The design of fluorescence tools is an ever-expanding research area in the chemical and biological sciences. The exploration, understanding and control of biological processes in a spatiotemporal manner relies increasingly on fluorescence, but the chemical tool kit assembled to date is still unfit to address the multitude of issues arising from the highly complicated nature of living systems. Thus, there is currently a high demand for more sensitive, more versatile and more suitable fluorescent compounds for in vivo applications. Through this proposal, we precisely intend to provide new, alternative and complementary sets of fluorogenic tools that are non- (or weakly) fluorescent in their native forms (OFF state) and whose turn-ON fluorescence is triggered by light excitation. Of particular note is that our attention will be focused on turn-ON fluorescence via two-photon excitation, which exhibits remarkable advantages in view of in vivo applications (ie, in-depth tissue penetration, low phototoxicity, very fine spatiotemporal control). Starting from the observation that the o-hydroxycinnamyl platform is quite underestimated notwithstanding its promising feature as a fluorescent phototrigger, we first ambition to raise value of this platform through improving its performance in terms of optical, photochemical as well as physicochemical properties. Furthermore, we would like to examine the potential of the o-hydroxycinnamyl platform as a light-activatable sensitizer for luminescent lanthanide complexes. The advantages of the so-designed novel class of turn-ON fluorogenic tools include i) near-infrared fluorescence emission, ii) long-emission lifetime, and iii) high resistance to photobleaching. From a synthetic viewpoint, another significant advantage of the o-hydroxycinnamyl platform is its simple and rational accessiblity in few steps from commercially available and quite cheap materials. Finally, the properties of all envisaged fluorogenic tools will be optimized to such an extent that they will be implemented to cancer biology as cell imaging probes and drug-delivery systems with fluorescence reporting. In that purpose, multicellular tumour spheroids will be utilized as original and pertinent three-dimensional models of cancer cells. If successful, this chemical biology-oriented project will supply both imaging and drug-delivery circles with appropriate fluorescent tools.

This project focuses on the assessment of genotoxic and membrane effects of radiofrequency waves on 3D multicellular models of microtissues thanks to the development of a metrology ensuring the calibrated and systematized conditions of application of electromagnetic fields. RF microdevices, associated with 3D models of multicellular spheroids allowing the detection by fluorescence of the activation of DNA damage response pathways or membrane permeabilization, will be developed and tested while varying different RF parameters (frequency, power, modulation type of electromagnetic waves...). This work will provide precise and quantitative data on the potential impact of electromagnetic fields for public and daily uses (Buetooth applications, including wireless), and also military "DREP" related issues.

Catalysis is widely accepted as one of the leading factors for promoting sustainable development. Although high selectivities have been achieved by tailored made catalysts, the synthesis of the latter is often obliterating the sustainability of the overall process. Our project aims at tackling this issue by developing hybrid catalysts made of a peptidic scaffold and a transition metal complex core. Libraries of peptidic scaffolds will be produced by both chemical synthesis and protein engineering techniques. Peptide sequences will be selected according to their ability to form amyloid aggregates (robust support allowing recyclability), then against their ability to interact with metal (either by direct coordination, supramolecular interaction with a metal complex or though bioconjugation) to form a hybrid catalyst that can promote synthetically relevant catalytic events generating molecular complexity through the formation of one C–C bond or more. Among the reaction proposed, the benchmark Friedel & Craft type addition of indole to enones will allow a direct comparison with other hybrid catalysts. Others reactions will be based on carbene addition (cyclopropanation and ylid formation-rearrangement sequences). Additionally fluorescence-inducing-reactions will be developed in order to facilitate HTS of libraries obtained by protein engineering. Overall, these sustainably produced hybrid catalysts are expected to open new dimension in fine chemical synthetic processes.

