CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Global distributions of diazotrophs Gamma-A nifH genes abundance - Depth integrated values computed from a collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present data set presents depth integrated values of diazotrophs Gamma-A nifH genes abundance, computed from a collection of source data sets.

Doença enxerto-versus hospedeiro: determinação da expressão de genes e alterações de vias associadas ao sistema imunológico usando metodos da biofísica

O transplante de medula óssea (TMO) é um procedimento terapêutico importante em casos relacionados à pacientes com leucemia ou linfoma. Em decorrência desse processo, uma reação conhecida como doença enxerto-versus-hospedeiro (GVHD) pode ocorrer em pacientes susceptíveis como conseqüência da presença de células imunocompetentes do doador. Entretanto, não existe um modelo para descrever completamente as ações relacionadas ao mecanismo imunológico da GVHD desde a fase que inicializa a doença até a fase efetora. O Objetivo geral deste estudo é a investigação da resposta imunológica considerando-se o sistema HLA (antígenos leucocitários humano) em pacientes que desenvolveram a GVHD em decorrência do TMO. O National Cancer Institute (NCI) – Pathway interaction Database e Reactome foram usados como bases de dados com o objetivo de se estudar a expressão de genes e vias relacionados às Classes I e II do sistema HLA (antígenos leucocitários humano). O estudo considerou a mudança de expressão de genes relacionados às 17 vias do sistema imunológico com potencialidade para se expressar em pacientes que desenvolveram a GVHD associada à TMO. Dados referentes aos transcriptomas foram obtidos utilizando-se a plataforma GPL570 Affymetrix Genoma Humano U133 Plus. A atividade relativa foi usada para determinar as alterações das vias em amostras de GVHD em relação ao controle. As análises foram realizadas utilizando-se o software Via Complex e Bioconductor. Observou-se aumento significativo da expressão de genes ralacionados às vias do sistema imune adaptativo, antígenos associados às Classe I e II do HLA, fosforilação de CD3 e CD247, sinalização dos receptores de células T em CD4+ nativas e ativação de NF-kapa β nas células B. Também observou-se alterações significativas na mudança de expressão dos genes associados às vias relacionadas à super família de moléculas B7:CD28\CTLA-4 quando comparadas ao controle. Isso pode indicar a necessidade de geração de um segundo sinal co-estimulador em GVHD, acionado pelas moléculas dessa super família. O aumento da expressão do gene CD69 nas amostras experimentais caracteriza a ativação celular e, portanto, a sinalização de estímulos em GVHD. Os achados obtidos neste estudo contribuem para melhor elucidar o mecanismo imunopatogênico associado à GVHD. P

Identificação e expressão de genes relacionados à via esteroidogênica e resposta antioxidante após a exposição à Atrazina em Poecilia vivipara (Poeciliidae, Cyprinodontiformes)

O herbicida Atrazina (ATR) é um agrotóxico utilizado há cerca de 50 anos, responsável pelo controle seletivo de plantas daninhas em cultivo de arroz, milho e cana-de-açúcar, principalmente. Estudos recentes apontam diversos efeitos desse herbicida em invertebrados e vertebrados, através da contaminação do solo, bem como da lixiviação para os ecossistemas aquáticos. Foi demonstrado que a ATR é um desregulador endócrino, além de causar efeitos como estresse oxidativo, imunotoxicidade e distúrbios no metabolismo energético. No presente estudo, a espécie nativa Poecilia vivipara foi utilizada como modelo experimental para identificar e analisar a expressão de genes atuantes na via esteroidogênica (StAR e Cyp19a1) e genes atuantes no sistema de defesa antioxidante enzimático (SOD-1 e CAT), frente a exposição à diferentes concentrações de ATR. Sequências parciais dos genes-alvo foram obtidas e comparadas com sequências disponíveis de espécies próximas. Foram analisadas a expressão órgãoespecífica para cada um dos genes isolados, bem como a expressão dos genes frente à exposição ao herbicida atrazina. Os animais foram expostos a ATR em concentrações de 2, 10 e 100 µg/L e a expressão dos genes em gônadas e fígado desses animais foram analisadas em 24 e 96 horas de exposição. As sequências obtidas dos genes StAR, Cyp19a1, SOD-1 e CAT apresentaram 821, 80, 954, 350 pares de bases respectivamente, com identidades que variam de 86 a 100% com espécies filogeneticamente próximas a P. vivipara. Os animais apresentaram uma maior expressão dos genes StAR e Cyp19a1 nas gônadas e no fígado, enquanto a menor expressão se mostrou em órgãos como intestino e baço. Já os genes SOD e CAT apresentaram uma maior expressão no fígado, e menor expressão no intestino. Em relação à expressão gênica frente à exposição à ATR, os resultados apontaram para uma indução dos genes StAR, SOD e CAT em 24 horas, nas gônadas e no fígado, enquanto 8 que a expressão do gene Cyp19a1 foi aumentada apenas após 96 horas de exposição. Foi demonstrado que o herbicida ATR, mesmo em baixas concentrações, é capaz de desregular a expressão de genes que codificam tanto para proteínas componentes da via de síntese de hormônios esteróides, quanto para enzimas atuantes na resposta antioxidante celular de P. vivipara. 

