Mycobacterium tuberculosis (Mtb) survival and replication within, or escape from, its phagocytic host requires molecular exploitation of host proteins and lipids. However, there are still large gaps in our understanding of bacterial survival and escape, particularly regarding quantitative analysis of interactions between host and Mtb derived proteins or lipids. Super-resolution fluorescence imaging has begun to yield unprecedented insight into biological processes including protein distribution at the nanometer scale and live dynamics of proteins, lipids and the cytoskeleton. However, these techniques have rarely been applied to Mtb research. Here, super-resolution imaging complimented with proteomics will allow the spatiotemporal analysis of membrane associated proteins, including pathogen derived proteins which interact with the intra- and extracellular milieu of primary human macrophages and neutrophils. Work will also utilise live-cell imaging sensitive to membrane composition to elucidate how the pathogen modulates host cell lipids for survival and replication. Additionally, how Mtb escapes its host for transmission is poorly understood. Mtb modulation of the host’s cytoskeleton, membrane and linker proteins using high- and super-resolution imaging, will clarify this process. Quantifying the distribution or disruption of host proteins and lipids through the host-pathogen lifecycle, and investigating whether Mtb proteins themselves present on the host plasma membrane will translate to application of therapeutics. This work will generate novel hypotheses for the Mtb field, and other pathogens, in the context of host immunity and disease equilibrium.

Insect herbivores and their food sources comprise ~50% of biodiversity, and their interactions impact community structure, species’ geographical ranges, and food security. How insects choose their food sources, and how plants can defend themselves, are therefore questions of substantial ecological, evolutionary and economical relevance. However, the astounding diversity of herbivorous insects and their food sources renders them challenging to answer. We seek to address this problem by investigating an aspect of plant feeding that all herbivores have in common: the mechanical breakdown of tough plant material. Plant fracture requires the application of a force, the magnitude of which depends on the morphology of the fracturing tool, and the plant material properties. This force is generated by muscle, in a process that consumes energy. Feeding on plants is therefore both mechanically challenging and metabolically costly, to an extent defined by muscle physiology, mouthpart morphology, and plant tissue mechanical properties. We will build on this common, physical foundation to develop a mechanical and energetic framework that can be used to derive first-principles predictions of herbivore behaviour. Developing such a framework will require advances in the fields of solid mechanics and muscle physiology. We will expand fracture theory to describe the mechanics of thin sheet cutting, taking into account tool geometry, large deformations, and multi-axial stress states that occur during plant tissue fracture; and we will develop thermal imaging as a novel method to study muscle energetics. We will apply this framework to the leaf-cutter ants, iconic, polymorphic herbivores that feed on a diversity of plants and exhibit a complex foraging behaviour, making them a rich and multi-faceted model system. We will predict food preferences and cutting strategies in individual workers and ant foraging parties, and use machine learning and computer vision to test these predictions in large-scale, ecologically relevant experiments. This international, inter-disciplinary collaboration will not only (i) provide a quantitative theory to study individual as well as group feeding behaviour, (ii) advance our understanding of insect-plant co-evolution, but also (ii) have impact in other fields, from inspiration for small-scale cutting tools to developments in motor control theory.

Over the last decade, humans have made extraordinary advances in our ability to synthetically adapt biology. We are in the midst of developing the tools that can reverse genetic disease by altering DNA, and we are creating proteins that do not exist in nature to perform new functions. Yet our understanding of the basic function of proteins and how this is influenced by design is extremely primitive. Even the basic understanding of binding-based signal transduction in proteins, allostery, is arguably far from complete: we are still a long way from being able to mimic nature’s sophistication in creating functional proteins and engineering allosteric functions into proteins – the difference between a mere description and a true understanding. Our mechanistic understanding of allostery has been hindered by a lack of experimental techniques that can probe single proteins within an ensemble and the gap between experimentally observed and computationally predicted quantities. We will tackle this problem by adopting new single molecule tools, in conjunction with protein design and multiscale theoretical modelling. To understand how protein design impacts allostery, we will use and further develop a combination of theoretical (based on Ising formalism, elastic network models (ENMs), and sequence coevolution analyses) and experimental (nanoplasmonic tweezers, extraordinary acoustic Raman (EAR) spectroscopy, and molecular characterization of folding/binding/function) methods. We choose as model system tandem-repeat proteins because of their simplicity and modularity, structural malleability and adaptability to new functions allosterically driven by alterations in interfacial interactions and assembly state. Using both natural and designed TR proteins (and their mutants), we seek to close the gaps between design, theory/computations and function. The goal is a deeper understanding of the biophysics of allostery that will inform our traverse into synthetic biology while also having a specific impact on TR proteins and their essential roles in cellular pathways. Notably, an important interdisciplinary impact of this work will be to quantify the interaction of electromagnetic waves with proteins in the >10 GHz frequency range, precisely where new 5G cellular phone standards are being developed and proteins are susceptible to disruption due to their elastic resonances.

