Fluorescence has become an essential observable in Biology and Medicine. The discrimination of a fluorescent label usually relies on optimizing its brightness and its spectral properties. Despite its widespread use, this approach still suffers from important limitations. First, extraction of a fluorescent signal is challenging in light-scattering and autofluorescent samples. Second, spectral deconvolution of overlapping absorption and emission bands can only discriminate a few labels, which strongly limits the discriminative power of emerging genetic engineering strategies, and falls short from the several tens needed for advanced bioimaging and highly multiplexed diagnostic assays. Our consortium of chemists, physicists, and biologists introduces the HIGHLIGHT concept (PHase-sensItive imaGing of reversibly pHotoswitchable Labels after modulatIon of activatinG ligHT) to achieve chromatic aberration-free highly multiplexed fluorescence imaging with only single and dual wavelength channels in emission and excitation. HIGHLIGHT aims at expanding the discriminative dimensions of fluorophore sets much beyond spectral and concentration information such as classically implemented in multicolor labeling approaches. In HIGHLIGHT, label discrimination will not necessitate anymore singular spectroscopic signatures, sophisticated reading-out instruments, or delicate data processing for signal unmixing. In contrast, it shifts towards designing reactive schemes and observables to selectively promote and retrieve the response of a targeted label. HIGHLIGHT exploits reversibly photoswitchable fluorescent proteins (RSFPs) as labels. Increasingly exploited in super-resolution microscopy and dynamic contrast, they are not only fluorescent but as well engaged in rich photocycles. The HIGHLIGHT protocols exploit their specific fluorescence responses to light modulation under well-designed conditions, which provides several dimensions of dynamic contrast to overcome the limitations encountered with spectral discrimination; These responses will serve as readouts either alone or combined using statistical machine learning strategies, which will enable us to perform real time multiplexed imaging of more than ten spectrally similar fluorescent labels and discriminate more than one hundred hues created by mixing these labels in variable amounts and cell territories. As a proof of principle, we propose to challenge HIGHLIGHT in two types of contexts where the paucity of spectrally distinct fluorescent markers has until now been a major hindrance: the analysis of the lineage of retinal cell subtypes and that of their connectivity. In this project, we will namely (i) design and implement a suite of transgenic tools enabling to express varied combinations of 6-12 RSFPs within a population of cells; (ii) design HIGHLIGHT protocols for wide-field and scanning microscopies as well as relevant barcoding strategies to discriminate different cells; (iii) evaluate the photoswitching properties of several tens of RSFPs with one- and two-photon excitation under various environments; (iv) validate HIGHLIGHT for its implementation in a commercial confocal microscope and in state-of-the art Single Plane Illumination scanning Microscopes to push forward acquisition depth and speed; and eventually (v) perform multiplexed clonal analysis in the vertebrate retina, and single-neuron tracing and analysis of axonal convergence. Eventually, the tools and protocols introduced in this project will have near-universal applicability in Biology for multiplexed fluorescence-based observations within biological samples.

Adapting selection tools and objectives to efficiently manage French cattle meat and milk productions is a major challenge of the next years. Genomic selection provides a fantastic opportunity to reorient bovine selection towards a more sustainable breeding. The Gembal project aims at developing a multi-breed genomic selection to extend its use to all beef and dairy breeds, including the small ones. Special attention will be paid for functional traits and maternal traits: calving ease, fertility and longevity of cows in both beef and dairy breeds. At national level, this project should be a common foundation for all breeding schemes, thus avoiding a multiplication of too small and inefficient initiatives. The core of the project is the making-up of the technical basis for the development of multi-breed genomic selection in beef and dairy cattle. The basic idea is that a sample - so-called imputation population - will be genotyped with a high density chip in each breed, whereas most other individuals will be genotyped at a lower cost for a medium density chip. The condition required to build the imputation populations is an extensive use of a new molecular tool, a high density chip with 800,000 SNP developed by Illumina with a consortium including INRA and UNCEIA. Task 2 is dedicated to this technical part of the project. In Task 3, the large multi-breed resource cattle population generated in Task 2 will be the basis for academic researches aimed at characterizing the genetic diversity across breeds and the history of each population submitted to its own context, i.e. drift and selection. This task will also be useful to detect the conserved chromosomal segments across breeds that can be used in multi-breed genomic selection as it will be envisioned in Task 5. Task 4 corresponds to imputation, i.e the statistical procedure to infer missing genotypes in most individuals from the complete genotype information in a limited imputation sample. We will study the quality of the imputation according to breed effective and imputation sample size. We will also develop more computationally efficient algorithms, as imputation will be very demanding with the fast development of genomic selection. Then, a genomic prediction model, using linkage disequilibrium information across breeds, will be developed in Task 5. The methodological challenges are the development of powerful and robust statistical approaches as well as and computing tools for the prediction in a multi-breed context, especially for functional traits with correlated direct and maternal genetic effects. The applications regarding functional traits will be carried out in Task 6 and Task 7 for dairy and beef breeds, respectively. In Task 6, the existence of three breeds in France for which reference populations of reasonable to very large size are available and for which genomic selection programs are already implemented will allow us to undertake reliable comparisons of within vs multi-breed genomic evaluations, hopefully revealing what are the underlying conditions for a successful implementation of multi-breed evaluation. An alternative strategy will consist in checking whether the conserved genome fragments corresponding to favourable haplotypes of QTL detected in any large breed are also segregating in the smaller breeds. Then a genomic evaluation based on these haplotypes could be implemented for the smaller breeds. In Task 7, the multi-beef breed reference population will be composed of the 2,300 bulls that also constitute the beef imputation populations. If a sufficient number of QTL are commonly detected across beef and dairy breeds, a QTL detection and a computation of prediction equations from the beef and dairy pooled reference populations will be undertaken for maternal functional traits.

