Brain metastasis (BrM) represents the most common brain malignancy, predominantly arising from non-small cell lung cancer, breast cancer and melanoma. We hypothesize that the tumor microenvironment (TME) plays an essential role in the establishment and progression of BrM. The brain is constituted by a unique TME, which includes tissue-resident cell types such as astrocytes and microglia, and critically, the blood-brain barrier (BBB). Cancer cells are able to exploit the brain vasculature for their own benefit, by forming the blood-tumor barrier. This specialized vasculature is an essential TME compartment, as it may suppress anti-tumor responses by blocking immune cell infiltration, specifically of cytotoxic T cells. Indeed, a new perspective to target the tumor vasculature in combination with immunotherapy has recently emerged from studying other cancers; however, this concept has not been explored in BrM to date. Therefore, in the proposed project, I will investigate the biology of two major cellular components of the BBB: endothelial cells (ECs) and pericytes in BrM arising from distinct primary tumors. For this purpose, I will perform transcriptomic analysis of these cell types in patient BrM samples originating from lung, breast or melanoma primary tumors. I will combine this analysis with different experimental approaches available in the host lab, including immunocompetent mouse BrM models and in vitro and ex vivo cell cultures that have been optimized to model the brain TME. This integrated and complementary experimental strategy will facilitate the identification of innovative therapies for BrM to be used alone or in combination with immunotherapies.

Pain sensation, though unpleasant, is considered protective as it provokes aversive behavior from noxious stimuli. Studies show that sensory neurons, which generate the perception of pain and touch, interact with another important protection mechanism, the immune system. Ablation of sensory neurons can alter the skin-resident immune cell response. However, many aspects of this interaction remain uncharacterized.
Cells and antigens from barrier sites, like the skin, are drained and sampled by the lymph nodes (LN). Interestingly, LN are innervated by fibers expressing the sensory neuron derived-peptides Substance P and Calcitonin gene-related peptide. Some of these fibers are found in close proximity to leukocytes. However, their direct impact on immune cell activity is unknown.
This project aims to characterize the effects of sensory neurons in the skin and LN on immune cell behavior. To this aim, we propose to combine chemogenetics to locally activate sensory neurons, with a broad analysis of immune cell responses. Mice expressing Cre under the control of sensory neuronal markers, including the broadly expressed gene Advillin and genes selective for subsets of sensory neurons, will be injected intradermally or in the LN with a viral vector encoding the Cre-dependent expression of the hM3D(Gq) activating receptor. Once validated, this technique will be used to locally activate sensory neurons and characterize subsequent immunological changes using flow cytometry, histological and live imaging techniques. This project can provide new insights into how sensory innervations, which regulate behavior by creating the sensation of touch and pain, shape local immune responses.

A key brain region involved in accurate temporal refinement of motor and also cognitive behaviours is the cerebellum. Cerebellar granule cells (GCs) receive multisensory information from outside the cerebellum via mossy fibres (MF). The prevailing hypothesis is that, in order to learn temporal sequences, the cerebellum requires a rich diversity of GC activity patterns that as a population represent a distributed temporal representation of sensory inputs. But this has never been directly demonstrated. Unpublished network models from the hosting laboratory strongly suggest that their previous findings (input specific diversity of MF-GC synaptic dynamics) are sufficient to generate a diverse GC firing patterns that act as a temporal basis for cerebellar learning. We will use novel high speed two-photon in vivo imaging of GC firing patterns in behaving animals, and examine whether the diversity of GC firing patterns requires functional diversity of MF-GC synapses, and whether this mechanism is specific for different sensory modalities, using optogenetic activation of specific input types. Finally, we will optogenetically inhibit specific MF types to show their influence over specific timescales of eyeblink conditioning, then image the temporal representation in the GC layer before, during and after a temporal learning task. Expected results will included identification of the underlying cellular and circuit mechanism of temporal learning required for fine tuning motor and cognitive processes.