Plants are sessile organisms whose growth and development rely on finely-tuned signaling mediated by plant hormones (phytohormones). One of the pivotal phytohormones, gibberellic acid (GA), promotes a wide range of developmental processes in plants, such as seed germination, root and shoot elongation, fiber and cambium development, flowering time and fruit patterning. It is therefore crucial for plants to tightly regulate the distribution of GA throughout their lifespan. Our original HFSP team, in parallel to colleagues in the field, demonstrated that a class of nitrate/peptide transporters (NPFs) actively transport GA and play major roles in GA delivery and response. However, we still do not know the mechanism by which the GA transporters select and move their substrates. One of the major impediments is the lack of high-resolution structures. Also, functional redundancy severely hampers genetic studies to investigate the contribution of each transporter in plant growth. To understand the mechanisms of active GA transport in plants, our team will address the following three objectives: 1) Uncover the molecular mechanisms of key novel NPF GA transporters using a combination of structural and functional studies; 2) Investigate the physiological functions of active GA transport in root development and fiber formation using genetic, cellular, and systems biology approaches; and 3) Identify novel GA transporters, beyond the NPF family. These objectives will require innovative techniques, for instance, we will overcome functional redundancy using our unique multi-targeted forward-genetic cell-type-specific transportome-scale screening. Our comprehensive studies on GA delivery at multiple levels—from the molecular and cellular mechanisms of individual transporters to the GA-trafficking protein network in the plant body—will greatly facilitate the design of GA-transporter specific modulators and deliver crucial knowledge on the mechanisms of active GA transport in plants. As genetic manipulation of GA biosynthesis and perception drove the first Green Revolution in world agriculture, controlling GA delivery through manipulation of GA transporters has the potential to generate a second Green revolution to improve agricultural traits.

Hybrids are offspring produced from mating events between two different species. Once thought to be rare, we now know that hybridization is quite common, and evidence for it is found across the tree of life, including in our own species. There has been a longstanding debate about the importance of hybridization because it can have both beneficial and harmful effects. Hybridization can help organisms adapt, but it can also drive extinction by generating harmful combinations of genes. Past work has proposed that in stressful environments, the benefits of hybridization may outweigh the costs, but this idea has not been clearly tested. How this tug-of-war between the positive and negative impacts of hybridization resolves is unknown and is an outstanding puzzle in several fields of biology. Moreover, understanding these dynamics has become more pressing as rates of hybridization have increased due to the stresses of rapidly changing environments. We will combine research approaches from mathematics to environmental chemistry to address this fundamental question. We will ask how hybrids respond to multiple environmental stressors, and what genetic tools they use to do so. To characterize the costs of hybridization we will develop new computational tools to identify gene combinations that may be harmful or helpful to hybrids. Finally, we will integrate results to build mathematical models to predict the possible consequences of hybridization. Together, this work will reveal the consequences of hybridization and allow us to predict its impacts in our changing world.

