Animals have to constantly make decisions about what to do next. Especially, when navigating in the natural world, they have to cope with an abundance of potentially conflicting cues. Hence, in order to interact with the environment in a meaningful way, animals have to extract the currently relevant sensory features and select an appropriate, experience- or internal state-based, behavioral action. How this is achieved by the nervous system is poorly understood in any sensory modality and organism.

Landmark studies in primates have already revealed some principles of decision making but, due to technical limitations, could only focus on confined brain regions. However, understanding the complex process of how an animal selects the right behavior requires complete explorations of the hierarchical and parallel processing steps within neuronal assemblies in different parts of the entire brain.

The larval zebrafish is becoming the model of choice for studying brain-wide sensory processing as it offers a unique combination of tools: Larval zebrafish possess a variety of innate and robust behaviors in response to sensory stimulation, while giving access to state-of-the-art rapid whole-brain imaging techniques with cellular resolution.

Adapting successful concepts used in classical primate literature, I will use larval zebrafish as a modern model to investigate general principles of sensory integration, stimulus competition, decision making, and action selection. This will permit unprecedented detailed explorations of how neuronal circuits in an entire vertebrate brain extract features from a complex sensory world in order to decide what to do next.

The cohesin-complex mediates sister chromatid cohesion from S-phase until mitosis and is involved in the formation of higher-order chromatin structure. To fulfill these vital functions, cohesin is loaded and positioned in the genome by mechanisms that are only poorly understood. In vitro, loading of cohesin on DNA only requires ATP and a loading-complex formed by Scc2-Scc4, while loading in vivo on chromatin is regulated by additional factors. For example, in Xenopus laevis oocytes, cohesin loading strictly depends on pre-replication complexes (pre-RCs), which are formed in telophase/G1.
Mechanistic studies are required to understand how cohesin-loading occurs at the molecular level. I will first determine the mechanism by which Scc2-Scc4 loads cohesin on DNA. Using single-molecule FRET and optical tweezers, I will monitor the effect of Scc2-Scc4 on conformational changes of cohesin as it is loaded on a DNA template. After characterizing this minimal loading reaction, I will reconstitute cohesin-loading during telophase/G1 using a purified system. With these experiments I will address why and how loading of cohesin is regulated by the formation of pre-RCs.

The capacity of cells to move and migrate is fundamental to their viability, whether they originate from multicellular or single-celled organisms. This is exemplified in the process of infection, such as that by the the protozoan parasite Plasmodium, the causative agent of malaria disease in humans. During an infection, cell migration for both the human immune cell or the malaria parasite both rely on force generation from structures within each that link them to, and propel them across, the extracellular environment. For the immune cell, its amoeboid-like movement is the product of polymerising actin filaments combined with force generation from a myosin motor, which together drive changes in cell shape propelling the cell at speeds of several micron/min. In contrast, while relying on very same actin-myosin proteins, the malaria parasite does not change its shape, yet can move at speeds of >1 micron/sec, an order of magnitude above our fastest cells. Whilst a great deal is understood about amoeboid migration, we know little about how malaria parasites achieve directional motility or such great speed.
Underpinning Plasmodium cell migration across its lifecycle, whether in the liver, the blood circulatory system or mosquito, is an unconventional myosin (XIV), lacking many of the canonical features associated with myosin motors. Together with dynamic parasite actin filaments these somehow generate a force that drives the parasite forwards, however, the mechanics of how this actually works is far from understood. Here, building on insights we gained into actin regulation and organisation in the malaria parasite from our first HFSP program, we turn our attention firmly on myosin to understanding how it interacts with actin inside the cell to produce directional cell migration. Combining the state-of-the-art in biochemical methods, molecular and cellular parasitology, biophysics and structural biology (including cryoelectron microscopy), we aim to dissect at every level - from single molecule to whole cell - how motor organisation inside the malaria parasite leads to directional, fast cell movement. This will uncover profound insights into the workings of an ancient, supremely fast cell migration machine, and may potentially reveal weaknesses that could be targeted to cure one of mankind’s greatest diseases

