Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis (CF), the most common life-shortening genetic disease among Caucasians. Although general features of the structure of CFTR have been predicted from homology models, the conformational changes that result in channel opening and closing have yet to be resolved. We created new closed- and open-state homology models of CFTR, and performed targeted molecular dynamics simulations of the conformational transitions in a channel opening event. The simulations predict a conformational wave that starts at the nucleotide binding domains and ends with the formation of an open conduction pathway. Experimentally confirmed changes in side-chain interactions are observed in all major domains of the protein. We also identified unique-to-CFTR substitutions that may have led to channel activity in CFTR. Molecular modeling and simulations are used to compare the effects of these substitutions against a canonical ABC transporter, and suggest that gain of channel function in CFTR may have risen from loss of ATPase function at its NBDs. The models and simulation add to our understanding of the mechanism of ATP-dependent gating in this disease-relevant ion channel.

Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane which prevents the accumulation of anticancer, antiviral, antifungal and antibacterial drugs in target cells. Three human ABC transporters are particularly involved in MDR phenotype: P-gp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2. Among the different approaches used to overcome the resistance linked to these transporters, the development of non-substrate drugs MDR-ABC transporters has been described. Here, new class of HIV-1 protease inhibitors not recognized by P-gp/BCRP were identified, promising to be attractive candidates to HAART therapy. Since the determination of the X-ray structures in different conformations is a key point to understand how MDR-ABC transporters translocate drugs across the plasma membrane, the crystal structures of three inward-facing conformations of mouse P-gp were resolved. One structure has a camel nanobody bound to the C-terminal side of the first nucleotide-binding domain, revealing a unique epitope on P-gp and freezing a new open-inward conformation. Finally, the enzymatic characterization of two inhibitors co-crystallized with the mouse P-gp has allowed to localize two main binding sites by which drugs efflux occurs. These results bring new findings of the drug-efflux mechanism and offer the possibility to target more precisely those sites to develop modulators of this pump

Mytilus edulis est un mollusque bivalve de grand intérêt économique et écotoxicologique. Cette espèce sentinelle est connue pour sa résistance aux contaminants chimiques et biologiques. Néanmoins, depuis quelques années la moule bleue est touchée par des mortalités dans les élevages des Pertuis Charentais ayant pour dénominateur commun la présence de bactéries virulentes de type Vibrio. Le premier axe de cette thèse décrit les interactions des isolats de V. Splendidus avec la moule bleue au niveau cellulaire et physiologique. Les infections expérimentales ont permis la sélection de deux isolats bactériens affiliés à V. splendidus/V. hemicentroti : une souche virulente codée 10/068 1T1 et une souche inoffensive codée 12/056 M24T1. Ces deux bactéries ont été marquées à la GFP et validées en tant que modèles authentiques d’exposition à travers leurs caractéristiques de croissance et de virulence. Par ailleurs, V. hemicentroti 10/068 1T1 est capable d’altérer différentes fonctions hémocytaires incluant la motilité, l’adhésion, l’internalisation, la production de ROS, la maturation du phagosome et la viabilité contrairement à la bactérie non virulente. Les produits extracellulaires bactériens semblent être toxiques et inhibent certaines réponses cellulaires (internalisation et production de ROS). Enfin, nous avons reproduis avec succès l’infection des animaux par le pathogène via un modèle expérimental de cohabitation. Le suivi de l’infection montre que V. hemicentroti 10/068 1T1 a pour cible principale les branchies. Le deuxième axe explore