During the central nervous system (CNS) inflammation, substantial number of T cells start to infiltrate into CNS and attack the neurons or myelin in several neurological disorders such as multiple sclerosis, suggesting their potent role during CNS inflammation. For T cells to exhibit their effector function, it is necessary to be re-stimulated and regulated in the inflamed region by tissue-resident cells. However, the CNS-resident cells that regulate the effector T cell (Teff) during CNS inflammation are still unclear. Interestingly, recent reports suggest that astrocytes are crucial participants in CNS inflammation. Furthermore, infiltrating Teffs are known to physically contact with astrocytes. Since astrocytes express vast range of immune-regulatory surface molecules such as MHC class II (MHCII), co-stimulatory molecules (B7) and immune checkpoints, it is plausible that astrocytes might utilize those molecules to regulate the infiltrating CNS-reactive Teffs. Based on this idea, we hypothesize that astrocytes contact with Teffs via surface proteins to regulate the Teffs during CNS inflammation.

Cells need to maintain metabolic homeostasis to survive and proliferate. Many biochemical systems have evolved to coordinate levels of cellular metabolites in response to changes in extracellular cues. While we know a lot about regulatory processes involved in cellular metabolite homeostasis, how organelles maintain their metabolite availability is poorly understood. This is particularly relevant for mitochondria, as the major oxidative organelle; but whether mitochondria can sense the levels of metabolites and accordingly regulate their availability is unknown. Studying such compartmentalized metabolite sensing mechanisms is challenging due to lack of technologies to measure and modulate organellar metabolite levels. Recent studies from the host lab discovered SLC25A39 as a mitochondrial transporter for glutathione (GSH), the major antioxidant thiol in mammalian cells. Remarkably, SLC25A39 protein levels are negatively regulated specifically by mitochondrial GSH availability. This observation raises the possibility that mitochondria harbor a sensing system for GSH availability. Building upon these observations, I will identify the mechanism by which mitochondria sense GSH availability and test whether it is required for tumor formation. My first aim is to determine the precise mechanism and molecular players involved in mitochondrial GSH sensing. Using a combination of biochemical tools and genetic screening approaches, I will identify the molecular players and critical domain(s) and residue(s) of the transporter essential for the GSH sensing. Additionally, my preliminary work shows that SLC25A39 expression is associated with poor prognosis and decreased survival of breast cancer patients, and SLC25A39 protein levels are strongly upregulated in breast cancer cells colonizing to the lung compared to those in mammary tumors. Building upon this, my second aim will determine whether mitochondrial GSH availability and its sensing is necessary for breast cancer progression. To test whether mitochondrial GSH sensing is required for metastatic colonization, I will generate mutant forms of the transporter deficient in GSH sensing. In these mouse models, I will determine the precise molecular pathways impacted by mitochondrial GSH loss, resulting in reduced metastasis. To further test the sufficiency of mitochondrial GSH availability for tumor progression, I will also engineer a mitochondrially-targeted bacterial enzyme called GSHf, which produces GSH in specific compartments, and express it specifically in the breast tumor cells. Collectively, these studies will provide a unique opportunity to uncover how mitochondria sense and regulate GSH and how compartmentalized redox sensing shapes breast cancer cell function. This will also provide a proof of principle for potential organellar sensing mechanisms for other metabolites.

