No description available.

Les systèmes de sécrétion de NO3– de la membrane plasmique des cellules végétales jouent un rôle important dans l'activité stomatique et la réponse des plantes à des stress biotiques et abiotiques. Malgré quelques avancées récentes, ces systèmes restent mal connus sur le plan moléculaire. Mon travail de thèse a consisté à caractériser le rôle physiologique de NAXT2, un membre du sous-groupe NAXT (NitrAte eXcretion Transporter) de la famille des transporteurs NRT1/PTR chez Arabidopsis thaliana. Mes résultats montrent que NAXT2 est un transporteur de NO3– et qu'il est principalement exprimé dans les cellules du péricycle de la racine, au voisinage des vaisseaux xylémiens. Sous contrainte saline et comparativement aux plantes sauvages (WT), un mutant knock-out pour NAXT2 (naxt2-1) présente un défaut de distribution du NO3– vers les feuilles et de sécrétion du NO3– dans la sève xylémienne, qui se traduisent par une diminution des teneurs en NO3– foliaires. NAXT2 est donc impliqué dans la charge du xylème en NO3– sous contrainte saline. Aucune des différences phénotypiques mises en évidence entre le mutant et WT sous contrainte saline n'a été observée sous stress osmotique ou en condition standard, suggérant que NAXT2 est essentiellement impliqué dans la réponse à la composante ionique du stress salin. Enfin, après un traitement salin prolongé, la biomasse foliaire de naxt2-1 est inférieure à celle de WT, ce qui indique que NAXT2 joue un rôle important dans l'adaptation des plantes aux contraintes salines modérées. Dans l'ensemble, ce travail suggère que NAXT2 est impliqué dans une fonction physiologique majeure, la translocation du NO3–, point de contrôle de la distribution du nitrate, et dans l'adaptation de la plante aux contraintes salines. / NO3– secretion systems at the plasma membrane of plant cells play an important role in stomata activity and plant response to biotic and abiotic stresses. Despite of few recent advances, these systems are still poorly known at the molecular level. During my thesis, I worked on the characterization of the physiological role of NAXT2, a member of the NAXT (NitrAte eXcretion Transporter) sub-group from the large NRT1/PTR transporters family in Arabidopsis thaliana. The results presented here show that NAXT2 is a NO3– transporter and that it is mainly expressed in root pericycle cells, close to the xylem vessels. Under salinity constraint and relatively to wild type plants (WT), a NAXT2 knock-out mutant (naxt2-1) displayed a defect in NO3– distribution towards the shoots and in NO3– secretion into the xylem sap, which lead to a decrease in shoot NO3– content. Thus, NAXT2 is involved in NO3– xylem loading under salinity constraint. None of the phenotypic differences described in this work between WT and mutant was observed under osmotic stress or standard culture conditions, suggesting that NAXT2 is specifically involved in response to the ionic component of salt stress. Finally, after a prolonged salt treatment, naxt2-1 shoot biomass was lower than that of WT, indicating that NAXT2 plays an important role in plant adaptation to mild salinity constraint. Altogether, this work suggests that NAXT2 is involved in a major physiological function, the NO3– translocation, control point of nitrate distribution and in plant adaptation to salinity constraint.

