My project aims to develop an in vitro platform to express and study gene expression in model and non-model bacteria, both cultivable and non-cultivable, from the human gut microbiome. Non-cultivable organisms represent the vast majority of gut bacteria and are poorly understood. The diversity of growth conditions, behaviors, and cell compositions makes standard gene expression and protein production protocols ineffective with these non-model bacteria limiting our understanding of these organisms. I propose to use cell-free systems and build a collection of plasmids designed to emulate the transcription machinery of human gut microbiota organisms. The cell-free systems consist of a mixture containing a cell lysate supplemented with an energy buffer and amino acids. As the transcription and translation processes remain active, proteins can be produced directly by adding DNA to the above mixture. The protocol requires large volumes of cell culture that cannot be obtained with difficult to culture or non-culturable organisms in the laboratory. However, the cell-free system can produce transcription machinery from non-model organisms, which will be able to express transcription units from organisms of interest. The objective of this platform is to express complete or partial genomes (natural, PCR amplified or synthesized) in a systematic and controlled manner. I will measure transcriptomes expressed under a wide range of conditions (different sigma factors, transcriptional regulators). This project will lay the foundation for functional genomics on organisms for which only the genetic sequence is known.

Transition management focuses on complex adaptive systems that involve fundamental nonlinear changes in cultures, structures and practices. Transition management represents a conceptual framework that encompasses many faces of transitions like the management of transitions in social-ecological systems (SES) as well as the transitions in governance. Transition management has not been analyzed and conceptualized through a modeling lens to the best knowledge of the authors. Our main research hypotheses is that mathematical tools may help the operationalization of transition management and that it may contribute to the debate on building up sustainable transition management. Based on these research hypotheses, the objective of this project is to move away from conceptual results to operational tools for policy-makers for the sustainable transition management of social-ecological systems. More specifically, we seek to propose operational tools (based on mathematical theory, such as viability theory) that can be nested in the transition management framework. Such approach has been seldom attempted yet to the knowledge of the authors. We will build on recent works developed that gave first insights for connecting governance to effective mathematical models. We naturally want to go further by adding an additional brick to this work: focusing on transitions for going further in the search of operational tools for sustainable transition management. This mono-team project will involve all the seven members of LISC (Laboratory of Engineering for Complex Systems).

MALCOM-X aims at elucidating allelopathic compounds from the moss Physcomitrium patens. These compounds are used by the plant to control both its interaction with neighbours and its own development. In particular, two types of compounds will be searched, based on their signalling roles in strigolactone (SL) or KAI2-Ligand (KL) pathways. Previous work suggest that several PpKAI2Like genes encode potential receptors for these compounds, and mutants in these genes will guide the study, along with a SL synthesis mutant. Metabolite analyses using UPLC of wild-type and mutant moss exudates will be used to spot these compounds. Biological assays, on moss and root parasitic plants will guide the analysis and address the specific role of these allelochemicals. These assays, along with the synthesis of novel standard molecules, will facilitate the purification of the allelopathic molecules. The discovery of allelopathic compounds of interest is expected.

Hemp is a sustainable, multi-use crop that shows fast growth and uses little pesticides. A strong gain of interest in the cultivation and utilization of industrial hemp has been seen over the last decades. Hemp seed and its more processed forms are emerging as a novel source for alternative plant-based foods thanks to their interesting nutritional properties. The increased exposure of the population to different forms of hemp has already revealed allergies. So far, however, most studies have focused on the allergenic risk of hemp leaves and flowers, and little attention has been payed to hemp seed. In light of the rapidly increasing exposure to different forms of hemp, ARCANA aims evaluate the potential allergenic risk of hemp seed consumption by using a multi-disciplinary approach. First of all, the clinical cross-reactive potential of hemp seed will be evaluated using two clinical cohorts in order to establish the prevalence of hemp seed sensitization, as evaluated by skin-prick tests. Prevalence will be assessed in a general allergic population and in populations allergic to cannabis or nuts, which might be at a higher risk for hemp seed allergies according to the small amount of available literature. We will also collect blood for basophil activation tests and serum to characterize the molecular determinants of hemp seed IgE reactivity. Secondly, we will assess the potential of hemp seed to de novo sensitize, by using an innovative human in vitro model and an in vivo mouse model. Together, the work conducted in ARCANA will be an important first step in the evaluation of the potential risk that the consumption of hemp seed, an emerging novel and sustainable food, poses to consumer health. ARCANA will also establish an innovative tool that can help to predict allergic sensitization potential of novel food proteins.

African Swine Fever (ASF) is a deadly epizootic disease affecting mostly domestic pigs and wild boars. Its re-emergence in Europe and Asia in recent years represents a significant threat for the livestock industry. Indeed, there is currently no prophylactic or therapeutic treatment widely available, leaving affecting countries with only one option, the mass culling of infected herds. Highly virulent strains of African Swine Fever Virus (ASFV) display mortality rates close to 100% in pigs. In contrast, attenuated ASFV strains harbor large genomic deletions that greatly reduce the severity of symptoms. Live Attenuated Vaccines (LAVs) based on naturally occurring or gene-deleted ASFV strains constitute the best hope to develop an effective vaccine against ASFV. Elucidating the mechanisms of ASFV virulence and attenuation would thus be key to understand the protection offered by ASFV vaccine candidates, and to better orient vaccine development. While it is known that attenuated ASFV strains induce a different cytokine response than virulent ones, the molecular mechanisms responsible for these differences remain poorly understood. We hypothesize that virulent and attenuated ASFV strains interfere with the pig’s innate immune response in distinct ways, resulting in different pathogenesis and clinical outcomes. The main goal of the MECHAVIR project is to characterize the key steps of the innate immune response to ASFV strains of different virulence levels. First, we will identify which Pattern Recognition Receptors (PRRs) recognize ASFV to kick-off the innate immune response. We will be assess whether PRRs other than cGAS/STING are at play, and whether crosstalk between sensing pathways may influence this process. We will also study how ASFV activates (or conversely, disrupts) the NFkB and IRF3 innate immune signaling pathways, and how this affects the downstream cytokine response. To this end, we will use cutting edge microscopy and medium throughput transcriptomic techniques. Finally, we will investigate how interferon (IFN) blocks the ASFV viral life cycle. We will implement an original single cell RNA sequencing (scRNAseq) approach to discover which IFN Stimulated Genes (ISGs) are blocking the replication cycle of ASFV. We will compare virulent and attenuated ASFV strains throughout this project in order to characterize the molecular mechanisms of virulence and to provide the knowledge required for the rational design of novel Live Attenuated Vaccine (LAV) candidates for ASFV.