The objective of this project is to design, characterise and functionally test new nanoscale agents based on single domain antibodies (nanobodies, Nbs), coupled to DNA scaffolds (DNAnobodies) for applications in immunology. The function of immune cells is tightly regulated by biomolecular recognition and signaling at cell-cell contacts, but emergent factors like geometry, multivalency and mechanical forces intervening at this interface are still poorly understood. The multiscale structures from single ligand-receptor bonds to supramolecular architectures, as well as the entanglement of biochemical and physical mechanisms at the nanoscale render functional studies difficult and the design of new therapeutic agents targeting this interface haphazard. We hypothesize that new nanotools are required to advance knowledge in those fields. DNAnobodies will help systematise fundamental studies as well as therapeutic design, since they offer a high control of specificity, reactivity, stoichiometry, architecture, and mechanics at the immune cell surface. This new family of hybrid nano-objects has a great potential for applications in both fundamental biology and therapeutics.

The balance between excitation and inhibition is critical to the proper function of neural circuits. Aberrant activity is characteristic of numerous neurological disorders, including autism spectrum disorder, Rett syndrome, schizophrenia, and epilepsy. Inhibitory interneurons are embedded in almost all central neuronal networks where they act to influence cell excitability, spike timing, synchrony, and oscillatory activity. GABA, the main inhibitory amino-acid neurotransmitter in mature neurons, is a remarkably multi-functional neurotransmitter: it can bind to either ionotropic GABAA (mediating fast neurotransmission) or metabotropic GABAB receptors (mediating slow neurotransmission) that may be localized extra-, peri-, pre- and postsynaptically. The GABAergic phenotype in vertebrates and invertebrates has been defined classically by the presence of three key players in the presynaptic neurons: (i)glutamic acid decarboxylase (GAD), the enzyme needed to synthetize GABA from glutamate, (ii)the H+-coupled transporter (VGAT) that packages GABA in synaptic vesicles, and (iii)the Na+-coupled transporter (GAT) that recaptures GABA at the nerve terminal after its release in the synaptic cleft. The Caenorhabditis elegans nervous system can be considered as a “microcosm” of the GABA universe as it is much smaller, simpler, and experimentally more accessible than a vertebrate nervous system. For over 20 years, the C. elegans GABAergic nervous system was thought to be composed of only 26 out of the total 302 neurons. However, during my post-doc, I have performed an in-depth revision of the GABAergic nervous system in C. elegans. After optimizing immunohistochemistry techniques in C. elegans for GABA staining and generating several fluorescent reporters, I have significantly given new perspectives on what really define a GABAergic neuron in this model organism. In particular, my work has shown that additional neurons contain GABA but do not always express GAD/unc-25, VGAT/unc-47 and GAT/snf-11, the landmark gene portfolio for classical GABAergic neurons. Indeed, I have identified 22 new GABA-positive cells that do not conform to this classical definition and can be categorized into 4 different types of neurons expressing different combinations of these factors. Two of these types show evidence of alternative modes of GABA transport because they lack expression of known GABA transporters, VGAT/unc-47 and/or GAT/ snf-11, and they do not synthetize GABA. Moreover, in vertebrate dopaminergic neurons, similar observations hint towards the presence of alternative mechanisms for GABA transport too Deciphering these new mechanisms of GABA transport will shed light into the regulation of neural circuits through inhibition. I propose to first take advantage of C. elegans, a powerful genetic model organism, to identify and characterize new presynaptic determinants of the GABAergic neurotransmission, focusing mainly on putative and known transporters. Then, we will test their vertebrate orthologues given that the already known components are very well conserved between mammals and worms. New function for already characterized vertebrate transporters could be uncovered as it happened for the glutamate vesicular transporter 1 (VGLUT1) alias BNPI. To achieve this goal my research project will be organized around two aims: AIM1-Novel actor(s) for GABA packaging (Identification and characterization of alternative GABA transporters for packaging GABA in synaptic vesicles) and AIM2-Novel actor(s) for GABA reuptake (Identfication and characterization of new GABA transporters at the plasma membrane, that provide an alternative supply mechanisms for GABA) Altogether, this project aims to extend our knowledge of the cellular mechanisms underlying inhibitory neurotransmission.

