La voie pulmonaire suscite un intérêt grandissant pour l’administration systémique des peptides et protéines thérapeutiques, aujourd’hui encore administrés essentiellement par voie parentérale. Un microsystème a été conçu pour permettre de faciliter et accélérer les études in vitro de criblage de différentes biomolécules actives et de sélectionner les formulations les plus adaptées à leur pénétration à travers l’épithélium pulmonaire, en vue de sélectionner les meilleurs candidats à une administration par voie pulmonaire. Organisé en deux configurations distinctes, ce microsystème permet dans un premier temps d’obtenir des barrières épithéliales pulmonaires polarisées et jointives (cellules Calu-3) en seulement 7 jours dans des micropuits de 1mm², sans avoir à renouveler le milieu nutritif ni avoir recours à un appareillage externe associé au microsystème. Grâce à la mise au point d’une technique simple de fabrication, des plateformes de culture contenant jusqu’à 12 micropuits en parallèle sont aujourd’hui fabriquées de manière standardisée. L’évaluation du passage de molécules est ensuite réalisée sous une deuxième configuration dédiée à la mesure de la perméabilité des barrières épithéliales cultivées en micropuits. La capacité de différents candidats (nanoparticules et biomolécules) à traverser l’épithélium pulmonaire a été étudiée. Le passage de nanoparticules de PLGA revêtues de chitosane ainsi que le passage de l’insuline ont été démontrés avec succès. Enfin, l’électrophorèse capillaire couplée à une détection par fluorescence induite par laser (EC-LIF), compatible avec les faibles volumes manipulés dans ce microsystème, a été exploitée pour la détection et la quantification de l’insuline après passage des barrières pulmonaires miniaturisées. A cette fin, l’insuline a soit été marquée par le FITC, soit complexée à un anticorps ou a un aptamère fluorescents. A l’heure actuelle, seule la méthode développée pour le marquage de l’insuline par le FITC est utilisable à des fins de quantification, mais le recours à un aptamère a montré des premiers résultats encourageants. / The pulmonary route is of increasing interest for the systemic administration of therapeutic proteins and peptides, still largely administered parenterally. A microdevice was designed to facilitate and accelerate the in vitro screening studies of various active biomolecules and to select the most suitable formulations for penetration through the lung epithelium, in order to select the best candidates for an administration via the lungs. Organized in two distinct configurations, this microdevice allows as a first step the culture of tight polarized bronchial epithelial barriers (Calu-3 cells) in 7 days in 1 mm² microwells, without the need for medium renewal or the use of an external apparatus. A simple manufacturing technique was developed and glass culture platforms containing 12 parallel microwells can be obtained in a standardized manner. The ability of molecules to cross the pulmonary barrier is then performed in the second configuration of the microdevice, which is dedicated to the permeability measurement of the tight epithelial Calu-3 barriers cultured in microwells. Among the different candidates studied (nanoparticules and biomolecules), the pulmonary barrier permeability regarding PLGA nanoparticules coated with chitosan and regarding insulin has been successfully demonstrated. Finally, capillary electrophoresis with laser induced-fluorescence (CE-LIF), a technique compatible with the low volumes handled in this microdevice, has been exploited for insulin detection and quantification after its transport across the miniaturized pulmonary barriers. To this end, insulin was either FITC-labeled or complexed with a fluorescent antibody or aptamer. Currently, only the derivatization method can be used for a quantification purpose, but the use of an aptamer to indirectly quantitate insulin has shown encouraging results.

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.