Pancreatic ductal adenocarcinoma (PDAC) remains an incurable disease with a high death rate. PDACs are exceptionally difficult to treat because of high genetic plasticity and encapsulation by cancer-associated fibroblasts (CAFs). Either are due to an altered epigenetic landscape where PDACs and CAFs show a switch towards global chromatin hypo-methylation, and concomitant hyper-methylation of tumor-suppressor genes. As CAFs do not show any mutagenesis, the molecular cause must be found in the tumor microenvironment.

The nutrient microenvironment represents a novel but important aspects of tumor biology. Nutrients are converted into the basic building blocks that fuel cellular proliferation, but also regulate epigenetic modifications. S-Adenosyl-Methionine (SAM) represents a nodal point in one carbon metabolism and is required for methylation of histones and DNA. We therefore propose that metabolism cannot sustain the high SAM requirement of both PDAC and CAF, thus shunting away carbons from epigenetic maintenance. This project therefore aims to map the link between nutrient availability, one carbon metabolism, availability of SAM, and the methylation profile of chromatin. Furthermore, we will establish a genetic sensor and CRISPR-based screening system to visualize the regulation of SAM compartmentalization, and attempt to normalize the epigenome of PDACs and CAFs through engineering of SAM metabolism (EpiSAMe). These studies will shed new insight into the crosstalk between metabolism and epigenetics in PDAC and provide novel molecular for the treatment of this devastating disease.

Genomic instability is a common feature of cancers and can have an important impact on cancer progression and response to treatment. However, the mechanisms underlying cancer genomic instability are not completely understood. While DNA repair defects are thought to be a major cause of this trait, other factors seem to be involved. In the last decade, metabolism has received a lot of attention within the cancer research community. Most of the work has focused on understanding the metabolic requirements of cancer cells for survival and proliferation. However, metabolism does not only provide building blocks for biosynthesis, but it also generates reactive by-products that can damage cellular components such as DNA. Thus, the metabolic alterations characteristic of cancer cells might not only support cancer development by supporting biosynthesis but also by promoting genomic instability.

The aim of this research proposal is to identify changes in the metabolism of endogenous genotoxins in cancer and to elucidate the role of these alterations, particularly in the context of genomic instability. In addition, the potential exploitation of this aspect of metabolism for cancer therapy will be explored. To achieve these goals a diverse range of techniques and study systems will be used, including transcriptomics, metabolomics, CRISPR-based genetic screens, cancer cell lines and mouse models.

In 2014, the Nobel Prize in Physiology was awarded for the discovery of place and grid cells that process spatial cues in the mammalian hippocampal formation;, the key structure for both navigation and episodic memory1. Place and grid cells, and additional cell types form the central building blocks in the current circuit models of navigation in mammals. However, a cohesive picture of how these circuits compute space and enable navigation has not been achieved. In fact, emerging evidence suggest that these navigation circuits are highly diverse in cellular phenotypes and functionality; they do not only map aspects of space but also elements like sound, time, and reward. How neural systems of spatial cognition have evolved outside of the mammalian clade is not clear, and comparative studies are critically needed to gain insights to basic functional constraints and structural requirements underlying these neural circuits.
The international research team of four PIs proposes a broad comparative systems neurobiological approach using teleost fish for integrative studies on higher navigation circuits. These fish are ideal models because they have conquered diverse spatial ecologies and show highly specialized sensory adaptions. Also, their brains exhibit an overall lower complexity to mammals, and are highly accessible to experimental manipulation. To establish systematic research on teleostean spatial cognition, the project combines neuroethological, electrophysiological, neuroimaging, and computational methodologies. Introducing a powerful electrophysiological recording technology in freely-moving fish and generating long-needed anatomical atlas resources, the project analyzes four teleost species with differing spatial ecologies. The team will uncover how different sensory modalities like vision, perception of depth, and active electrolocation are integrated during spatial navigation tasks, thereby investigating how top-down mechanisms modulate sensory integration of spatial learning.
Finally, the team will test specific hypotheses developed in small-scaled laboratory setups in an unconstrained natural environment. Here, the group will measure the activity of neurons in freely moving fish that explore a coral reef habitat. This will be the first ever attempt to analyze brain activity underlying navigation in the wild. Altogether, the project will provide new perspectives on the evolution, function, and mechanism of memory systems in animal navigation.

