The capacity to aggregate in a nest is at the core of insect societies, but the underlying sensory and neural mechanisms are unknown. Yet, recent studies with the clonal raider ant (Ooceraea biroi) imply the presence of an odor cue (a pheromone) that mediates nest formation. Theoretical modeling suggests that this aggregation pheromone scales with group size. Group size in turn influences many aspects of eusocial insect behavior and colony organization, including division of labor and collective responses in O. biroi. The underlying mechanisms are poorly understood, but the missing link, never tested before, might be that ants compute group size through sensory perception of a hitherto uncharacterized aggregation pheromone, and use this sensory channel to guide their social behaviors in the nest. With the proposed project I aim to: 1) develop behavioral assays to establish whether aggregation behavior is induced by an odor cue, and whether this is colony- or species-specific in O. biroi, 2) identify the pheromone’s chemical basis through GC-MS analyses, 3) establish whether aggregation pheromone concentration conveys group size information and modulates previously implicated collective responses (e.g. nest evacuation upon temperature perturbation) and division of labor (e.g. social task selection), 4) characterize the neural correlates of aggregation pheromone perception and related behaviors using an established genetic calcium indicator (GCaMP) transgenic line. I will focus on how neural coding of this pheromone might vary depending on its concentration (i.e. for group size computation) and on ant age, which is the prime correlate of behavioral division of labor in ants. I hypothesize that 1) there is an odor cue that mediates aggregation behavior in O. biroi, 2) the chemical cue is fixed/species-specific, triggering an innate attraction response, 3) the concentration of this pheromone modulates social behaviors, and 4) neural activity elicited by this pheromone is similar across individuals, but already at the first olfactory processing station the intensity/pattern of the neural response scales with concentration (group size), and changes with ant age, contributing to natural variation in behavioral propensities inside the nest. O. biroi lack queens and reproduce asexually, making this species particularly amenable to genetic manipulation, perhaps uniquely among social insects. With a further hint at an aggregation pheromone, this opens up unprecedented possibilities to gain a mechanistic understanding of social behavior. In conclusion, by investigating which cue induces nest aggregation in O. biroi and how this is processed in the brain and affects social decisions, this project will elucidate how particular modes of perceiving the world can sustain the sophisticated behavior of animal societies.

Photosynthesis, the “engine of life”, feeds oxygen to the biosphere and constitutes the basis of heterotrophic life on Earth. The primary steps of the conversion of sunlight energy into biomass occur in the thylakoid membranes inside plant chloroplasts. Increasing temperatures, an unavoidable effect of the current climate crisis, can damage membrane components and lead to a sustained decline in photosynthetic efficiency. Thylakoids demonstrate an exceptional capability to adapt to environmental challenges, but the factors controlling their tolerance to heat stress are currently unclear. This proposal aims to identify the interplay between membrane composition and its chemical properties that is required to protect light-harvesting functions under high temperatures. The first objective aims to determine how membrane fluidity affects the stability and function of light-harvesting proteins. State-of-the-art plant growth facilities will be used to grow wild-type and heat-resistant Arabidopsis thaliana mutants with altered thylakoid lipid composition, displaying decreased membrane fluidity. A novel combination of fluorescence recovery after photobleaching and Fourier transform infrared spectroscopy will provide a detailed picture of how photosynthetic processes are affected at high temperatures under different lipid backgrounds. The heat-resistance action of specific carotenoids such as zeaxanthin, a yet unexplored field, will be assessed and the nature of their protective role (lipid stabiliser/antioxidant/quencher of unused light energy/membrane fluidity modulator) will be disentangled. By applying artificial membrane modulators, I will quantify the impact of membrane fluidity on light use efficiency. The second objective aims to determine how the membrane protein composition affects the tolerance of thylakoids to heat stress. I will test my hypothesis that proteins crucial for the rapid regulation of light harvesting during light intensity fluctuations can also improve the heat tolerance of plants. A cutting-edge approach will combine multi-photon microscopy with chlorophyll fluorescence lifetime measurements, to link structural and functional aspects of plant adaptation to high temperatures. This will be used to gain unprecedented detail about the 3D organisation of the chloroplasts inside the leaf and its temperature dependence as well as detailed insights into thylakoid ultrastructure. Time-resolved fluorescence, performed at various temperatures, will explore the dynamics adaptations of light-harvesting protein to heat stress. The unique approach of this proposal, combining aspects of plant physiology, biochemistry, spectroscopy and microscopy, will result in the identification of targets for the genetic engineering of heat-resistant crop varieties. Moreover, the universal nature of temperature responses of biological membranes will allow translating the fundamental knowledge gained in this proposal to other areas of human and animal research.