Hematopoietic stem cells (HSCs) are responsible for the production of all blood cells throughout life. In vivo, HSCs reside within heterogeneous microenvironments or niches able to promote their function. Understanding the mechanisms responsible for the niche ability to support HSCs is required to expand our knowledge of HSC-niche interaction in vivo in healthy conditions and after niche injury induced by chemotherapy. Additionally, it will pave the way to create in vitro niches allowing for HSC reprogramming, maintenance and expansion for therapeutic purposes. Using zebrafish, a powerful model due to its similarity to human developmental processes, hematopoietic master regulators and disease inducing mutations, I will: Aim 1: Unravel the cellular and molecular mechanisms providing niche cells the ability to support HSC using live imaging and single-cell multiomics. a- Characterize the 3D structure and the cellular dynamics of the HSC niche using live imaging of a newly generated multicolor zebrafish reporter line in healthy conditions and after cell-specific ablations. b- Decipher the niche transcriptional code responsible for HSC support combining single-cell RNA-seq, single-cell ATAC-seq and spatial transcriptomics. Aim 2: Characterize the clonal dynamics of HSCs and niche cells in the marrow in perturbed conditions using genetic lineage tracing. a- Analyze the clonal origin of HSCs and niche cells in the healthy marrow using the scGESTALT barcoding technology. b- Expose animals to sublethal irradiation and characterize the clonal dynamics of niche recovery after injury.

Plants have a remarkable ability to undergo transitions during their life in a robust and synchronized fashion. They are able to achieve this despite the environment they are in being highly variable, and not having sophisticated information processing systems like brains. A striking example of how plants use variable signals such as temperature to time a critical developmental transition is the breaking of bud dormancy in in long-lived trees that grow in boreal and temperate regions of the world. In these trees, growth stops in the autumn and a state of dormancy is established that prevents reactivation of leaf formation which is timed to coincide with the advent of spring. The timing of dormancy break so the growth can be restarted is highly critical decision since a premature release from dormancy exposes plants to damage from sudden frosts in the early spring, whereas restarting growing too late will compromise fitness and productivity due to reduction in the length of their growing season. How trees accurately time the break of bud dormancy so that growth can start again in spring is question that has puzzled biologists for over 100 years, and will be addressed in this project. This will be achieved by exploring parallels between how human engineered computers process information, and the cells in tree buds process temperature. The genes which control bud dormancy have recently been described, yet how they operate within the multicellular context of a bud tissue remains unknown. We propose that controlled communication between cells plays a central role in the release from bud dormancy, and their ability to generate robust responses. A synergistic combination of biological experiments, computer modelling, physics and 3D image analysis, will provide unprecedented insight into how tree buds process temperature information. The project will lead to the identification of algorithms invoked by trees and the molecular and cellular logic used by trees to control bud dormancy, thereby revealing the operating system used by a plant to control its growth over the course of its life cycle.

All multicellular organisms are genetic mosaics, composed of different cell populations that have acquired somatic mutations due to mitotic errors. It is generally assumed that this intra-organismal genetic heterogeneity is detrimental for organismal fitness and results in disease such as cancer and senescence. The goal of ADAPTASEX is to unite studies on the dark side of such genetic heterogeneity with an exploration of possible beneficial adaptive effects. In doing so, we are analyzing parallels in modular organisms that occur frequently among the plant and animal kingdom. Corals and seagrasses, as examples, grow asexually to very large size, resulting in clones (=genets) of hundreds of m2. Our research is motivated by preliminary, genetic marker based observations of within-genet heterogeneity (corals, seagrasses), its transfer to the germline, and the increasing recognition of the importance of genomic heterogeneity for predicting cancer etiology, dynamics and treatment. We will describe somatic genetic heterogeneity at the genome level of model coral and seagrass species, and compare patterns to available cancer data. For the free-living species, we perform experimentation to assess fitness effects of somatic genetic variants, and study the transfer of somatically generated variation to the sexual cycle. We hypothesize that the emergent multi-level selection contributes to adaptive dynamics and may purge deleterious genetic variation at the cell population level, explaining delayed senescence and reduced mutational load in modular species. Our interdisciplinary team will develop a joint modeling approach to follow adaptive dynamics of the observed intra-organismal genetic heterogeneity in diverse asexually evolving systems ranging from cancer to modular plant and animal genets and gain synergistic insights into the role of cell population competition and spatial structure for asexual selective sweeps. We will develop protocols to validate low frequency genetic variants, and to predict adaptive dynamics depending on localization, level of selection, and effects of somatic mutations. We expect to contribute to the unification of biological theory by establishing a framework for the study of somatically derived genetic variation from humans to plants, its evolutionary dynamics and fitness effects.