Rationale During development, sensory structures change and grow in a way that can impact stimulus perception and motor decision. However, we do not know the mechanisms by which sensorimotor circuits adjust for continuous change to ensure adequate behavioral responses. The lateral line is essential for survival of fishes: it responds to small changes of water flow to adjust body orientation, and to large changes to elicit escape responses. During my PhD in Zoology, I used electrophysiology to study hair cell signaling across a diversity of species. I uncovered a novel mechanism of anion efflux driven signal amplification that depends on the ionic strength of the surrounding water. While my work focused on sensory signaling at the cellular level, I now want to decipher at the organism level how sensorimotor circuits adapt upon change to sensory organs. I discovered the gelatinous encapsulant atop lateral line hair cells, referred to as the cupula, unexpectedly grows in height at a rate of ~12 µm / h in larval zebrafish. A 20-µm increase in cupula height theoretically shifts the detection cut-off frequency of hair cells by an order of magnitude (Van Trump and McHenry 2008). My observation triggers a crucial question: how does the animal accurately detect and adequately respond to flow despite indeterminate growth of its sensors? Specific Aims I will elucidate how the effects of sensor growth on gain are modulated and how motor command neurons ensure adequate behavioral responses: Aim 1. Dissect the mechanisms and kinetics of cupula growth on the entire organism Aim 2. Measure impacts of cupula growth on sensory function and motor output Aim 3. Investigate the peripheral and central mechanisms that adjust sensorimotor gain Central Hypothesis Based on spontaneous activity of the sensor, homeostatic inhibition of neurons in the anterior and posterior lateral line nucleus scales the gain of the sensorimotor circuit. Strategy To test this hypothesis, I will join the lab of Claire Wyart to learn high-throughput behavioral analysis, high speed volumetric population imaging and 3D optogenetics to reveal how the sensorimotor circuit adjust its gain to compensate for the growth of sensory organs: 1) I will monitor the growth of cupulae across neuromasts of the head and tail; 2) I will investigate how cupula size impacts behavioral choice to graded mechanical stimuli; 3) I will investigate by volumetrically imaging of neuronal activity combined with optogenetics in afferent and inhibitory efferent neurons as well as motor command neurons in the brainstem. Conclusion This project will leverage a unique example of sensory change to reveal mechanisms of regulating gain and behavioral output. The HSFP Long-term Fellowship provides an ideal opportunity to reach my long term goal: building a lab using optical and genetic tools to reveal unifying principles of sensorimotor fidelity across aquatic species.

Multicellular organisms gradually develop from a homogenous cell population into well-organized and highly complex shapes. The key concept underlying this developmental process, still far from being fully understood, is self-organization – the emergence of complex well-ordered structures in an autonomous manner. The emerging field of synthetic developmental biology aims to study self-organization by reproducing it “from the bottom up”; that is, engineering cells that don’t usually self-organize to acquire specific and predictable shapes and patterns. While this approach was successful in reproducing simple patterns, its utility in reproducing more complex naturally-occurring developmental processes has yet to be demonstrated. A fundamental step in vertebrate development is the embryonic formation of segments and their corresponding structures along the spine (e.g., vertebrae and ribs). This process is based on a segmentation clock: a self-sustained oscillator with a period of a few hours, which resides in the cells of the embryo’s tail bud. This clock depends on a morphogen gradient that decreases in concentration from the tail tip towards the more frontal areas, until it becomes too low and the clock “arrests”. The phase of the oscillator at the specific time and place of arrest determines the edge of the newly-formed segment. The importance of this strategy goes beyond vertebrate development, as similar mechanisms were suggested in insects, plants, and recently even bacteria. Here, I propose to engineer a segment generator in a tissue-culture setup, based on a design which resembles the natural segmentation clock. As this fundamental and well-studied system consists of several regulatory modules, it can serve as a good model for understanding the emergence of complexity by combination of simple sub-systems. We hypothesize that the module combination will be sufficient to facilitate the emergence of robust segmentation. This will be done by introducing the cells with suitable orthogonal genetic constructs which together will form the regulatory circuits controlling self-organization. Specifically, the design will combine a synthetic oscillator, a short-range cell-cell coupling and longer-range morphogen signaling system. In addition, issues of cell fate commitment and robustness will be addressed. This research is expected to shed new light on the design principles governing segmentation specifically, and developmental processes as a whole. For example, it will allow to address questions such as: What is the relative importance of the different modules? How can the size and number of segments be tuned? How does cell proliferation interact with segmentation? Moreover, expanding the scope of synthetic developmental biology toward complex and modular designs can have immense implications for fields such as tissue engineering and regenerative medicine, where induction of predictable and reproducible self-organization is of essence.