Macrophages play beneficial roles in protective immunity. However, they also favor the progression of several pathologies when they massively infiltrate diseased tissues. A present challenge in cancer, for instance, is to control macrophage tissue infiltration, which involves the mesenchymal motility. This motility is characterized by the ability of the cell to form protrusive structures called podosomes. A podosome constitutes a submicron core of actin filaments surrounded by a ring of integrin-based adhesion complexes. Our working model postulates a mechanical connection that counterbalances the actin-rich protrusive core by traction at the adhesive ring, likely embodied by acto-myosin contractile cables. Therefore core and cables would form a unique two-module protrusive force generator that balances forces at the level of a single podosome to ultimately contribute to the mechanics of macrophage migration.
Our objective is to build a sound experimental corpus to substantiate this two-module mechanism of force generation and determine its implications for macrophage 3D motility. To this end, we will characterize podosome molecular architecture and formulate its relationship to force generation. Furthermore, we will identify the mechanical role of key podosome components in cell migration.
We assembled a multidisciplinary team that combines cutting-edge expertise in: i) macrophage 3D migration, ii) 3D nanoscale imaging, iii) live super-resolution imaging, iv) cryo-electron tomography technologies allowing resolution of actin filaments within cellular networks, v) measurement of podosome protrusion forces and vi) mechanics of 3D cell migration in custom microfabricated environments.
Our ambitious research plan will deliver the nanoscale localization of podosome molecular components (applicants A1, A3), determine how the architecture evolves along with force dynamics (A2), investigate the mechanics of 3D migrating cells (A4) and reveal the role of podosome components in these uncharted features (A1-4). Thanks to this groundbreaking study, we will articulate mechanical and architectural insights into an integrated portrait of podosomes from the molecular scale up to the biological context of 3D cell migration and thus identify molecular means for the modulation of pathological macrophage tissue infiltration.

Inorganic polyphosphate (polyP) is a biopolymer that serves multiple critical functions in biology. PolyP has been mainly studied in bacteria and shown to be involved in such diverse processes as the bacterial stress response, biofilm formation, plasmid uptake and antibiotic resistance. In comparison, its functions in mammals are significantly less well understood. Even so, polyP has been linked to multiple important physiological phenomena, such as blood clotting, chaperone function, posttranslational protein modification and neuronal signaling.
While in bacteria the enzymes that generate polyP are known (polyphosphate kinases Ppks), the mammalian enzymes remain elusive to date. In fact, we know that polyP is present in different cell types and that it is enriched in different organelles, but we do not know how and where it is made and how it is shuttled. Moving polyP research forward will critically require identification of the elusive enzymes that make and traffic polyP. Currently, a dearth of chemical tools has precluded such deeper insight into mammalian polyP physiology.
Our proposed research will culminate in new technologies required to make, modify, analyze, transport and deliver polyP both ex and in vivo. We will develop and combine multiple novel synthetic approaches to generate a range of modified polyP analogs that are not yet available. Furthermore, we will develop technologies to deliver and release those synthetic analogs in a controlled way outside and inside of cells. Together, these technologies will provide valuable novel insight into how cells make, regulate and distribute polyP. We will use these tools to identify the mammalian enzymes involved in polyP turnover, investigate how polyP levels are affected by other signaling molecules such as inositol pyrophosphates, and try to understand how polyP affects different biological processes. For example, an in-depth evaluation of the role of polyP in blood clotting will be achieved using our novel approaches. These studies will lay the foundation for further research into polyP physiology. By virtue of the combination of synthetic organic chemistry, drug delivery technology, and cell and animal biology, we will be able to develop and apply powerful novel tools dedicated to the elucidation of polyP physiology in mammals.

Understanding the mechanisms of longevity can provide ways to achieve healthy ageing. Naked mole rats (NMRs) are the longest-lived rodents that remain healthy until the end of their lives and are resistant to age-related diseases including cancer. One causative agent of this remarkable trait is the acquisition of high molecular mass hyaluronic acid (HMM-HA). The catalytic domain of the HA synthase gene HAS2 is highly conserved across several phyla, exhibiting two amino acid changes unique to NMRs that result in the synthesis of HMM-HA. I propose to create transgenic mice using CRISPR/Cas targeted genome editing that will contain the unique NMR AA changes engineered into the murine HAS2 gene. An orthogenic mouse model of the NMR HAS2 gene will tell whether HMM-HA can delay aging and prevent cancer in a species other than NMR. To identify genes involved in HMM-HA pathway and novel tumor suppressor mechanisms we will generate a whole-genome NMR CRISPR KO library. Primary NMR skin fibroblasts expressing SV40-LT antigen and Ras-V12 will be transduced with the library and malignantly transformed colonies will be selected. Unpublished data from the Gorbunova lab show that NMR hematopoietic stem cells (HSCs) are biased towards erythroid lineage. I will apply the NMR KO library to NMR bone marrow (BM) cells revealing genes responsible for the prevalence of erythroid development inherent to NMR HSCs. The genome scale NMR KO library will prove an invaluable tool for researchers utilizing NMR, while the discovery of new tumor suppressors and the molecular understanding of the longevity and cancer resistance of this remarkable rodent have great potential for biomedical applications.

