The endocannabinoidome (eCBome) is a signaling system that heavily regulates many physiological processes and disorders via multiple signaling molecules, catabolic/metabolic enzymes, and receptors, including cannabinoid receptors 1 and 2 (CB1; CB2). CB1 and CB2 regulate numerous functions, notably appetite, adipogenesis, inflammation, and inflammatory pain, and are activated by two primary ligands stemming from arachidonic acid: the N-acyl-ethanolamine (NAE) anandamide (AEA) and the monoacylglycerol (MAG) 2-arachidonoyl-glycerol (2-AG). There is well-documented connection between the eCBome and the gut microbiome. Thus, there is evidence of their co-modulation. E.g., compounds produced by commensal bacteria can modulate eCBome receptors, while other bacteria-derived metabolites produced by the oxidation of linoleic acid have proven beneficial against obesity and related disorders. Metabolites, provided from the intestinal flora, may yield new eCBome modulators, some with unknown physiological effects. Thus, we believe that eCBome and/or gut metabolites are the primary link between the two. Cyclooxygenase-2 and lipoxygenases metabolize 2-AG and AEA into their oxidated counterparts, except 5-lipoxygenase. Due to steric hindrance, the MAG and NAE headgroups cannot enter the 5-lipoxygenase active site and therefore 5-oxygenated metabolites of MAGs and NAEs should not exist. However, leukocytes have been found to produce novel 5-oxygenated MAG and NAE congeners of fatty acids utilizing a novel biosynthetic pathway. Therefore, we hypothesize that leukocytes and possibly other cells can produce novel oxygenated metabolites that modulate eCBome receptors and have modulatory effects on inflammation, some of which arising from the interaction of host cells with the gut microbiota. The aims of this project are to provide evidence of this pathway via the 1) biosynthesis, characterization, and 2) in vivo identification of novel oxygenated metabolites, and 3) define the cellular receptors by which they exert their biological effects. Suspected metabolites will be synthesized using previously published methods (PMID 33915294). Gut microbiota-derived metabolites will be used to synthesize an array of MAG and NAE congeners, and we will validate their existence in human blood and tissue samples from the IUCPQ tissue bank. Cellular targets of novel metabolites will be screened at GPCRs using the PRESTO-Tango assay, at TRP channels using the Fluo-4 AM assay, and at PPARs using luciferase reporter assays. The expected outcome of this project is to validate the existence of a novel biosynthetic pathway in leukocytes and possibly other cells, identify the scope of the metabolites it can generate, and obtain evidence of their physiological functions via their cellular targets. These data will provide basis for, and shape, the direction of future projects including investigation of host-gut interactions and metabolite variation between obese and non-obese samples.

Proper regulation of gene expression is critical to biological function, and dysregulation is widely implicated in disease. Typical genes are controlled by transcription factors that bind both at promoter-proximal elements as well as distal enhancers as far as hundreds of kilobases away and tend to be highly cell-type specific. Improper enhancer-promoter interactions can cause devastating effects, with a prime example being oncogenes’ hijacking of other genes’ enhancers in tumorigenesis. While enhancer-promoter associations were traditionally thought to involve stable long-range looping of chromatin to physically bridge regulatory elements, convincing alternatives have emerged from recent studies on loop-extrusion and biomolecular condensates. Therefore, the precise molecular mechanism governing enhancer-promoter interactions remains unsettled. The major roadblocks hindering our understanding of enhancer-promoter interaction mechanisms are technical. First, the close spatial proximity of many enhancers to their cognate promoters cannot be resolved by typical methods (e.g., Hi-C or FISH). Second, dynamic mechanisms cannot be resolved with static methods (e.g., Hi-C or FISH). Here, to overcome these limitations, we will combine two cutting-edge techniques developed in the host lab: region capture Micro-C (RC-MC) and Super-Resolution Live Cell Imaging (SRLCI). Whereas RC-MC enables the visualization of the local genome architecture around select target loci with unprecedentedly high resolution in space to catalog all potential enhancer-promoter pairs, SRLCI complementarily tracks defined enhancer-promoter interactions over time to chronicle dynamic reorganization. As a novel addition, we aim to further visualize nascent transcription simultaneously by using PP7 technology. Finally, in collaboration with biophysicist Dr. Leonid Mirny (also at MIT), we will build a 3D polymer-simulation model recapitulating dynamic enhancer-promoter contact to reconcile experimental results with competing hypotheses. These experiments will be carried out using a differentiation system producing three different lineages. By focusing on a master transcription factor for one specific lineage, we will dissect the cis-regulatory circuits promoting nascent transcription and ultimately lineage specification– a central open question in developmental biology. In terms of perturbations to this system, we will modulate the composition of loop-extruding cohesin complex and transcription proteins. My project integrating bioengineering, biophysics, computational modeling, genomics, and genetics will therefore answer two major questions: “how do enhancers mechanistically regulate transcription” and “how does dynamic 3D rewiring of enhancer-promoter contacts at a key transcription factor locus drive lineage specification.” Consequently, these fundamental results will advance our understanding of physiology and disease, impacting the broader scientific community.