le fonctionnement du système MXR (MultiXenobiotic Resistance) chez la moule bleue. La séquence codante complète d’un nouveau transporteur ABCG2 a été établie et la protéine résultante a été identifiée. La caractérisation moléculaire montre la présence du transcrit dans les hémocytes ainsi que dans les branchies et son homologie avec les autres protéines appartenant à diverses espèces. L’utilisation des sondes fluorescentes bodipy prazosin et pheophorbide A, combinées avec des bloqueurs spécifiques, démontre l’activité d’efflux de ce transporteur et son hétérogénéité dans les tissus et cellules. Par ailleurs, il est également démontré que l’expression des trois transporteurs ABC (abcb, abcc, abcg2) identifiés chez la moule bleue est modulée par les contaminants chimiques. Les animaux exposés au BaP au laboratoire ou prélevés sur un terrain contaminé montrent une surexpression des transcrits abc dans les branchies et une sous expression dans les hémocytes. La saisonnalité, sur le terrain, a également un effet sur les niveaux des transcrits et interfère avec les réponses liées aux contaminants. Seul le transporteur abcb exprimé dans les branchies n’est pas affecté par des variations saisonnières et montre une surexpression dans le site contaminé tout au long de l’année. En conclusion, nos résultats démontrent la vulnérabilité de la moule bleue face un pathogène. L'impact immunotoxique des xénobiotiques et le rôle que peuvent jouer les transporteurs ABC dans le fonctionnement du système immunitaire des moules reste à explorer. / Mytilus edulis is a bivalve mollusc representing an economic and ecotoxicological interest. This sentinel species is known for its resistance to chemical and biological contaminants. However, for few years, abnormal mortality events have been reported for farmed blue mussels in France where different Vibrio strains were isolated. The first section of this thesis describes cellular and physiological interactions of V. Splendidus isolates with the blue mussel. Experimental infections allowed the selection of two isolates affiliated to V. splendidus/V. hemicentroti type strains: a virulent 10/068 1T1 and an innocuous 12/056 M24T1. These two strains were GFP-tagged and validated for their growth characteristics and virulence as genuine models for exposure. V. splendidus 10/068 1T1 is capable to alter different functions of hemocytes including motility, adhesion, internalization, ROS production, phagosome maturation and viability, unlike the avirulent strain. Furthermore, bacterial extracellular products appeared toxic and inhibit cellular responses (internalization and ROS production). Finally, we successfully reproduced experimental infection by water tank cohabitation assays with septic animals. Infection monitoring shows the targeting of gills by bacteria. The second section explores the MXR (MultiXenobiotic Resistance) system functioning in the blue mussel. For the first time, a complete ABCG2 amino acid sequence was established. Molecular characterization shows the presence of the abcg2 transcript in hemocytes and gills and its homology with other proteins from various species. The combination of the fluorescent probes bodipy prazosin and pheophorbide A with specific blockers demonstrate the transporter efflux activity and its heterogeneity in tissues and cells. Moreover, the expression of three ABC transporters (abcb, abcc, abcg2) identified in the blue mussel has been shown to be modulated by chemical contaminants. Mussels exposed to BaP in the laboratory or collected from contaminated mussel beds in the field show upregulated abc transcripts in gills whereas these mRNA undergone a downregulation in hemocytes. Season had also an effect on mRNA levels and interacted with site effects. Only the abcb gene displayed a more abundant mRNA level in gills dissected from animals collected in the more polluted area all over the diachronic study. In conclusion, our results demonstrate the vulnerability of the blue mussel towards a pathogen. The immunotoxic impact of environmental xenobiotics and the precise role of ABC efflux pumps in the immune system of the mussel has yet to be explored.