Background: The hippocampus is critical for learning and memory. Hippocampal place cells, i.e., neurons that selectively fire when the animal traverses a specific location in the environment, play an essential role in spatial memory. Memories are thought to be stored in the hippocampus as changes in synaptic connectivity between cells that were coactive during learning. Hippocampal subfield CA3, with abundant recurrent synaptic connectivity, is hypothesized to store memories as stable attractor states. A widely studied phenomenon reflecting such attractor dynamics is replays, i.e., internally generated events wherein place cell assemblies are briefly reactivated during resting periods in the same sequence as during prior experience. Replays play a key role in learning and memory. However, due to limitations in performance of stable recording of population activity at high temporal resolution and in the identification of synaptic connectivity in behaving animals, little is known about the synaptic mechanisms underlying replay formation and long-term dynamics. Aims: 1) Demonstrate the capability to infer synaptic connectivity in behaving mice using novel ultrafast voltage imaging techniques. 2) Identify hippocampal replays based on voltage imaging from large neuronal populations and study their long-term stability. 3) Study the changes that occur during learning in synaptic connectivity between cell assemblies that participate in the same replay events. Central hypothesis: The hippocampus is pre-configured, providing a repertoire of cell assemblies that can be recruited for storing new memories. Based on this hypothesis, I predict that pairs of CA3 neurons that show pre-existing synaptic connectivity will have a higher probability of participating in the same replay events following a learning experience compared to unconnected neurons. Experimental system: Voltage imaging techniques were recently shown to allow stably recording hippocampal population activity at the resolution of single action potentials, yielding >50 simultaneously recorded cells at ~1,000Hz. Furthermore, unlike Ca2+ imaging and dense electrophysiology, voltage imaging enables measuring subthreshold changes in membrane potentials. Thus, voltage imaging can be utilized to infer synaptic connectivity by time-locking the subthreshold responses of a given cell to spiking activity in a candidate pre-synaptic cell within the same imaged field-of-view. In my research, I plan to apply these novel approaches to study the synaptic mechanisms underlying the formation and long-term dynamics of hippocampal replays. Conclusion: Achieving these aims could settle long-standing controversies about the mechanisms that support memory encoding, storage, and retrieval. More broadly, it will help relate measurements of functional connectivity to synaptic connectivity and open the door to studying how this relationship supports cognition, a fundamental goal in neuroscience.

Introduction Collective cell extrusion, through which excess cells are removed from epithelial tissues, is crucial in developmental, homeostatic and pathological processes such as cancer metastasis. In many cases, cell collectives exhibit nematic order and behave like active liquid crystals (LCs). It has shown that cell extrusion depends strongly on the LC order of cellular nematics. In traditional LC droplets, the curved surfaces deform the director field and induce topological defects. The effect of surface curvature on cellular nematics remains largely unexplored. It is of particular interest in the context of intestinal epithelia for which cells are experiencing stereotyped out-of-plane negative (crypts) and positive (villi) curvatures, while extrusion occurs at the tip of villi only. Although little is known about cell fate on curved substrates in vitro, surface curvature such as convexity and concavity has proven to induce unexpected behaviors in epithelia such as cell spreading, migration and architecture. Research aim Understand the effects of surface curvature on cell fate and extrusion. Control the dynamics and topology of cellular nematics through curved surfaces. Central hypothesis Surface curvature plays an important role in determining cell fate and extrusion. Methods I will grow intestinal organoid cells on PDMS or hydrogel microstructures with curved surfaces. I will then track the movements and orientations of individual cells and construct 3D velocity and nematic orderings fields along curved surface by combining multiplane optical microscopy with 3D optic flow algorithms and neuronal networks. I will also characterize the cellular flows and stresses over curved structures through high-resolution live cell imaging. As an active LC, I will characterize the order parameter of the cellular nematic and the spatiotemporal correlation between cells. Based on these measurements, I will quantify the dependence of cell dynamics and orientations on surface curvature and examine if surface curvature can favor cell extrusion at defined locations and if curvature changes can promote collective cell extrusions. I will also collaborate with theoreticians to develop a new active LC model to explain how local curvature affects the behavior of cellular nematics. Special attention will be paid on the formation and dynamics of topological defects. I will investigate the role of curvature in the dynamics of these defects within curved epithelial tissues. Conclusion Collective cell extrusion is important to morphogenesis of cellular nematics. In vivo systems, cells are usually self-organized into curved structures and cell extrusion depends on the local surface curvature. I will try to understand and control the cell fate and extrusion through curved surfaces by combining microscopy, soft matter theories, non-equilibrium statistical methods, molecular biological techniques, advanced data analysis and physical modeling.

The interplay between the force-generating cytoskeleton and the deformable membranes underlies diverse biological phenomena ranging from cell division to motility. The goal of this project is to reconstitute a model system in which one can visualize, characterize and control an active force generating network and study its interactions with a deformable soft interface. Once understood such interactions will be used to develop synthetic droplets that exhibit autonomous motility. This biomimetic system will provide an experimental platform to quantitative test theoretical models of the coupling between cytoskeletal active stresses and interfacial deformations. Once validated in a simplified setting, such theoretical models have the potential to deepen our quantitative understanding of diverse membrane-based cellular phenomena. The first aim is to visualize the 3D structure of dynamical kinesin-driven microtubule (MT) networks with very high spatial and temporal resolution. Existing techniques have employed fluorescence-based confocal imaging, which is limited by slow speed, photobleaching, and phototoxicity. I aim to build on the technique developed during my doctorate - dielectric tensor tomography. Specifically, using the MT birefringence, I will visualize the 3D spatial structure and temporal dynamics of kinesin-driven reconfigurable MT networks in a non-invasive manner. Dielectric tensor tomography relies on elastic light scattering and thus does not require fluorescence labeling. The full 3D structure can be imaged with very high spatial resolution and a temporal resolution of tens of milliseconds. Fast imaging of dynamical 3D MT networks will provide invaluable insight into mechanisms by which they generate extensile active stresses and drive their chaotic flows. In the second aim, I will merge the above-described MT-based active network with a phase-separating binary polymer mixture of poly(ethylene glycol) and dextran. Active MT-network partitions into the dextran phase where it continues to generate active stresses that strongly deform the soft PEG/Dextran interface. By simultaneously visualizing the instantaneous network structure and the associated interface deformation I aim to correlate the two phenomena and develop appropriate theoretical models. Subsequently, by using recently developed optically controlled kinesin clusters I am to create spatial patterns of active stress to deform soft interfaces predictably. In particular, I am to control the wetting angle, which the active droplets make with the hard substrate. By controlling spatial patterns of the active wetting, I am to generate spontaneous droplet motility, which has been predicted by simplified theoretical models. The described projects build on the optics skills I developed during my doctoral work while introducing me to reconstituted biomimetic materials. I expect that the proposed research will broaden our understanding of cytoskeletal active matter and cell motility.

