Living matter represents spatially structured soft materials with internal energies far from the thermal equilibrium. The inherent order and fluidity in such systems lead to dynamic, anisotropic properties spanning orders of scales. Spontaneous symmetry breaking in structured matter nucleates local singularities called topological defects. Despite reported evidence of topological defects in living systems, their biological functions have remained grossly unexplored, denying us, to date, a holistic biophysical picture. With a focus on microbial species, I propose to develop the first experimental framework to understand topology-induced microbial physiology, based on a combination of cutting-edge methods including advanced imaging, spectroscopy and machine learning. Specifically, by tracking defect genesis and dynamics in bacterial colonies of species with diverse phenotypes (morphology and motility traits), I will quantify biological activity in the defect vicinity over short to generational timescales. Using fluorescence confocal and electron microscopy (to track molecular transport, exudates and ROS levels), and nanoSIMS technique (to quantify nutrient uptake), I will obtain seminal data linking defects to microbial physiology, and harness this mapping relation to construct machine learning algorithms that will ultimately predict the biological role of defects in multi-species communities. By discerning the biological functions of topological defects, I will be in robust position to pioneer a quantitative approach to this uncharted problem, heralding a unifying framework to predict more complex scenarios, thereby advancing our general understanding of physics of life.

The small intestine is the major site of nutrient absorption after food intake and malabsorption (impaired nutrient uptake) affects millions of people worldwide. However, there is an unmet need to study malabsorption with physiologically relevant in vitro models. Glucose-galactose malabsorption (GGM) is a genetic disorder that affects the SGLT1 protein, which results in diarrhea and life-threatening dehydration upon glucose and galactose consumption. In my research I aim to establish a physiologically relevant platform to assess nutrient bioavailability employing intestinal organoids (biovanoids) to model GGM. GGM biovanoids will be generated by introducing a disease-associated mutation in SGLT1 (C292Y) using CRISPR/Cas9-mediated base editing. Some aged GGM patients describe an acquired glucose tolerance, which accelerated upon probiotic consumption of certain bacteria. The influence of SGLT1 mutations on host-microbe interactions and possible metabolic adaptation through e.g. age-associated hormonal changes will be studied using GGM biovanoids. This may offer an opportunity to introduce glucose tolerance in young GGM patients. Overall, biovanoids offer a promising platform to generate essential knowledge about malabsorption. To elaborate further applications, biovanoids will be validated for their potential to predict intestinal absorption and gut wall metabolism of drugs. Poor bioavailability contributes to high attrition rates of oral drugs during clinical phases of drug development and currently applied models fail to reliably predict bioavailability in humans. Biovanoids could improve these predictions to eventually increase successful drug approval to the market.

A high level of oxygen in the atmosphere is taken for granted as it is indispensable for human and most animal life. Yet, life (bacteria and unicellular eukaryotes) evolved for over 2 billion years in a low oxygen environment. Oxygen became widely available only in the last 600 million years and was likely the main factor behind the rapid diversification of most complex multicellular life forms. For elaborate multicellular tissues to form, their overall shapes however must still satisfy oxygen demands of individual cells. Oxygen consumption will rapidly reduce the oxygen level within a few cell depth into the tissue. This becomes a problem in early embryonic development before respiratory and circulatory systems are established. Many cells still need to differentiate and migrate for long distances and place themselves at the right destination. It is largely unknown what makes the varying oxygen environment and the execution of their precise differentiation and navigation within a tissue compatible. The project aims at elucidating the common basis of oxygen dependency in morphogenesis by studying how the social amoeba Dictyostelium detects and reacts to oxygen at the molecular level and how this dictates cell migration and morphogenesis. Dictyostelium is an organism evolutionarily close to the branches of multicellular evolution, and presents enormous advantages to be easily cultured in the laboratory and reversibly switched from unicellular to multicellular states in less than a day. Amoeboid cells are able to change their state (by differentiation) and place themselves in more favorable conditions for foraging and respiration (by migration). An emphasis is placed on clarifying this most fundamental and primordial characteristic with a focus on the cross-scale quantitative relationship between oxygen, metabolic states, cytoskeletal machineries and migratory modes. New screening methods based on aerotaxis responses, oxygen sensors and 3D microscopy will be combined with molecular genetics of oxygen response pathways. The role of the new mechanism unearthed will further be tested for their roles in neural crest cell migration in zebrafish. They will be formulated into multi-scale mathematical models to clarify how the coupling between oxygen consumption, sensing and migration of individual cells gives rise to robust and collective morphogenetic movements.