Understanding how consciousness arises from neural activity in the human brain remains one of the greatest scientific challenges of modern times. Two of the most influential theories of the neural correlates of consciousness (NCC) are Global Neuronal Workspace (GNW) theory and Integrated Information Theory (IIT). GNW posits that what we experience as a conscious state is the global broadcasting of information in an interconnected neuronal network. IIT instead defines a measure for integrated information, known as phi (f), which can theoretically be used to locate this physical substrate within the brain. The empirical research on these theories have been focused on the visual modality alone, even though the theories make predictions of the full conscious experience, where all sensory modalities necessarily must be included. For example, very little attention has been directed towards the auditory modality. These theories have also been developed separately without much interaction between them and the empirical research has been focused on testing each theory individually, rather than contrasting them to evaluate their explanatory and predictive power. The research question of this project is which of these two theories better explains the NCC, using an adversarial and multi-modal approach designed to cover a maximal space of the testable predictions of these models. It is adversarial in the sense that the strongest advocators for both theories are involved in the project, and it is multi-modal in the sense that all experiments are tested in both the visual and the auditory modality.

Sexual imprinting is a process of instinctive learning occurring during early postnatal development, through which many animal species acquire memories of the odor, vocalizations, and other characteristics of their parents (or siblings), and then utilize this information to select their mates as adults. Sexual imprinting has evolved in many taxa, and yet surprisingly almost nothing is known about the neural mechanisms controlling this type of learning. What neural circuits are involved in this process? How is information from multiple sensory modalities integrated, and do they have synergistic effects on female preferences? Laboratory mice have proven to be useful models to investigate sexual imprinting, but do these findings generalize to outbred, wild house mice? To answer these questions and decipher whether sexual imprinting represents a general mechanism guiding mate selection in females, we will take a multidisciplinary approach using behavioral, neuroanatomical and advanced optical imaging techniques. We will conduct cross-fostering experiments, and apply whole brain activity pattern reconstruction and in vivo functional imaging to address several specific aims: i) experimentally test the synergistic effects of acoustic and olfactory cues on sexual imprinting; ii) reconstruct the brain regions activated by sexual imprinting; and iii) functionally trace the circuits that influence female mating preferences. To address these aims we created a novel collaboration between three research groups with very different expertise. The multidisciplinary research capitalizes on recent advances in optical imaging and neuroanatomy, including light-sheet whole-brain imaging and highly innovative in vivo technology for simultaneous imaging on multiple brain regions. We aim to reveal the organization of the circuits that are shaped during postnatal development to form memories of conspecific that are recalled in adults, which enhance inbreeding avoidance and reproductive success.

The gut-brain axis allows for information transfer between gut microbiota and the brain. The microbiome influences brain health, with links to autism, anxiety, and depression. However, pathways of communication between gut and brain have not been fully established. This study focuses on neural information transfer along the gut-brain axis. The enteric nervous system of the gut consists of parallel nerve plexi running along the gut. The enteric nervous system sends and receives signals through the vagus nerve which connects to the brain, thus providing a route of communication between gut microbiota and the brain. The exact pathways of information travel, regarding locality and extensivity of given microbial cues, are not well understood. This study utilizes a tissue engineered neural probe, consisting of a 3D network of recording sites within a cell-seeded collagen gel. These structures are designed to integrate into the gut, allowing for long-term implantation and recording. To determine locality of signals originating from the gut microbiome, probes will be tested in vitro on a population of gut epithelial cells followed by ex situ implantation into a gut explant model. After improving device efficacy, probes will be implanted into live mice. Bacteria will be injected into the gastrointestinal tracts of mice, and neural signals will be recorded from multiple locations along the gut to determine signal locality and route. Overall, this study focuses on a quickly developing area of research in examination of mechanisms by which the gut communicates with the brain, while generating a new form of neural probe for integrating with the nervous system.