La présence croissante de nanoparticules (NPs) dans les produits de la vie quotidienne (alimentation, médicaments, cosmétiques, textiles…) soulève de sérieuses inquiétudes quant à leurs potentiels effets nocifs pour la santé humaine. Les NPs de dioxyde de titane (TiO2) sont produites à l’échelle industrielle et peuvent déjà être trouvées dans plusieurs produits commerciaux tels que les peintures, les cosmétiques ou dans les systèmes de décontamination de l’eau ou de l’air. Dans le passé, les NPs de TiO2 étaient considérées comme inertes, mais, très récemment, l'Agence Internationale pour la Recherche sur le Cancer les a classées comme possiblement cancérogènes (groupe 2B) pour l’homme. De nombreuses études in vitro et in vivo ont démontré la potentielle neuro-toxicité des NPs de TiO2, mais très peu d'études se sont concentrées plus spécifiquement sur la barrière hémato-encéphalique (BHE), protégeant le cerveau. Aujourd'hui, en dépit des avancées constatées, la bio-cinétique et la bio-accumulation des NPs de TiO2 ainsi que les conséquences sur la physiologie de la barrière hémato-encéphalique (BHE) in vivo restent très peu documentées. De plus, dans l’évaluation du risque lié à l’exposition aux NPs, des facteurs de risque tel que l’âge ont jusqu’ici été quasiment ignorés. Dans ce contexte, l’objectif de ce projet est donc d’évaluer chez le rat adulte et âgé, l’impact d’une exposition aux NPs de TiO2 sur les fonctions de la BHE et sur le métabolisme cérébral. Nos résultats ont montré que les NPs de TiO2 s’accumulent dans certains organes et tissus (principalement dans les poumons, la rate et le foie) et ne sont pas distribuées au système nerveux central (SNC) que ce soit après injection intra-veineuse (IV) ou après une inhalation subaiguë à un nano-aérosol de TiO2. Après administration IV, une interaction directe entre NPs et les cellules endothéliales microvasculaires conduit à des altérations fonctionnelles au niveau de la BHE. Malgré l'absence de translocation vers le SNC, la bio-persistance du titane dans les organes périphériques semble être la cause de modulations de perméabilité de la BHE et d’une inflammation cérébrale. L'implication de médiateurs circulants faisant le lien entre la bio-persitance de titane dans les organes périphériques et les modulations observées au niveau cérébral a été démontré en utilisant un modèle in vitro de BHE. Une réponse exacerbée en termes de neuro-inflammation et de modulation de perméabilité de la BHE établit la vulnérabilité du cerveau âgé à la toxicité des NPs inhalées. Ces résultats ont démontré que malgré l'absence de translocation cérébrale, l'exposition aux NPs de TiO2 induit des altérations fonctionnelles de la BHE et une neuro-inflammation qui pourraient conduire à des troubles neurologiques. L’identification des médiateurs et la description des effets neurotoxiques restent encore à préciser. / The overwhelming presence of nanoparticles (NPs) in products including foods, medications, cosmetics, or textiles raises serious concerns about their potential harmful effects on human health. In the wide diversity of NPs, titanium dioxide (TiO2) NPs are among those produced on a large industrial scale and can already be found in several commercial products such as paints, cosmetics or in environmental decontamination systems. In the past, TiO2 NPs was considered inert, but, very recently, the International Agency for Research in Cancer (IARC) has classified TiO2 as possibly carcinogenic (group 2B) to human beings. Numerous in vitro and in vivo studies have shown the potential neuro-toxicity of TiO2 NPs, but very few studies focus on the central nervous system (CNS), Nowadays, notwithstanding the reported advances, the biokinetic and bioaccumulation ofTiO2 NPs and the consequences on the physiology of the blood-brain barrier (BBB) in vivo are unknown. In addition, NPs effect on susceptible population such as the elderly have been mostly ignored. In this context, the target of the present studies is to evaluate the in vivo impact of exposure to NPs on the BBB physiology and brain inflammation which could promote neurotoxicity in young adults and aging. Our results have shown that TiO2 NPs bioaccumulate in organs and tissues (lungs, spleen and liver especially) and don’t translocate to the brain either after IV or subacute inhalation exposure. In IV administration case, the direct interaction between NPs and brain endothelial cells induces BBB functional alterations. Despite the lack of CNS translocation, the biopersistence of titanium in peripheral organs may be indirectly the cause of BBB permeability alteration and brain inflammation. The involvement of circulating mediators linking titanium biopersitence in peripheral organs and brain impact has been demonstrated using an in vitro BBB model. An exacerbated response in term of neuro-inflammation and BBB permeability modulation has established the vulnerability of the aging brain to inhaled NPs toxicity. Taken together, our findings demonstrated that despite lack of brain translocation, exposure to TiO2 NPs induce BBB physiology alteration and neuro-inflammation that may lead to CNS disorders. Thereafter, identification of mediators and description of the neurotoxic effects may complete the assessment of the impact of TiO2 NPs exposure on the brain.

The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...