The skin epithelium forms a primary barrier against inflammation-inducing environmental agents and is maintained by the epithelial stem and progenitor cells (EpSCs). Stem and progenitor-cells (SCs) adapt to various conditions via crosstalk with their local microenvironment or "niche". The EpSC niche is comprised of fibroblast, neuronal, immune, and other cells that provide context-specific signals to direct SC function. However, how the EpSC niche is affected by inflammation is poorly understood. In addition to actively participating in inflammation, EpSCs also retain a memory of inflammation long thereafter, which enables more rapid responses to subsequent assaults. However, the contribution of niche factors to maintaining EpSC memory is entirely unknown. To address this knowledge gap, I propose to systematically chart the cellular constituents of the EpSC niche before, during, and after acute inflammation. Armed with this high-resolution map of the EpSC niche, I will then perform functional studies to identify niche factors that control EpSC inflammatory function and memory. Understanding inflammation-induced niche rewiring and identifying the key inflammatory niche regulators of EpSCs may illuminate mechanisms of inflammatory epithelial pathologies such as psoriasis, chronic non-healing wounds, and cancers that are rooted in SC dysfunction.

The world around us is constantly changing – new elements appear all the time. How does the brain decide what novel things to explore? Animals exist in a constant push and pull between being adventurous and conservative. My objective is to understand how the brain controls the behavioral response to novel stimuli under naturalistic settings, and how those brain functions have been modified by evolution and ecology. During my fellowship I will approach the neurobiology of novelty from an interdisciplinary perspective that merges the evolution of behavior with its neurophysiology. I chose an ideal model system to study comparative neurophysiology: the deer mouse (genus Peromyscus). Deer mice are the most abundant mammal in North America (comprised of >50 species). Different Peromyscus species have evolved distinct behavioral repertoires. I will combine evolution and ecology with novel tools such as fiber photometry and wireless electrophysiological recording to understand the computations performed in the deer mouse brain under ethological conditions in response to novel stimuli. Specifically, I will study the brain of a variety of Peromyscus species with distinct behaviors under different novelty challenges. Overall, I will focus my research program on testing three general hypotheses: (i) different species or populations of Peromyscus will present differential response to novel objects, and (ii) dopaminergic neurons projecting to the tail of the striatum, controlling novelty responses behave differently in these species (iii) dopaminergic neurons projecting to the tail of the striatum shape neophobic behaviors in the wild.

Gene regulatory networks (GRNs) represent the wiring diagrams of cells and can be envisioned as the blueprint for organism development. Transient interactions of transcription factors (TFs) and DNA as well as chromatin structure fine tune gene expression and allow cells with the same genome to give rise to a myriad of cell types and tissues. My overall-goal is to elucidate this fundamental process live and investigate, how exactly transient interactions, dynamics, and variations in absolute concentrations of TFs on the molecular level shape GRNs, impact on vertebrate embryo development, and can be used to infer cellular identity. Our current understanding of gene regulation arises mostly from static measurements employing sequencing technologies and in situ hybridisation approaches. However, extracting quantitative clues such as TF concentrations and their changes over time is very challenging yet crucial to shed light on the organising principles. I propose to use quantitative imaging, fluorescence fluctuation spectroscopy (FFS), and endogenous labelling of TFs to measure absolute, native concentrations and interaction dynamics directly during live zebrafish development. Using these readouts as a new handle on cellular heterogeneity, I will elucidate how specific TFs regulate hindbrain establishment and by employing engineered TFs as reporters for DNA accessibility, how overall chromatin organisation evolves in different developmental contexts. Together with the design of an open-source microscopy-spectroscopy platform tailored for in vivo FFS, this project will result in previously unattainable, quantitative insights into vertebrate embryogenesis.

