The recent rise of high-resolution and depth imaging techniques like photoacoustic microscopy (PA) stimulates novel research areas in biology. In vivo tracking of immune cells, signaling inflammation and severe pathologies thereof, is one of them and attracts great interest. The AZOTICS project thus aims at addressing the current PA microscopy limitations by fabricating innovative biocompatible elastomeric nanolabels relying on azo photochromes. Photostimulated actuation mechanisms will help amplify the PA contrast based on thermal expansion. The photoinduced mechanical deformations of single nano-objects will be assessed at the nanoscale using atomic force microscopy in order to propose a rationale for the performance of photoacoustic probes beyond their sole optical absorption ability. Their PA imaging capability will be validated through an in vitro, in cellulo and in vivo continuum of studies involving macrophage staining, microfluidic systems mimicking microvasculature, and models of acute inflammatory activated in mice. The interdisciplinary AZOTICS consortium gathers experts in chemistry, physics and optics from Nantes and Grenoble, having already tightly worked together and being keen to share their knowledge in order not only to address unexplored fundamental questions but also to propose innovative photoacoustic systems for in vivo imaging.

Alzheimer’s disease (AD) consists of a continuum from an asymptomatic preclinical form to the disease objectivated by cognitive and memory tests. Differential diagnosis is difficult during the asymptomatic preclinical phase of the pathology. Alzheimer's disease is defined by the association of a progressive dementia syndrome and of two main characteristic brain molecular lesions: extracellular senile plaques composed primarily of amyloid peptides, and neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau aggregates. Recently a major role for extracellular pathological forms of Tau proteins, Tau oligomers, has been shown in the propagation mechanism of the pathology within the brain by contaminating neuron after neuron. Cognitive impairment becomes manifest when lesions reach the hippocampus, with abundant neocortical Tau inclusions and extracellular amyloid deposits. The development of new therapeutic strategies to eliminate the pathological forms of tau protein requires their evaluation during preclinical and clinical trials in well-characterized patient groups. Furthermore, early diagnosis is required for implementing appropriate therapy. Our project concerns the development of nuclear imaging tools to detect the early abnormal forms of Tau in the human brain with Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) in order to improve the diagnosis and the follow-up of AD hallmarks. Our strategy is to develop radiolabeled nanobodies (Nbs) directed against the oligomeric form of pathologic Tau protein. Nbs represent the smallest possible (15 kDa) functional immunoglobulin-like antigen-binding fragments, possess nanomolar affinity and are by nature present in camelids. We produced a Nb (2C5), with both good affinity and specificity for human tau protein oligomers. We significantly increased the passage of the blood-brain barrier (BBB) from 2C5, as demonstrated in vitro in an artificial BBB model, by improving its isolectric point and lipophilicity and by associating it with peptides facilitating the BBB crossing. The purpose of our project is to validate in vivo this new ligand of early pathological forms of tau. The vectorized Nb will be radiolabeled, with 99m technetium (99mTc) by coordination according to the method developed in the laboratory for other Nbs. Binding properties of this radioligand will be evaluated in vitro as previously for 2C5. Then, its biodistribution will be assessed in wild-type mice to determine the radiotracer retention in the various organs. The radiotracer will then be validated in transgenic mice along SPECT imaging studies. Secondarily, the development of radiolabeling of this Nb with 68 Gallium (68Ga) will be carried out and pharmacokinetic and PET imaging studies conducted as for the 99mTc-ligand. The 99mTc and 68Ga radiolabeled ligands will finally be compared to 18F-AV1451, the most currently used NFTs radiotracer: The main goal of this project is to provide a radiotracer specific for abnormal forms of Tau usable in Nuclear Medicine facilities for SPECT and/or PET imaging.

The gastrointestinal tract involves many biological, chemical and physical phenomena to secure the absorption of nutrients from our food. Also, specific sites of the digestive mucosa are gateways to our immunologic system which pave the way for the development of innovative oral therapeutic strategies. These strategies are based on the encapsulation of drugs in nano- or micro- particles or the administration of bacteria, which would target these sites in order to induce an immune response. However, a major scientific barrier is to be able to predict the flow of these "micro-particles" and thus control the dose absorbed by our body. The objective of TransportGut is to develop a predictive and comprehensive modelling of the transport of microparticles in the gastrointestinal system. The challenge of such a model is to account for the different specificities of the physical environment of the digestive tract on the phenomena of transport and mixing. On the one hand, transport and mixing are controlled by the mechanical activity of the smooth muscles of the intestinal mucosa, on both macroscopic and microscopic scales. On the other hand, this activity varies according to the time scales considered. Several scales are thus relevant: the microstructures of the mucosa, the isolated organ and along the digestive system. Mixing at large scales are probably controlled by mixing at small scales. There is currently no particle transport model that takes into account these different scales. TransportGut is an integrated and interdisciplinary project that draws on the complementary expertise of three teams in biorheology, theoretical biophysics and physiology. The team of the Laboratoire Rhéologie et Procédés (LRP) has significant experience in the development of experiments in complex fluid mechanics at macroscopic and microscopic scales, as well as in numerical modeling of flows in the gastrointestinal tract. The team of the Laboratoire Jean Perrin (LJP) has expertise in theoretical modeling of the transport of bacteria and their interactions with the immune system of the digestive tract. Finally, the team from Techniques de l’Ingénierie Médicale et de la Complexité (TIMC-IMAG) develops experimental systems and original technologies for understanding the physiology of smooth muscles. Based on experiments at the interface of physiology and fluid mechanics and numerical simulations of flows, we propose to develop an analytical model of transport connecting these different scales. We will develop experiments on animal models to study the transport of particles along the digestive system and in the vicinity of microstructures of the intestinal mucosa. These experiments will be used to simulate numerically the coupling between flows at microscopic and macroscopic scales in order to understand the role of active and microstructured interfaces on the transport and mixing of microparticles. All of these data from experiments and numerical simulations will make it possible to build analytical and simplified models of the transport and mixture of particles at different spatial and temporal scales. This model would predict the spatiotemporal dispersion of particles in order to be a decision-making tool for the pharmaceutical industry, but also to understand the fundamental mechanisms that govern the spatial structure of the intestinal microbiota.