Prolonged developmental plasticity is a hallmark of plant biology. This feature relies on the function of cell clusters with self-renewal and flexible differentiation capacity, which collectively form different types of “meristems”. It is still unclear if a specific molecular makeup support a “meristematic cell”, and how hundreds of cells within meristematic tissues act in coordination to derive various morphogenetic outcomes. Understanding the cellular- and tissue- level mechanistic principles that underlie specification of the multipotent tissues during early development are crucial to answering these questions. Emerging single-cell methodologies provide an unbiased approach for mapping meristematic processes, but their scalable implementation remain challenging due to the rigid-cell wall, laborious tissue handling and the minute sample size. In addition, moving from instantaneous single-cell profiles to understanding tissue-level dynamics remains non-trivial. Liam Dolan's group is utilizing genetics and modern imaging tools to study the early development of the liverwort M. polymorpha from a single spore. This process, which occurs in isolation and can be massively co-cultured, stands as a unique and accessible model to study how self-organization of a single ancestral cell gives rise to different structures, and in specific to understand how a confined multipotent niche is formed and maintained. We propose to perform in-depth temporal dissection of early spore development and to formulate testable mechanistic hypotheses on the principles that guide formation of plant stem-cell niches and regional specialization of cells in those niches. Given the lack of a comprehensive framework to define meristems or meristematic processes, we speculate those may be non-intuitive and cannot be spelled out a-priory. To achieve this, we plan an initial tri-partite profiling experiment: First, we will utilize recent pipeline (Meir et al, 2021) to derive bulk transcriptomes and images from hundreds of individual sporelings. Second, we will perform single-cell RNA sequencing that will draw an exhaustive manifold of cell states. To understand tissue-level differentiation dynamics, we will de-convolute the single-sporeling bulk transcriptomes based on the single-cell map. This analysis will provide rigorous temporal models for flows between cell states, based on observed data. The resulting model, assisted by spatial reporters that will be derived from it, will be then re-evaluated in perturbed genetic backgrounds. This research will (i) discover the acting cellular states within a minutely sized embryonic-like plant differentiation process that was thus far unreachable, (ii) couple molecular to morphogenetic progression, and (iii) test for mechanistic principles that facilitate initial meristem formation. We speculate such discovery can lead to better understanding of the multi- and pluripotent tissues that support the great developmental plasticity of plants.

Microbes synthesize proteins that mediate biochemical reactions, communication, and competition in their environments. DNA sequencing of environmental samples has allowed us to catalog proteins in nature. However, small open reading frame-encoded proteins (SEPs) have been overlooked due to technical challenges. SEPs, despite size, impact vital cell functions and can act as antimicrobial agents or signaling molecules. However, the way in which SEPs evolve and their diversity in the environment are unclear. Considering that life evolved from the ocean, where diversity is influenced by greater environmental variability, we can gain insight into the origins, evolution, and ecological relevance of SEPs through a systematic investigation of ocean microbiomes. Here, I will develop a computational approach to leverage sequence and structural information by machine learning algorithms to identify novel bioactive SEPs in the most extensive genomic resource for ocean microbiomes. Next, by integrating multi-omic data across various spatial and temporal scales, I will conduct an in-depth analysis encompassing the following aspects: 1) Identification of SEP functional clusters within ocean microbiomes; 2) Comparative analysis with other biomes; 3) Examination of genetic and environmental factors influencing SEP evolution; 4) Exploration of co-evolution patterns. Finally, I will verify the antimicrobial efficacy of newly discovered SEPs that could help tackle the looming antibiotic resistance crisis. This project will expand the functional landscape of ocean microbiomes and highlight evolutionary mechanisms of SEPs as ecological modulators. This has the potential to lead to significant discoveries about interactions in microbial communities and to inspire innovative biotechnological applications.