Carbon dioxide (CO2) has been present in the atmosphere and dissolved in water throughout the almost four billion years over which life has evolved. Over this time, CO2 has emerged as the primary carbon source for all life on earth through its assimilation by plants and bacteria to form carbohydrates and other organic molecules that are the building blocks of life. Consistent with its abundance in the atmosphere and its ability to diffuse through membranes, many organisms are well known to sense and respond to CO2. Despite the clear physiological importance of CO2 and its prevalence within the environment, remarkably little is known about the potential of CO2 to directly regulate protein function and how it may thereby serve as a metabolic signaling molecule within organisms. We hypothesize that reversible carboxylation of specific lysine residues by CO2 within various proteins, in a manner that is sensitive to cellular CO2 levels, can alter protein function in vivo and thereby serve as a mechanism for organisms to sense and respond to varying levels of CO2. We believe that several difficulties have limited progress in addressing this fundamental question. Principally, the rapidly reversible nature of lysine carboxylation makes it notoriously difficult to detect CO2 modified residues and to characterize the functional consequences of this protein modification. Here we propose to work as a team to develop a series of technologies to enable investigation of protein carboxylation by CO2 and its role in sensing within the microalgae Chlamydomonas. Chlamydomonas is an ideal model system because it shows clear physiological responses to CO2 and can be readily genetically engineered. Using these new research tools we will test the hypothesis that fluctuations in CO2 levels are sensed by Chlamydomonas through modification of the lysine residues of various cellular proteins. Finally, we will use targeted genetic engineering of Chlamydomonas to validate our understanding of this system.

The decline in regenerative potential that follows aging is associated with organ failure ultimately leading to frailty and death. Understanding the mechanisms underlying the loss of regeneration with age may unlock novel therapies for treating age-related disease. The freshwater cnidarian Hydra vulgaris is capable of whole-body regeneration and appears to lack senescence. The closely related Hydra oligactis displays these features when cultured in normal conditions, but chemical treatment or temperature reduction induces an aging phenotype, which is accompanied by a decrease in regenerative potential. Interestingly, H. vulgaris does not age or lose regenerative potential when subjected to the same treatments. Thus, these two Hydra species present a valuable opportunity to use comparative approaches to uncover the mechanisms linking aging to a loss of regenerative potential. To accomplish this, I will use RNA-seq to profile regenerating tissue in aging and non-aging H. oligactis, and in H. vulgaris. In addition, I will profile aging H. oligactis treated with the anti-aging drug Rapamycin which promotes regeneration in aging H. oligactis. Together, these experiments will allow me to identify genes that are activated in regenerating H. vulgaris and non-aging H. oligactis, but not in aging H. oligactis after injury. I hypothesize that these genes are essential for regeneration, which I will test by knocking them down in H. vulgaris and assaying regenerative potential. The results of this proposal will contribute to understand the mechanisms of regeneration during aging and uncover potential molecular targets for the development of new anti-aging therapies.

Exocytosis is a conserved vesicle trafficking pathway responsible for the delivery of biomolecules to the cell surface and to the extracellular media. While regulated exocytosis is present only in secretory cells (i.e. neurotransmission or hormone release), constitutive exocytosis is generally present in all eukaryotes, where it regulates cell growth, cell progression, cell polarity and it is involved in multiple human pathologies including cancer and neurological disorders. Our goal is to time-resolve the molecular mechanism of constitutive exocytosis (i.e. exocytosis). Understanding complex and multi-step processes such as the exocytosis requires a complete overview of the structure-function relationships and the dynamics of the protein machinery involved. Exocytosis is regulated by an extensive network of macromolecules organized in protein complexes, regulatory proteins and membranes with specific compositions. Despite it has been long studied, the complexity and dynamism of the mechanism of exocytosis could not be reconstituted in vitro. This and other technical constraints limited the characterization of the cellular machinery in charge of performing this function. To investigate this further, we have gathered an international team of scientists to resolve the global mechanism controlling exocytosis through the concerted application of advanced imaging techniques. The team includes experts in biology, mathematics and engineering of instrumentation for microscopy. We intend to decipher the molecular mechanism of exocytosis by addressing the system with all of its complexity, including its topology, dynamics and function. We will combine live-cell imaging, the development of imaging instrumentation and advanced mathematical computation to build an enhanced setup capable of reconstructing the cellular machinery beyond current technical constraints. Together we will embark on a comprehensive approach to solve the long-standing question of how the cell is capable to coordinate such a massive and dynamic protein machinery in charge of driving exocytosis.