Red flesh fruit in european pear (Pyrus communis): genetic sources, QTLs, candidate genes and tools for breeding

Red flesh fruit is a character which interest is increasing in several commercial species. Following a review of the research on the biosynthesis and accumulation of anthocyanin in pears (Chapter 1) the general aim of the project is reported in Chapter 2. Chapter 3 reports the results of a molecular analysis of 33 red-fleshed pear accessions, genotyped with 18 SSR markers with the aim of improving germplasm conservation strategies to support ongoing breeding programs. The molecular profiles revealed both cases of synonymy and homonymy and 6 unique genotypes were identified. The S-allele were established to highlight the genetic relationships among these landraces. Four of the unique genotypes have been clustered based on pomological data. In the Chapter 4, the work was directed to identify the putative genomic regions involved in the appearance of this character in pear fruit. A crossing population (‘Carmen’ x ‘Cocomerina Precoce’) segregating for the trait was phenotyped for 2 consecutive years and used for QTL analysis. A strong QTL was identified in a small genomic region related to the red flesh fruit trait at 27 Mb from the start of LG5. Two candidate genes were detected in this genomic region: ‘PcMYB114’ and ‘PcABCC2’. SSR marker SSR114 was found able to detect the red flesh phenotype segregation in all the red-fleshed pear accessions and segregating progenies tested. Chapter 5 focuses on examining the trend of anthocyanin synthesis and accumulation during the fruit development, from fruit set to ripening time. Three different trials were planned: qPCR and HPLC methods were performed to correlate the genes expression with the anthocyanin accumulation in ‘Cocomerina Precoce’ and six progenies. Total transcriptome sequencing was used to compare the differential genes expression between red and white-fleshed fruit. Chapter 6 reviews and analyses all the earlier study findings while providing new potential future perspectives.

Treinamento resistido reduz inflamação em músculo esquelético e melhora a sensibilidade à insulina periférica em ratos obesos induzidos por dieta hiperlipídica

OBJETIVO: Investigar em ratos obesos o efeito da prática de exercício resistido sobre a sensibilidade à insulina e sobre a expressão de citocinas pró-inflamatórias e de transportador de glicose em músculo solear. MATERIAIS E MÉTODOS: Ratos Wistar alimentados com dieta hiperlipídica (grupos obesos) foram submetidos ao protocolo de exercício tipo jump squat. A sensibilidade à insulina e a expressão gênica de Tnf-α, SOCS3 e GLUT4 foram comparadas entre os grupos obesos sedentários (OS) e exercitados (OE) e controles sedentários (CS) e exercitados (CE). RESULTADOS: A sensibilidade à insulina estava reduzida no grupo OS e elevada no OE. Os conteúdos de RNAm de Tnf-α e de SOCS3 estavam aumentados no músculo esquelético do grupo OS e reduzidos no OE. O conteúdo proteico e de RNAm de GLUT4 não diferiu entre os grupos. CONCLUSÃO: O exercício resistido reverte o quadro de resistência à insulina periférica e de inflamação no músculo esquelético de obesos induzidos por dieta.

Coordinated plant defense responses in Arabidopsis revealed by microarray analysis