Our goal is to unveil evolutionary and ecological roles of horizontal gene transfer (HGT) in natural ecosystems. HGT is believed to be promiscuous in nature and one of the major driving forces for microbial evolution. Yet, little is known about the frequency of HGT and its contribution to structures, interactions, and functions of natural microbial communities. HGT is often studied under artificial laboratory conditions using simple donor-recipient pairs of microbes. Furthermore, HGT events in natural communities are typically inferred from comparative genomics without experiments. Here, we propose to exploit the characteristic gut microbiota of honey bees as a versatile model to study the impact of HGT in a natural microbial community. Honey bees harbor remarkably stable and simple bacterial gut communities consisting of only eight bacterial species. Each species is represented by a number of divergent strains that can coexist. Preliminary genomic data suggests that HGT occurs within and across species in the community. In this proposal, we hypothesize that HGT confers robustness on the community level to hedge against environmental perturbations by two mechanisms: (I) HGT allows the community to maintain beneficial functions by increasing their redundancy, (II) HGT contributes to maintaining species diversity by spreading conditionally critical functions. To test these hypotheses, we will combine genomics, experimental, and theoretical approaches and investigate dynamics, functions, and consequences of HGT in the bee gut microbiota. Using in vivo screening and population genomics, we will first establish a comprehensive gene catalogue of horizontally transferred elements. We will develop an ex vivo system that will enable us to track HGT and community structure changes at the single-cell resolution in real-time in the actively maintained bee gut. Genomics and ex vivo data will be used to develop theoretical and computational models of eco-evolutionary dynamics that account for how HGT alter quantitative ecological interactions. Ultimately, we will test HGT-deficient communities of bee gut symbionts in bee colonization experiments to show the influence of identified HGT on community structure and symbiotic functions in vivo. The project will lead to novel insights into eco-evolutionary dynamics by incorporating HGT into community dynamics.

Vocal learning is a fundamental trait of spoken language and yet the neuro-molecular mechanisms underpinning this trait are poorly understood. The capacity for vocal learning has only been identified in a handful of non-human mammals; cetaceans, elephants and bats. The first evidence for some vocal adaptations in non-human primates is only now emerging. Despite intense interest, these limited experimental options mean that the neural underpinning of mammalian vocal learning has been massively understudied. Some birds, such as parrots, hummingbirds and songbirds, are also vocal learners. Work in songbirds has provided valuable insights, but the evolutionary divergence of avian brain structure is a barrier for direct extrapolation to mammals. Furthermore the lack of a mammalian animal model means that it is unclear whether or not mammals and birds use similar mechanisms during vocal learning. As a result, fundamental questions in the field remain unanswered, such as: how is vocal learning encoded in the mammalian brain, how do neurological and genetic substrates contribute to this trait, and are these mechanisms similar across divergent species?
We will establish bats as an ideal model system because they are mammals that have robustly shown vocal learning and have remarkable capacity for combining vocal phonemes to create new types of vocalisations. Bats can be maintained in laboratory colonies, and there is already a wealth of information regarding the neuroethological mechanisms by which they produce and perceive their vocalizations. We aim to establish bats as the first mammalian model of vocal learning by designing innovative communication paradigms coupled to comprehensive neurological and genetic interrogations. This proposal integrates psychophysical, anatomical, electrophysiological & genetic research in an innovative way to begin to address the encoding of vocal learning in the mammalian brain.
This complementary and coordinated research effort, focuses on a tractable and very promising animal model, addresses a fundamental gap in the field and is likely to significantly advance our knowledge about the origins of vocal learning and ultimately human speech. We believe that this work will have important repercussions across sensory ecology, neuroscience, genetics, evolutionary biology and linguistics.