Prochlorococcus, the smallest photosynthetic organism, has diversified into many ‘ecotypes’ with finely tuned distinct ecological niches. The small and variable genomes of these cyanobacteria encode genes unique to particular habitats. A driving force for gene transfer and evolution in cyanobacteria is phage-mediated. While numerous reports describe lytic cyanophages that infect Prochlorococcus, surprisingly there is only indirect evidence that lysogenic phages – those that spend a portion of their life cycle incorporated into the host genome – exist. The central goals of this proposal are i) to analyze the occurrence of lysogens in wild populations of Prochlorococcus, and ii) to establish a robust protocol for gene editing allowing precise manipulation of the Prochlorococcus genome. The second goal will exploit, but is not dependent upon, advances from the first.
This will be the first study to assess lysogeny across natural Prochlorococcus populations and to develop phage-mediated genetic systems for engineering cyanobacteria. The development of efficient tools for genome editing would be extremely beneficial for understanding Prochlorococcus metabolism. It would allow functional studies for thousands of unannotated genes that influence the relative fitness of different ecotypes in different environments. A unique aspect of my proposal is the employment of a novel synthetic biology strategy to build engineered phages with broad host specificity, and to use them as vehicles for genome editing systems such as CRISPR/Cas9. This technique will be useful for several applications and will further establish Prochlorococcus as a model organism in environmental microbiology.

Phosphatidyl-inositol 3,4,5 triphosphoshate-dependent Rac exchanger (P-Rex) are a group of proteins involved in the activation of the small GTPase Rac, by acting as a guanidine exchange factor (GEF). Both Rac and P-Rex proteins are known to be overexpressed and altered in several cancers, consistent with their role in both cell mobility and their numerous interactions with both the mTOR axis and the PI3K/PTEN pathway. P-Rex1/2 specifically, are known to interact with mTOR and PTEN, among others, placing them at the nexus of regulatory pathways for cell metabolism, proliferation and mobility.
Despite their central role in cell signaling, very little is known about the interplay between such interactions. No structural information is available for the full-length P-Rex proteins and how different interaction partners affect its structure and function. I will therefore set out to better characterize the emerging properties of the multidomain P-Rex proteins by purifying both P-Rex1/2 and determining their structure using cryo-EM and orthogonal biophysical characterization methods. I then aim to define the interaction between P-Rex proteins and its regulatory partners, PTEN and the mTOR complexes 1 and 2. I will define their domains of interactions and determine the structure of the complexes, using a combination of cryo-EM and cross-linking and mass-spectrometry.
I expect to provide new insights into the functions of P-Rex proteins and their crosstalk with core cellular signaling pathways. Structures of full-lengths P-Rex alone or in complex with its interaction partners will serve as a platform for drug discovery, thereby providing new cancer therapeutic avenues.

The brain comprises billions of neural cells interwoven in a highly precise way and it is ultimately responsible for how we perceive, react to, and remember the world around us. We are fascinated by how the intricate structure of the brain is assembled step-by-step at an early stage of life (“embryonic development”), starting as a relatively shapeless mass within the nascent embryo and culminating in a highly complex organ in an adult. Specifically, we hypothesize that blood vessels that permeate the early brain have important roles in fostering brain assembly, and we further predict that they produce important cues that actively shape a specific early step of brain development. This is of great significance, because blood vessels were once thought only to passively supply oxygen and nutrients to various organs. We instead suggest an active role in which blood vessels function as a ‘signaling center’ to actively control the arrangement of neural cells during brain assembly.
We have assembled an intercontinental team to tackle this question via a three-part, integrated effort. First, we will create a map of where and when arteries and veins (two major types of blood vessels) first enter the nascent brain. Second, we will intermingle neural and blood vessel cells in a Petri dish and study their interactions. Third, we will block specific functions exerted by blood vessel cells and propose that this will have a commensurate impact on the spatial arrangement of nearby neural cells. These activities are innately highly collaborative and will involve interactions between the partner laboratories in each nation.
Taken together, we propose to explore how the brain is assembled during embryonic development and we will test whether blood vessels act not as passive support for, but rather as active executors of, this intricate process at early steps. If true, this has important ramifications. Not only will it shed light on how the early brain is assembled, but it adds to the emerging idea that neural cells and blood vessels share an intimate functional and spatial relationship, starting from the embryo and lasting into the adult — an idea pertinent to early brain assembly; our efforts to engineer this process in a Petri dish for regenerative medicine; and finally, the origin of certain neurological disorders that are caused in part by blood vessel dysfunction.