Disease resistance is associated with a plant defense response that involves an integrated set of signal transduction pathways. Changes in the expression patterns of 2.375 selected genes were examined simultaneously by cDNA microarray analysis in Arabidopsis thaliana after inoculation with an incompatible fungal pathogen Alternaria brassicicola or treatment with the defense-related signaling molecules salicylic acid (SA), methyl jasmonate (MJ), or ethylene, Substantial changes (up- and down-regulation) in the steady-state abundance of 705 mRNAs were observed in response to one or more of the treatments, including known and putative defense-related genes and 106 genes with no previously described function or homology, In leaf tissue inoculated with A. brassicicola, the abundance of 168 mRNAs was increased more than 2.5-fold, whereas that of 39 mRNAs was reduced. Similarly, the abundance of 192, 221, and 55 mRNAs was highly (>2.5-fold) increased after treatment with SA, MJ, and ethylene, respectively. Data analysis revealed a surprising level of coordinated defense responses, including 169 mRNAs regulated by multiple treatments/defense pathways. The largest number of genes coinduced (one of four induced genes) and corepressed was found after treatments with SA and MJ. In addition, 50% of the genes induced by ethylene treatment were also induced by MJ treatment. These results indicated the existence of a substantial network of regulatory interactions and coordination occurring during plant defense among the different defense signaling pathways, notably between the salicylate and jasmonate pathways that were previously thought to act in an antagonistic fashion.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Variation in coumarin 7-hydroxylase activity associated with genetic polymorphism of cytochrome P450 2A6 and the body status of iron stores in adult Thai males and females

The relationships between catalytic activity of cytochrome P450 2A6 (CYP2A6), polymorphism of CYP2A6 gene, gender and levels of body iron stores were analysed in a sample group of 202 apparently healthy Thais, aged 1947 years. Eleven individuals were found to have high activity of CYP2A6, judged by the relatively large amounts (11.2-14.6 mg) of 7-hydroyxcoumarin (7-OHC) excreted 3 h following administration of 15 mg of coumarin. Ten individuals, however, did not excrete any 7-OHC. Of these 10, four were found to have no CYP2A6 gene (whole gene deletion; CYP2A6*4 allele). The frequency of the CYP2A6 alleles; *1A, *1B and *4 in the whole sample group was 52, 40 and 8% while the frequency of the CYP2A6 gene types; *1A/* 1A, *1A/* 1B, *1B/* 1B, *1A/* 4, *1BI* 4, *4/* 4 was 29, 41, 16, 7, 5 and 2%. Subjects having CYP2A6* 1A/* 1B gene-type group were found to have higher rates of coumarin 7-hydroxylation compared with those of the CYP2A6* 1B/* 1B and CYP2A6* 1A/* 4 gene types. The inter-individual variability in CYP2A6 catalytic activity was therefore attributed in part to the CYP2A6 genetic polymorphism. Variation in CYP2A6 activity in this sample group was not associated with gender but, interestingly, it did show an inverse association with plasma ferritin; an indicator of body iron stores. Higher rates of coumarin 7-hydroxylation were found in individuals with low body iron stores (plasma ferritin < 20 μg/l) compared with subjects having normal body iron store status. Subjects (n = 16) with iron overload (plasma ferritin > 300 mug/l) also tended to have elevated rates of coumarin 7-hydroxylation. These results suggest an increased CYP2A6 expression in subjects who have excessive body iron stores. Further investigations into the underlying factors that may lead to increased expression of CYP2A6 in association with abnormal body iron stores are currently in progress in our laboratory. Pharmacogenetics 12:241-249 (C) 2002 Lippincott Williams Wilkins.

Generation of CTL responses using Kunjin replicon RNA

The Kunjin replicon was used to express a polytope that consisted of seven hepatitis C virus cytotoxic T lymphocyte epitopes and one influenza cytotoxic T lymphocyte epitope for vaccination studies. The self-replicating nature of, and expression from, the ribonucleic acid was confirmed in vitro . Initial vaccinations with one dose of Kun-Poly ribonucleic acid showed that an influenza-specific cytotoxic T lymphocyte response was elicited more efficiently by intradermal inoculation compared with intramuscular delivery. Two micrograms of ribonucleic acid delivered in the ear pinnae of mice was sufficient to elicit a detectable cytotoxic T lymphocyte response 10 days post-vaccination. Further vaccination studies showed that four of the seven hepatitis C virus cytotoxic T lymphocyte epitopes were able to elicit weak cytotoxic T lymphocyte responses whereas the influenza epitope was able to elicit strong, specific cytotoxic T lymphocyte responses following three doses of Kun-Poly ribonucleic acid. These studies vindicate the use of the Kunjin replicon as a vector to deliver encoded proteins for the development of cell-mediated immune responses.

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

Modern cancer epidemiological research: genetic polymorphisms and environment

Individual cancer susceptibility seems to be related to factors such as changes in oncogenes and tumor suppressor genes expression, and differences in the action of metabolic enzymes and DNA repair regulated by specific genes. Epidemiological studies on genetic polymorphisms of human xenobiotics metabolizing enzymes and cancer have revealed low relative risks. Research considering genetic polymorphisms prevalence jointly with environmental exposures could be relevant for a better understanding of cancer etiology and the mechanisms of carcinogenesis and also for new insights on cancer prognosis. This study reviews the approaches of molecular epidemiology in cancer research, stressing case-control and cohort designs involving genetic polymorphisms, and factors that could introduce bias and confounding in these studies. Similarly to classical epidemiological research, genetic polymorphisms requires considering aspects of precision and accuracy in the study design.