Lipids are one of the most essential nutrients for biological activity, playing a variety of roles such as energy storage, construction of cell membranes, and intracellular signal transduction including developmental biology and cancer. Depending on their chemical structures, there are more than 1000 different types of lipids in a typical eukaryotic cell. However, it is still not well known “how the structural diversity of lipids function differently in vivo context?” Although lipid research has made progress in the identification of lipid species using mass spectrometry, it falls behind protein research, especially in terms of imaging. This is because, unlike proteins, the observation of lipid species under light microscopy at high resolution using gene editing or chemical reactions without perturbing their structures and functions has not been achieved. Thus, the high-resolution visualization of lipids with their native structures and functions should develop this lipid field and pioneer a new field in biology. Recently, a technique called bifunctional lipid probes has been developed using photo-activation and click chemistry techniques. This technique enables us to observe the localization of near-native lipid species in cells with high resolution fluorescence microscopy. This study aims to investigate the biological significance of the structural diversity of lipids in terms of how they function and are involved in biological contexts by combining a novel lipid visualization technique with super-resolution microscopy observations and mass spectrometry. To demonstrate the feasibility of this approach in monitoring lipid dynamics, I will apply the visualization system to the small intestinal organoids model. The small intestine is one of the major lipid processing organs such as uptake, transport, subcellular conversion, and secretion (lipid flux). This organoid model exhibits intracellular transportation of lipids and offers convenient imaging accessibility. I will test the hypothesis that lipid structure, e.g. head group, chain length, and saturation, determines trafficking routes and kinetics within intestinal cells. This study will reveal the spatiotemporal dynamics and hidden biological functions of structurally diverse lipids with unprecedented resolution. It will provide a novel platform for the analysis of lipid flux at the intercellular and tissue level and an important resource to reveal the function of specific lipids in homeostasis and disease. After this proof-of-concept experiment, I will practically apply the imaging system to the mouse primordial germ cell (PGC) culture system, where I established an imaging system. PGC development has unique and interesting dynamics of lipids. For example, PGCs have transiently conspicuous lipid droplets during the migration to future gonads, however, their function is still unknown. Thus, this future study will provide novel insights into lipid function during germ cell development.

I aspire to study the neural basis of social cognition, especially the processes that implement Theory of Mind (ToM) in humans, which likely exist in other species to varying extents. ToM is the ability to put oneself in someone else’s shoes. It lets us adapt our behaviour based on norms, read between lines and keep unity in a complex society. People with autism spectrum disorder often struggle with this, which may lead to constant social stress and higher comorbidity risks. Thus, understanding the neural basis of social cognition is imperative. I propose to study the neurocognitive processes of social decisions in rhesus macaques using an innovative closed-loop approach in free-foraging individuals. Macaques are evolutionary much closer to humans than rodents and the best studied species for higher neurocognitive functions, making them the most advanced animal model for theoretical and clinical neuroscience. Still, to what extent their cooperative behaviour compares to that of humans remains debated. Also, due to technical limitations, most neurophysiological data come from experiments using highly restrained behaviours. In contrast, natural social interactions are dynamic mutual exchanges between agents. I aim to fill these gaps by recording neural data from macaque pairs during free social foraging in a novel and unique environment with multiple experimentally controlled dyadic feeders. I hypothesize that for macaques to behave cooperatively, there must be dynamic interactions between the foraging reward distribution and the pairs’ social and spatial states (e.g., hierarchy, past interactions, how far the feeders/peers are). I predict, 1) the level of cooperation will vary based on these states and reward distribution, and 2) the neural activity in social, spatial, and reward-oriented brain areas will distinctly reflect respective states. I will combine wireless neural recording, AI-based action recognition, and cortical microstimulation to analyse and causally manipulate the activity of target areas and behaviour. I expect to 1) establish the link between cingulate areas and social states, retrosplenial cortex and spatial states, and prefrontal areas and reward states, and 2) test their causality by stimulating in one area while recording in the rest. My project will explore the fundamental yet previously inaccessible decision variables in an ethologically relevant setup with rigorous experimental control, closing the loop of brain and behaviour as an organic unit. With my PhD on value-based motor decisions, I gained a solid foundation on decision neuroscience. Through this project, I will expand my electrophysiology skills to wireless recording in multiple unrestrained agents, learn the latest multi-agent tracking methods, and grow new collaborative networks, while contributing my computational modeling expertise and perspectives of the evolution of organisms as control systems. With that, I am advancing toward my ambition: to study ToM.

Dendrites, the elaborate tree-like structure of neurons, are the key sites of neuronal computations by processing synaptic inputs and transmitting information to the cell body of neurons. Their significance is underscored by their observed degeneration and dysfunction in numerous neurodegenerative diseases. While interdependent somatic and dendritic activity has been suggested to implement functions that determine the input-output relationships in individual neurons, it is still technically impossible to capture dendritic computations in isolation from the influence of somatic activity in the intact brain. Therefore, we still do not know how soma and dendrites operate independently or collaboratively to achieve cognitive functions in behaving animals. To address this question, it is necessary to monitor and manipulate the activity of soma separately from dendrites in behaving animals. In my post-doctoral work, I will combine in vivo dendritic voltage imaging, intracellular recordings, and manipulation of somatic membrane potentials in hippocampal pyramidal neurons to elucidate the potential roles of dendritic computations in neuronal plasticity underlying learning and memory. My research will leverage the formation and subsequent stabilization of spatial representations in hippocampal CA1 pyramidal neurons as a model of neuronal plasticity associated with spatial learning. The plasticity rule behind this process, known as behavioral timescale plasticity (BTSP), is characterized by dendritic plateau potentials and somatic burst spiking and suggested to involve somatic and dendritic plasticity, the precise roles of which remain enigmatic in vivo. I will first reveal the relationships between dendritic computations, somatic spikes, and dendritic plateau potentials throughout spontaneous place field formation and subsequent refinement of the spatial information associated with sharp-wave ripples (SWRs), the high-frequency oscillations in the hippocampal local field potentials (LFP). To achieve this, I will conduct two-photon voltage imaging of dendritic activity with ultrafast acousto-optical microscopy and simultaneous whole-cell patch-clamp recordings from their soma, together with hippocampal LFP recordings while mice explore a novel environment. Next, I will manipulate somatic membrane potential to prevent soma from emitting spikes and study dendritic activity. This revolutionary approach will clarify for the first time whether and how somatic spikes and plateau potentials are causally related to dendritic computation and propose a potential role of dendrites in spatial memory formation. The innovative combination of technologies that I am pioneering will open up a whole new world in understanding soma-dendrite computation in general and may provide novel insights into the pathologies caused by its abnormalities.