Vocal learning is a fundamental trait of spoken language and yet the neuro-molecular mechanisms underpinning this trait are poorly understood. The capacity for vocal learning has only been identified in a handful of non-human mammals; cetaceans, elephants and bats. The first evidence for some vocal adaptations in non-human primates is only now emerging. Despite intense interest, these limited experimental options mean that the neural underpinning of mammalian vocal learning has been massively understudied. Some birds, such as parrots, hummingbirds and songbirds, are also vocal learners. Work in songbirds has provided valuable insights, but the evolutionary divergence of avian brain structure is a barrier for direct extrapolation to mammals. Furthermore the lack of a mammalian animal model means that it is unclear whether or not mammals and birds use similar mechanisms during vocal learning. As a result, fundamental questions in the field remain unanswered, such as: how is vocal learning encoded in the mammalian brain, how do neurological and genetic substrates contribute to this trait, and are these mechanisms similar across divergent species?
We will establish bats as an ideal model system because they are mammals that have robustly shown vocal learning and have remarkable capacity for combining vocal phonemes to create new types of vocalisations. Bats can be maintained in laboratory colonies, and there is already a wealth of information regarding the neuroethological mechanisms by which they produce and perceive their vocalizations. We aim to establish bats as the first mammalian model of vocal learning by designing innovative communication paradigms coupled to comprehensive neurological and genetic interrogations. This proposal integrates psychophysical, anatomical, electrophysiological & genetic research in an innovative way to begin to address the encoding of vocal learning in the mammalian brain.
This complementary and coordinated research effort, focuses on a tractable and very promising animal model, addresses a fundamental gap in the field and is likely to significantly advance our knowledge about the origins of vocal learning and ultimately human speech. We believe that this work will have important repercussions across sensory ecology, neuroscience, genetics, evolutionary biology and linguistics.

The phenotype of an animal cannot be explained entirely by its genes. It is now clear that factors other than the genome contribute to the development and dynamic homeostasis of animals. Two fundamentally important factors are epigenetic regulations and the microbial communities associated with the animal. Unlike the genes and regulatory regions of the genome, epigenetics and microbial composition can be rapidly modified by environmental cues, and may thus represent mechanisms for rapid acclimation to a changing environment. At present, the individual functions of epigenetics, microbiomes, and genomic mutations are largely studied in isolation, particularly for species in marine ecosystems. This leaves significant questions open for how these mechanisms intersect in the acclimation and adaptation of organisms.
The aim of this research is to determine how epigenetic regulations and microbial communities participate in thermal acclimation of a coastal marine species residing in a dynamic temperature environment, and how these non-genetic factors interact with each other. The model species used for this study, the sea anemone Nematostella vectensis, enables us to carry out unprecedented functional experiments to dissect the interactions between microbes and epigenetic mechanisms in the acclimation of the holobiont.
We will first monitor the physiological, transcriptomic, epigenetic and microbial changes associated with thermal acclimation. We will then separate the effects of microbial and epigenetic effects in a series of bacterial transplantation experiments. Finally, we will carry out gene knockdown and over-expression experiments to elucidate the function of host genes in epigenetic regulations and the plasticity of the microbiota. We hypothesize that changes in the microbial community improve the thermal tolerance of the host, and the epigenetic landscape is responding both to shifts in temperature and altered microbial composition.
Studying these interactions will require a concerted effort drawing on the diversity of expertise across our three research groups. Our results will have important consequences for our understanding of the response of marine species to climate change, and more broadly they will shed light on many unanswered questions regarding the role of epigenetic regulations and microbes in animal ecology and evolution.

Humans and monkeys are very good at interpreting intent from other’s body movements and facial expressions. This visual recognition is achieved seemingly effortlessly. This visual recognition is also highly accurate, even though the possible number of body movements and facial expressions is extremely large. While much is now know about the different brain mechanisms involved in the recognition of objects, scenes and faces, little is known on how the visual system recognizes other agent’s intent. We will use functional Magnetic Resonance Imaging (fMRI) in humans and fMRI and single cell recordings in monkeys to identify the neural mechanisms responsible for this recognition. The neuroimaging data will be analyzed using sophisticated machine learning algorithms. These methods of analysis will be used to identify the regions of interest (ROIs) involved in this visual recognition of intent, determine their interaction, and study the image or higher-level features used in each ROI. We hypothesize that, as in object and face recognition, there is a hierarchy of areas with increasing level of abstraction. Finally, we will use the results of our experimental data to derive a neurophysiologically plausible model and optogenetics to test for the causal role of these computations.