Brown algae (Phaeophyceae), or brown seaweeds, are multicellular macroalgae that belong to the larger eukaryotic Stramenopile supergroup, which also includes microalgae (e.g., diatoms) as well as heterotrophic protists (e.g., oomycetes). Like plants and animals, brown algae are one of the small number of lineages that evolved complex multicellularity. Yet, the evolutionary process leading to multicellularity in brown algae has been quite distinct, leading to the acquisition of some unique characteristics which are absent in the other lineages. This project aims to deepen the understanding of the molecular processes underlying brown algae development and acquisition of multicellularity through an analysis of the functional regulatory roles of long non-coding RNAs (lncRNAs), key actors in cellular regulation across the Eukarya domain of life. It will employ a multidisciplinary approach, for which the three partners involved have demonstrated expertise, involving comparative genomics and trancriptomics, computational biology, functional biology and epigenomics to characterize how lncRNAs control gene expression programs that define distinct development states in brown algae. Ectocarpus sp. and Saccharina sp. will be used as preferred model systems since they show distinct developmental morphologies but are evolutionary close. On a fundamental level, the BrownLincs project will provide new knowledge on brown algal development regulation and acquisition of complex multicellularity. On a more applied level, this project has the potential to provide novel molecular tools to enable the engineering of high biomass production in brown algae that can be explored to produce new generation, added value materials for food, feed and other industrial applications, in a blue economy context.

Photosynthesis in the ocean is as important to the planetary climate as that of plants; and is performed by a wide range of cyanobacteria and eukaryotic algae, which possess chloroplasts derived through endosymbioses. Previously, I have used phylogenomics and in vivo localisation to show that chloroplasts of secondary red endosymbiotic origin, which form over 85% of total eukaryotic algal abundance in the modern ocean, are differentiated from plant and other chloroplasts via complex sets of nucleus-encoded and chloroplast-targeted proteins, derived from multiple sources including the endosymbiont, host, and horizontal acquisitions. I will use experimental and computational approaches to identify how the mosaic composition of the secondary red chloroplast proteome underpins its success in the modern ocean. This will include next-generation proteomic (LOPIT) characterisation of dinoflagellate chloroplasts, the least-studied secondary red chloroplast group; phylogenomic and spatial reconstruction of chloroplast proteomes across the algal tree of life, and environmental data from the Tara Oceans expedition; and functional characterisation of key proteins via CRISPR/CAS9 mutagenesis of the model diatom Phaeodactylum. I am particularly interested in characterising novel proteins connected to marine primary production; and temperature adaptations. In preliminary work, I have identified a chloroplast-to-mitochondria metabolite transporter unique to secondary red chloroplasts that underpins photo-acclimation under Fe limitation conditions; and a complete chloroplast glycolysis pathway specific to diatoms, which regulates physiology under conditions associated with high oceanic latitudes (continuous illumination, and low temperature). Future projects may seek to identify chloroplast proteins associated with specific oceanic regions (e.g., the Arctic), and modelling how chloroplast physiology will underpin algal responses to oceanic warming caused by anthropogenic climate change.

One of the most remarkable features of vertebrate morphology is the complexity of the head that harbours a “big” brain, many sensory organs, and an assemblage of bones, cartilages and muscles organized in a much-elaborated way, and its evolution remains an unanswered question. Concerning muscles, their developmental origins are diverse, (some derive from the somites (tongue), others from the paraxial unsegmented pharyngeal mesoderm, whereas the extraocular muscles develop from the prechordal plate). Vertebrates belong to the chordate phylum, with the tunicates and the cephalochordates. Both tunicates and cephalochordates (i. e. amphioxus) are filter feeders and share a pharynx adapted to this mode of nutrition, little sensory organs and reduced anterior centralized nervous system, as well as relatively simple oropharyngeal/atrial muscles. It has been proposed that vertebrates acquired their complex head during evolution as an adaptation to a predatory life style and Partner1 proposed a scenario for the evolution of the head muscles of vertebrates from a chordate ancestor with a mesoderm organization similar to that of cephalochordates. However, how anterior mesoderm derived muscles and associated motoneurons (MNs) co-evolved is still a major question. Thus, the aim of this project is to provide clues about the evolution of the motoneurons associated with anterior mesoderm derived muscles. In other words: are the motoneurons contacting non-myomeric mesodermal anterior muscles of the three chordate clades homologous? To achieve this, we will use cutting-edge techniques (scRNA-seq/ATAC-seq, transgenesis, retrograde labelling, etc.) applied to three chordate models (amphioxus, ascidian and mouse) to provide the information needed for an exhaustive comparison of anterior muscle/neuron systems, in order to define the co-evolution scenario of these structures in relation to the appearance of the complex vertebrate head.