Signal transmission in a neuron has a defined polarity as it proceeds from dendrites and the cell body to the axon. Santiago Ramon y Cajal proposed this principle, the “law of dynamic polarization”, over a century ago. It has been validated by functional, anatomical, and molecular studies across various types of neurons. Surprisingly, the host laboratory has recently discovered that midbrain dopamine neurons have an action potential initiation mechanism in distal dopamine axons in the striatum. Axonal firing is triggered by local cholinergic interneurons and leads to dopamine release independent of ascending activities from the cell bodies in the substantia nigra pars compacta (SNc). These findings go against core principles of the dynamic polarization law, and establish that dopamine neurons contain specialized machinery for initiating signal transmission in distal axons. The architecture of this machinery, and how far axonally-induced action potentials can propagate, has remained unexplored. Remarkably, dopamine neurons release dopamine not only from axons in the striatum, but also from somatodendritic compartments in the SNc. Building on these features, I hypothesize that there is a reverse information flow in dopamine neurons: action potentials initiated in distal dopamine axons in the striatum can backpropagate to the SNc to trigger somatodendritic dopamine release. Alternatively, these action potentials might selectively regulate striatal activities without backpropagation, accounting for different spatiotemporal signaling dynamics between axonal and somatodendritic compartments. I propose to dissect the directionality of information flow in dopamine neurons and the underlying signaling structures with a three-pronged approach. In aim 1, I will determine the molecular and functional architecture of somatodendritic dopamine release sites. I will combine electrophysiological recordings and mouse genetics with newly developed morphological methodologies that combine cryo-fixation and expansion microscopy. In aim 2, I will dissect the protein machineries for action potential initiation in striatal dopamine axons. I will assess whether there are non-uniform and co-organized distributions of three key components: vesicular release sites of cholinergic interneurons, nicotinic acetylcholine receptors on dopamine axons which depolarize their membrane, and voltage-gated sodium channels on these axons which initiate and propagate action potentials. In aim 3, I will test whether action potentials induced in distal dopamine axons reach the soma and trigger somatodendritic dopamine release in vivo. I will utilize optogenetics, in vivo imaging with genetically encoded dopamine sensors and electrophysiology in mice. My work is likely to revise fundamental principles on the directionality of information flow in neurons and will define the molecular machineries for signaling polarity reversal.

G protein-coupled receptors (GPCRs) are important for cells to sense their external environment. Plenty of studies on their structure, pharmacology and so on has led to successful therapies of GPCR-related diseases. However, there are still hundreds of "orphan" GPCRs whose function remains unknown. Revealing the function and ligands of orphan GPCRs, called “deorphanization”. Deorphanization of these receptors could have significant implications for human physiology and disease treatment, but efforts to find their activating molecules have slowed down over the past decade. The lack of an activator makes it difficult to study downstream signaling pathways and to develop assays for probe molecule discovery of orphan GPCRs. My research aims to develop new approaches to illuminate the function of orphan GPCRs, particularly those expressed in the central nervous system. To overcome these challenges, I propose a new approach to purify orphan GPCRs and use them as ”bait” to find endogenous hormones or surrogate activators for orphan GPCRs, combining biochemistry, native mass spectrometry, antibody development and structural biology. As a prototype of screening of orphan GPCR activators, I chose GPR85, the most conserved GPCR in all vertebrates. Knock out of GPR85 leads to a 20% increase in brain mass, while overexpression studies implicate it as a negative regulator of hippocampal neurogenesis and spatial learning. But the lack of ligand of GPR85 hinders understanding of the precise function and signaling of GPR85. My research has two aims. The first aim is to identify endogenous ligands for GPR85 using mass spectrometry and NMR techniques. And I will use extracts from the hippocampus to identify other molecules that bind tightly to GPR85. The identified molecules will be used to determine the cellular signaling pathways modulated by GPR85. The second aim is to use combinatorial antibody libraries to find new surrogate ligands for GPR85. The approach will identify molecules that bind to GPR85 in a specific conformation and have the potential to induce or inhibit GPR85-mediated signaling. The candidate molecules will be screened for activity against a panel of cellular signaling pathways. Orphan GPCRs represent a huge opportunity for drug discovery, but progress has been slow. A new approach could shed light on the function of these mysterious receptors and pave the way for similar approaches to tackle other orphan receptors. The ultimate goal is to illuminate the function of one of the most intriguing orphan GPCRs in the genome and pave the way for similar approaches to numerous other orphan receptors. This approach could lead to the discovery of new therapies for recalcitrant diseases. The support from the Human Frontier Science Program would enable a high-risk, high-reward approach to a longstanding problem in sensory biology.