The repressor element silencing transcription factor (REST)-mediated transcriptional repression requires the inhibition of Sp1.

The terminal differentiation of neuronal and pancreatic beta-cells requires the specific expression of genes that are targets of an important transcriptional repressor named RE-1 silencing transcription factor (REST). The molecular mechanism by which these REST target genes are expressed only in neuronal and beta-cells and are repressed by REST in other tissues is a central issue in differentiation program of neuronal and beta-cells. Herein, we showed that the transcriptional factor Sp1 was required for expression of most REST target genes both in insulin-secreting cells and neuronal-like cells where REST is absent. Inhibition of REST in a non-beta and a non-neuronal cell model restored the transcriptional activity of Sp1. This activity was also restored by trichostatin A indicating the requirement of histone deacetylases for the REST-mediated silencing of Sp1. Conversely, exogenous introduction of REST blocked Sp1-mediated transcriptional activity. The REST inhibitory effect was mediated through its C-terminal repressor domain, which could interact with Sp1. Taken together, these data show that the inhibition of Sp1 by REST is required for the silencing of its target genes expression in non-neuronal and in non-beta-cells. We conclude that the interplay between REST and Sp1 determines the cell-specific expression of REST target genes.

Sensing, uptake and response to c4-dicarboxylic acids in pseudomonas aeruginosa pao1