Microbial communities play a central role in ecosystems and make up to 20-30% of the total world biomass. They engage in different types of interactions, from mutualism to competition. Several studies have shown that these interactions are plastic and can change when biotic (e.g. species composition) or abiotic (e.g. nutrients) factors are modified. Manufactured chemicals such as pesticides are a major long-term stress to which microbial communities are exposed. These compounds can be detected in soil, water and even in our body up to years after the exposure and are close to exceed the planetary boundaries. However, it is still unclear how microbial molecular interactions and the genetic composition of microbial consortia evolve during co-adaptation to pesticides at an evolutionary time-scale and how this can be generalised to different communities and conditions. Moreover, emergent properties of microbial consortia such as the rate of pesticides degradation or co-adaptation can not be predicted from the response of single species. In this project, we propose to go beyond single-species adaptive evolution and investigate how microbial species co-evolve during long-term exposure to pesticides. To this aim we will use a combination of bottom-up and top-down approaches evolving artificially-assembled microbial communities and natural ones isolated from soil, water and the human gut. We will expose the selected microbial communities to representative chemicals of a library containing ~1000 pesticides and track how species composition changes during the co-evolution. We will dissect which genomic alterations are responsible for the adaptation during the experiments by performing genome-sequencing of the co-evolved communities and of single strains isolated from them. This approach will enable us to unveil how emerging strains carrying new genetic variants interact with the microbial community and whether these new interactions lead, for instance, to co-adaptation or community collapse. The genomic analysis will be coupled with a metabolomic study of some of the evolved communities to understand how genetic alterations translate in an altered metabolic network. Then, we will evaluate if patterns of polymicrobial convergent co-adaptation emerge across different conditions by comparing replicates of the co-adapted communities. Finally, we will use the evolved communities to assess ecological co-selection when microbial consortia co-adapted to similar or different pesticides are brought together (coalesce). These experiments will tell us how interspecies interactions co-evolve during adaptation to manufactured chemicals and whether co-adapted microbial communities influence each other during community coalescence. This project aims at expanding the frontier of our understanding of co-adaptation of multi-species microbial populations and will provide an unprecedented view of how microbial consortia co-evolve, stabilise and build community resilience.

Background of the proposed research project : To position organelles and transport vesicles and mRNAs inside the cell, cargoes recruit molecular motor proteins that walk unidirectionally along microtubules. Polarised microtubule arrays span the entire cell. Cytoplasmic dynein is the major retrograde motor, while more than a dozen different kinesins transport cargo towards the cell periphery. Active transport in both directions is essential, especially for neurons. Therefore, mutations in molecular motors cause neurological and neurodegenerative diseases. Recent progress, in structural cell biology and biochemical reconstitution experiments, enabled an understanding of how the activity of motors is triggered by binding to cargo adapter proteins. However, how these mechanisms control cycles between autoinhibited and active states of motor proteins in the context of the cell remains to be elucidated. This is particularly interesting for bidirectional motility which enables cargoes to avoid traffic jams on microtubule ends and other obstacles and to rapidly search the cytoplasm for their destination. Central Hypothesis : We hypothesize that cargoes bind to motors of opposite polarities at the same time, and mechanical or biochemical regulatory mechanisms trigger switching the transport direction. This enables efficient cargo transport in either direction, a swift response to obstacles, and efficient redistribution of motors. Aim of the proposed research project : To test this hypothesis, we aim to directly visualize individual molecular motors in cells, obtain their detailed life histories, and understand their engagement with microtubules and cargoes. Specifically, we aim to determine: 1) duration and fraction of time that individual motors bind to a cargo/move along microtubules, 2) how motor life histories change when we deplete/overexpress motor regulators, 3) whether motor engagement is regulated by the cargo. Experimental system : We will tag kinesin-3 KIF1C and dynein with fluorogenic nanobody arrays or inject fluorocube-labelled motors for long-term imaging in human cells using lattice light sheet microscopy. We will co-label cargoes, endosomal markers, or cargo adapters such as Hook3 to which both KIF1C and dynein can bind. From these data, we will extract detailed life histories of single motor molecules and will be able to correlate this to the transport of different cargoes. From the difference in cargo behavior in response to the recruitment of a labeled motor, we aim to infer the total number of active motors on each cargo. Conclusions: Discovering how motors behave in cells is key to understanding the mechanism of fundamentally important transport processes. We will use state of the art technology to establish new single-molecule live cell imaging tools to understand how motors engage with cargoes to bring about their observed motility in a physiological and pathological context.