Background: Hosts and parasites exist in a delicate balance: many of these associations are ancient, the result of an equilibrium of adaptation and counter-adaptation in the Red Queen's Race. The host-parasite interface is thus the site of rapid evolution, and of nature's most elaborate weapons and ruses. The shapeshifting of Plasmodium falciparum, the most lethal malaria parasite, provides an example: infection with sporozoites (PfSPZ), the stage of which moves from bite through blood to the liver does not induce sterilizing immunity. Vaccines and therapeutics for this stage must contend with its immune evasion. PfSPZ's surface is heavily covered with circumsporozoite protein (PfCSP), which is critical for mammalian host cell invasion. PfCSP includes a central region comprised of repeating tetrapeptides: these copious repeats have been proposed as an immune-evasive feature, drawing unproductive responses from the host. Aims of the proposal: To determine whether the central repeat or other PfCSP epitopes are immune-distracting and whether immuno-focused approaches can teach a host to evade the parasite's own evasive maneuvers. 1. Develop preclinical models expressing human PfCSP antibodies. Antibodies will either bind only a single PfCSP epitope (in/out of the repeat region), or will be dual-specific to the central repeat and at least one other PfCSP epitope. 2. Investigate which epitopes impart a robust response. Humanized mice will be vaccinated with immunogens containing various segments of PfCSP. 3. Generate mini-repertoires to mimic the host environment. B cells are in competition for antigen: to determine whether responses to the central repeat crowd out other responses by B cell competition, we will generate mice with multiple humanized B cell lineages, expressing precursors to antibodies to various parts of PfCSP. Central hypothesis: Humoral immune responses to the central repeat will not be protective: furthermore, immuno-focused responses excluding the central repeat will outperform broad responses. The experimental system: The Batista lab has developed an approach to rapidly generate mice with B cells bearing fully humanized B cell receptors (BCRs), as well as state-of-the-art immunocharacterization approaches to trace of a single B cell lineage. This proposal advances those methods by generating models capable of expressing multiple PfCSP antibodies, recreating the competitive environment in humans in miniature. Conclusions: Whether the central repeat performs an immune-evasive function will be determined by both direct measurement of the humoral responses (identifying which cells are activated, the amount and specificity of the circulating antibody response), and, perhaps more tellingly, how vaccinated mice respond to a malaria challenge. This will provide either confirmation or refutation of this proposed mechanism by which parasites overcome acquired immunity not by avoiding a response, but by eliciting a harmless one.

Ultrasound (US) is an exciting tool for biomedical purposes due to its ability to non-invasively interact with matter deep inside our body with relatively high precision. These interactions can be exploited at low intensities for imaging, or at high intensities for destruction of tissue. The intermediate US intensity range, however, presents a grand challenge, since it affects biological cells through a complex interplay of several forces, such as the acoustic radiation force (ARF), acoustic interaction force (AIF), forces from acoustic streaming (AS) and cavitation. These US-induced forces can lead to significant biological responses, enabling for example neuromodulation, biomarker release, sonoporation, and angiogenesis; however, despite their enormous potential these forces remain poorly understood due to the entanglement of the underlying mechanisms, as well as the fast time- and small length-scales making experimental analysis challenging. Neuromodulation with US (USNM) has been demonstrated in vitro and in vivo and is particularly interesting, as it could yield significant breakthroughs in neuroscience and neurological treatments. Potential uses include suppression of epileptic seizures or symptoms of psychiatric disorders. In the proposed project, I plan to first discover which acoustic forcing mechanisms are responsible for USNM and to what extent, and then determine the magnitude and nature of the involved forces. Currently, several mechanisms are hypothesized to be responsible for USNM, specifically: pressure and velocity variations on the microsecond time-scale, ARF, AIF and AS. These mechanisms are in the typical focused US acting concurrently. However, standing-wave US fields, combined with varying the position of neurons, media properties and US parameters, could enable a decoupling of the mechanisms. The experiments developed in the project will be based on standing-wave lab-on-a-chip acoustofluidics and will provide well-defined acoustic environments for neurons to interact with, each promoting a different isolated mechanism of acoustic forcing and thus revealing its role in USNM. The forces on neurons at the cellular level will be investigated through particle image velocimetry, traction force microscopy, and fluorescence resonance energy transfer force sensors. Computational models of a neuron in US for quantification of forces will be developed and calibrated through extensive material characterization of neurons and the surrounding media. The project aims at discovering the main underlying mechanisms of USNM and could therefore provide the missing foundation, which would have an enormous impact on the development of USNM and its translation to clinics and neuroscience research. Furthermore, the developed methodology could pave the way towards understanding how US interacts with biological cells in the context of other, equally promising biological responses, such as angiogenesis or release of cancer biomarkers.