Symmetry breaking is a process during which a homogeneous system adopts asymmetry and is fundamental during initial cell fate determination in early development and regeneration. Single-cell interactions and information exchange with the environment allows for such large-scale coordinated behaviors. Identifying mechanisms driving symmetry breaking is not trivial due to frequent nonlinear relationships between the system’s lower- and higher-order dynamics. Employing approaches from cell-, systems- and biophysical fields, I will study symmetry breaking in intestinal organoids. In a uniform growth-promoting environment, only a subset of intestinal stem cells differentiates into secretory cells. Recent evidence implicates an upstream regulatory pathway of extracellular signal-regulated kinase (ERK) in contributing to proper patterning of the stem cell niche. The proposed research aims at understanding whether and how ERK mediates symmetry breaking during intestinal organoid formation. To address this fundamental question, I will: 1) analyze the spatiotemporal-specific role of ERK signaling during symmetry breaking, 2) identify ERK-mediated tissue-level coordination focusing on biochemical and mechanical regulation, 3) determine how single-cell interactions are integrated into large-scale collective behaviors using theoretical modeling and optogenetics. This research project will determine the spatiotemporal-specific role of ERK during symmetry breaking with unprecedented multiscale resolution and provide key insights into the emergence of complexity and collective behavior fundamental to processes such as mammalian development, cancer invasion and wound healing.

Cardiovascular disease (CVD) is the leading cause of global morbidity and mortality. While psychological stress is a known cardiovascular risk factor, the mechanism by which the brain translates stress into CVD is poorly understood. The underlying cause of CVD is a chronic inflammatory disease called atherosclerosis, the progression and exacerbation of which have been strongly associated with the immune system, including B cells. B cells perform many functions including production of antibodies that provide immunity against disease and cytokines that modulate leukocyte function. However, it remains unknown whether B cells mediate the effects of stress on CVD. Recent studies from the host lab show that stress profoundly affects the number and distribution of B cells in the body. This occurs via a mechanism dependent on the activation of the hypothalamic-pituitary-adrenal axis. In this proposal, I will test the hypothesis that stress aggravates atherosclerosis by modulating B cell function. To recapitulate stress, I will employ a combination of optogenetic and chemogenetic approaches available in the host lab to locally activate specific regions in the brain and characterize the subsequent impact on the phenotype and functional diversity of B cells. I will then identify the B cell-specific mechanisms that mediate the effects of stress on the progression of CVD. These studies will not only delineate potential therapeutic targets for immunomodulation of B cells in prevention and treatment of atherosclerotic CVD, but also provide a direct mechanistic link between stress and chronic inflammation, a general concept with implications beyond atherosclerosis.

The flow of information between different regions of the brain during decision making is a largely unknown process. This is mainly because there are no tools available to disentangle the complex interaction of myriad cell types in the brain, governed by gene expression, connectivity, spatial organization, and other properties. This proposal aims to investigate the anatomical substrate of inter-regional communication at single neuron level. We hypothesize that a specific group of neurons that project directly between brain areas provide the “scaffolding” for the correlation between structural and functional connectivity. Tools developed in the proposed host group, as well as their previous work on the topic, make this question accessible for investigation. We aim to identify this privileged subpopulation of neurons by combining two-photon imaging of activity in single neurons with anatomical projections extracted using Barcoded Anatomy Resolved by Sequencing (BARseq). Successful completion of this project would generate unprecedented datasets that bridge information at all levels–anatomical, genetic, physiological and behavioural. Gaining knowledge of the flow of information in the brain would push the frontiers of what it is one of the grand challenges of our time–to understand how behaviour arises from neural circuits.

With their multiple arms, three hearts, suckered tentacles, camera eyes, and fast colour-changing skin, cephalopods seem to come from an imaginary world. These iconic invertebrates play a major role in balancing the entire marine ecosystem, yet the 4 million metric tons caught annually have led to their global decrease. Cephalopod diet and feeding systems remain enigmatic. They have a ‘beak’ composed of two jaws with no direct contact, a radula, and specialised masticatory muscles. Unravelling the secrets of their feeding system can inform us about their diet and potentially have huge global impact by aiding their survival. In this project, I will bring together for the first-time expertise in cephalopod biology and biomechanics to quantitatively characterize the anatomy and function of the feeding system in different species representing all cephalopod families. I will use a range of techniques based on material characterization and computational modelling to test 4 hypotheses. I will investigate the (1) material content and mechanical properties of the jaws and correlate these with their bite force and diet; (2) shape and surface properties of cephalopod jaws and its potential correlation with their diet; (3) muscular jaw articulation of cephalopods and the mechanisms that control its motion considering a range of diet; (4) impact of overall jaw and muscular joint morphology on the level of mechanical strain across each component. A major part of cephalopod biology will be unravelled in this project, providing new foundations for cephalopod conservation and other areas such as bioinspired soft robotics.