Pathogenic RNA viruses pose a serious health concern. Influenza A virus (IAV) is a single-stranded negative-sense RNA virus with a segmented genome that causes seasonal flu epidemics and circumvents vaccination strategies as a result of genetic reassortment and antigenic drift. Thus, developing effective therapies requires understanding and targeting the conserved biology of the virus. IAV is an obligate intracellular parasite that depends heavily on host machinery for its replication and propagation. While it encodes a polymerase for RNA synthesis, it must make effective use of host RNA-binding proteins and ribonucleoprotein (RNP) complexes to coordinate splicing, nuclear import and export, and translation of each viral RNA segment in space and time. I hypothesize that the RNA lifecycle nuclear localized negative-sense viral RNA genomes (vRNA) and the cytoplasmic positive-sense viral mRNAs (vmRNA) are governed by entirely different sets of host RNPs that are recruited to their client RNAs in a spatiotemporally regulated manner. This study will identify the molecular architecture of the specific RNPs that assemble on each viral RNA segment in a strand-resolved manner to shape the fate of viral RNA. To address this challenge, I will leverage my graduate training in RNA biology and mass spectrometry (MS) to systematically characterise the dynamics of RNP formation on each IAV RNA segment using state-of-the-art MS methodologies. I will utilise RNA antisense purification mass spectrometry (RAP-MS) to capture each viral RNA segment in its positive and negative-sense orientation along with its direct protein interactors. These directly bound proteins will constitute the first layer of the IAV vRNA interactome. Next, I will identify proteins linked to direct RNA binders via protein-protein interactions by combining RAP with in situ cross-linking and mass spectrometry (in situ CLMS). In situ CLMS utilises cell-permeable cross-linking reagents to introduce a covalent bond between nearby residues on proteins in the native context of a virus-infected cell. This enables the identification of RNP complex members and also maps protein-protein interactions at the peptide level by MS-based identification of cross-linked peptide pairs. Hence, combining RAP with CLMS will provide not only the composition of vRNA-associated RNPs, but also reveal information on their 3D conformation and stoichiometry. Upon identifying key proteins and amino acid residues, I will dissect the structure-function relationship of identified RNPs using genetic means. I will perturb proteins and the interacting residues to assess their impact on viral RNA transport/localisation and vRNA replication. This project will yield fundamentally new insights into the host dependency of viral RNA biology and reveal the composition and structure of regulatory relevant vRNA-associated RNPs, which will lay the foundation for rationally designing novel antivirals.

Background:Eusocial insects are characterized by a remarkable system of division of labor within the same colony.The C. floridanus ant has two worker castes that differ in both morphological and behavioral characteristics. Major workers have larger body size and defend the colony, whereas Minor workers provide nursing and foraging.Recently it has been shown that young Majors can be reprogrammed to display increased foraging behavior like the Minor foraging state. Strikingly, this can be done using injection of Trichostatin A (TSA), a histone deacetylase inhibitor.Interestingly, this behavioral plasticity has an optimal time window at 5 days after hatching, and then at 10 days reprogramming efficiency abruptly decreases.In another form of behavioral changes of worker ants, Majors show a natural gradual increase in foraging behavior with age, whereas Minors forage less. Therefore, strict behavioral caste identity is compromised with time. Aims:We propose to investigate the epigenetic mechanisms that affect the aging-like characteristics of behavioral caste reprogramming in ants and that are altered in separate castes during natural ant aging. Central hypotheses:Epigenetic mechanisms are the driving force behind the qualitative division into different castes, and caste reprogramming has a limited time window that is determined by early adult-associated decrease in brain plasticity.On the other hand, the process of animal aging is associated with epigenetic alterations and aging-associated behavioral changes in ants are similar to loss of cellular identity during aging.Because of the parallels between the two processes, we hypothesize that the epigenetic mechanisms which underlie the time-limited plasticity of behavioral reprogramming are general mechanisms related also to ant aging, and may be similar in humans. The experimental system:We will characterize the transcriptional changes that are associated with Major and Minor aging focusing on gain and loss of foraging respectively, as well as the changes in TSA-injected young Major ants between d5 and d10 after hatching, when the reprogramming time window closes. Based on these results, we will find key responsible epigenetic factors that parallel, and then genetically perturb them to alter the aging-related behavioral phenotypes.In particular, we postulate that similar perturbation will extend the reprogramming window in Majors in early adult life, and will extend foraging in Minors during aging. Conclusions:The molecular mechanisms that will be revealed by this project may shed light on human related processes.These include psychiatric diseases (like depression) that are associated with loss of brain plasticity and which peak around late adolescence (the parallel of Major-induced reprogramming), and human psychological changes and neurological diseases associated with aging (the parallel of behavioral changes during ant aging) and could be cross-correlated with the relevant human GWAS studies.