Prenatal exposure to psychotropic substances, such as cannabis, greatly increases offspring vulnerability to psychiatric disorders. However, the molecular, cellular, and circuit level mechanisms that predispose exposed children to psychiatric disorders are poorly understood. I recently found that cariprazine, a novel antipsychotic and antidepressant medicine, primarily binds to a subregion of the ventral striatum called the "Islands of Calleja" (IoC). By developing a fluorescent-ligand-based labeling approach for super-resolution microscopy, I observed that cariprazine preferentially binds to D3 dopamine receptors on IoC neurons, suggesting this understudied brain area has substantial psychiatric significance. The IoC has also been linked to neurodevelopmental processes, but the impact of early drug exposure on IoC development and how molecular processes in the IoC increase susceptibility to psychiatric disorders remain unknown. My specific hypothesis is that maternal substance abuse elicits maladaptive changes in the dopaminergic system within the IoC, which may contribute to offspring vulnerability. Importantly, the prevalence of maternal cannabis use is steadily increasing with its worldwide trend for legalization. However, prenatal exposure to ?9-tetrahydrocannabinol (THC), the psychoactive substance in cannabis, leads to a hyperdopaminergic state in offspring in preclinical models, and longitudinal studies demonstrate a strong association between maternal cannabis use and the risk of psychiatric disorders. Thus, prenatal THC (pTHC) treatment is a suitable model with timely translational relevance to address my hypothesis. Prof. Ken Mackie is one of the leaders in cannabinoid research. His group has recently established in vivo pTHC mouse models and developed an innovative microfluidic system in collaboration with Dr. Feng Guo, enabling high-throughput pharmacological tests on human brain organoids in a standardized manner. His state-of-the-art approaches, combined with my prior experience in super-resolution imaging, offer a unique opportunity to investigate drug effects within the microenvironment of the developing brain, providing testable hypotheses for in vivo studies. My proposed strategy is to integrate microfluidic technology with IoC-containing striatal organoids and use fluorescent ligand-based super-resolution imaging of the dopaminergic system to determine the molecular and cellular effects of THC exposure on IoC circuit maturation in organoids. I will test if the most important findings can be replicated in the developing brain in vivo and use interrogation experiments to elucidate which molecular and cellular changes lead to behavioral abnormalities. The proposed research will not only lead to a better understanding of the adverse effects of maternal cannabis use but will also provide a conceptual and methodical framework for future studies on a wide range of perinatal stressors and their causal link to psychiatric disorders.

Thyroid hormones (TH) are essential for development and metabolism in all vertebrates. TH imbalance severely compromises the well-being of about 10% of the world population, and represents a yearly financial burden of over several billion dollars for the health system in a country like the United States of America. However, the molecular mechanism behind their regulation is far from being understood, as so their tuning in vivo. TH are synthesized via iodination of their protein precursor thyroglobulin (TG) within the extracellular lumen of thyroid follicles, then released into the bloodstream to reach target tissues in the organism (e.g., brain, muscles, liver). TG endocytosis is a key step for TH release and a membrane protein receptor (TGR) has been shown to mainly promote TG internalization from the extracellular lumen. How the TG recruitment and internalization machinery works in different cellular conditions remains an outstanding question in the field. We propose to fill this knowledge gap, unraveling the mechanistic aspects of TG endocytosis by TGR via an integrative structural biology approach. First, using single particle cryo-electron microscopy (cryo-EM), we aim to unravel the first structure of human TG receptor in isolation and in complex with TG, identifying the determinants of iodinated TG recognition by TGR, mapping the effects of disease mutations. Secondly, using a combination of proteomics, gene editing and cellular endocytosis assays, we aim to unravel the complete machinery of the TG internalization complex. Finally, we seek to visualize it in situ, recapitulating the internalization process directly in cells. We expect to decipher the rules of TG endocytosis in the thyroid, opening a new front for targeted TH control in organisms. Since TGR is also expressed in many other tissues and mediates the transport of other organ-specific cargoes this study will have ample application to understand key trafficking routes in other tissues.

Dendroclimatology, the science of extracting past climate records from the distinct properties of annual tree-rings plays a critical role in climate research. The science has been widely applied in the temperate and boreal regions, where the strong yearly cycles in temperature and light availability induces annual ring formation in trees. In the tropics, however, its application has been limited as tree-ring formation in many tropical tree species can be complex due to mainly the low seasonal climatic changes and the high climatic fluctuations within seasons in the region. Consequently, paleo-climate records in tropical countries, particularly in Africa are scarce and the continent’s climate science is currently the least advanced in the world. Nevertheless, tree-rings are the widely available climate-proxy data sources in Africa, indicating the need for improved tree-ring detecting and characterizing technologies. Hyperspectral imaging (HSI) provides an efficient method for the characterization of physical and chemical features in objects at the molecular level. HSI systems capture vast amounts of spatially resolved spectral data and provide rich information about the composition and characteristics of objects in an image. On the other hand, deep learning (DL) provides an effective HIS data processing technique owing to its ability to automatically learn and extract complex patterns and features from images. In this context, the aim of the proposed project is to test a novel approach to dendroclimatology using a combination of these cutting-edge analytical technologies on two selected tropical tree species. To achieve the aim, we will collaborate between a fully equipped HSI in Norway with an emerging tropical dendroclimatology lab in Ethiopia. The proposed project will involve research in both HSI and DL techniques. Different HSI systems, sample preparation and instrumental setup techniques, and light conditions appropriate for detecting and characterizing tree-rings from different sample types will be explored in laboratory environment. Subsequently, advanced DL data processing algorithms will be explored to obtain good spectral signal from the samples and quality data for robust prediction models. Finally, a hyperspectral camera with relevant wavelength bands will be applied outdoors for field analysis of wooden samples.