Despite the central role operons play in prokaryotic genome organization, their origin, evolution, function and unique characteristics are not well-understood. The primary difference between operons and the comprehensively-studied monocistronic mRNAs is that operonic cistrons frequently overlap with one another. These overlaps mandate that the translation initiation of a distal cistron must occur within the coding region of the upstream proximal cistron. This process does not allow for independent translation initiations, a fact inadequately explained by the current paradigm for translation initiation, the 30S binding mode, as it necessitates constant interference between elongating and initiating ribosomes. Recently, a new mode of translation-initiation, the 70S scanning mode, was experimentally observed, and it offers a challenge to the existing paradigm. However, it remains unknown which initiation mode occurs at each cistron, nor how to perform measurements that differentiate between the two modes and capture their genomic distribution inside a living cell. Moreover, it is not known what processes are involved in the ribosomal initiation mode-decision or its mechanism of regulation. Herein, we propose an experimental framework based on ribosomal titration assays followed by ribosome profiling, which will enable us to identify the genome-wide distribution of initiation modes, and measure and map their ribosome-mRNA affinity landscape. Using this data, we will study and characterize the mechanisms that govern the mode of initiation, and build synthetic operons to validate our findings and enable precise prediction and utilization of the operon unique characteristics.

The mitotic spindle is a dynamical apparatus consisting of microtubules (MTs) and associated proteins. Mitotic spindle bipolarity is vital for faithful separation of chromosomes during cell division. The bipolar spindle architecture can be conceptualized by a nematic network of antiparallel MTs at the spindle center and, in many cell types, polar (or astral) MT arrays radiating from the spindle poles. Although most spindle components are known, how they work together when spindles self-organize it is still not clear. It would be illuminating to understand what is the critical minimal set of activities that is necessary and sufficient to form a spindle-like active MT network. This project addresses this question by exploring the morphogenetic potential of active MT networks in 3D space using computer simulations. The project will explore the conditions allowing the coexistence of nematic and polar MT network states as observed in spindles. We will investigate networks organized by antagonistic motors, passive crosslinkers, and regulators of MT nucleation and MT stability. We will study how spatial confinement affects active network organization. We will determine a predictor of network topology based on the biophysical parameters of the underlying components. The computational work will be critically informed and validated by in-vitro reconstitution experiments and theoretical understanding will guide the design of in-vitro reconstitutions. The extracted design principles for self-organizing minimal systems will then form the basis for understanding more diverse and complex spindle networks in living cells.

A biological neural network is formed by living neurons with massive communication links between them. Neurons communicate by delivering electrical signals through insulated axonal fibers, providing the communication speed of up to 100 m/s. Although this biological neural network enables powerful human computation, the more powerful and efficient computation can be made if the neural network equips a faster mode of communication. Inspired by the revolution in the modern communication industry from electrical cables to optical fibers, we aim to explore whether a living neural network can be engineered to communicate with optical signals. Compared to electrical signals, optical communication is faster by orders-of-magnitude and selective by wavelength, which could significantly improve both communication speed and bandwidth. Specifically, we will first construct light-emitting neurons using a ‘cell laser’ technique and light-receiving neurons constructed by optogenetics. Based on these living optical elements, we will develop optical logical circuits on a chip for logical computational operations. We will further incorporate the time-varying property of living neurons by using the light-controlled expressions of photoactive proteins. To successfully achieve the research goal, we have assembled a multidisciplinary team of a physicist (Dr. Humar) for microlasing and optical configuration, a biomedical engineer (Dr. Choi) for neural engineering, and an electrical engineer (Dr. Im) for on-chip integration and analysis. Successful completion of this project will bring a new concept of optically communicating neural circuits that could realize the fastest communication mode in biology.