The oceans are teeming with microbial life, which impacts global biogeochemistry, climate and human health. Omics methods enable us to identify "who is there" and their genetic potential, but understanding how these organisms interact in nature and how they affect biogeochemical processes remains a major open challenge. A crucial component of this puzzle, at the heart of the cycling of nutrients in the biosphere, is the coupling between organisms that fix carbon using solar energy (phytoplankton) and microbes that rely on organic carbon for energy and growth (heterotrophs). The diversity of microbial species and the richness of their metabolism make the problem of predicting how each pair of species will interact impossible to address using traditional approaches. We propose to tackle this challenge through a tightly integrated combination of genome scale modeling and laboratory experiments, to identify genomic traits dictating how environmentally-relevant microbes interact. We will generate a library of ~100 heterotrophic bacteria representing major marine lineages and fully sequence and annotate their genomes. We will implement genome-scale (dynamic flux balance) models of each of these organisms, as well as of four key phytoplankton species, calibrating these models using high-throughput measurements of growth under different conditions. We then grow binary phytoplankton-heterotroph co-cultures in the lab and compare the results with in-silico models of the interacting genomes, studying key interactions in more detail. Recognizing that microbes in the ocean are seldom in steady-state, our work will encompass exponential growth, stationary stage and culture death. Most importantly, missing or incorrectly predicted interactions will give us an opportunity to revisit our models, and suggest the mediation of alternative processes such as allelopathy or other types of chemical signaling. This study will provide the first detailed "roadmap" linking genomic traits (genes and metabolic pathways) and rate measurements with species interactions in environmentally-relevant microbes. Our approach, which - we anticipate - will be embedded in a not-so-distant future in global-scale models of the Earth System, will provide a critical stepping-stone towards predicting how marine microbial systems will evolve in a changing world.

The oceans are teeming with microbial life, which impacts global biogeochemistry, climate and human health. Omics methods enable us to identify "who is there" and their genetic potential, but understanding how these organisms interact in nature and how they affect biogeochemical processes remains a major open challenge. A crucial component of this puzzle, at the heart of the cycling of nutrients in the biosphere, is the coupling between organisms that fix carbon using solar energy (phytoplankton) and microbes that rely on organic carbon for energy and growth (heterotrophs). The diversity of microbial species and the richness of their metabolism make the problem of predicting how each pair of species will interact impossible to address using traditional approaches. We propose to tackle this challenge through a tightly integrated combination of genome scale modeling and laboratory experiments, to identify genomic traits dictating how environmentally-relevant microbes interact. We will generate a library of ~100 heterotrophic bacteria representing major marine lineages and fully sequence and annotate their genomes. We will implement genome-scale (dynamic flux balance) models of each of these organisms, as well as of four key phytoplankton species, calibrating these models using high-throughput measurements of growth under different conditions. We then grow binary phytoplankton-heterotroph co-cultures in the lab and compare the results with in-silico models of the interacting genomes, studying key interactions in more detail. Recognizing that microbes in the ocean are seldom in steady-state, our work will encompass exponential growth, stationary stage and culture death. Most importantly, missing or incorrectly predicted interactions will give us an opportunity to revisit our models, and suggest the mediation of alternative processes such as allelopathy or other types of chemical signaling. This study will provide the first detailed "roadmap" linking genomic traits (genes and metabolic pathways) and rate measurements with species interactions in environmentally-relevant microbes. Our approach, which - we anticipate - will be embedded in a not-so-distant future in global-scale models of the Earth System, will provide a critical stepping-stone towards predicting how marine microbial systems will evolve in a changing world.

Monomeric actin is readily detectable in the nucleus of mammalian somatic cells. Whether this pool of actin forms filaments has been debated for decades. Recent advancements have established that, indeed, nuclear actin can assemble dynamic filamentous structures in response to external stimuli. However, how nuclear actin filaments exert their biological function remains an unanswered question in cell biology. This is particularly relevant given the highly dynamic nature of nuclear actin filaments, which reside within the intricately organised nucleus that harbours the genome in the form of chromatin. Thus, through this collaboration, we aim to address this by developing and applying novel approaches for optogenetics, advanced cell-imaging and next generation sequencing. Accordingly, we will employ tools to spatiotemporally control nuclear actin filaments with light, and determine their role in regulating chromatin organisation and nuclear structure. Further, we will determine the cell and biological consequences of actin filament-driven chromatin organisation/re-organisation in relation to cell division and cellular reprogramming. By completing these studies, we envisage to advance our understanding of the biological functions of nuclear actin filaments in a manner that will encompass nuclear actin polymerisation as a key mediator of chromatin organisation and regulation. Beyond this, findings from our studies may have translational potential in areas of regenerative medicine, and may delineate new mechanisms that underlie ageing and degeneration.