Sporadically, novel and potentially devastating pandemic influenza A viruses (IAVs) are generated through genome reassortment between human and animal co-infecting IAVs. Such pandemic viruses emerge as a consequence of the segmentation of IAVs genome into a bundle made of 8 distinct viral RNAs (vRNAs). However the molecular mechanisms of vRNA intracellular transport and assembly into vRNAs bundles, which are critical for reassortment, remain largely unknown. Our project aims to elucidate these fundamental aspects of IAV life cycle by developing innovative approaches.
We challenge the original model that newly synthesized vRNAs, in the form of viral ribonucleoproteins (vRNPs), are transported across the cytoplasm on Rab11-dependent recycling endosomes. Based on our recent work, we hypothesize that the concomitant transport and assembly of vRNPs is driven by their physical association with remodelled endoplasmic reticulum (ER) membranes and Rab11-dependent transport vesicles distinct from recycling endosomes.
We will set up a cellular system which resembles the natural respiratory tissue targeted by IAVs, while being amenable to simultaneous imaging of the endogenous Rab11 protein and tagged vRNAs. We will develop two cutting-edge and complementary imaging methods: dual-color single molecule fluorescence in situ hybridization (FISH) in live cells for the tracking of distinct vRNAs that diffuse concomitantly, and cryo-Focused Ion Beam combined with electron microscopy in situ hybridization (EMISH) to image individual vRNPs and their transport vesicles at molecular resolution. We will further assess the role of cellular ER-shaping proteins by performing CRISPR/Cas9-mediated knockdowns, and by monitoring changes in the viral-induced remodelling of ER and biogenesis of vRNP transport vesicles by live fluorescence imaging and cellular EM.
The proposed research will require the very close collaboration between three partners with distinct but complementary expertise. The approaches developed jointly are poised to revolutionize our understanding of IAV multi-RNA genome transport and bundling, and thus help in the broader goal of achieving better prevention and treatment of influenza disease. Additionally, the proposed technical developments in live cell FISH and cellular EM, will have impact on other fields of studies well beyond the scope of the proposed project.

Glycolysis is a fundamental energy metabolic pathway which consists of ten enzymatic steps. Unlike the mitochondrion, which is a membrane-bound organelle, glycolytic enzymes are soluble proteins in the cytosol. Based on biochemical evidence, glycolytic enzymes have long been hypothesized to form functional complexes to sustain the rates of glycolysis. This purported complex, called the glycolytic metabolon, was a subject of intense study and debate thirty years ago. Still today we do not yet understand how these purported complexes are organized in cells to sustain local energy metabolism, or their physiological importance. This gap in knowledge results from the fact that when glycolysis was being rigorously examined forty years ago, the techniques did not exist to conclusively answer these questions. The main challenge in addressing these questions lay then, as now, in the ability to both examine the localization of the glycolytic enzymes in living cells, while understanding the biophysical and biochemical mechanisms of their association, and its implications in cellular physiology. We have established a collaboration to address these fundamental questions by making use of our joint expertise in vitro reconstitution and C.elegans physiology, using the energy demands of the C.elegans synapse as a model system.