DNA mutations occur randomly throughout the genome; then natural selection removes mutations from the important regions. This long-standing premise of evolution has been challenged, as studies revealed the mutation occurrence itself varies among genomic loci (mutation bias). Studies using Arabidopsis thaliana have demonstrated that essential genes mutate less, and those genes have distinct chromatin modifications, in particular H3K4me1. This observation leads to a hypothesis; the chromatin modifications that cover the genes locally repress DNA mutations, potentially influencing the genome evolution. However, this hypothesis is untested, and the mechanisms behind mutation bias remain unknown in plants. Here I propose the first two steps and one backup plan on a long road to understanding the mechanisms of mutation bias using model system A. thaliana. As a first step, the causal relationships between chromatin modifications and mutation bias will be experimentally tested. In mutant plants of selected chromatin modifications such as H3K4me1, I will quantify the mutation bias. To this end, I will employ genome sequencing and experimental evolution systems developed by the host researcher. This approach will identify the chromatin modifications (hereafter modification X) responsible for biasing the mutation. In the second step, I will examine the molecular mechanisms of how 'modification X' affects local mutation rates using genetic screening. The screening utilizes the fact that DNA stress is inducible by radiation or drugs, and the plants susceptible to the stress exhibit a scorable phenotype. For example, if the lack of modification X enhances double-strand break(DSB), then the root of the mutant-of-modification-X will be shorter than Wild Type (WT) in the presence of phleomycin, a drug that induces DSB. In the pool of the mutant-of-modification-X, I will screen for the second site mutations that suppress or enhance the DNA-stress-induced phenotype. Characterizing the enhancer and suppressor genes will map out the molecular black box that generates mutation bias. As a backup plan, in case the first step demonstrates any of the tested chromatin modifications have minimal effect on the mutation rate, I will explore the evolutionary universality of the mutation bias phenomena by examining the genomes of other organisms. This approach may provide new insights into the cause of mutation bias. A deeper understanding of the biology underlying mutation biases has the potential to refine our view of mutation’s role in evolution. This study investigates 'chromatin-directed mutation bias hypotheses', providing clarity into how organisms can influence evolution through mechanisms that affect intragenomic variability in mutation rate. New research projects may arise, such as investigating the fate of the genome in the presence of mechanisms to bias mutation and how selection acts on mechanisms that control mutation rates.

