Unlocking the mysteries surrounding nutrient fate within the human body has remained an enigmatic pursuit. Our organs and tissues crave specific nutrients, especially in physiological and pathological conditions. Yet, the intricate journey these nutrients embark upon within our bodies remains largely unclear. The aim of my ambitious project is thus to create an innovative chemical-spatial workflow capable of (1) providing the anatomical coordinates of nutrients of interest across tissues (and organs) and (2) understanding the biochemical route these nutrients undergo in the respective tissue areas of the target organ. For this, I will use a complementary approach relying on advanced techniques such as tracer metabolomics, mass spectrometry imaging (MSI) and Stimulated Raman spectroscopy (SRS). I will benchmark this technological platform onto a model of acute kidney injury (AKI) to identify and localize the predominant biochemical pathways. The amalgamation of SRS and MSI with tracer metabolomics is groundbreaking given the current technological landscape and provides an innovative and feasible solution to the posed challenge.

It is common knowledge that an ethernet connection is safer than a wireless connection, and cellular organelles share this approach. To communicate directly among each other, organelles are thought to use Membrane Contact Sites (MCS), the “optical fibers” at the intracellular level. Organelles are in a prime position to sense and communicate stress signals due to their tight integration into the cell’s metabolic networks. Inter-organelle communication is thus crucial to pass on the message to coordinate cellular stress responses and maintain homeostasis. I hypothesise that MCS allow fast and efficient communication of stress signals between intracellular organelles (e.g. mitochondria and ER) to coordinate the cellular stress responses. However, the organelle tethering proteins, the stimuli driving MCS formation and the signalling molecules mediated through these MCS remain elusive in plants. In INTERCOM, I will characterise ER-mitochondria communication in response to stress in Arabidopsis thaliana. For that, I will leverage the host lab’s previously established ER-mitochondria communication model system and establish a proteomic screening setup to identify novel proteins involved in ER-mitochondria MCS in plants. In addition, I will develop a high-throughput platform to study inter-organelle interactions in vivo and to identify stimuli driving MCS dynamics. Finally, I will employ genetically encoded biosensors to pinpoint ROS and calcium inter-organelle signalling events. To reach my goal, I will combine my expertise in intracellular signalling and bioimaging with cutting-edge interactomics, live-cell imaging and high-content technologies available at the host institute. This innovative and interdisciplinary approach will allow me to shed light on plant inter-organelle communication, a field still in its infancy in plant biology with the potential to pave the path for the biotechnological engineering of plants more resilient to harmful environmental conditions.

Fertilization is essential for a species to survive. Mammalian sexual reproduction requires the fusion between the haploid gametes sperm and egg to create a new diploid organism. Although fertilization has been studied for decades, and despite the remarkable recent discoveries of Izumo (on sperm) and Juno (on oocytes) as a critical ligand:receptor pair, due to the structure of Izumo and Juno, it is clear that other players on both the sperm and the oocytes must be involved. While the focus of our laboratory over the years has been in understanding apoptotic cell clearance by phagocytes, we accidentally noted that viable, motile, and fertilization-competent sperm exposes phosphatidylserine (PtdSer). PtdSer is a phospholipid normally exposed during apoptosis and functions as an ‘eat-me’ signal for phagocytosis. Further, masking this PtdSer on sperm inhibits fertilization in vitro. Based on additional exciting preliminary data, in this ERC proposal, we will test the hypothesis that PtdSer on viable sperm and the complementary PtdSer receptors on oocytes are key players in mammalian fertilization. We will test this at a molecular, biochemical, cellular, functional, and genetic level. From the sperm perspective — we will ask how does PtdSer changes during sperm maturation, and what molecular mechanisms regulate the exposure of PtdSer on viable sperm. From the oocyte perspective — we will test the genetic relevance of different PtdSer receptors in fertilization. From the PtdSer perspective — we will test PtdSer induces novel signals within oocytes. By combining the tools and knowledge from field of phagocytosis with tools from spermatogenesis/fertilization, this proposal integrates fields that normally do not intersect. In summary, we believe that these studies are innovative, timely, and will identify new players involved in mammalian fertilization. We expect the results of these studies to have high relevance to both male and female reproductive health and fertility.

Asthma is the most common chronic lung disease. In most asthmatics, allergen-specific type-2 CD4 T cells orchestrate allergic airway inflammation by producing type-2 cytokines that ultimately drive asthma pathology. Studies suggest that a subset of long-lived memory Th2 cells that reside in the lung, known as tissue-resident memory Th2 cells (T2RM), play a significant role in driving asthma chronicity and persistence. Therapies aimed at depleting T2RM from the lung represent a novel approach to treating asthma, however factors that regulate the generation and persistence of lung-T2RMs remain poorly understood. I hypothesize that distinct and druggable molecular pathways control lung-T2RM survival and re-activation. This work aims to decipher molecular and cellular mechanisms that regulate the maintenance and re-activation of T2RM within the asthmatic lung niche. The host lab has recently developed a novel TCR transgenic (Tg) mouse line (1DER-CD5), containing a monoclonal population of house dust mite (HDM) specific CD4 T cells with high T2RM differentiation potential, which has been confirmed in preliminary experiments performed since joining the host lab. In WP1, I will perform single cell transcriptomic and proteomic analysis on 1DER-CD5 T cells in the lungs of mice in the memory phases of HDM-induced asthma to predict molecular and cellular networks that regulate lung-T2RM. In WP2, I will utilize targeted spatial transcriptomics, paired with confocal microscopy, to define the lung-T2RM niche and validate molecular and cellular interactions predicted in WP1. In WP3, I will use an in vivo CRISPR-screening approach with Cas9+ 1DER-CD5 T cells to pinpoint key druggable pathways that regulate lung-T2RM survival and re-activation. This work is expected to identify molecular pathways critical in regulating the persistence and re-activation of lung-T2RM, potentially leading to the development of novel therapies to deplete pathogenic T2RM from the airways of asthmatics.