Multidrug resistance (MDR), a phenomenon in which tumors that were initially sensitive, recur and start showing resistance not only to the initial chemotherapeutic agent but also to various anticancer drugs that are structurally and functionally different from the initial drug, constitutes one of the main reasons for the failure of chemotherapy. An important mechanism of MDR is the enhanced cellular efflux of anticancer agents due to an overexpression of ATP-binding cassette (ABC) transporters (i.e. efflux transporters), especially P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1) and Breast Cancer Resistance Protein (BCRP), in cancer cells. In order to reverse this resistance, there has been a lot of emphasis on the development of Pgp, MRP1 and BCRP inhibitors. Although this search has been ongoing for three decades, there are still no clinically available efflux transporter modulators. Tyrosine kinase inhibitors (TKIs) are a novel, rapidly growing class of anticancer agents that have a target-based mechanism of action, and their use transformed cancer chemotherapy due to higher specificity and enhanced safety profiles compared to conventional chemotherapeutic agents. Despite their tremendous success in treating various types of tumors, patients develop resistance to TKIs over time. Most of the FDA- approved TKIs are substrates of Pgp and/or BCRP, and as a result, these efflux transporters are also an important cause of conferred resistance against TKIs in cancer cells. Additionally, none of the 31 approved TKIs have an indication for use in brain tumors and interestingly, this may also due to the presence of Pgp and BCRP at the blood-brain barrier (BBB) and in the tumor cells, which prevent the TKI from crossing the BBB and reaching its target tumor site. Since Pgp- and BCRP- mediated TKI efflux has been shown to be involved in TKI resistance, the inhibition of these transporters could represent a potential TKI resistance reversal strategy. Over the last three decades, a large number of Pgp and/or BCRP inhibitors have been identified, but none of them have successfully made it to the clinic. It was observed that most drugs identified as inhibitors were either unable to achieve Pgp and BCRP inhibitory concentrations in-vivo without imparting severe toxicity, or did not possess adequate bioavailability and tissue distribution profiles in order to reach the tumor site. From these identified candidate inhibitors, after much thought and consideration, we chose to investigate TKIs and methylated flavones as modulators of efflux transporter-mediated TKI resistance. The overall goal of this project was to investigate the promising chemosensitizing potential of TKIs and methylated flavones in efflux transporter-mediated TKI resistance, both in-vitro and in-vivo. To identify potent efflux transporter inhibitor TKIs, we evaluated the effect of various TKIs on the accumulation of afatinib, the model TKI substrate, in Pgp- and BCRP- overexpressing cell lines. Afatinib was chosen as the model TKI substrate for our study because it undergoes very minimal metabolism in several species. Afatinib is a substrate of both Pgp and BCRP, but is not a substrate of uptake transporters. Therefore, it was anticipated that an in-vivo efflux transporter-mediated interaction with afatinib would most likely not be confounded or masked by other factors influencing its disposition. From the in-vitro cell uptake studies, we found that nilotinib is a potent inhibitor of both Pgp and BCRP, and it reversed Pgp- and BCRP- mediated afatinib efflux. Subsequently, an in-vivo study was carried out in mice to investigate the interaction between afatinib and nilotinib; and also the impact of nilotinib on the pharmacokinetics and tissue distribution of afatinib. Afatinib exposure in the plasma and in most tissues, namely liver, lung, kidney, heart, muscle, fat, and skin, was found to be significantly increased when nilotinib was coadministered with afatinib. Further, the nilotinib concentrations in most mice tissues was above that needed for Pgp and BCRP inhibition. These results showed that nilotinib could be a potent chemosensitizing agent for Pgp- and BCRP- mediated TKI resistance. Additionally, a significant increase in afatinib brain exposure was observed in the mice which were administered afatinib in combination with nilotinib. This is an interesting and important finding that could potentially be very useful in the treatment of primary and metastasized brain tumors. We also developed a physiologically based pharmacokinetic model of afatinib to characterize its tissue disposition in mice organs, and this model was then scaled up to humans. The developed model accurately predicted afatinib plasma exposure in healthy volunteers and patients with solid malignant tumors, renal impairment, and hepatic impairment. To investigate the chemosensitizing potential of methylated flavones in efflux transporter-mediated TKI resistance, the Bcrp1 inhibitory effect of 5,7-DMF and its effect on sorafenib accumulation was evaluated in-vitro. 5,7- DMF was found to be a potent inhibitor of Bcrp1 and consequently, its impact on the pharmacokinetics and tissue distribution of sorafenib was evaluated in mice. Results showed that co-administration with 5,7-DMF led to significantly greater sorafenib exposure in plasma and in most tissues collected. This indicated that 5,7-DMF may represent a promising chemosensitizing agent for Bcrp1-mediated TKI resistance due to its low toxicity and potent Bcrp1 inhibition. Our results may have important clinical implications as TKIs are currently the most widely used anticancer agents. 5,7-DMF may show great potential in reversing MDR in tumors expressing BCRP. On the other hand, TKI-TKI combination therapy, especially with nilotinib as the perpetrator, is an attractive strategy to combat both Pgp- and BCRP-mediated TKI resistance. Additionally, since nilotinib has a wide volume of distribution and can reach various tissues at concentrations sufficient enough to inhibit Pgp and BCRP; it could potentially be used as a chemosensitizer in the treatment of numerous types of cancers. Furthermore, its chemosensitizing potential could particularly be useful in the treatment of primary and metastatic brain tumors. Further studies are warranted to assess the chemosensitizing effect of nilotinib in tumor xenograft models.

No description available.

Au cours de cette thèse, nous montrons que l’obésité est un facteur de variabilité pharmacodynamique et pharmacocinétique de la morphine. En particulier, l’absorption et l’exposition à la morphine orale augmentent de façon significative après chirurgie de type bypass gastrique. Nous démontrons le rôle du contenu entérocytaire en transporteur d’efflux P-gp, dans la détermination de l’absorption et de l’exposition à la morphine. / In this thesis, we show that obesity is a factor of pharmacodynamic and pharmacokinetic morphine variability. In particular, absorption and exposure to oral morphine increases significantly after gastric bypass surgery . We demonstrate the role of enterocyte content of efflux transporter P-gp in determining the absorption and exposure to morphine.