten Signaltransduktion des IL-4 Rezeptors vorgestellt. Dazu wurde ein Vektorsystem etabliert, bei dem ein von dem Drosophila-Transkriptionsfaktor Antennapedia abgeleitetes 16 AS langes Peptid benutzt wird. Dieses Antennapediapeptid kann Plasmamembranen lebender Zellen energie- und rezeptorunabhängig durchqueren und dabei andere hydrophile Moleküle mittransportieren. Stat6 bindet über eine SH2 Domäne an phosphorylierte Reste von IL4Ra und bildet, nachdem es selbst phosphoryliert ist, mit anderen Stat6-Molekülen aktive Dimere. Ein aus der Stat6-Bindestelle des IL-4Ra abgeleitetes phosphoryliertes Peptid (Stat6BP) wurde mit Hilfe des Antennapediapeptids in verschiedene humane und murine Zellinien transportiert. Für Stat6BP konnte mit Hilfe von spezifischer Immunpräzipitation und Western-Blot gezeigt werden, dass es IL-4 induzierte Phosphorylierung und Aktivierung von Stat6 transient hemmen kann. Durch zusätzliche Applikation des Tyrosinphosphataseinhibitors Natriumpervanadat gelang es, die hemmende Wirkung von Stat6BP zu verlängern. Unter gleichen Bedingungen konnte auch gezeigt werden, dass Stat6BP spezifisch die Aktivierung von Stat6 hemmt, da die durch IL-4 oder IL-3 induzierte Phosphorylierung des eng verwandten Stat5 völlig unbeeinträchtigt bleibt. Ferner wurde durch das Peptid die Expression eines Stat6 kontrollierten Reportergens gehemmt. Im Rahmen dieser Arbeit wurde außerdem die Rolle der Src-Typ Kinasen p56lck und p59fyn in der IL-4 Signaltransduktion in unterschiedlichen T-Zellinien untersucht. Es zeigte sich, dass die Aktivierung der beide Kinasen stark von der getesteten Zellinie abhängt. In einigen T-Zellinien aktiviert IL-4 eher p56lck, in anderen eher p59fyn. / Interleukin-4 (IL-4) is the major factor in the development of allergic diseases like hay fever or asthma. The most important cytoplasmic event following stimulation with IL-4 is the activation of the transcription factor Stat6 (signal transducer and activator of transcription 6). Stat6 binds via a single SH2 domain first to tyrosine-phosphorylated motifs in the IL-4Ra-chain, and then to another Stat6 molecule, which results in the formation of active dimers. Since Stat6 is exclusively used by the IL-4 receptor, it is a promising approach to specifically disrupt IL-4 signal- transduction by inhibiting Stat6 activation. A vector system was established for the delivery of hydrophilic agents into living cells. To this purpose, a 16 amino acid membrane-permeable peptide derived from the Drosophila transcription factor Antennapedia was used. The Antennapedia peptide has been shown to internalize into living cell in a receptor- and energy-independent manner. In this thesis it is shown that a peptide derived from the Stat6-binding region of IL-4Ra (Stat6BP) is an effective inhibitor when it is delivered into cells by coupling with the Antennapedia peptide. Stat6BP completely inhibited IL-4 dependent phosphorylation of Stat6 in different human and murine cell lines, while IL-3 and IL-4 dependent phosphorylation of Stat5 was not affected. The inhibitory effect of Stat6BP was transient, but could be prolonged by treating the cells with the phospatase inhibitor sodium pervanadate. Transcription from a reporter gene construct with a Stat6-dependent promoter was inhibited by Stat6BP as well, indicating that the peptide is a suitable inhibitor for cellular responses downstream from Stat6 phosphorylation. Another aim of this study was to investigate the role of the src-kinases p56lck and p59fyn in IL-4 signaltransduction. The results indicate, that the activation of both kinases is celline dependent. In some T-cellines p56lck was activated dominantly, in others p59fyn.

No abstract available

The oxidation of 4-t-butyltoluene in glacial acetic acid by hydrogen peroxid in a process catalysed by cobalt(II) acetate tetrahydrate and sodium bromide has been studied with the aim of increasing the selectivity towards 4-t-butylbenzaldehyde.

