Bioenergetic chains that fuel cellular metabolism have experienced dramatic changes since life appeared on Earth, with a diversification of environmental energy sources and the evolution of numerous dedicated enzymes. The mechanism of energy conservation, however, has remained nearly constant. It predominantly involves ATP synthases operating on a transmembrane proton motive force created mainly by the diffusion of liposoluble H+/electron carriers (quinones/quinols (Q/QH2)) in the membrane between bioenergetic enzymes carrying Q/QH2-binding sites. These Q/QH2 have diversified over the course of evolution and exhibit today a wide chemical and redox variability. The evolutionary history of this variability and the adaptations of enzymes to the different Q/QH2 are far from understood. Our hypothesis proposes that bioenergetic enzymes have adapted over time their Q/QH2-binding sites either for a specific Q/QH2 or for several Q/QH2-types, depending on the thermodynamic constraints of their redox reactions. When the thermodynamic constraint of the co-reaction with Q/QH2 and environmental substrate allows for it, the enzymes would have evolved their Q/QH2-binding site to accommodate any type of Q/QH2, whereas when the co-reaction doesn’t allow for it, enzymes would have evolved by shaping their Q/QH2-site for a specific Q/QH2. Here we aim at testing this hypothesis by working on four structurally distinct enzymes, i.e. the respiratory nitrate reductase Nar, the cytochrome bd oxidase, the Rieske/cytb complex and the alternative arsenite oxidase Arx. The two first ones are furthermore thermodynamically not constrained while the two last ones are. They embrace therefore the enzymatic diversity in bioenergetics. The choice of these four enzymes maximizes the expected information that can be obtained in the time allocated to this proposal. By combining cutting-edge bioinformatics, biochemistry, biophysics, molecular modeling, enzyme engineering and organic synthesis we will address four objectives: (1) establish the level of wild-type protein specificity towards Q/QH2-types, (2) identify amino acids which are part of wild-type Q/QH2-sites and those interacting with Q/QH2, (3) reveal the evolutionary link between Q/QH2-site structure and Q/QH2-type availability and (4) change the Q/QH2-specificity of the enzymes by protein engineering. The first output from ADAPT2Q is the synthesis of new hydrophilic and spin-labelled Q/QH2-analogs that will benefit the entire community working on Q/QH2-enzymes. The second output from ADAPT2Q is an unprecedented global view of the evolution events in the Q/QH2-biogenesis pathways across the prokaryotic world that will be useful to all researchers interested in bioenergetics. But the major benefit from ADAPT2Q will be a comprehensive molecular view of the interplay between Q and their partner enzymes, as well as the evolutionary history of this interplay over the past 3 billion years.

Angiogenesis is the process by which new blood vessels form from pre-existing vessels. The vascular germination which takes place simultaneously at several points of one or more vessels makes it possible to grow a set of neovessels. These come into contact two by two at their ends to form a bridge or vascular loop allowing blood to flow, this is the process of anastomosis. This process is fundamental since it allows blood flow to gradually establish itself through the vascular network in a dynamic way: that is, by redistributing itself as new connections are made. This helps make the network functional and efficient in delivering oxygen (and nutrients) to cells in hypoxic distress when the tissue is damaged. Despite its importance, the process of anastomosis in angiogenesis is still not fully explained due to its complexity which involves understanding how cells perceive and respond to their mechanical environment and how they remodel it dynamically. This reciprocity between the adaptation of the cell to its environment and the remodelling of the environment by cells is a numerical challenge since all the structures to be considered, cells and matrix, constantly evolve in time. As part of this project, our ambition is to explore this bi-directional interaction through a computational model that makes it possible to assess the relative importance of the different mechano-chemical mechanisms and environmental parameters in deciphering the anastomosis. The model will be based on the vast body of knowledge available in the literature on the ability of cells to adapt to the properties of their environment. It will then be calibrated and validated quantitatively by a series of dedicated experiments that we will carry out specifically in this project. We will develop a hybrid and multiscale computational model to study how endothelial cells – i.e. the cells forming the blood vessels – can communicate at a distance via the extracellular matrix to finally meet one another and form the contact required for successful anastomosis. To that end the model will be tightly combined with in vitro experiments. The project is organized into three workpackages. The first workpackage aims at applying well mastered methods for cell characterization on 2D polyacrylamide biogels coated with collagen. This will bring new knowledge on the endothelial cell type that will be used to calibrate the computational model. The endothelial cell morphodynamics and ability to generate forces will be specifically quantified, as well as its matrix remodelling potential (proteolytic activity). The second workpackage aims to validate the model in the more realistic 3D matrix environment made of a network of collagen fibres. Here we make the assumption that the data collected in 2D and characterizing the cell biomechanical properties remain mostly valid in 3D. Generalization of the model in 3D will allow to predict the cell behaviour in the 3D environment. Additionally, imposed matrix deformations will be tested to numerically predict the cell-matrix reactions that will be verified with a posteriori experiments. Finally in the third workpackage, the model developed will be used to identify the optimal conditions that lead to successful anastomosis, i.e. to higher occurrence of cell-cell encounters. From a societal point of view, a better understanding of the mechano-chemical conditions leading to anastomosis is fundamental for tissue engineering and the optimization of tissue reconstruction. The model that we will develop through this project will be the perfect tool to achieve such an optimization.