Humans and animals learn schemas – common structures in prior experiences – and apply them to novel problems to efficiently guide their decisions. Prefrontal cortex (PFC) has been identified as a key brain area that encodes schemas. However, how such schemas are implemented in neural circuits is unclear yet fundamental for a mechanistic understanding of flexible behavior. Emerging research in the host laboratories has begun to provide insight into this question; in a sequence learning task in mice, they revealed how PFC neurons encode the learned schema and map it to new tasks. Critically, the data suggest a mechanism for building the schema representation that, if true, would generalize to complex real-life schemata and reveal a novel general mechanism for structuring behavior. Testing this requires tracking individual neurons in PFC from the onset of learning to expert behavior – exactly the skillset I developed during my PhD. In this project, I aim to characterize how prefrontal cortex forms schema representations with learning. First, I will develop a head-fixed version of the sequence learning task to enable simultaneous two-photon calcium imaging. I will use this design to follow the activity of PFC neurons in task across learning to identify how and when the schema representation arises. Finally, I will investigate the role of inputs from key areas including entorhinal cortex (EC) and hippocampus (HC) and dopamine signals in the formation of schema representations. I hypothesize that: 1) at the start of learning, PFC neurons map progress through individual goal-directed actions (termed phase cells); 2) these cells split into groups tracking the progress of each action in the schema (i.e. subgoals), allowing generalization across tasks; 3) this learning and subsequent generalization relies on inputs from EC and HC (providing schema content), and dopamine signals (enabling remapping). I will use virtual reality technology to create a spatial sequence learning task for mice that are head-fixed on a track ball. I will then make medial PFC accessible for two-photon imaging with a microprism implant and transduce neurons in PFC with a fluorescent calcium indicator, utilizing the expertise from my PhD. This will enable chronic recording of cellular and subcellular calcium activity in behaving animals throughout learning. Next, two-photon imaging of a dopamine sensor will allow me to follow dopamine signals in PFC during learning and generalization. Lastly, by expressing a calcium indicator in EC and HC neurons, I will image their axon terminals in PFC and record the information communicated to PFC before, during and after learning. With this, I plan to observe what inputs PFC neurons integrate during learning to elucidate what changes in neuronal computations lead to schema representations. In conclusion, my project will shed light on how prefrontal cortex forms models of tasks to efficiently and flexibly guide behavior in novel situations.

During embryonic development the cytoplasm separates into distinct compartments prior to cell division. This partitioning occurs spontaneously and in the absence of external stimuli or physical boundaries (i.e., the plasma membrane). Regulated by microtubules, the partitioning forces can lead to large-scale cytoplasmic flows. These flows, in turn, redistribute cytoplasm and nuclei, which define the new cell centers. Meanwhile, the constituents of the cytoplasm are free to move. Yet, they must relocate to a suitable position before the subsequent cell division. Reliant on the capacity of the cytoplasm for self-organization, the coordination of this redistribution results from the interplay between chemical and physical processes. While spatially synchronized chemical oscillators determine the timing of the cell cycle, active processes facilitate flow patterns that robustly redistribute the constituents within the cytoplasm. The potential for chaos has been hypothesized to pose an inherent restriction to development, which relies on maintaining order in the chemical oscillations as well as cytoplasmic flows. While simulations of the cell cycle oscillator confirm its potential for chaos, experiments have yet to confirm this possibility. Additionally, the consequences of a perturbed cell cycle oscillator on the spatially reorganization of the cytoplasm are unknown. Recent studies showed that active fluids can transition to turbulent flows even in the absence of inertial forces. Unpublished results in the host lab confirmed this for flows in reconstituted cytoplasm of Xenopus laevis. In this proposal, I aim to experimentally investigate the role of both cellularization and coupling of active flows with the cell cycle oscillator as strategies to avoid chaos. I hypothesize that the mechano-chemical coupling between the cytoplasm and the cell cycle actively regulates the transition to chaos. First (i), using microfluidics, I will generate droplets containing cycling cytoplasmic extract. By varying droplet size and boundary properties (e.g., stiffness), I will explore the effects of boundary interactions on internal flows. Second (ii), I will extend my investigation to a multiple droplet system to explore the effects of direct or indirect coupling (e.g., chemical or mechanical) of adjacent droplets and its effect on internal flows. Third (iii), I will perturb timing and spatial coupling with the cell cycle in (i) and (ii). I will evaluate the robustness of these systems by analyzing their response to external perturbations (e.g., mechanical deformations or localized temperature increases). In summary, this proposal aims to investigate the competition between the mechanisms of self-organization and the antagonizing transition to chaos in cytoplasmic flows. This research has the potential to explain why some embryos develop as syncytial masses while others divide into individual cells.

We aim to understand the fundamental principles that lead to the emergence of collective intelligence (CI) in groups of humans. We will work toward this understanding by modeling and perturbing the emergent dynamics of teams of humans as they interact and collaborate to accomplish tasks on an online gaming platform. Ours will be an interdisciplinary effort that utilizes our combined expertise in behavioral science, machine learning and artificial intelligence, neuroscience, and complex systems theory: We will model the behavior of individual participants using reinforcement learning (RL) agents informed by cognitive science, and we will quantify the interaction patterns of participant groups during gameplay using tools from network science and information theory. The incorporation into our RL agents of different artificial neural networks that each resemble cognitive functions of the individual human mind, coupled with a precise network characterization of group dynamics, will allow us to compare collaboration patterns of humans and RL agents and isolate underlying cognitive features that may be operative in both groups. We will first create a set of experiments that validate that our online gaming platform can be used to study CI, and that RL agents can be used to model human behavior on this platform. We will establish which patterns of interaction among agents (both human and RL) and which individual cognitive features of RL agents are especially correlated with CI. We will then perturb group behavior through the manipulation of several factors we hypothesize have a significant impact on human CI. Specifically, we are interested in understanding how a continuous change of individuals impacts CI and if the creation of generations could optimize the learning process of the collective. We will also study how CI is modulated by predators and other threatening factors, the overall diversity of the group in terms of participant ability within the game, and participants’ anticipation of each other’s behavior. The intersecting nature of our expertise will allow us to perform both types of experiments (human & AI), compare emergent group behavior, and uncover the underlying cognitive processes present in the individuals. Our findings may also have implications for fields such as decision-making in organizations, swarm robotics, and social behavior in animals.