The epidemiology of infectious diseases, and especially of emerging and re-emerging diseases, can benefit from evolutionary and ecological approaches, notably when they deal with the mechanisms of host response to parasitism. A response to parasitism like the transfer of antibodies from mother to offspring has also broad potential implications in evolutionary ecology, from the adaptive value of maternal effects to the role of transgenerational plasticity in host-parasite interactions. Recent contributions have addressed key issues such as environmental factors affecting the amount of antibodies transferred and whether maternal antibodies affect offspring immunity, but little is still known about factors driving the evolution of the maternal transfer of antibodies and its eco-epidemiological implications. In the current project, we propose to extend our previous work on this topic by addressing a series of key related issues. To do so, we will use complementary approaches, from theory to field and laboratory experiments. The first objective (Task 2) will be to develop theoretical approaches on the evolutionary issues and epidemiological implications of the maternal transfer of antibodies. This will allow us to investigate feedback loops between ecological and evolutionary processes, notably in relation to the dynamics of parasite transmission within host populations. Second, we will investigate predictions about among and within-species variability in traits related to the transfer of maternal immunity, namely the rates of transfer of antibodies and the temporal persistence of antibodies in the offspring. Recent results we obtained show that strong variability among species exists for at least one of these traits. The potential genetic basis of the transfer of maternal antibodies and its associated potential costs will be investigated by developing a selection experiment with poultry (with the possibility to assess correlated responses to selection on the propensity to transmit antibodies while controlling statistically for the mother capacity to produce antibodies) (Task 3). Third, we will ask whether specific benefits of the transfer of maternal antibodies occur in natural situations (Task 4). This will be done by using a novel approach (the injection of antibodies into the egg yolk) to specifically address the potential protective effects of maternal antibodies in a natural host-parasite system involving the kittiwake gull Rissa tridactyla, seabird tick Ixodes uriae and Lyme disease agent Borrelia burdgorferi s.l. Finally, we will address how social interactions and spatial structure can empirically affect the relationships between disease agent circulation and maternal antibody transfer (Task 5). This will be done by conducting a specific experiment on allosuckling and maternal antibody transmission in a social mammal, and by investigating factors affecting the prevalence of eggs containing antibodies against zoonotic agents in spatially structured populations of wild birds. Overall, the expected results could have important basic implications on the evolutionary ecology of host-parasite interactions, but could also lead to interesting applications in terms of immuno-therapeutics, poultry production, eco-epidemiology and conservation.

The current biopsy procedure is to introduce a needle inside the patient towards a given target using echography imaging for control of the position. Reaching the target at the right position is a real issue for diagnosis, therapy and also prognosis, for example concerning tumors or abscess. The NOCT project aims at developing two apparatus, one for imaging and one for navigation, integrated in a complete clinical application of real-time echography navigated biopsy, which would be the first of its kind in the world. We will build an optical imaging system with a needle-like probe that could perform virtual “optical biopsy” prior to the excision of the sample by revealing in vivo the fine microstructures of the tissue. Full-Field OCT is the best-suited technique for this purpose. This technology is now commercialized in a microscope for ex vivo imaging, and we plan on adapting it in a system with a thin rigid probe, with emphasize on ergonomic constraints such as the diameter and length of the needle. We will create a precise surgical navigation system that will be adapted to the clinical ambulatory context, so that it would become in the next decade a reference system for computer assisted medical interventions. This project is a translational research between physics, informatics and medicine, where a key point is to adapt novel technologic apparatus to specific clinical needs. With the aim of the future clinical application we will characterize the preclinical and clinical performance and pay attention on risk management and authorization from the CPP, ANSM and HAS. We will meet these challenges as a consortium of five partners: two laboratories specialized in optics and in computer assisted medical interventions, Institut Langevin-ESPCI and TIMC-IMAG, one clinical investigation center specialized in interventional radiology and computer assisted medical interventions, CIC-IT, and two private companies that will industrialize the final resulting systems, LLTech and Surgiqual Institute.