Plants live in an ever-changing environment which is not always compatible with their survival. A major life threatening condition is drought stress. While most plants can deploy an array of physiological countermeasures to endure remarkable levels of stress, there is a huge variability in the different strategies that plants choose to adopt to deal with drought stress. Many plants evade detrimental stress conditions by activating their reproductive development (flowering) earlier compared to non-stress conditions, a strategy known as drought escape (DE). Notably, even in the most water-rich environments plants face unpredictable dry periods, yet how this information affects the floral network at the molecular levels is unknown.
We will address this question with a blend of molecular and genetics-based approaches. We will leverage known mutants with altered DE to identify candidate mechanisms that are drought sensitive and can act as molecular switch to activate flowering. To comprehensively define DE mechanisms utilized under natural conditions we will assess the variability of the DE response in natural plant populations and in crops, which were selected by human intervention for the different field scenarios. Our targeted large-scale screens will allow us to identify naturally occurring variants in the DE process and decipher the molecular mechanism responsible for DE activation. This information will provide breakthroughs in our understanding of novel regulatory mechanisms that play a role in driving developmental adaptations across extremely variable environmental conditions, their natural genetic variation and the selective forces that maintain such variation in populations, an important aspect for predicting and dealing with the effects of climate change. Finally, because flowering time is a major component of yield potential in crops, the defined mechanisms will help us develop breeding strategies targeted for sub-optimal irrigation scenarios to produce crops with ameliorated performances under drought conditions.

The endocannabinoid system (ECS) is involved in a variety of brain functions, including reward and aversion, mainly through type-1 cannabinoid receptors (CB1). The Nucleus Accumbens (NAc) is a key brain region in the control of positive and negative reinforcement. ECS impairments in the NAc lead to negative emotional states. By responding to neurotransmitters with intracellular Ca2+ increases and releasing gliotransmitters that modulate synaptic transmission and animal behavior, astrocytes play active roles in neural information processing. While CB1-mediated astrocyte-neuron communication has been shown in several brain regions, it is unknown whether this communication exists in in the NAc. Mitochondrial CB1 receptors (mtCB1) directly regulate mitochondrial energetic activity, synaptic transmission and behavior, but the role of astroglial mtCB1 in these processes is unknown. Combining electrophysiology, subcellular-specific live Ca2+ imaging and behavior, this proposal aims at defining the contribution of astrocytic CB1 (plasmatic and/or mitochondrial) to synaptic plasticity in the NAc and their behavioral impact. The specific goals of this proposal are to define 1) whether astrocytes in the NAc express functional CB1; 2) how astrocytic CB1 regulate synaptic transmission and plasticity in the NAc; 3) how astrocytic CB1 impact reward and aversion and 4) how astrocytic mtCB1 influence astrocyte activity, synaptic plasticity in the NAc and behavior. The expected results will shed light onto the involvement of astrocytes in brain motivational systems through the ECS, and would reveal astrocytes as potential targets for treatment of motivational disorders.

Compartmentalization into functional units is the key principle of cellular life. In addition to conventional membrane-bound organelles, cells utilize membrane-less biomolecular condensates to locally concentrate proteins and nucleic acids. Prominent examples of such condensates are nucleoli, P granules or centrosomes, which play central roles in diverse cellular functions. Recent studies have demonstrated that membrane-less condensates assemble by liquid-liquid phase separation of proteins that are characterized by multi-valency, intrinsic disorder and low complexity sequences. The molecular mechanisms that control assembly and disassembly, shape and size of such membrane-less organelles remain key unanswered questions of modern cell biology. To address this fundamental gap in our knowledge, I propose an interdisciplinary research programme that combines powerful in vitro reconstitution with systematic screening technologies. Based on my discovery that the chromosome surface protein Ki-67 acts as a surface-active agent (surfactant) for one of the largest membrane-less assemblies, the mitotic chromosome, I will explore the radically new concept of surfactants for the global regulation of other membrane-less organelles. Starting from defining the molecular properties, mechanisms and regulatory basis for the action of Ki-67, I will systematically identify and characterize the surfactants of other membrane-less organelles in human cells. This ambitious research programme has the potential to provide novel paradigms of spatial and temporal control of membrane-less cellular assemblies and thereby substantially advance our understanding of cellular organization.