The molecular and genetic mechanisms that underlie the formation of a complex brain with extensive cognitive abilities remains one of the biggest biological mysteries. Decades of research investigating the connection between embryonic development and the adult brain suggest a correlation between advanced cognitive behavior and the complexity of early neurogenesis, as the latter provides greater cell diversity and brain size. While vertebrate models have provided key insights into neurodevelopmental mechanisms, little is known about the development of the largest invertebrate nervous systems: those found in coleoid cephalopods (octopus, squid, and cuttlefish). The astounding intelligence of cephalopods has fascinated researchers for decades, yet modern molecular tools, including high-quality genome assemblies and CRISPR-mediated gene manipulation, have only recently been developed. With brains that rival those found in vertebrates in both size and complexity, cephalopods stand out among invertebrates and provide a unique opportunity to investigate the fundamental principles that build a centralized nervous system remarkably similar, but convergently evolved, to vertebrates. Here, I will harness newly developed tools to provide a comprehensive molecular, genetic, and morphological description of the key stages in early cephalopod neurogenesis, including the (i) specification of neuroectoderm and neural progenitors, (ii) the early patterning of different brain regions, and (iii) the morphogenetic mechanisms shaping the embryonic brain. To accomplish these aims, I will leverage recently established tools in the longfin inshore squid Doryteuthis pealeii and the bobtail squid Euprymna scolopes, including multicolor fluorescent in situ hybridization, single cell RNA sequencing, and live cell imaging to identify and detail the cellular and molecular events in neural progenitor specification and neuronal patterning. I will also develop new methods for use in cephalopods based on my previous work in zebrafish, such as lineage tracing and cell transplantation, to reveal key mechanisms of neurogenesis. Furthermore, through genetic manipulation using the CRISPR/Cas9 system, I will then identify and functionally characterize specific molecular factors that are essential for different steps of neurodevelopment. In summary, this project will elucidate the molecular mechanisms underlying early neurogenesis in cephalopods. This work will reveal elements that are either cephalopod-specific, which will provide insights into alternative ways to build a complex brain, or that are similar to mechanisms described in vertebrates, thereby identifying convergent mechanisms that are essential for the development of a complex brain and lay the foundation for enhanced cognitive behaviors.

To flexibly navigate their environments, animals create internal maps of the world. Such internal maps have been proposed to be crucial not only to spatial navigation, but also to abstract higher order cognitive inferences in humans. Seminal works in rodents have uncovered diverse representations of space in the brain, which likely implement these internal maps. However, the sheer complexity of mammalian brains has hampered our understanding of how microscopic circuits of neurons construct these internal maps. Larval zebrafish, with its small size, optical accessibility, and genetic tractability, is an ideal vertebrate model to study circuit mechanisms underlying neural representations of space. A recent work in larval zebrafish has discovered a group of neurons innervating a highly conserved brain structure called interpeduncular nucleus (IPN) whose population activity keeps track of the animal's heading by integrating its intended turns in an angular coordinate. However, it still remains unclear how this heading representation is used for flexible behaviors, as well as how the circuitry surrounding these compass-like neurons implement angular integration. My postdoctoral project will address these two key questions. First, to explore potential behavioral functions of the compass-like neurons, I will establish novel place learning assays that exploit ethologically relevant, naturalistic behaviors of larval zebrafish, such as positional homeostasis or hiding in the presence of visual threats. Using targeted laser ablation and optogenetic manipulation, I will then aim to demonstrate that the heading representation in the compass-like neurons is involved in such spatial behaviors. Second, I will attempt to identify the sources of turn-related motor signals provided into the compass neuron network. To this end, I will first morphologically identify neuron populations that innervate both the compass neurons and the IPN using light-inducible gene expression systems and/or photoactivatable fluorescent proteins. I will then functionally characterize the activity of these identified neurons with two-photon calcium imaging to see if any of them encode intended turning of fish. In parallel, I will perform electron micrograph reconstruction of IPN-projecting neurons to find circuit motives that can support angular integration. Overall, these experiments will uncover the functions and mechanisms of a conserved circuit that computes heading directions, shedding a new light to how internal maps of the world are constructed in vertebrate brains, including ours.