The resistance to tamoxifen, a drug used in the adjuvant therapy for hormone sensitive breast cancer, represents a major clinical obstacle. Although various mechanisms leading to tamoxifen resistance have been described and intensively studied, a significant number of patients still become resistant to the treatment and eventually relapse. Tamoxifen therapy has been shown to enrich tumors with cancer stem cells (CSCs), which are naturally resistant, and have self-renewal ability and the potential to form secondary tumors. Metabolic rewiring, altered iron metabolism and upregulation of ATP-binding cassette (ABC) transporters have been shown to be important in the maintenance of CSC phenotype. Therefore, we investigated these mechanisms as possible contributors to tamoxifen resistance in vitro in two tamoxifen resistant (Tam5R) cell lines that we established. We show that Tam5R cells have dramatically disassembled and less active mitochondrial supercomplexes (SCs) and higher level of mitochondrial superoxide, together with a fragmented mitochondrial network. Such dysfunction of mitochondria results in the AMP-activated protein kinase (AMPK) activation and metabolic rewiring towards glycolysis. Importantly, cells lacking functional mitochondria are significantly more resistant to tamoxifen, supporting...

La surexpression de pompes d’efflux par les cellules cancéreuses permet l’élimination d’agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : ABCB1 (aussi appelé P-gp), ABCC1 (ou MRP1) et ABCG2 (ou BCRP, MXR, ABCP). Du fait de leur implication dans le phénotype de « MultiDrug Resistance », il est essentiel de mieux comprendre le fonctionnement de ces transporteurs. Une étude par mutagenèse dirigée a montré que les boucles intracellulaires, ICL0 et ICL1 sont impliquées dans le transport des substrats. Deux résidus sont particulièrement intéressants : W379 qui agirait comme un filtre des substrats ; et H457 qui participerait à la reconnaissance ou à la fixation des substrats. Par ailleurs, il est important de moduler cette chimiorésistance. Dans ce contexte nous avons développé une nouvelle classe d’inhibiteurs d’ABCB1 et ABCG2 non compétitifs basés sur un motif dipeptidique. Les composés les plus efficaces, CT1347 pour ABCB1 et CT1364 pour ABCG2, s’avèrent, d’une part peu ou pas cytotoxiques à fortes concentrations, abolissent d’autre part la résistance induite par ABCB1 ou ABCG2 et se comportent comme des inhibiteurs non compétitifs du Hoechst 33342 et de la daunorubicine. De plus, CT1364 inhibe l’activité ATPasique d’ABCG2 et induit une diminution rapide de l’expression de la protéine. Enfin, les 1ers tests in vivo de ce composé montrent que l’association avec l’irinotécan ralentit la croissance des xénogreffes de petite taille chez des souris / Resistance to chemotherapy is partly due to efflux pumps expressed in the plasma membrane which prevent the accumulation of anticancer drugs in the tumour cells. Three human ATP-binding Cassette (ABC) transporters are particularly involved in this phenotype: P-gp/ABCB1, MRP1/ABCC1, and the last discovered BCRP/ABCG2. Because of their involvement in chemoresistance, it is critical to understand the mechanism by which those ABC transporters recognize and transport drugs. The mutagenesis study of the intracellular loops, ICL0 and 1 shows that these loops are involved in this mechanism. Two amino acids were particularly remarkable: W379 which act as a substrate filter and H457 which can be involved in substrate recognition and binding. In order to restore the cancer cell sensitivity to chemotherapeutic drugs, we have developed a new class of peptide inhibitors, specific to one transporter. A structure-activity relationship study has been performed and made it possible to develop a second generation of molecules. The most efficient compound inhibiting ABCB1 (CT1347) or ABCG2 (CT1364) have none or limitated cytotoxic effects. These compounds restore the activity of chemotherapeutic drugs and act as non competitive inhibitors. Moreover, CT1364 inhibits the ATP hydrolysis activity and lead to a rapid reduction of ABCG2 expression. Initial in vivo tests that have been carried out with CT1364 associated with irinotecan allow to observe a growth reduction of small mice xenografts