Background: Adult neural stem cells (NSCs), a group of quiescent cells that possess the potential to generate specialized neurons and glial cells, are present in the brain of vertebrates including humans. NSCs are embedded in dedicated niches that are composed of heterogeneous cells, which provide a unique combination of signaling molecules and physical properties to support NSC function. Important contributions of niches cells have been identified under physiologic conditions. But how niche cells remodel to promote complex NSC activation for repair and act as cohesive unit still remains elusive. The host lab previously identified quiescent NSC-like cells in the adult brain of genetically versatile fruit flies, which activate upon injury for regeneration suggesting an important role for integration of multiple niche features. Aims: My proposal aims to genetically manipulate the neurogenic niche cells in vivo for the first time and characterize their coordinated functioning in the transition of NSCs from quiescence to an active proliferative state during brain tissue regeneration. Specifically, we will 1) develop a genetic toolkit that allows to mark NSC-interacting niche cells in homeostatic and regenerative conditions in the Drosophila adult brain and access molecular fingerprints, 2) manipulate identified biological and physical properties of the niche microenvironment to determine the effect on NSC activation and 3) interrogate whether changed niche properties modulate the type of cells that are regenerated using cutting-edge lineage tracing. Central hypotheses: The group of cells that surrounds NSCs, known as the niche, exhibits spatial and biological organizations that remain poorly understood. My hypothesis is that the niche functions as a unit that can be remodeled to intervene in the states of quiescence, proliferation, or fate of NSCs. Experimental system: To characterize and manipulate NSC niches in vivo, I will use adult NSC-like cells in the fly brain expressing the HES1-like transcription factor deadpan as a bait to mark and visualize directly interacting cells via activation of a transgenic expression system in the neighboring cells without interfering with niche cell function. This cell-cell interaction tracing system will be activated in homeostatic and injured brains to isolate niche cells and analyze their features by transcriptional and proteomic analyses. Overall, this approach will enable specific manipulation of the niche cell population for genetic experiments to elucidate the function of identified molecules. Conclusions: How niche cells integrate injury signals and coordinate NSC activity in the brain is still not well understood. By genetic manipulation of the niche in vivo I aim to generate profound insight into molecular and cellular mechanisms governing niche dynamics during regeneration that is likely applicable to other species and tissues and will shed light on fundamental aspects of tissue repair.

The lining of the uterus (endometrium) is the most powerful regenerative system in the human body. It undergoes monthly shedding during menstruation and subsequent regeneration, reaching up to 400 cycles over the human reproductive lifespan. Studying menstruation is not only critical for hundreds of millions of people affected by its associated disorders, but it can also provide crucial insights into human tissue regeneration. However, the molecular mechanisms of endometrial shedding and regeneration remain poorly understood[1]. The endometrium is compartmentalized into two layers: the functionalis, which sheds during menstruation, and the basalis, which remains and proliferates to regenerate the functionalis[1]. This compartmentalization is essential for physiological endometrial regeneration, as damage to the basalis is associated with non-regenerative outcomes (fibrosis)[2–4]. However, we currently lack a detailed molecular definition of functionalis and basalis that explains their functional differences. Here, I propose a dynamic and multimodal characterization of endometrial compartments throughout the menstrual cycle. My studies will leverage the African spiny mouse (Acomys cahirinus), a rodent that was recently discovered to menstruate[5], which provides a unique platform for experimental dissection of menstruation. In Aim 1, I will use spatial transcriptomics to identify gene expression signatures that distinguish the functionalis and basalis within the cycling endometrium. In parallel, I will assess two potential functional differences between the functionalis and basalis (Aims 2 and 3). In Aim 2, I will test a longstanding but largely untested hypothesis in the field: that secretion of matrix metalloproteinases (MMPs) within the functionalis initiate endometrial shedding by degrading the extracellular matrix holding the tissue together[6,7]. I will assess MMP protein expression and activity profiles across endometrial strata and evaluate whether MMP activity is essential for menstrual shedding using specific MMP inhibitors. In Aim 3, I will test whether differing membrane potentials between functionalis and basalis cells contribute to their distinct behaviors during menstruation. Cell membrane potentials are emerging as important determinants of cellular behavior[8], and endometrial cell membrane potentials are known to fluctuate throughout the menstrual cycle[9]. I will assess membrane potentials in the endometrial strata over the menstrual cycle by intravital imaging and perturb ion fluxes with specific ion channel inhibitors to assess their functional contributions to endometrial cell behaviors. Taken together, this multimodal characterization of the cycling endometrium will identify the unique properties of its layers and provide novel perspectives for the holistic understanding of its regenerative processes. These findings will hold tremendous potential for future applications in regenerative medicine and reproductive health.