Determining how mechanistically distinct elements of brain processing combine to perform integrated functions is one of the foremost challenges of neuroscience. A new technology called hemogenetic imaging addresses this need by permitting brain-wide studies of neural signaling by genetically targeted cell populations for the first time in living animals. Hemogenetic imaging is performed using reporter proteins called NOSTICs. These probes are calcium-activated enzymes that produce the gaseous vasodilator NO in stimulated cells; this induces artificial neurovascular signals that can be detected by virtually all noninvasive functional neuroimaging methods, permitting these methods for the first time to monitor resolved elements of neural processing. In initial studies, hemogenetic probes were applied for first-of-their-kind circuit-specific functional magnetic resonance imaging (fMRI) studies in rat brains. Although such studies are already powerful, improvements to the probe technology could produce critical quantitative and qualitative enhancements that I propose to pursue in this project. Aim 1. Develop second-generation NOSTIC probe for multiplexed hemogentic imaging. Understanding brain mechanisms requires discerning how functionally distinct neural populations in the brain interact. In this aim, I approach this problem by designing new NOSTIC reporters that display differential sensitivity to substrates and inhibitors that will allow functional imaging signals from different reporters to be distinguished from one another and from endogenous activity signatures in the brain. I will validate the reporters by fMRI in rats. Aim 2. Develop NOSTIC-based probes to study gene regulation events. Existing NOSTIC probes function as probes for intracellular signaling dynamics, but it is of great interest to engineer variants that can monitor other parameters of cellular function in the brain. In this aim, I will generate NOSTICs that are activated by exogenous designer drugs. Because these drug-induced activatable NOSTICs (diaNOSTICs) can be constitutively expressed and are only turned on in the presence of the drug, comparison of image intensities obtained before vs. after diaNOSTIC activation will act as a sensitive basis for resolving reporter expression profiles, potentially repeatedly and in the same animal. Central hypothesis: I hypothesize that next-generation NOSTIC probes can be effectively engineered using protein chemistry techniques and that these probes will permit multiplexed brain-wide imaging studies of cellular signaling and gene expression in live animals. Experimental system: My work will be carried out using invitro molecular biology, biochemistry, and cell biology techniques, followed by invivo imaging in rodents. Conclusion: Collectively, the work I plan will establish revolutionary new technology for studying the brain-wide patterns of activity and gene expression that underlie mammalian brain function in diverse contexts.

For most of my clinical training in physiotherapy, the sensorimotor system was depicted as a primitive “motor” that moves the body in a reflexive and subconscious manner, separate from the mind. Yet, my recent research has upended this dichotomy, revealing that high-level decisions and low-level action execution jointly enable successful motor control. That is, cognition and action are inseparable. Here, I aim to push this envelope to a new frontier, asking how sensorimotor representations in the brain are shaped by semantic cognition. Specifically, I will use gestural movements to investigate whether the primary motor cortex (M1) carries semantic meaning. By pairing powerful neuroimaging with representational similarity analyses, I will tackle three fundamental questions in cognitive neuroscience: Year 1: are semantic representations embedded in low-level sensorimotor processing; Year 2: how are M1 representations transformed by semantic-motor experience; and, Year 3: how is semantic information transmitted to M1. In one extreme, semantic and sensorimotor representations may inhabit orthogonal representational subspaces, with comprehension and production requiring distinct representational geometries. Alternatively, the “embodied” nature of gesticulation may have a multiplexed representation, with semantic and sensorimotor information embedded in the same brain regions. A cross-disciplinary approach will be necessary to answer these questions: Together with my host Prof. Tamar Makin, and collaborators Profs. Mairead Macsweeney and Matt Lambon Ralph, (Directors of UCL’s Deafness, Cognition and Language Research Centre, and the MRC Cognition and Brain Unit, respectively), I will design novel paradigms to elicit a range of semantic processes, quantify gestural production with markerless tracking, unveil geometric features of brain representation using advanced neuroimaging methods with high temporal and spatial resolution (MEG/fMRI). To examine the effect of neuroplasticity, I will compare how representations differ before and after semantic-motor training, and between patient groups with semantic cognition and sensorimotor control deficits. The training enabled by this proposal will position me at the forefront of a new intersection between semantic cognition and sensorimotor control. Together, this research offers a paradigmatic shift in human cognitive neuroscience – from a traditional view in which semantic and sensorimotor representations operate in isolation, to one where semantic content may prove vital for successful sensorimotor control, and vice versa. Not only will this work offer a more holistic view on these two essential abilities, but it may also improve the capability of brain-computer interfaces to harness semantic and sensorimotor content, and thus augment rehabilitation for patients with language and speech deficits.

• Background The aim of cancer immunotherapy is to engage the immune system against tumor cells but spare normal tissues. Tumor-specific ‘neoantigens’ generated by mutations present only in cancer cells, make an ideal target for immune-mediated tumor control, yet how the immune system engages directly with nascent cancers expressing neoantigens is poorly understood. Understanding the dynamics of immune cell engagement with tumor cells as they become ‘visible’ to the immune system may help define new ways to improve immune-based cancer treatments. Genetically modified mice (GEMM) are key model systems for understanding immune-cancer interactions, particularly at the early stages of tumor development; However, the lack of neoantigens produced during murine tumor development limits their utility. My proposal aims to use new in vivo base editing tools to allow temporal control of antigen production in early and evolving pancreatic tumors, in vivo. • The aims of the proposed research project and central hypotheses To improve upon existing pre-clinical immunogenic mouse models, we will use CRISPR base editing technologies to induce and generate neoantigens at defined times during tumor development. This will allow us to explore early interactions between tumor cells and the immune system and antigen-specific T cell immune responses. • The experimental system We will use a novel mouse model, developed by the Dow lab, that carries an inducible and regulated optimized cytosine base editor (iBE) that enables temporal regulation of a base editor in murine tissues. The inducible and transient expression of the BE can drive high base editing efficiencies in multiple mouse tissues and can be used to build in vivo models that harbor specific cancer-associated single nucleotide variants. The model allows us to generate highly multicomplex mutational profiles using organoids which can be studied ex vivo and then transplant into immunocompetent mice. In addition, we can drive tumor growth in vivo using established Cre-based cancer models and induce the expression of neoantigen via base editing at defined stages throughout tumor expansion. Specifically, we will use a BE-dependent reporter (GO) (Katti et al) to initiate expression of neoantigens. As a proof of concept, I will use the ovalbumin peptide SIINFEKL which expression will be selectively induced when the BE is activated. In addition, and in contrast to other inducible antigen systems that contain a limited number of antigens, this system has the capacity to generate multiple endogenous neoantigens derived from single nucleotide variants. This will be important to interrogate the impact of intratumoral heterogeneity of neoantigens. • Conclusions Our model will offer the possibility of spatial-temporal regulation of inducible neoantigens expression in vivo in immunocompetent mice enable to study a physiologic response by endogenous T cells during the initiation and evolution of cancer.