Engineering and imaging multicellular tumour spheroids to study 3D cell proliferation. SPH-IM-3D stands for SPHeroids and IMaging in 3D, that are the three keywords associated with this project aiming at developing imaging approaches and at engineering original multicellular tumor spheroid models to explore cancer cell proliferation dynamics in 3D. This project is organized into three complementary objectives. First, we will aim at the development of a new 3D imaging technology, the SPIM (Single Plane Illumination Microscope), a non-commercial device which there are only a few prototypes in the world. We will conduct innovative developments on the use of adaptive optics, on the implementation of photomanipulation devices, and on signal and image processing. In parallel, we will develop new and original models of 3D growth in which cellular tumor heterogeneity will be considered. Multicellular tumor spheroids (MCTS) will be genetically engineered to express fluorescent biosensors and markers specific to a function or a parameter of interest. Finally, we will demonstrate that these complementary technological and biological innovative tools allow monitoring the exploration of the biology of cell division and tumor proliferation in integrated 3D models, giving much attention to the spatial regulation of mitosis and the response to genotoxic stress. This work opens up various perspectives, in particularly in the use of the models and technologies developed in this project for the identification of new putative therapeutic targets and for the assessment of anti-tumor efficacy of new compounds. This project is supported by two academic groups with complementary skills and expertise in cancer biology, cell imaging, optics and mathematics and by a SME specialized in adaptive optics. It is coordinated by the imaging group of the newly created multidisciplinary Institute for Advanced Technologies in the Life Sciences (ITAV – Centre Pierre Poitier).

Responses of biological tissues to mechanical stress are increasingly assessed, owing to their demonstrated link to many biological functions and the onset and progression of various pathologies such as fibrosis or ischemia. The study of these phenomena implies the need of new technologies enabling concomitant stimulation and characterization of the tissue. Materials that can be tuned by an external stimulus have been thus increasingly assessed for applications linked to biology. Among them, stimuli-responsive hydrogels are essential, owing to their naturally high content of water. In this topic, the GELLIGHT project aims at developing biocompatible hydrogels, the stiffness of which can be reversibly tuned by light and which will be used as active sample holders for photonic microscopy of 3D-cultured cardiac cells. For this, dual chemical and reversible physical (based on host-guest interactions) crosslinking will be used and the biological tissue encapsulated directly inside the hydrogels. The originality of GELLIGHT stands at different levels: - the possibility to obtain large stiffness variation in a biocompatible hydrogel, typically over 30%,, in a photo-tunable and reversible manner, as demonstrated in our preliminary results. - influence of mechanical stresses on living cells in a 3D imaging set-up, interplay of responsive hydrogel and cell culture in 3D - the yet unattained level of hydrogel specifications regarding the long-term application to microscopy - the concept of using a kind of sample holder as a basis for biological tissue manipulation, enabling future development of new observation techniques, all in an all-optical set-up for manipulation and observation The project will be organized in 4 scientific work packages: - Synthesis of adequate photochromic dyes which will be responsible of the reversible crosslinking following their shape change. - Formation of the hydrogels, including optimization by design of experiment procedure - Physical characterization of the hydrogels by rheology, dynamic light scattering and atomic force microscopy, specifications tests linked to the use in microscopy (transparency, refractive index compatibility...), - Use of hydrogels as actuators around 3D cell culture for photonic microscopy. This part will examine all links to biology, including cytotoxicity of chemical compounds, and finally confrontation of cardiomyoblasts and cardiomyocytes to the stimulating hydrogel inside the microscopy set-up. This interdisciplinary project joins together 4 academic laboratories with expertise in material (IMRCP in Toulouse, IMMM in Le Mans), photo-responsive systems (IMRCP), microscopy (ITAV in Toulouse), cell biology (ITAV, I2MC in Toulouse) and clinical sciences (I2MC). The geographic proximity of three partners and the already existing collaborations by pairs of all 4 partners ensures a maximum efficiency for the project. This project will lead to the development of new visible-responsive hydrogel formulations, which will open applications for soft actuators for biology or responsive drug delivery systems. It will also provide new tools to simultaneously manipulate and observe biological tissue. This is expected to constitute a basis for next generation mechanobiology set-ups with a diversification of the studied pathologies.