En raison de leur résistance aux agents chimiothérapeutiques, les transporteurs ABC de phénotype MDR ont attiré l'attention de la communauté scientifique. Notre projet vise à trouver des conditions dans lesquelles les transporteurs ABC restent fonctionnels en solution pour aboutir à la cristallisation de ces protéines dans une conformation active. Dans ce but, nous avons conçu et développé une nouvelle classe de détergents, à base de calix[4]arène, qui stabilisent ces protéines. Afin de résoudre la structure 3D à résolution atomique du transporteur ABC bactérien "BmrA", responsable de la résistance aux antibiotiques, nous avons utilisé une approche classique utilisant des détergents commerciaux en parallèle à nos détergents innovants. En présence de la Foscholine 12, nous avons obtenu des cristaux diffractant jusqu’à 5 Å de résolution. Cependant, les données de diffraction n’étaient pas suffisantes pour déterminer la structure tridimensionnelle complète de la protéine, seuls les domaines transmembranaires ont été résolus. D'autre part, nous avons atteint l'objectif de l'extraction, la purification et la stabilisation de ce transporteur à l'aide des détergents à base de calix [4] arène. Nous avons également montré que ces détergents promeuvent et améliorent la cinétique de cristallisation de BmrA, une étape que nous sommes en train d’optimiser, pour obtenir des cristaux de meilleure résolution, pour résoudre la structure 3D de BmrA qui sera utilisé pour concevoir des inhibiteurs adaptés / Due to their preponderance in the resistance to chemotherapies, the MDR ABC transporters have drawn the attention of the scientific community. Our project aimed at finding conditions in which ABC transporters are active in solution to lead the crystallization of these proteins in an active conformation. In this purpose, we conceived and developed a new class of detergents, based on calix[4]arene ring, that stabilize these proteins. In order to solve the 3D-structure to atomic resolution of bacterial ABC transporter “BmrA” responsible for antibiotic resistance, we used a classical approach with commercial detergents in addition to the innovative ones. We have crystallized the protein in presence of Foscholine 12 with a diffraction resolution up to 5 Å. The data was incomplete; solving partially the structure of the transmembrane domains. On the other hand, we have reached the objective of extraction, purification and stabilization of this transporter by using calix[4]arene-based detergents. We have also shown that these detergents promote and enhance the kinetics of crystallization of BmrA, a step that we are improving, to get crystals of better resolution, for resolving the BmrA 3D-structure which will be used to design adapted inhibitors

A eficácia das estatinas em reduzir o risco de eventos coronarianos não é completamente explicada por seus efeitos em diminuir colesterol de lipoproteína de baixa densidade (LDL-C). Um dos seus efeitos adicionais pode ser decorrente da modificação na concentração de lipoproteína de alta densidade (HDL), reconhecida como ateroprotetora, principalmente por seu papel no transporte reverso do colesterol (TRC). Os transportadores de membrana do tipo ATP-binding cassette, ABCA1 e ABCG1, e o scavenger receptor BI (SRBI) são proteínas importantes envolvidas no TRC e seus genes são regulados por vários fatores de transcrição, entre eles os liver-x-receptors (LXRs). Com a finalidade de avaliarmos os efeitos dos hipolipemiantes sobre expressão dos transportadores ABC e do receptor SRBI, a expressão de RNAm do ABCA1, ABCG1, SCARB1, NR1H3 (LXR&#945;) e NR1H2 (LRX&#946;) foi avaliada por PCR em tempo real em células das linhagens HepG2 (origem hepática) e Caco-2 (origem intestinal) tratadas com atorvastatina ou sinvastatina (10 µM) e/ou ezetimiba (até 5 µM) por até 24 horas. Além disso, a expressão desses genes também foi avaliada em células mononucleares do sangue periférico (CMSP) de 50 pacientes normolipidêmicos (NL) e 71 hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4semanas, n=48) ou sinvastatina e/ou ezetimiba (10mg/dia/4 ou 8 semanas, n=23). A possível associação entre os polimorfismos ABCA1 C-14T e R219K e a expressão de RNAm em CMSP também foi avaliada por PCR-RFLP. O SCARB1 foi o gene mais expresso nas células HepG2 e Caco-2, seguido por NR1H2, NR1H3, ABCG1 e ABCA1 em HepG2 ou por ABCA1 e ABCG1 em Caco-2. O tratamento com estatinas (1 ou 10 µM) ou ezetimiba (5 µM), por 12 ou 24 horas, aumentou a expressão de RNAm do ABCG1, mas não de ABCA1 e SCARB1, em células HepG2. Ainda nesta linhagem, o aumento na transcrição dos genes NR1H2 e NR1H3 foi observado somente com a maior concentração de atorvastatina (10 µM) e, ao contrário, o tratamento com ezetimiba causou redução na transcrição de NR1H2, sem alteração de NR1H3. Em células Caco-2, o tratamento com atorvastatina ou sinvastatina por 12 ou 24 horas reduziu a quantidade do transcrito ABCA1 e não alterou a expressão do SCARB1 e do ABCG1, embora, para este último, tenha havido uma tendência à diminuição da expressão após tratamento com sinvastatina (p=0,07). Após tratamento com ezetimiba isolada (até 5 µM) nenhuma alteração de expressão de RNAm foi observada em células Caco-2; no entanto, após 24 horas de tratamento com sinvastatina e ezetimiba, foi reduzida a taxa de transcrição de ABCA1 e ABCG1, mas não de SCARB1. Ao contrário das linhagens celulares, em CMSP os genes NR1H2 e ABCG1 foram os mais expressos, seguidos pelos genes SCARB1 e ABCA1 e, finalmente, pelo NR1H3. Indivíduos HC tiveram maior expressão basal de NR1H2 e NR1H3, mas não de outros genes, quando comparados aos NL (p<0,05). Além disso, nos indivíduos HC, a expressão basal de ABCA1 foi maior em portadores do alelo -14T do polimorfismo ABCA1 -14C>T quando comparados aos portadores do genótipo -14CC (p=0,034). O tratamento com estatinas, com ezetimiba ou com a terapia combinada diminuiu a transcrição de ABCA1 e ABCG1. Para o SCARB1, NR1H2 e NR1H3, nenhuma alteração de expressão de RNAm em CMSP foi detectada após os tratamentos in vivo. Após todas as fases de tratamento, ABCA1 e ABCG1 e também NR1H2 e NR1H3 foram significativamente correlacionados entre si, mas nenhuma correlação com perfil lipídico sérico foi relevante. Coletivamente, esses resultados dão indícios de que os hipolipemiantes analisados (estatinas e ezetimiba) têm um importante papel na regulação da expressão de genes envolvidos no transporte reverso do colesterol e sugerem a existência de regulação tecido-específica para os dois transportadores ABC. Além disso, o efeito das estatinas ou da ezetimiba sobre a expressão do ABCA1, do ABCG1 ou do SCARB1 não sofreu influencia de alterações diretas da transcrição dos LXRs. / The efficacy of statins in reducing the risk of coronary events is not completely explained by their effects in decreasing cholesterol low-density lipoprotein (LDL-C). One of their additional effects may result from the change in concentration of high-density lipoprotein (HDL), recognized as atheroprotective, mainly for the role in reverse cholesterol transport (RCT). The membrane transporters, as ATP-binding cassette, ABCA1 and ABCG1, and scavenger receptor BI (SRBI) are important proteins involved in the RCT and their genes are regulated by various transcription factors, including the liver-X-receptors (LXRs) . In order to evaluate the effects of lipid lowering on expression of ABC transporters and SRBI receptor, the mRNA expression of ABCA1, ABCG1, SCARB1, NR1H3 (LXR&#945;) and NR1H2 (LRX&#946;) was assessed by real time PCR in HepG2 (hepatic origin) and Caco-2 (intestinal origin) cells treated with atorvastatin or simvastatin (10 µM) and/or ezetimibe (up to 5 µM) for 24 hours. Furthermore, the expression of these genes was evaluated in peripheral blood mononuclear cells (PBMC) of 50 normolipidemic (NL) and 71 hypercholesterolemic (HC) patients treated with atorvastatin (10mg/d/4 weeks, n = 48) or simvastatin and/or ezetimibe (10mg/d/4 or 8 weeks, n = 23). The possible association between ABCA1 C-14T and R219K polymorphisms and mRNA expression in PBMC was also evaluated by PCR-RFLP. SCARB1 was the most expressed in HepG2 and Caco-2 