The role of metabolism during developmental processes remains largely unexplored due to challenges in measuring spatiotemporal dynamics of metabolic activities. In the mammalian post-implantation embryo, pluripotent cells differentiate into three germ layers, the ectoderm, mesoderm and endoderm that undergo specific morphogenetic movements during a process known as gastrulation. While it is known that metabolites influence cell fate by modifying the epigenetic and transcriptional states, the question, whether metabolism plays a functional role in germ layer specification, has not been addressed in an in vivo context. The two main aims of the proposed project are to characterize the metabolic profiles that accompany the emergence of germ layers and their possible function during germ layer segregation i.e. symmetry breaking that establishes the primary body axis. To accomplish both aims, I will use gastruloids, aggregates of embryonic stem cells that recapitulate hallmarks of gastrulation, as a model system. The gastruloid system will allow me to combine metabolic measurements, live imaging of biosensors and metabolic state reconstruction, to uncover emerging differences in metabolism among cells of the different germ layers. Further specific manipulations with inhibitors and optogenetic tools will test if the activity of certain metabolic pathways regulates cell fate decisions and coordinates tissue scale cell movements and thus probe possible molecular mechanisms. The findings of this project will reveal fundamental mechanisms underlying the interplay between metabolism and cell fate determination and thereby change our view of how gastrulation is robustly regulated.

Broadcast spawning is a method of reproduction used by many species of coral, where the corals release their gametes in a perfectly timed, synchronous manner to maximise fertilisation. The timing and molecular mechanism behind this extraordinary event are not understood. Coral bleaching and the death of reefs is something that is now firmly anchored in the public consciousness. However, another threat is looming for coral, the asynchrony of spawning. This marginalised ecosystem now faces an even bigger threat, the possibility of not being able to reproduce, spelling disaster not just for marine life, but also for the communities that depend on them. An understanding of this asynchrony is key and I have therefore designed a research plan which examines the molecular mechanisms behind the spawning event. In particular, I aim to study the light biology of coral, as they use multiple light stimuli, including moonlight, to regulate reproduction. To this day, we do not know what photopigments corals employ, where these are located and whether the algal symbiont is key in initiating different spawning steps. Moonlight is the critical signal, however, the full moon is present every 29 days, so another gating mechanism must exist. I aim to find out exactly what this is by varying different parameters in a lab setting. Using temporal differential gene expression analysis around this critical moonlight signal will provide a basis for understanding the key cellular responses driving spawning. Pioneering a deep understanding of reproductive asynchrony will allow the implementation of improved conservation measures and help save the world’s reefs from destruction.

The group setting forms the basis for most social interactions. However, despite the importance of group social behavior in almost every aspect of animal’s lives, very little is known about its neuronal substrates. This can be attributed to challenges associated with reductionist approaches to capture the complexity of group social interactions. Thus, a paradigm shift is necessary. Specifically, we identify these major gaps where most studies have: (1)focused on one brain at a time, (2)often did not consider individual differences and (3)neglected the long-term dynamics of group social behavior, especially in the natural context. To overcome these gaps, I propose an approach that leverages the rich social interactions in groups of bats, a new animal model for group social interactions in neuroscience. Utilizing cutting-edge methodologies that enable simultaneous wireless recording and manipulating of neural activity across the brains of group members, I will study the neural mechanisms of group social interactions as these naturally unfold. I will begin by focusing on two core aspects of group social interaction and their manifestation in the frontal cortex of socializing bats: (1)neuronal representations of individual’s identity as related to social vocal communication, (2)interbrain coupling across group members. Thus, I will study the relationship between group social and neural dynamics extending from the single neuron level in the individual brain to the relationship in neural dynamics across the brains of group members. Combined, this project will allow, for the first time, a unique mapping of the group social network onto the neuronal network of its members.

