We present a phase-shifting digital holographic microscopy technique, where a digital micromirror device enables to perform a precise phase-only shift of the reference wave. By coupling the beam into a monomode fiber, we obtain a laser mode with a constant phase shift, equally acting on all pixels of the hologram. This method has the advantage of being relatively simple and compatible with high frame rate cameras, which makes it of great interest for the observation of fast phenomena. We demonstrate the validity of the technique in an off-axis configuration by imaging living paramecia caudata.

Food transition requires the replacement in human diet of animal-based proteins by alternative sources of proteins including plant-based proteins. This calls for a detailed knowledge of the functional properties of plant-based proteins, including their surface activity. In this framework, we provide here a comparative study of the interfacial properties of two plant proteins, extracted respectively from wheat and sunflower. We combine time- and concentration-dependent measurements of the surface tension and the surface rheology, as measured with a pendant-drop set-up, and of the surface excess concentration, as measured by ellipsometry, of plant protein interfacial films. We demonstrate a time-concentration superposition principle for the surface pressure and surface excess concentration, showing that the kinetics for the building of the interfacial films is essentially governed by the diffusion of the proteins from the bulk to the interface. We find that the rheological and structural properties of the interfacial protein films show markedly different behaviors for the two classes of protein, which is encoded in the structural features of the individual proteins: wheat proteins are more surface active than sunflower proteins, are keen to compress and re-arrange at an air-water interface, whereas sunflower proteins do not. This work provides qualitative and quantitative analysis of the comparative interfacial behavior of flexible and rigid plant proteins extracted respectively from wheat and sunflower, and demonstrates that a combination of several experimental techniques is necessary to obtain insightful information on the interfacial properties of any species.

Mucus is a viscoelastic aqueous fluid that participates in the protective barrier of many mammals' epithelia. In the airways, together with cilia beating, mucus rheological properties are crucial for lung mucociliary function, and, when impaired, potentially participate in the onset and progression of chronic obstructive pulmonary disease (COPD). Samples of human mucus collected in vivo are inherently contaminated and are thus poorly characterized. Human bronchial epithelium (HBE) cultures, differentiated from primary cells at an air–liquid interface, are highly reliable models to assess non-contaminated mucus. In this paper, the viscoelastic properties of HBE mucus derived from healthy subjects, patients with COPD and from smokers are measured. Hallmarks of shear-thinning and elasticity are obtained at the macroscale, whereas at the microscale mucus appears as a heterogeneous medium showing an almost Newtonian behaviour in some extended regions and an elastic behaviour close to boundaries. In addition, we developed an original method to probe mucus adhesion at the microscopic scale using optical tweezers. The measured adhesion forces and the comparison with mucus-simulants rheology as well as mucus imaging collectively support a structure composed of a network of elastic adhesive filaments with a large mesh size, embedded in a very soft gel.

Background: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air–liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery.

Nous avons conçu un prototissu biomimétique comme modèle de tissus cellulaires qui permet d'identifier le rôle individuel des différents constituants cellulaires qui jouent un rôle dans le comportement rhéologique des tissus. L'objectif final est de caractériser le comportement d'écoulement de ce prototissu sous confinement microfluidique. La première partie de la thèse se concentre sur la conception et la synthèse du prototissu à partir de l'assemblage de Vésicules Unilamellaires Géantes (GUVs). Le système ligand-récepteur que nous avons utilisé pour l'assemblage est la paire streptavidine-biotine. Nous avons démontré qu'en modifiant le rapport streptavidine-biotine, le nombre de vésicules en solution et la concentration de biotine dans la membrane des vésicules, il est possible de contrôler la taille des agrégats et la compacité du tissu. Nous avons également modifié la morphologie du tissu biomimétique en changeant la méthode d'incubation, passant ainsi de formes 3D à une structure monocouche 2D. Un autre système d'adhésion basé sur des complémentarités de séquences d’ADN a également été évalué. Il s'est avéré efficace pour contrôler l'adhésion entre les vésicules, et a permis de concevoir des prototissus avec un niveau élevé de compaction. Dans la deuxième partie de la thèse, la rhéologie de ce tissu biomimétique a été testée au moyen d'une configuration microfluidique. Plus précisément, une pression contrôlée a été appliquée et la déformation de l'agrégat lors de son écoulement à travers une constriction a été suivie. Le changement de taille et de forme de l'agrégat a été calculé pour les petits agrégats, ce qui a contribué à élucider la nature de leur comportement élastique. Pour les agrégats plus grands, le mouvement vers l'avant du front de l'agrégat dans une constriction microfluidique en fonction du temps a été mesuré. Il a été possible d'observer un comportement viscoélastique, que nous avons comparé à celui observé dans les tissus épithéliaux. Le modèle de prototissu et les outils que nous avons développés pour caractériser sa rhéologie peuvent être mis en oeuvre à présent pour étudier les propriétés mécaniques des tissus cellulaires en faisant varier ses propriétés clés : l'adhésion entre les cellules individuelles, les propriétés mécaniques du cytosquelette et l'activité cellulaire.