Single domain antibodies (sdAb or nanobodies) based imaging agent directed at the inflammatory marker Vascular Cell Adhesion Molecule 1 (VCAM-1) have recently been validated by the LRB (partner 1) for the non-invasive nuclear imaging of atherosclerosis and liver inflammation in mice. The lead compound (cAbVCAM1-5) has been produced according to good manufacturing practice and a phase I/II clinical trial is underway at Grenoble-Alpes University Hospital. The aim of the present project is to further evaluate its capabilities using preclinical mice models in order to pave the way of future clinical evaluations. To fulfill this objective, we will evaluate its ability to detect and quantify chronic inflammation in various pathological settings and organs. Moreover, we will evaluate the potency of anti-VCAM-1 sdAb for magnetic resonance imaging (MRI), as an alternative to nuclear imaging, using biodegradable microparticles developed by partner 4 (PhIND). cAbVCAM1-5 will therefore be evaluated in a mouse model of spondyloarthritis in collaboration with the T-Raig team (Lab TIMC, partner 2), in mice models of myocardial infarction with HP2 laboratory (partner 3) and in mice models of neuroinflammation in collaboration with PhIND. Upon completion of this research project, the consortium will have investigated the potential of the anti-VCAM-1 sdAb in 3 pathological settings that could potentially benefit from improved diagnostic and prognostic imaging biomarkers for the management of patients. The results will therefore allow determining relevant strategies for the evaluation of this novel imaging agent, in particular for the design of phase II clinical trials. Moreover, it will provide a better understanding of the tissue and subcellular distribution of this imaging agent, and will explore the possibility to extend its field of application using MRI as a second imaging modality.

In aerobic biological hydroxylations, mono-oxygenases activate molecular oxygen to a peroxy intermediate, using bound cofactors such as heme, flavin or metal ions (Fe, Cu). In anaerobic biological hydroxylations, molybdopterin mono-oxygenases activate a water molecule. We have identified a new type of mono-oxygenases that do not depend on molecular oxygen nor water. The UbiU-UbiV proteins are bacterial enzymes possessing a [4Fe-4S] cluster and perform three hydroxylation reactions on the ubiquinone ring during its anaerobic biosynthesis. Based on strong preliminary results suggesting that the UbiU iron-sulfur cluster activates prephenate, we will combine genetics, biochemistry, biophysics, and organic synthesis to elucidate the unprecedented chemistry of such anaerobic hydroxylation reactions in which prephenate is the sole source of oxygen atom. We will also evaluate the possibility to target UbiU-UbiV proteins from pathogenic bacteria, such as Pseudomonas aeruginosa, for future antimicrobial approaches. Indeed, these proteins do not exist in humans and we propose to synthesize specific UbiU-UbiV inhibitors based on the results of our study of the active site and mechanism of these proteins. These inhibitors will also refine the understanding of the enzymatic mechanism of anaerobic hydroxylation.

The SSP-IQA-"Sustainable, Secure, and framework for Air Quality" project proposes to design, implement and validate a low-cost IT infrastructure with the necessary functionalities for collecting, transmitting, processing and analyzing data relating to outdoor and indoor air quality, particularly in public buildings such as schools/universities or hospitals. The infrastructure will make it possible to link up different platforms existing locally at the consortium partners, with the specification and implementation of efficient solutions at different levels (Fog and Cloud) and integrating security aspects at the level of data collection and transmission. The aim is to enable efficient, scalable handling of large volumes of data on air quality in general, and indoor air quality in particular. Estimating individual exposure inside buildings is a major challenge, due to the variability of temporal and spatial exposure, linked to individual behavior (e.g. studying in classrooms or eating in cafeterias), but also to the dynamics of the surrounding environment, which can also influence indoor air quality (e.g. meteorological conditions, nearby polluting factories, car traffic in the vicinity, occupational context). In this way, the specific environmental context of each consortium partner will enable rich and varied data to be collected, taking into account the multi-dimensional nature in time and space of the data needed in studies of air quality in internal and external environments. Information sharing (open data) is an important aspect of our project. In the medium and long term, the infrastructure can be used to share the data collected and enrich the data concerning air pollution. The creation of structured data, under controlled conditions (taking into account the context) and with real data, is one of the current gaps in the study of air pollution, particularly inside buildings. An important point is that this proposal also addresses international collaboration, in particular with Latin America (Mexico) and Africa (Cameroon), two countries for which the issue of air quality and its influence on human health is a priority area of research. Also, building on each partner's IoT platform the idea is to integrate our contributions and move towards a multi-platform vision for the constitution of a large volume of rich, structured data. Such an infrastructure will not just collect multi-dimensional data in time and space on the qualitý of the air, but also data on the state of computing, storage and transmission infrastructure resources and their energy costs. All to ensure intelligent operation covering sustainable development objectives specific to partners in Europe, America and Africa.