In order to gain something, sometimes you have to lose something. This is true in our lives, and it is also true for cells to gain special functions. The individual component of the phloem sieve tube, the sieve element (SE), loses the cytoplasm, organelles, and even part of the cell wall to be able to transport photosynthetic products and signaling molecules over long distance. However, the molecular mechanisms behind these dramatic changes have been shrouded in mystery for more than 60 years since the discovery. The main reason for this is due to the small size of SEs and their deep location in the tissue, which makes them extremely difficult to observe with confocal live imaging. In this proposal, I aim to reveal the autolytic mechanisms of SEs by establishing a novel SE induction system that overcomes such technical hurdles. This research provides new insights into the coupling between autolysis and cell specialization. It also has the potential to expand the field of plant science, as plant development have rarely been studied in terms of degradation control.

Protein quality control systems have been perfected through evolution to guard the proteome. Their capacity, however, decreases during ageing, which in turn threatens proteostasis and exposes humans to plethora of age-related disorders. Currently, a vast majority of these diseases - including neurodegeneration - remain incurable. The next scientific frontier is to convert the broad protein quality control knowledge into tangible benefit to humans by harnessing this intrinsically protective cellular system to foster cellular and organismal resilience to pathogenesis. An unbiased search for compounds that promote cell survival in stress pointed at the phosphorylation of the eIF2a translation initiation factor as a potent modulator of cellular fitness. Thus, eIF2a phosphorylation emerges as a key player in cellular resilience with its manipulation being potentially beneficial in many conditions. This cross-disciplinary proposal aims at using thorough and unbiased set of orthogonal approaches to delineate the role of phosphorylated-eIF2a in human pathogenesis. While exploiting human and mouse genetics, cell biology and pharmacology, I will: i) study the mechanistic bases of diseases genetically linked to eIF2a phosphorylation and the cellular consequences of its alterations; and ii) genetically modulate levels of phosphorylated-eIF2a in neurodegeneration to unambiguously define its role in disease pathogenesis. Together, this project will provide a fundamental, mechanistic framework to define how to manipulate the cellular cost-benefit ratio of eIF2a phosphorylation in pathogenesis and point at conditions that may profit from its modulation.

Over the past decade, programmable gene editing has been transformed by the discovery and development of prokaryotic adaptive immune systems known as CRISPR-Cas. These systems, which evolved to eliminate foreign genetic elements such as plasmid and phage via RNA-guided nucleic acid degradation, have been powerfully redeployed for many gene editing applications. Recently, these systems have been expanded with the discovery of nuclease-deficient CRISPR-Cas systems that direct RNA-guided transposition. Transposon-encoded CRISPR–Cas systems can insert large genetic payloads into bacterial genomes without relying on DNA double-strand breaks or host repair factors, eliminating the major limitations of current integration approaches that rely on CRISPR-Cas9. Nonetheless, the molecular mechanisms of key steps in this newly discovered RNA-guided transposition pathway remain uncharacterized, restricting the applications of this potentially powerful technology. Specifically, the mechanisms through which the transposase machinery is recruited following DNA targeting, the integration orientation of donor DNA is selected, and the megadalton protein-DNA transpososome complex is disassembled, are yet to be elucidated. To determine these mechanisms, I propose to employ a powerful combination of genetic, biochemical, and single-molecule biophysical approaches that is made uniquely possible by integrating the expertise in the Sternberg and Gonzalez laboratories. The results of my studies will transform RNA-guided transposition into a highly tractable, precise, and efficient RNA-guided DNA integration tool for next-generation genome engineering.

Although diseases are clearly harmful, many organisms carry genetic variation that causes disease. Why is this the case? My research proposes to combine techniques from field ecology and population genomics to investigate the forces maintaining genetic variants that cause cancer in swordtail fishes. In Mexico, it was recently discovered that some populations of swordtail fish have high frequencies of genes causing melanoma. Yet, these populations show no signs of losing these genes. I propose to conduct a large field experiment where I will manipulate the two major forms of selection - natural and sexual selection - to identify mechanisms that lead to the preservation of melanoma-causing genes in these populations. I hypothesize that reduced survival due to melanoma is countered by with increased growth rate and attractiveness to mates. My experiment will involve the construction of artificial streams where we can manipulate factors such as exposure to predators, mate choice, and parasitism. Using cutting edge techniques in population genetics, I will quantify directly how our manipulations change selection on genes. I anticipate that the results of this interdisciplinary experiment will yield novel and surprising insights into the forces that maintain disease-causing genetic variation in natural vertebrate populations.