Chromosome segregation is a fundamental yet poorly understood cell cycle process allowing cells to transmit genetic material to their progeny. In all organisms it is tightly regulated and highly accurate to avoid the loss of genetic information (aneuploidy, chromosome breaks). In spite of an apparent diversity, in particular in the bacterial world, sister chromosome segregation follows conserved rules. In this project, we will use the E. coli model to study the mechanisms underlying one of these rules in bacteria: the pairing of newly replicated sister chromatids and the controlled release of this pairing. We will build up on our recent observations suggesting that sister chromatid pairing involves the establishment of specific chromatin, particular dynamics of paired loci and their segregated neighbors, and transient restructuring of the whole genome. Our working hypothesis is that these features reflect the dynamics of sister chromatid linkages, mainly topological linkages called precatenates and catenates that entangle sister chromatids, and the mechanisms responsible for their release. Based on these results, we will characterize the pairing and segregation mechanisms (WP1), the functioning of the terminal region of the chromosome as a hub dedicated to the control of decatenation (WP2) and we will develop in silico models allowing a quantitative interpretation of the experimental data (WP3). Technically, SISTERS proposes to develop complementary assays to characterize the paired state, of which the monitoring of chromosome loci and the dynamics of sister chromatids by fluorescence microscopy as well as population-based genomic studies and the physical characterization of catenated states. SISTERS also relies on the development of new polymer physics models adapted to the study of braided molecules. This dialogue between modeling and experiments will allow us to evaluate the relevance of our working hypotheses and to feed new research routes. The experiments and models planned during SISTERS will establish the mechanistic basis of the control of sister chromatid pairing and will pave the way to understanding the integration of this process in the bacterial cell cycle.

Invasive fungal infections kill an estimated 1.6 million people each year – as many deaths as are caused by tuberculosis or malaria. Currently, only four drug classes are available to treat these infections (polyenes, azoles, flucytosine and echinocandins). This limited repertoire of antifungal drugs, combined with an alarming rise in drug-resistant fungal strains, has created an urgent need for novel therapeutic agents. In the developed world, the most common fungal disease among hospitalized patients is invasive candidiasis. Among Candida species, C. albicans and C. glabrata rank first and second in isolation frequency, respectively, accounting for ~70% of all systemic candidiasis. This project investigates new antifungal strategies that target the BET family of transcriptional regulators in these Candida species. RATIONALE: The fungal BET protein Bdf1 contains two bromodomains (BDs) and an extra-terminal domain (ET). We recently established the proof of concept that small-molecule inhibition of Bdf1 BDs compromises the viability and virulence of C. albicans (Mietton et al., Nature Communications 2017 and patent WO2018022802A1) and C. glabrata (unpublished data). Bdf1 BD inhibitors thus hold great promise for development as a novel class of antifungal drug. In addition, preliminary data suggest that the Bdf1 ET domain may also constitute a potential therapeutic target. OBJECTIVES: Specific aims of this project are: (i) to generate Bdf1 BD inhibitors with high potency, high selectivity relative to human BDs and demonstrated antifungal activity in a mouse model of disseminated candidiasis; (ii) to validate the Bdf1 ET domain as an antifungal drug target and identify novel ET inhibitory molecules; (iii) to develop an innovative nanoparticle-based delivery strategy that enhances the potency of Bdf1 inhibitors in C. albicans and C. glabrata. NOVELTY: Small-molecule inhibitors targeting chromatin signaling pathways ("epi-drugs") have recently entered the clinic or are in clinical trials to treat cancer and other diseases. By contrast, epigenetic targets have largely remained unexplored in the fungal infection field. Thus, our investigation of Bdf1 as a therapeutic target to combat invasive candidiasis represents a novel and highly promising area of exploration in the antifungal field. BIOMEDICAL RELEVANCE: Candida species annually account for >750,000 cases of systemic infection, with a mortality rate above 40% for patients under antifungal therapy. Hospital costs for invasive candidiasis treatments are exorbitant, estimated at over 40k€ for an adult patient and 130 k€ per child. The translation of Bdf1 inhibitors into new antifungal therapies will have a substantial impact on the health of individuals at risk of an invasive fungal infection and in the long term lead to a significant reduction in national health care costs. CONSORTIUM: This project is a collaborative effort involving four French, one American and one British research groups. It mobilizes a collective expertise in genetics, medicinal chemistry, structural biology, biochemistry, nanotechnology and medical mycology.