C4-dicarboxylates are one of the preferred carbon and energy sources for the growth of P. aeruginosa, a ubiquitous and metabolically versatile bacterium. However, despite their importance, C4-dicarboxylates sensing and uptake systems were poorly understood in P. aeruginosa and only little information was available in the literature. In our work, the C4-dicarboxylate transport (Dct) system in P. aeruginosa was found to be composed of a novel two-component system, called DctB/DctD, regulating together with the sigma factor RpoN the expression of two newly identified C4-dicarboxylate transporters: DctA and DctPQM. Inactivation of the dct A, dctB or dctD gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating their major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of redundant C4-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the kdctA mutant, combined with a screening for growth on succinate, led to the identification of a second Dct system, the DctPQM transporter, belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. AdctAAdctPQM double mutant showed no growth on malate and fumarate albeit residual growth on succinate suggested that additional transporters for succinate are present. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at μΜ concentrations, whereas DctA was the major transporter at mM concentrations. For the first time, high- and low-affinity uptake systems for succinate (DctA and DctPQM) are reported to function co-ordinately to transport C4- dicarboxylates. Most probably, the presence of redundant uptake systems contributes to the versatility of this bacterium. Next, the regulation of the Dct system was investigated. While performing a parallel study about the carbon catabolite repression (CCR) phenomenon in P. aeruginosa, a link between the CCR cascade (CbrAB/CrcZ/Crc) and the Dct system was observed. Crc is a translational repressor acting when preferred carbon sources (like C4-dicarboxylates) are present. CrcZ is a small RNA acting as a functional antagonist of Crc and induced by the CbrA/CbrB two-component system when non preferred carbon sources (like mannitol) are utilized. Novel targets of the CbrAB/CrcZ/Crc system in P. aeruginosa were identified using transcriptome analysis; among them dctA and dctPQM were detected. CCR is regulating the dct transporter genes expression depending on the succinate concentrations in the medium of growth; this modulation of CCR is possible because, at the same time, succinate concentrations tune CCR. In a medium containing high succinate concentrations, CrcZ levels were low and therefore Crc inhibited the translation of mRNA targets. Whereas in a medium containing low succinate concentrations, the subsequent increase of CrcZ levels sequestered Crc, inhibiting its activity. This model shows for the first time that CCR possesses a feedback-based circuitry, a very important type of regulatory loop that confers the best adaptive response under changing environmental conditions. The expression of the dct transporter genes is also found to be regulated by the RNA chaperone protein Hfq. Hfq has the same post-transcriptional effect than Crc at high concentration of succinate, i.e. inhibiting dctP and dctR and indirectly favouring dctA expression. Moreover, an additional indirect positive regulation of dctP expression by Hfq was found. Finally, a metabolome approach was performed to investigate the internal signals modulating CCR via induction of CbrA activity in P. aeruginosa PAOl and P. putida KT2442. The results of the analysis are currently under study in the laboratory. - Les acides C4-dicarboxyliques font partie des sources de carbone et d'énergie préférés de P. aeruginosa, une bactérie versatile et ubiquitaire. Néanmoins, malgré leur importance, comment la présence des acides C4-dicarboxyliques dans le milieu est sentie par la bactérie et comment ils sont transportés dans la cellule chez P. aeruginosa n'étaient pas connus. De plus, peu d'informations sur ces procédés ont été répertoriées dans la littérature. Grace à notre travail, le système de transport des acides C4-dicarboxyliques (Dct) chez P. aeruginosa a pu être caractérisé. En effet, il est composé d'un nouveau système à deux composants, nommé DctB/DctD, qui régule, en combinaison avec le facteur sigma alternatif RpoN, l'expression des deux nouveaux transporteurs des acides C4-dicarboxyliques: DctA et DctPQM. L'inactivation des gènes dctA, dctB or dctD cause un défaut de croissance des souches mutantes dans un milieu minimum contenant du succinate, fumarate ou malate; confirmation de leur rôle dans le Dct. Cependant, une croissance résiduelle du mutant dctA dans ces milieux suggérerait une redondance des transporteurs d'acides Grdicarboxyliques. Une expérience de mutagenèse dans la souche AdctA, utilisant le transposon Tn5, combiné avec un criblage génétique sur la croissance dans le succinate, nous a permis d'identifier le deuxième transporteur DctPQM. DctPQM appartient à la famille des transporteurs TRAP (tripartite ATP-independent periplasmic). Un double mutant AdctAAdctPQM ne pousse pas dans du malate ou fumarate mais par contre présente une croissance résiduelle dans le succinate suggérant l'existence de transporteurs supplémentaires pour le succinate. En réalisant des expériences de compétitions nous avons démontré que le transporteur DctPQM est plus efficace que le transporteur DctA pour l'utilisation de succinate à une concentration de l'ordre du μΜ. Par contre, DctA est le transporteur le plus important pour une concentration de succinate de l'ordre du raM. Pour la première fois, deux systèmes de transport, un avec une forte- et un avec une faible-affinité (DctA et DctPQM) pour le succinate, sont coordonnés dans leur activité de transport des acides C4- dicarboxyliques, probablement contribuant à la versatilité de la bactérie. Ensuite, nous avons étudié la régulation du system Dct. En effectuant, en parallèle, une étude sur le phénomène de la répression catabolique (RC) chez P. aeruginosa, un lien entre la RC et le système Dct a été observé. La cascade des régulateurs formant la RC est composée de CbrA/CbrB, CrcZ et Crc. Crc est un répresseur traductionnel qui agit quand des sources de carbone préférées (comme les acides C4-dicarboxyliques) sont présentes dans le milieu. CrcZ est un petit ARN non-codant qui agit comme antagoniste de Crc. L'expression de CrcZ est induite par le système à deux composants CbrA/CbrB lorsque une source de carbone non-préférée est utilisée (comme le mannitol). Des nouvelles cibles du système CbrAB/CrcZ/Crc chez P. aeruginosa ont été identifiées grâce à une analyse du transcriptome des souches mutantes des régulateurs de la cascade. Parmi les cibles identifiées, les gènes dctA et dctPQM étaient présents. La RC régule l'expression des transporteurs dct en fonction de la concentration de succinate dans le milieu de croissance. Cette régulation est possible parce que, en même temps, les acides C4- dicarboxyliques régulent la RC. Dans un milieu contenant une grande concentration du succinate, le niveau d'expression de CrcZ est faible, donc Crc peut inhiber l'expression de ces ARN messagers cibles. Par contre, dans un milieu avec une faible concentration de succinate, l'augmentation de l'expression de CrcZ titre Crc et inhibe son activité. Ce modèle de régulation rétroactive est très important pour le phénomène de la RC, parce qu'il permet à la bactérie d'accorder une meilleure réponse à un changement environnemental. L'expression des gènes codant pour les transporteurs dct sont aussi régulés par la protéine chaperonne d'ARN Hfq. Hfq semble avoir le même effet traductionnelle que Crc, lorsqu'il y a une forte concentration de succinate. Nous avons ainsi observé une régulation négative de l'expression du gène dct Ρ et dctR, qui code pour un répresseur de la transcription de dctA. Nous avons aussi observé une régulation positive de la transcription de dctP par Hfq, probablement de façon indirecte. Enfin, une analyse du metabolome a était utilisée pour chercher les signaux internes modulant la RC et, en particulier, l'activité de la protéine senseur CbrA chez P. aeruginosa PAOl et P. putida KT2442. Les résultats de l'analyse sont en cours d'étude dans le laboratoire.