Organisms need strategies to survive when conditions are hard. For mammals, winter is particularly difficult - they have to invest large amounts of energy into keeping warm, while food availability is low. For this reason, many mammals migrate or hibernate. However, what to do if you are too small to migrate long distance, burn your energy fast, and cannot hibernate? The common shrew is such a mammal and has evolved an astonishing strategy: each individual shrinks in winter by up to 20% and then regrows in the spring by about 13%. This size change, thought to allow shrews to survive on fewer resources because of the smaller size and linked lower energy requirements, include not just overall size, but specifically organs that do not usually change size in fully grown animals, such as the brain, heart and liver.
The process of neurological degeneration and regeneration is of great interest, since many central nervous system diseases (e.g., Alzheimer’s, multiple sclerosis) involve degeneration, but ongoing research for therapies to reverse this process has been of limited success. As one of only a few recorded examples of mammalian brain regeneration, understanding how the shrew regrows its brain can accelerate research that leads to future therapies.
To answer the question of how the shrew shrinks and then regrows its brain, we will establish this unusual species as a new model, by studying the biological, molecular, biochemical and genetic processes behind this reversible size change. Besides establishing a database of information that can be mined and researched in years to come to discover the pathways that generate this cycle in the shrew, we will test a metabolic model of neurological change by artificially blocking molecular access to fats. Thus, the cross-disciplinary study of this wintering adaptation may help us understand more about regeneration in mammals in general, and the brain in particular.

A plausible explanation for why mitochondria have their own DNA (mtDNA) is to ensure colocalization of redox-genes and redox-regulation to the same membrane-bound organelle. While this distributed control system seems logical, very little is known about how such autonomous regulation takes place. A variety of methods that are widely applicable to study genomes fail to work on mtDNA, and sequence-to-function mapping of the mitochondrial genome is missing. I plan to create a modified, hypermutating DNA polymerase gamma (polG), the designated mtDNA replicase. polG is encoded in the nuclear genome and can therefore be easily manipulated. Hypermutator polG will be integrated into the nuclear genome of S. cerevisiae, which will introduce mutations in mtDNA without altering the nuclear genome. I will then use selection assays followed by next generation sequencing to study the ability of mtDNA yeast variants to adapt to respiratory conditions. Such experiment will highlight regulatory hotspots in mtDNA that will be mechanistically evaluated using genome-wide methods like native elongating transcript sequencing (NET-seq) and mitochondrial ribosome profiling that are the ‘bread and butter’ of the Churchman lab. Clear view of the fitness landscape associated with mtDNA will inform research into many deep questions in mitochondrial biology, ranging from ‘how selection shapes mitochondrial genomes?’ to ‘why do certain mtDNA mutations cause cancer?’.

Trypanosoma brucei is the causative agent of sleeping sickness. In the mammalian host, parasites exist in two major niches: the blood, and the brain. Recently, Dr. Figueiredo’s lab found a previously undiscovered third reservoir of parasites in the adipose tissue which were transcriptionally distinct from blood stream forms, and the genes upregulated included various markers relevant to parasite metabolism. My proposed work in Dr. Figueiredo’s lab is to implement in vivo imaging methods to a) investigate the parasite’s mechanisms of homing and crossing of the vascular endothelium into the adipose tissue, and to carefully define the adipose tissue niche that acts as reservoir for the parasites; and b) to elucidate host vasculature, and parasite factors involved in allowing such unique phenomenon, including generating parasite mutants deficient in adenylate cyclises, flagellar proteins, and mechano-sensing proteins among others. Using in vivo techniques and biophysical and molecular methods, I aim to investigate the biological significance of parasite residence in the adipose tissue – a location which could be clinically relevant for interventions in human and veterinary medicine.