Conscious perception is a core quality of human cognition. However, a comprehensive empirical account for consciousness remains one of the greatest challenges in modern neuroscience. Theoretical descriptions put forward mechanisms, but their realization in neural tissue is scarcely tested. One gap in knowledge preventing progress is in knowing where exactly to investigate. That is, the understanding of which brain areas are necessary for conscious perception remains elusive. Are there specific brain areas always engaged for conscious awareness and do any contribute more than others? Our objective is to map the functional connectome for stimuli entering and being maintained in conscious awareness. We hypothesize there is a core set of brain areas necessary for the conscious awareness of perceivable stimuli. To test this hypothesis, we will employ electromicrostimulation with functional magnetic resonance imaging (EM-fMRI) in behaving monkeys. Subjects will receive state-of-the-art cranial implants for unprecedented whole-brain access for neural stimulation. This method allows us to (1) probe a vast array of brain areas to determine their role in conscious perception and (2) identify the minimum configuration of brain areas that activate when a stimulus is perceived. Notably, where human fMRI falls short in causal experimental control and monkey electrophysiology in the spatial scope of measurement, the unique combination of EM-fMRI allows for comprehensive evaluation of neural circuitry for conscious perception. Crucially, we design the study to (3) dissociate circuitry for consciousness from that for attention, oft tightly coupled but rarely experimentally distinguished, and (4) test predictions of theoretical accounts for conscious awareness. The task designs orthogonalize attention and conscious access through multimodal electrical and sensory stimulation at the threshold for awareness. Moreover, the results will be explored within theoretical frameworks, including the Global Neuronal Workspace Theory and the Integrated Information Theory and the data can be used to test other theories, because we will publish them open access. The project is highly feasible, given the experience of the Roelfsema Group in probing neural mechanisms of consciousness (e.g., van Vugt et al., 2018, Science) and EM-fMRI (Ekstrom et al., 2008, Science). Beyond local institutional support, Profs. Stanislas Dehaene (Neurospin, FR) and Wim Vanduffel (KU Leuven, BE) are active collaborators whose expertise in consciousness and EM-fMRI will enhance the project and enhance my training. My experience in monkey cognitive neurophysiology has prepared me to pursue these objectives, yet the project affords substantial scientific growth in the form of methodological and conceptual training. Altogether, this project is designed to yield impactful findings that will propel the field of consciousness research forward and prepare me for a career as an independent investigator.

Phytoplankton species account for half of the worldwide CO2 fixation and determine several biogeochemical cycles. Characterizing these organisms' gene expression control mechanisms is critical to understanding their ecological role. Importantly, ocean microbial communities are synchronized by the effect of light fluctuations during the day:night cycle. Environmental and laboratory-based studies have shown different phytoplankton components such as RNA levels, metabolites, and lipids, cycle following diel patterns. Interestingly, the diel pattern also dictates metabolic interactions between single-cell eukaryotes and heterotrophic bacteria. Notably, phytoplankton was shown to have mutualistic and antagonistic interactions with bacteria. However, it is unclear if these interactions are diel regulated and how gene expression is controlled depending on the community composition and the light status. While the application of RNA-seq based protocols has allowed quantifying phytoplankton response at the mRNA level, the translational regulatory layer remains unexplored. Studies in several eukaryotes have shown that mRNAs get translated at specific rates, allowing fine-tuning gene expression, but its role in phytoplankton biology is unknown. In this work, I aim to characterize the phytoplankton translational regulatory landscape focusing on the impact of two key factors, the diel cycle, and bacterial interactions. I plan to use the centric diatom Thalassiosira pseudonana and the pennate diatom Phaeodactylum tricornutum as models, representing two subdivisions of an ecologically relevant phytoplanktonic group (Diatoms). I will measure translation efficiency using ribosome profiling in a culture experiment simulating the day:night light changes and in co-cultures with Ruegeria pomeroyi, a mutualistic bacteria, or Croceibacter atlanticus, an antagonistic bacteria. However, because laboratory models do not represent most species, it is also required to study natural populations directly. Yet, we lack a protocol to measure translational efficiency in them. To solve this constraint, I will develop a new method termed Meta-Riboseq inspired by the principles of ribosome profiling and metatranscriptomic protocols to quantify translational efficiency in natural phytoplankton populations. This combined approach will quantify the extent of translational regulation present in single-celled marine eukaryotes in a controlled experiment and in their environment. Specifically, this setup will allow identifying dynamically translated genes across the diel cycle and specific profiles triggered by mutualistic or antagonistic bacteria-algae interactions. Comparing the two different diatoms models and the diversity within natural populations will allow inferring evolutionary trends in this regulatory layer. In addition, this work will provide a new method and a relevant dataset contributing to further exploration and generation of new hypotheses in the field.