Central nervous systems constantly detect environmental stimuli and integrate them into long-lasting behavioral changes. Adaptations to experience, for example associating a context with a positive or negative outcome represents an important form of learning. Such contextual learning is achieved in part by the modification of transcriptional programs in individual neurons. A broad range of evidence supports that immediate early gene transcription factor (IEG-TF)-dependent induction of gene expression controls various aspects of synaptic plasticity, and learning and memory. However, functions of the downstream activity-dependent gene products themselves are still poorly understood. Behaviorally-induced neuronal activity of hippocampal excitatory neurons was recently discovered to result in a bidirectional adaption of neuronal inhibition by parvalbumin- and cholecystokinin-positive interneurons. This requires a neuropeptide precursor SecretograninII (Scg2), downstream of the IEG-TF Fos, highlighting an unexpected role for a little-studied neuropeptide. In this research proposal I will use a combinatorial approach of molecular, electrophysiological, behavioral and imaging methods to identify how Scg2 mediates behaviorally-induced control of synaptic plasticity and connectivity in the mouse hippocampal microcircuit. In parallel, I will investigate the involvement of Scg2 in establishing neuronal assemblies for encoding memories and behavioral plasticity. Together, these studies will enhance our understanding how gene expression programs and, in particular, neuropeptide signaling regulate synaptic specificity and memory traces in neuronal circuits.

Neurons can be studied by monitoring intracellular calcium ion levels using fluorogenic calcium ion sensors. However, current sensors produce transient signals, potentially missing integral neuron activity and making it challenging to measure accumulated neuron activity over time. Alternatively, genetically encoded calcium ion signal integrators accumulate and integrate neuron activity over a defined time. However, these signal integrators are dependent on a light-assisted photoconversion process and only integrate neuron activity over a short time period. Furthermore, imaging cannot take place during irradiation and even irradiation must be ensured. We propose a calcium ion signal integrator that functions independently of photoconversion for monitoring neuron activity that has accumulated over extended time periods to address these issues. This novel tool consists of a genetically encoded protein sensor (GEPS) that undergoes a conformational change from an opened state to a closed state upon binding to calcium ions. After binding to calcium ions and adopting its closed conformation, the GEPS brings together two cysteines, which both react with a complementary and fluorogenic di-acrylamide probe. Labelling the closed GEPS with this fluorogenic probe slowly produces an irreversible and accumulating fluorescent signal independent of photoconversion but depends on calcium ion concentration. The integrated signal can be later imaged by fluorescence microscopy to identify activated neurons and their relative activity. This novel calcium ion signal integrator will greatly contribute to the future understanding of neuron development and diseases such as stroke.

As the causative agents of toxoplasmosis and malaria, the apicomplexan parasites Toxoplasma gondii and Plasmodium spp. are responsible for a massive burden of disease worldwide. However, our understanding of how these parasites function at a molecular level is incomplete, limiting development of therapeutic interventions. The apicomplexan ubiquitination system represents one such major gap in our knowledge. Ubiquitination is an essential eukaryotic post-translational regulatory system that mediates protein homeostasis and activity. In apicomplexans, ubiquitination is linked to many essential parasite processes, and represents a potential source of drug candidates. However, apicomplexans are far removed from humans and yeast, the species in which ubiquitination is best studied, precluding bioinformatic assignment of critical functions. Recent advances in T. gondii genome engineering provide the opportunity to systematically investigate the apicomplexan ubiquitination system. My goal is to systematically map the entire ubiquitination system of T. gondii and assess the contribution of each component to infection. I will further determine the interactome of components deemed essential, or conditionally essential during stress, and attempt to determine their specific role in regulating parasite growth and infectivity. This project will lay the foundation for research into potential treatments and improve our understanding of apicomplexan biology.