Multiple neurochemical species that play functionally distinct roles in neural circuitry interact to influence the large-scale activity of the brain. Understanding the spatial and temporal characteristics of such chemical signaling thus becomes crucial for building mechanistic models of brain function. The technology for measuring neurochemical activity in vivo is still incipient. Fluorescent probes allow several neurotransmitters to be imaged in optically accessible structures, but mapping the topography of neural signaling events in deep tissue is not possible with optical methods or with older point measurement techniques. Over the past few years, Alan Jasanoff’s lab has led efforts to create probes for mapping neurochemical events using molecular-level functional magnetic resonance imaging (“molecular fMRI”). The scope of such experiments has however been limited by the modest sensitivity provided by the existing probes, which must be applied at concentrations that substantially exceed physiological neurotransmitter levels. Thus, this proposal aims to provide a platform technology for noninvasive neurochemical imaging with improved sensitivity, initially targeting monoamine transmitters. Our idea is to develop a fundamentally distinct and more sensitive architecture for neurochemical imaging in the nervous system. The new probes are liposomal nanoparticle reporters (LisNRs), which give rise to substantial stimulus-dependent MRI contrast changes even at nanomolar concentrations. To achieve sensitivity to neurotransmitter targets, we will establish ligand-responsive LisNRs that employ peptide-based pores in the liposome membranes. Neurotransmitter-dependent contrast changes occur when protein domains block pore permeability in the absence of free neurotransmitters but not in their presence. In our proposed research, we will optimize this mechanism for the detection of behaviorally relevant interstitial dopamine and serotonin concentrations. Next, we will optimize the distribution profile of liposome-based dopamine and serotonin LisNRs when introduced into the brain using minimally invasive procedures, before then validating these probes by molecular fMRI in live rat brains. By creating sensors for neurochemical targets, optimizing their delivery to brain tissue, and performing functional imaging studies in rats, we will define stimulus-induced signaling profiles that are inaccessible to other experimental means and establish a platform that can be adapted for imaging additional targets in optically inaccessible tissue. Our experiments will set the stage for the broad application of LisNR probes for qualitatively novel characterization of neurochemical dynamics relevant to behavior and neural circuit function at a brain-wide scale in many species, potentially including humans.

Memory impairments are extremely common as part of healthy aging, but we still lack an understanding of how experiences are remembered or why this process can so often fail. Possible breakthroughs have been hindered by two major obstacles: the use of coarse neuroimaging tools that measure activity averaged across large neural populations and the use of highly simplified experimental paradigms. These practices have uncovered simple features of how memories are formed but cannot capture critical features of real-life experiences. Realistic life experiences consist of a continuous stream of information that is connected from moment to moment, but how do memories form amidst continuous stimuli fluctuations? How do we naturally make choices about segmenting this experience into individual events and how can we later remember them? Unfortunately, most of the detailed neural mechanisms underlying these processes are still unknown. Rodent studies have suggested potential mechanisms but cannot capture the full complexity of human behavior. Here we propose a unique experimental paradigm which will provide the first evidence in humans for the mechanisms by which streams of sensory experiences are segmented and stored by the human brain as discrete memories. We plan on collecting a dataset in which we will use intracranial electrodes to measure the activity of individual neurons, a type of recording which was not possible in humans until recently. This will allow us to study the precise firing of neurons during the creation of a memory trace and test competing hypotheses about detailed neural activity. We expect neurons to build up an event representation by rapidly replaying a sequence of information from that event, multiple times a second. To explore how that sequencing takes place we will examine the time, frequency and phase domain of the signal using recently developed tools (e.g., phase-amplitude coupling and circular statistics). The participants will be asked to perform a task in which they must watch a series of short movies and then perform a cued-recall task. By testing memory for extended experiences like movies, participants are required to build up complex models of the event unfolding in front of them over time, which parallels the demands the brain goes through while encoding real-life experiences. We hypothesize that mechanisms extensively observed in rodents such as theta cycle resets (4-8 Hz oscillations) and sharp-wave ripples (strong ~150-Hz oscillations) will support the above-mentioned sequencing of information allowing the brain to encode/recall a new event. Collaborating with the proposed host supervisor, who is one of the few researchers in the world currently with access to this unique type of human neural recordings and the expertise in the quantitative tools necessary to analyze it, would provide me with the opportunity to acquire the necessary skills to place myself as a lead researcher in the field.

The study of phage biology and infection mechanisms is an important field of research that, since the first half of the 20th century, has delivered major scientific outputs for both fundamental scientific fields notably molecular biology and applied domains through applications like phage therapy. Viral entry process that goes from phage fixation to viral DNA ejection inside the host plays a major role in the phage biology governing central aspects such as its infectivity and its specificity. It relies on the interaction between phage elements (notably phage tail & fibers) and bacterial ones including protein receptors and elements from the cell wall. These mechanisms have been investigated for decades using various molecular and structural biology approaches however, important knowledge gaps remain centered notably on quantitative aspects and non model bacteriophages. The proposed research project aims at unlocking the potential of recent advances in the fields of biophysics and quantitative biology for a deeper understanding of these mechanisms. The central goal is to reproduce the phage infection process in an “in vitro” setup, by engineering synthetic cells encapsulating gene expression mixtures to be infected by natural or engineered bacteriophages. The infection of highly controlled synthetic cells by viral particles would be a game changer in the study of phage infection mechanisms. It allows for the quantitative assessment of the role of each partner involved and of important environmental conditions on the various mechanisms supporting the process of cellular phage entry. To achieve that goal, state of the art methods will be applied to the engineering of both partners interacting : phage particles and synthetic cells. On the side of phage particles, their engineering and production will be performed on dedicated “in vitro” expression platforms using newly “in house” developed pipelines enabling the high throughput editing of phage candidates. In parallel, recent progress achieved on the engineering of custom lipid bilayers relying notably on the quartz crystal microbalance with dissipation monitoring (QCM-D) technology have enabled the tuning of synthetic cell membrane composition enabling the integration of various lipids and protein receptors. We expect that this project will lead to major advances in our knowledge of phage biology. The possibility to systematically study the determinants of infection mechanisms should accelerate the development of mechanistic models describing more precisely these phenomena. In the long term, such an approach may also allow the acceleration of research on non-model phages as well as phages infecting hazardous or non cultivable bacteria. Finally, in the long term, the details of this approach can also be transferred to eukaryotic cells to study the mechanisms governing their infection by viruses, opening new horizons for cell biology and virology.