Cyclic nucleotides stimulate the immune response to pathogens and disease by acting as second messengers. In human cells, the enzyme cyclic GMP-AMP synthase (cGAS) recognizes double-stranded DNA from pathogens or tumor cells and produces 2'–5'/3'–5'-cyclic GMP-AMP (2'3'-cGAMP). 2'3'-cGAMP controls antiviral and anti-tumor responses by activating the receptor stimulator of interferon genes (STING) (Sun et al., Science 2012; Ablasser et al., Nature, 2013). Surprisingly, recent studies reveal that prokaryotic and eukaryotic genomes encode various cGAS-like nucleotidyltransferases named cGAS-like Receptors (cGLRs) (Whiteley et al., Nature 2019; Slavik et al., Nature 2021). Animal genomes each encode several cGLRs, and four cGLRs in human cells have no known function. These uncharacterized cGLRs suggest fundamental gaps in our understanding of nucleotide second messenger signaling. I hypothesize that cGLR enzymes in human and animal cells produce diverse nucleotide second messenger signals and control novel immune signaling pathways. My proposed research will combine biochemistry and structural biology approaches with an unbiased cell-based screen to elucidate how uncharacterized animal cGLRs recognize distinct ligands and to discover how cellular receptors respond to distinct nucleotide signals. First, I will use X-ray crystallography and cryo-EM to determine the structures of uncharacterized human cGLRs and a Drosophila enzyme named cGLR1 known to respond to double-stranded RNA. These structural and biochemical analyses will define how animal cGLRs evolved a conserved ligand-binding groove to recognize the various ligands, including viral nucleic acid and pathogen-derived metabolites. Second, I will conduct a cellular activation screen using nucleotide signals produced by prokaryotic cGAS-like enzymes to identify novel protein receptors stabilized in the presence of nucleotide signals. Given that human STING recognizes both prokaryotic cyclic dinucleotides and the endogenous cGAS product 2'3'-cGAMP, I predict that prokaryotic nucleotide signals including pyrimidine-containing cyclic dinucleotides and larger cyclic trinucleotide signals will enable me to discover new nucleotide signal receptors in animal cells. Supporting this notion, we have completed a pilot screen in human macrophages revealing new candidate receptors specifically stabilized in the presence of nucleotide second messenger signals. Analogs of 2'3'-cGAMP are rapidly developing as promising new therapeutic drugs, suggesting the importance of understanding nucleotide second messenger signals in cells. My results will reveal novel signals and nucleotide receptors that control the innate immune response and provide the molecular mechanism of how animal cells respond to viral infection.

T cell activation starts with the engagement of the transmembrane T cell receptors (TCRs) with foreign antigen peptides loaded in the major histocompatibility complexes (pMHC) on antigen-presenting cells (APCs). Accumulating evidence in the literature indicates that the TCR acts as a mechanoreceptor, but it remains unclear how pMCH engagement generates forces that are converted to intracellular signalling. One possible hypothesis is that mechanical forces acting on engaged TCRs causes conformational changes within the TCR complex, leading to dissociation of its cytoplasmic zeta-chains from the inner leaflet of the plasma membrane and making them accessible for Scr kinases phosphorylation. However, to date, there are no experimental data that can directly depict this process due to technological three dimensional (3D) imaging limitations in the nanoscale. The overall goal of this project is to develop and implement new DNA- and plasmonic-based technology to quantify the threshold force required for TCR-complex conformational changes and T cell activation upon antigen binding. DNA molecular force sensors (MFS), developed to detect single molecule forces on the piconewton scale, have been recently adapted to be compatible with DNA-PAINT, a super-resolution fluorescence imaging technique capable of achieving sub-10 nm spatial resolution in 2D. This new technology, named tension-PAINT, has so far been only used to map integrin receptor pN mechanical events with 25 nm resolution. To examine the effect of mechanical forces on TCR conformational changes, in this project I will combine tension-PAINT with a new 3D super-resolution method based on metal-induced energy transfer (MIET) capable of achieving sub-10 nm axial resolution. MIET makes use of the distance-dependent coupling of a fluorescent emitter's evanescent field to surface plasmons in a thin metallic layer. Like FRET this coupling shows strong distance dependence with a larger dynamic range (of around 150 nm). This phenomenon can be exploited to obtain the axial position of single molecules with nanometre precision through fluorescence lifetime experiments using a wide-field TCSPC camera. The project has the following scientific aims: 1) Transduce the fluorescence lifetime of single fluorophores to their axial position via MIET using wide-field fluorescence lifetime imaging microscopy. DNA-PAINT compatible 3D nanostructured samples (i.e. DNA-origamis) will be used for calibration. 2) Combine tension-PAINT with MIET to simultaneously map pN mechanical events and molecular positions in 3D with sub-10 nm spatial resolution. Initial demonstrations will be performed using Jurkat T cells and pMCH decorated lipid bilayers to mimic artificial T cell synapse. 3) Use the developed technology to evaluate the impact of receptor-peptide binding affinity and ligand density on the required force to (i) provoke a conformational change within the TCR complex and (ii) to mount proper T-cell activation.