The experience of traumatic, life-threatening events gives rise to some of the most enduring forms of fear memories, which can degenerate into a devastating pathological state known as post-traumatic stress disorder (PTSD). Yet, surprisingly little is known about how long-lasting memory are formed and stored. Likewise, the neural and molecular mechanisms of how to best overcome remote fear memories remain unknown. The aim of this project is to identify the transcriptional and epigenetic changes occurring at a single cell level during remote fear memory attenuation, and to validate their functional relevance. To this end, I will use a mouse line in which the promoter of a neuronal activity marker (c-Fos) drives a tetracycline inducible system that allows for the identification of the neuronal subpopulation underlying fear memory attenuation. FACS cell sorting coupled with single cell RNA-seq, ChIP-seq and ATAC-seq will capture the genome-wide changes in gene expression and chromatin organization occurring within the tagged subpopulation of cells. Finally, spatiotemporally controlled CRISPR approaches will be used to dissect what gene-specific alterations, either transcriptional or epigenetic, are responsible for remote fear attenuation. I anticipate that this proposal has the groundbreaking potential to uncover the molecular mechanisms behind the overcoming of remote fear memories, providing precious insights for the treatment of PTSD. In extension, showing how the modulation of epigenetic marks in a cell type and locus-specific manner can impact neuronal function and memory capacity will represent a major step forward for the entire field of molecular neuroscience.

Our objective is to elucidate the mechanism, from subcellular to global scale, underlying the interdependence of plants and climate during Earth's current climate transition. This century, rising CO2 will force global temperatures to increase, pushing some vegetation systems into heat stress and potentially physiological collapse, irrespective of rainfall. Indeed, the most vulnerable regions are humid, high rainfall zones where 'wet-bulb' temperature (the lowest temperature that an evaporating object or organism can cool to) will rise to levels known to induce physiological stress and cellular death. Plants crucially affect climate through stomatal regulation of transpiration, which influences partitioning of net solar radiation into heating and evaporative cooling actions at the land surface. Plant vulnerability to heat stress can diminish this role and promote further climate warming, but little is known of the physiological and molecular mechanisms of stomatal regulation under heat stress, or of the combined physiological and climatic conditions that generate lethal leaf temperatures. This limits our capacity to predict and prepare for the long-term effects of global warming. This project will unlock mechanistic information from plant molecular genetics and heat stress physiology to formulate new theory describing stomatal regulation under temperature extremes, and apply this in an Earth system modeling framework to develop tools for predicting and adapting plant-atmosphere dynamics. Our central hypothesis is that previously unrealized breakdown of the stomatal regulation mechanism under heat stress, when expressed at the landscape scale, will add a significant and as yet unforeseen enhancing feedback to climate change this century. To test this, we will undertake the first coordinated integration and scaling of the stomatal control mechanism responding to heat under high CO2, from molecular signaling through to global fluxes, to quantify the full effects of plant heat stress on regional and global climate. Genetic and physiological methods will be used to develop and validate a new leaf-scale model of stomatal regulation at high temperature. This will be implemented in Earth system simulations to re-evaluate end of century climate scenarios in the most heat-vulnerable regions using improved surface energy partitioning calculations.

Biosensors are used extensively to transduce critical biological parameters such as biomarker concentrations into measurable signals. However, current biosensors are poorly suited for studies that benefit from long observation time or live hosts, especially those involving complex biological processes such as metabolism and neural signalling. This can be due to either low in vivo stability, limited sensitivity, short monitoring times, or high invasiveness. To overcome these limitations, I propose the design of a novel class of biosensor combining the specificity and stability of protein-based biosensors with the minimal invasiveness and long monitoring times of electrochemical sensing. These biosensors will be based on periplasmic binding protein (PPBP) scaffolds, which convert binding of an analyte into a conformational change. As local analyte concentrations fluctuate, this conformational change will be harnessed to move a redox sensitive probe, such as a ferredoxin, in relation to the surface of an electrode on which the sensor will be immobilized. As probe proximity will in turn be detectable through electron voltammetry (monitoring the transfer of electrons to/from the probe) and PPBPs bind their targets reversibly, the result will be a biosensor suited to continuous analyte monitoring. After optimization of the sensor in vitro, we will validate the sensors in a complex cellular environment by detecting the flux of glycine and other neurotransmitters in live brain slices. Altogether, these biosensors will be an important step towards the holy grail of biosensing; sensors capable of on-demand, continuous, and non-invasive sensing of any analyte of choice.