cells, followed by NR1H2, NR1H3, ABCG1 and ABCA1 in HepG2 or by ABCG1 and ABCA1 in Caco-2. The treatment with statins (1 or 10 µM) or ezetimibe (5 µM) for 12 or 24 hours, increased mRNA expression of ABCG1 but not ABCA1 and SCARB1 in HepG2 cells. Moreover, in HepG2 cells, atorvastatin also upregulated NR1H2 and NR1H3 only at 10.0 &#181;M, meanwhile ezetimibe downregulated NR1H2 but did not change NR1H3 expression. In Caco-2 cells, atorvastatin or simvastatin treatment for 12 or 24 hours reduced the amount of ABCA1 transcript and did not alter the ABCG1 and SCARB1 expressions, despite the tendency to decrease ABCG1 mRNA expression after simvastatin treatment (p = 0.07). After treatment with ezetimibe alone (up to 5 &#181;M) no change in mRNA expression was observed in Caco-2 cells; however, after 24 hours- simvastatin and ezetimibe treatments decreased the transcription of ABCA1 and ABCG1, but not of SCARB1. Unlike cell lines, in PBMC, NR1H2 and ABCG1 were the most expressed, followed by SCARB1 and ABCA1 and finally by the NR1H3. HC patients showed higher NR1H2 and NR1H3 basal expressions, but not of other genes, compared to NL (p <0.05). Moreover, in HC individuals, the ABCA1 basal expression was higher in individuals carrying -14T allele of -14C> T polymorphism when compared with -14CC carriers (p = 0.034). Treatment with statins, ezetimibe, or combined therapy downregulated ABCA1 and ABCG1 expression. For SCARB1, NR1H2 and NR1H3, no change in mRNA expression in PBMC was detected after treatments. After all phases of treatment, ABCA1 and ABCG1 as well as NR1H2 and NR1H3 were significantly correlated, but no correlation with serum lipid profile was relevant. Collectively, these results provide evidences that the lipid lowering (statins and ezetimibe) have an important role in mRNA expression regulation of genes involved in reverse cholesterol transport and suggest the existence of tissue-specific regulation for the ABC transporters. Furthermore, the effect of statins or ezetimibe on ABCA1, ABCG1 or SCARB1 expression was not directly influenced by changes of LXR transcription.

Molibdênio é um elemento traço envolvido na fixação de nitrogênio, enxofre e carbono. Oligopeptídeos estão envolvidos na nutrição bacteriana e diversos outros processos de sinalização intercelular. O objetivo do presente estudo foi investigar o papel funcional das proteínas ligadoras dos sistemas de captação de molibdato (ModA) e oligopeptídeo (OppA) em Xanthomonas axonopodis pv. citri em condições in vitro e in vivo. O mutante ModA mostrou uma produção diminuída de goma xantana, alteração do biofilme e adesão prejudicada em condições in vitro. In vivo a interação do mutante modA mostrou alterações nas lesões causadas em folhas de grapefruit possivelmente resultado da baixa expressão do gene gumB. O mutante na proteína OppA apresentou células mais co-agregadas alterando a estrutura do biofilme e consequentemente diminuindo sua capacidade de adesão. In vivo, a linhagem mutante não alterou o fenótipo de patogenicidade, mas a sua capacidade de crescimento foi afetada no início da fase estacionária sugerindo que o sistema Opp desempenha um papel nutricional. / Molybdenum is a trace element involved in nitrogen fixation, sulfur and carbon. Oligopeptides are involved in bacterial nutrition and several other intercellular signaling processes. The aim of this study was to investigate the functional role of binding proteins of molybdate (ModA) and oligopeptide (OppA) uptake systems of Xanthomonas campestris pv. citri in in vitro and in vivo conditions. ModA mutant showed a decreased production of xanthan gum, altered biofilm and adhesion impaired in vitro conditions. In vivo ModA mutant interaction showed changes in injuries on leaves of grapefruit possibly due to low expression of the gumB gene. The OppA mutant showed more cells co-aggregated by changing the structure of the biofilm and consequently reducing their capacity to adhere. In vivo, the mutant strain did not modify the phenotype of pathogenicity, but its ability for growth was affected at the early stationary phase suggesting that the Opp system plays a nutritional role.