Pain is a multidimensional sensory experience produced by complex activity in the nervous system triggered from the periphery through the spinal cord and on to the brain. Under healthy conditions, pain is essential for protecting us from harm, however, in the context of injury or disease of the nervous system, pain loses its defensive role and instead produces persistent suffering and disability, constituting a pathological disease state termed neuropathic pain that affects ~10% of the population worldwide (van Hecke et al., Pain, 2014). Neuropathic pain is categorized by both stimulus-evoked pain and spontaneous pain which possess distinct pathophysiological mechanisms. Of note, the majority of patients complain more of spontaneous pain than evoked pain (Bouhassira et al., Pain, 2005). However, to date preclinical studies are sharply geared only towards studying evoked pain. This is because while an evoked behavioral pain readout can be readily correlated with a stimulus, spontaneous neuropathic pain (SNeuP) arises without an external stimulus and possesses complex dynamics, rendering quantitative study very difficult, and in consequence the fundamental neurobiological mechanisms are essentially unknown (Mogil, Nat. Rev. Neurosci., 2009). I now aim to mechanistically interrogate the cellular circuits responsible for SNeuP in a rodent model of neuropathic pain developed in my host lab (Decosterd and Woolf, Pain, 2000). Importantly, we will leverage a novel behavioral readout in mice: brief awakenings from sleep, which is causally linked to, and a biomarker of SNeuP (unpublished data). We hypothesize that there are specific cortical areas whose neural activity are associated with brief awakenings and SNeuP. To test this, we will combine brain-wide functional imaging with miniaturized microscope-based neuronal calcium imaging in freely behaving mice to determine which cortical activity pattern is associated with brief awakenings. We will also examine whether this pattern is present only in specific cortical areas or occurs globally. Based on the spatiotemporal features of cortical activation, we will further classify the activity patterns in fully awake mice using support-vector machine algorithm, and determine which activation patterns are similar to those occurring during SNeuP-evoked brief awakenings in sleeping mice. Next, we will employ a spinal window to chronically image ascending neural pathways emanating from spinal cord nociceptive output circuitry that drive cortical activation. Electrophysiological alterations in the pathways will be studied to identify the source of the ectopic neural activity associated with SNeuP. Our goal is to link this neural activity to spontaneous pain behaviors, using optogenetics and machine learning-based behavioral analyses. In conclusion, this project aims, for the first time, to tease out the precise circuits underlying SNeuP, which will pave the way for exploration of novel therapeutic interventions.

Dreams during sleep are a case of cognition unconstrained by concurrent environmental information. Despite the unique potential regarding psychological diagnostics and theories of human experience, the cognitive neuroscience of sleep makes only slow progress of low reliability. One of the main reasons is that experimental stimulus control is prohibited by global sensory inhibition during sleep. I aim to circumvent this shortcoming via experimentally controlling the neurophysiology of sleep cognition via high-resolution, 3-dimensional neuromodulation. This possibility has been established recently with transcranial focused ultrasound (tFUS), which is based on mechanical effects of reversible displacements of neuronal membrane molecules by acoustic vibrations, in turn lead to the opening of ion channels, the influx of calcium ions and thus to the negativization of the membrane potential. The application of tFUS to human sleep is a novel approach. I propose to modulate two key brain structures during REM sleep as a high-yield starting point: (1) the amygdala as a subcortical structure, and (2) the frontopolar cortex. Empirical data on lucid dreaming suggest that triggering both areas in sequence might result in state insight during sleep, leading to volitional control over non-paralyzed muscle groups such as eye movement muscles. If successful, applying tFUS during sleep could solve the two main issues hindering robust progress in studying sleep cognition, namely the lack of stimulus control and missing behavioral measures. This would have far-reaching consequences for scientific dream research, therapies and our understanding of human experience.

Prion diseases are incurable neurodegenerative conditions. They are caused by PrPSc, an infectious form of the cellular prion protein PrPC. Upon ingestion, PrPSc causes misfolding of PrPC and successively propagates to the brain, where it causes a universally fatal spongiform encephalopathy. Inhibition of gut-to-brain transmission should theoretically prevent disease, but the detailed circuit by which PrPSc reaches the brain remains incompletely defined. We have recently used polysynaptic tracing in combination with single-nucleus RNA-sequencing to comprehensively map neuronal gut-brain pathways from the intestinal epithelium to the brain. We made the surprising discovery that a single such pathway, leading from enteroendocrine cells to the brainstem via the enteric nervous system, is characterized by expression of the endogenous prion gene Prnp in each component of the circuit. In this project, we aim at (I) determining the physiological function of PrP in this gut-brain circuit, as well as (II) elucidating if this pathway transmits PrPSc from the intestine to the brain and whether its inhibition can prevent prion disease. To achieve these goals, we will join forces between a lab that has pioneered the study of prion transmission (Aguzzi) and a lab that specializes in the investigation of gut-brain communication (Thaiss).

The astonishing capacity of the brain to process and store information crucially relies on properly functioning synapses. They provide the connecting entities within neural circuits and they properties define circuit function. The molecular composition of synapses can be very different, which has not been appreciated yet to the extent that would allow deeper insights into the molecular underpinnings of information processing in different circuits. While the role of proteins, as core components of the synaptic cell membrane and synaptic transmission has been addressed in certain detail it is unclear to what extent the molecular diversity of lipids can influence synaptic function. Based on new technologies allowing for the first time lipidomic studies at the level of different synapse types, the project aims at unveiling the molecular interactions between lipids and proteins. Newly developed analysis strategies as well as innovative lipid tagging and imaging approaches will enable us to determine the membrane composition of specific synapses with high resolution. This spatial lipidomics workflow will pave the way to studies on how aging and neurological diseases influence synaptic lipid composition with the prospect of therapeutic interventions.