Graduation date: 2002

2,4,6-trinitrotoluene (TNT) is a toxic and recalcitrant pollutant contaminating soils and groundwater. Therefore, characterization of microbial populations of TNT-contaminated soils and isolation of bacteria degrading this pollutant are of primordial importance. Comparison of hybridizations of 16S rRNA derived from uncontaminated and TNT-contaminated soil samples required the development of a functional ANOVA model. Specifically, a statistical tool was necessary to compare dissociation curves obtained from thermal dissociation analysis of RNA hybridizations to DNA microarrays, and to determine if the dissociation curves significantly differed. To test and validate the model, we used dissociation curves from in vitro transcribed 16S rRNA amplified from two environmental samples hybridized to a phylogenetic microarray. Detection and rejection of outlier curves was important for appropriate discrimination between curves. The identification of significantly different curves was more efficient with the model than approaches relying on measurements at a single temperature. This functional ANOVA analysis was used to improve discrimination between hybridizations of two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from an uncontaminated and a TNT-contaminated soil sample to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when values of Td (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. These differences in hybridizations between the two soil samples were likely the result from the presence of TNT. The effect of TNT on soil microbial communities was further investigated with additional uncontaminated and TNT-contaminated soil samples using 16S rRNA PCR-DGGE and cultivation-dependent techniques. In all contaminated soil samples, the amount of DNA extracted was lower than in the uncontaminated ones. Analysis of bacterial diversity by DGGE showed a predominance of Pseudomonadaceae and Xanthomonadaceae in the TNT-contaminated soil samples compared to the uncontaminated ones. Caulobacteraceae were also present in several contaminated soil samples. The culturable microflora of these soils was studied by plate counts on agar supplemented with dilute nutrient broth. The number of CFUs was lower in a TNT-contaminated soil inoculum than in an uncontaminated one. In the former, most of the CFUs belonged to Pseudomonadaceae, and to a lesser extent, to Caulobacteraceae. In addition to the above contaminated soil samples, a pristine soil was artificially contaminated with different concentrations of TNT and incubated for 4 months. The amount of DNA extracted decreased in the highly contaminated soil samples (1.4 and 28.5 g TNT/kg soil). After 7 days of incubation of these soil samples, there was a clear shift of their original flora to a population dominated by Pseudomonadaceae, Xanthomonadaceae, Comamonadaceae and Caulobacteraceae. When the TNT concentration was lower (140 mg TNT/kg soil), a moderate shift in the bacterial population was observed. These results indicate that TNT affects soil bacterial diversity and richness by selecting for a narrow range of bacterial species that belong mostly to Pseudomonadaceae and Xanthomonadaceae. TNT-contaminated soil samples probably contained TNT-degrading bacteria. In order to isolate bacteria that can denitrate TNT, enrichment cultures were carried out with TNT as sole nitrogen source and in the absence of oxygen. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum, in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. Concomitantly, TNT-reduced compounds were detected as well as unidentified polar metabolites. The concentration of nitrite released from TNT was proportional to the concentration of ferrihydrite in the medium. The release of nitrite was lower when the concentration of initially spiked TNT was reduced by one order of magnitude. Under these conditions, the concentration of nitrite peaked and then its concentration slowly decreased and production of ferrous ions was detected. A decrease of nitrite concentration and production of ferrous ion were observed when TNT was omitted and nitrite and ferrihydrite were provided. These results suggest that nitrite-reducing conditions were initially achieved, followed by iron-reducing conditions. When grown aerobically on a chemically defined medium, P. aeruginosa strain ESA-5 produced a greenish extracellular compound. This product was identified as phenazine-1-carboxylic acid (PCA). When purified PCA was incubated with TNT in the presence of NADH, nitrite was released. The concentration of nitrite released was dependent on the concentration of NADH and PCA. Denitration also occurred with two TNT-related molecules, 2,4,6-trinitrobenzaldehyde and 2,4,6-trinitrobenzyl alcohol. The release of nitrite was coupled with the formation of two polar metabolites and mass spectrometry analyses indicated that each of these compounds had lost two nitro groups from the trinitroaromatic parent molecule. The results obtained with the PCA mediated denitration of TNT in the presence of inhibitors of oxygen reactive species suggested the involvement of superoxide (O2.-). When exogenous PCA was added to a P. aeruginosa ESA-5 liquid culture containing TNT as sole nitrogen source, bacterial growth was significantly enhanced compared to cultures containing TNT without PCA.