Background Mosquitoes are often defined as the deadliest animals on earth for their potential to transmit dangerous pathogens to humans. Aedes aegypti is the prime vector for arthropod-borne viruses such as Dengue (DENV), Zika and Chikungunya. Arboviruses are (re)emerging diseases whose burden on public health is expected to increase concurrently with the global expansion of Aedes aegypti. Arboviruses and mosquitoes share one common target: humans. The transmission of arboviruses depends on the success of infected female mosquitoes in obtaining human blood meals, which they require to reproduce. Prior work has suggested that DENV may manipulate its mosquito host to enhance virus transmission, but this has never been mechanistically investigated. Hypothesis Arboviruses hijack mosquito physiology to promote more aggressive host-seeking and blood-feeding behaviour in mosquitoes, which in turn increases DENV transmission. Viral non-structural proteins may control host gene expression in the mosquito to heighten their sensitivity to humans, increase their motivation to bite, or shift their daily activity timeframes . Aims I will examine the effects of DENV infection on the mosquito brain and sensory systems. How mosquito activity is modified upon infection will be tested, while taking into consideration how the virus affects insect fitness. Molecular changes in gene expression in brain and sensory system will be studied to reveal host-virus interactions. Each individual viral non-structural protein will be functionally tested in-vivo to determine its role in changing mosquito gene expression and ultimately shaping the insect brain and sensory apparatus. Experimental system To test for behavioural changes in DENV-infected mosquitoes, I will take advantage of different behavioural assays: olfactometers, activity monitors and feeding assays. Mosquito survival, fecundity and fertility will be evaluated to quantify the impact of viral infection on the host fitness. Next, I will use RNA and smallRNA sequencing to produce an atlas of differentially expressed genes plus antiviral and regulatory small molecules in infected mosquito brains and antennae. Lastly, I will genetically engineer mosquitoes to express DENV non-structural proteins in brain and sensory neurons in a controlled and reversible way, mimicking a viral infection. This will allow me to make mechanistic linkages between the effect of DENV on gene expression in the host, and subsequent effects on the aggressiveness of the mosquito vector in finding and drinking the blood of human hosts. Conclusion My results will expand our understanding of the complex relationship between mosquito vectors and arboviruses and inform novel methods of vector control. In addition, I will generate transgenic mosquito lines containing single arboviral non-structural proteins: a useful resource for further study on mosquito-virus interactions.

Diverse ethological goals drive species-specific specializations in body morphology, biomechanics, and neural circuitry. However, how these features adaptively coevolve remains poorly understood. As an HFSP Long Term Fellow, I will address this important knowledge gap by comparing the biomechanics, neuroanatomy, and neural dynamics of morphologically and behaviorally divergent species of flies: Drosophila prolongata (‘Dpro’), and its closely-related sister species Drosophila rhopaloa (‘Drho’), and the commonly studied Drosophila melanogaster (‘Dmel’). Dpro males have massively enlarged forelegs (>5 times the volume) relative to Drho and Dmel males. They use these enlarged limbs to perform a species-specific high-frequency leg vibration during courtship. This courtship behavior and limb enlargement may have coevolved via sexual selection. Notably, Dpro males must also use their enlarged forelimbs to perform common behaviors like walking and grooming. Thus, coincident with this morphological adaptation, I hypothesize that during evolution, Dpro males (i) established new neural circuits to control high-frequency leg vibrations, and (ii) adapted existing neural circuits to control walking and grooming with massively enlarged forelimbs. To test these hypotheses, I will develop new transgenic, computational, and microscopy tools. Using these, I will measure and model the relationship between the evolutionary specializations in the body and nervous system of Dpro males. First, I will determine how motor commands have adapted to compensate for the morphological specialization of limb growth. I will quantify these species’ limb movements in 3D and model joint torque dynamics in novel Dpro and Drho neuromechanical simulations. Second, I will identify how motor neuron populations controlling the front limbs–but not middle or hind limbs–may have expanded in Dpro to enable a wider dynamic range of joint torques. I will do this by generating transgenic Dpro and Drho expressing photoactivatable fluorophores and using these for neural circuit mapping. Third, I will discover to what extent a dedicated pool of motor neurons, not present in Dmel or Drho, becomes active during Dpro leg vibrations but not during common walking and grooming behaviors. I will do this by generating transgenic Dpro and Drho expressing indicators of neural activity and imaging motor circuit dynamics during behavior. These studies will reveal fundamental principles of how body morphology and neural circuits coevolve to enable behavioral specializations. Evolutionary and comparative neuroscience is an exciting new research direction for me, my host lab, and the field of motor control. I will learn new experimental approaches and apply them in contexts that are new for my host lab. I envision that comparative studies of neural circuits and behavior will reveal general principles of how nervous systems evolve, pushing the frontiers of both neuroscience and evolutionary biology.

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.

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.

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.