Harmful algal blooms (HABs) have been long studied in marine and fresh water environments, yet most research to date has focused at the bulk scale. Despite the well-documented toxic ramifications of HABs on vertebrates and mammals, including humans, we still lack a biophysical understanding of the mechanisms by which toxins disperse during a bloom event. Using a combination of experiments and modelling, in this project I will explore the physico-chemical interplay underlying the release and transport of toxins at the scale of the microorganism. Towards this I will develop micro- and millifluidic experiments to generate ‘bloom-in-lab’ and visualize the dispersion of targeted toxins under three ecologically relevant cues: fluid flow, temperature and salinity. Specifically, I will study red-tide forming Heterosigma akashiwo, a globally distributed marine raphidophyte, known to release a range of toxins, including reactive oxygen-nitrogen species (RONS) during bloom conditions. Original data from high-speed and time-lapse microscopy on fluorescently labelled ‘target’ molecules will put forward first experimental quantifications of toxin release and transport under different micro-environmental cues, thereby informing a new mathematical model that will capture, at microscales, dispersion and auto-feedback dynamics during bloom events. Analysing the coupling between microbial biophysics, transport phenomena and biochemistry, this project will bring about a fundamental, mechanistic understanding of toxin transmission, and help develop accurate and comprehensive algorithms for predicting HAB formation, especially during the rapidly warming climate we encounter today.

Human medicine is on the verge of a new era where we face the possibility to precisely rewrite the genome to prevent, ameliorate and cure a wide range of immune-hematological diseases. Hematopoietic stem and progenitor cells (HSPC) have long been a preferred source for ex-vivo gene therapy, as gene correction in multipotent progenitors ensures a life-long supply of corrected progeny and polyclonal reconstitution of the bone marrow. Recently, programmable nucleases brought the possibility of genome editing (GE) within the reach of gene therapy by allowing locus-specific gene correction without the risk of aberrant transgene expression. However functional studies that accurately assess possible acute and long-lasting consequences of GE procedures in HSPC are needed to harness GE therapeutic potential. Indeed, despite recent advances in the generation of corrected HSPC by GE, the efficiency of the targeting process and the ability of edited cells to stably reconstitute the hematopoietic system upon transplantation remain limited, pointing to a loss of repopulating capacity of HSPC upon targeting. The goal of this project is to investigate the role of DNA damage response (DDR) pathways in GE strategies and uncover novel molecular players that could be exploited to increase the yield of long-term engrafting gene-edited HSPC. Our findings could be easily applied to gene correction strategies for a variety of inherited pathologies affecting the human immune-hematopoietic system and provide a foundation for more efficient modalities of editing HSPC at genomic sites of interest for both basic and translational research.

Areas of visual cortex integrate signals representing sensory input, ongoing motor actions, and internal states. While neurons in primary visual cortex (V1) were classically interpreted as feature detectors that signal the presence of a specific visual stimulus, recent evidence suggest that V1 circuits participate in the specific, topographical integration of sensorimotor signals. How sensory and sensorimotor computations are implemented in the circuitry of V1 and higher visual areas, and how these computations depend on the behavioral context, is unknown. I will combine novel circuit dissection tools, wide-field-of-view holographic stimulation, and virtual reality environments to approach these questions. I will selectively activate V1 ensembles with distinct functional properties during sensorimotor behavior using holographic stimulation and monitor the effects on the surrounding populations in V1 and higher visual areas. Furthermore, I will analyze the effects of holographic stimulation as a function of different behavioral parameters and during optogenetically induced variations in internal state. Finally, I will explore methods to target the expression of genetically encoded tools to distinct V1 ensembles, each with specific function during sensorimotor behavior, which will allow me to study if different ensembles are selectively connected to circuits throughout the brain, as well as to determine their selective impact on brain-wide dynamics. This project will shed new light onto the function of V1 during sensorimotor behavior, and yield fundamental insights into the mechanisms by which cortex can adapt its processing machinery to varying behavioral demands.