Like human babies learning speech, young songbirds learn to produce song by imitating adults. Both babies and young birds need to listen to adult vocalizations, extract the relevant information and store it in the brain and then improve their own ‘babbling’ sounds step by step towards the adult version. Experiments on vocal learning have focused mainly on sounds, although sight also seems to play an important role. Babies of only 8 weeks of age already associate speech sounds with the correct mouth position required to make that sound. Moreover, people report that they hear the syllable ‘ga’ when in fact a loudspeaker plays ‘pa’ while a video screen shows the face of a speaker mouthing ‘ka’. This perceptual phenomenon is known as the ‘McGurk' effect. It reveals that sensory information from the ear and eye are integrated in the brain to form a combined, multimodal percept.
Like human speech, birdsong can be heard and seen. Young birds see their fathers moving their beaks while hearing their song. There is substantial evidence to assume that the combined exposure to acoustic and visual cues enhances song learning not only in human but also in bird babies. We aim to uncover the role of this multimodal perception and its underlying neural architecture in vocal learning. Focusing on the zebra finch, the prevalent animal model for molecular, neural and developmental aspects of human speech we will adopt a robotic approach that allows full experimental control over auditory and visual cues.
Simultaneously recording acoustic and visual cues of singing adult birds, we will analyze synchrony between different song elements and the opening and closing of a bird's beak. We will use this to develop a robotic zebra finch that allows us to create matched and mismatched multimodal stimuli. Juvenile birds will be raised with various tutoring regimes to assess the relative roles of unimodal versus multimodal cues, life versus robot cues, and matched versus mismatched cues. Upon reaching adulthood, tutored birds will be tested on their song copy performance (males only), song preference (females only), or discrimination ability (all birds). Finally, we will use a subset of tutored birds to study underlying neural processes by using molecular tools that allow us to pinpoint where in the brain multimodal information is processed and how this is linked to vocal learning.

Like human babies learning speech, young songbirds learn to produce song by imitating adults. Both babies and young birds need to listen to adult vocalizations, extract the relevant information and store it in the brain and then improve their own ‘babbling’ sounds step by step towards the adult version. Experiments on vocal learning have focused mainly on sounds, although sight also seems to play an important role. Babies of only 8 weeks of age already associate speech sounds with the correct mouth position required to make that sound. Moreover, people report that they hear the syllable ‘ga’ when in fact a loudspeaker plays ‘pa’ while a video screen shows the face of a speaker mouthing ‘ka’. This perceptual phenomenon is known as the ‘McGurk' effect. It reveals that sensory information from the ear and eye are integrated in the brain to form a combined, multimodal percept.
Like human speech, birdsong can be heard and seen. Young birds see their fathers moving their beaks while hearing their song. There is substantial evidence to assume that the combined exposure to acoustic and visual cues enhances song learning not only in human but also in bird babies. We aim to uncover the role of this multimodal perception and its underlying neural architecture in vocal learning. Focusing on the zebra finch, the prevalent animal model for molecular, neural and developmental aspects of human speech we will adopt a robotic approach that allows full experimental control over auditory and visual cues.
Simultaneously recording acoustic and visual cues of singing adult birds, we will analyze synchrony between different song elements and the opening and closing of a bird's beak. We will use this to develop a robotic zebra finch that allows us to create matched and mismatched multimodal stimuli. Juvenile birds will be raised with various tutoring regimes to assess the relative roles of unimodal versus multimodal cues, life versus robot cues, and matched versus mismatched cues. Upon reaching adulthood, tutored birds will be tested on their song copy performance (males only), song preference (females only), or discrimination ability (all birds). Finally, we will use a subset of tutored birds to study underlying neural processes by using molecular tools that allow us to pinpoint where in the brain multimodal information is processed and how this is linked to vocal learning.

Engineering of recombinant pathways in microbial hosts is a powerful tool to provide access to sustainable building blocks for the chemical industry. Such a bottom-up synthesis route has recently been established for monomeric phenylpropanoids. However, the yield and diversity of products obtained are still lagging behind their natural plant biosynthetic pathways. The aim of the herein described project is to explore and develop tools for the reconstitution of plant metabolic pathways in bacterial hosts: namely, tools for the availability of bacterial strains as framework, for protein engineering and for identifying novel pathway enzymes for crucial product tailoring steps.

Therefore, I propose to investigate the applicability of Corynebacterium glutamicum as a bacterial chassis for plant metabolic pathways in general and the phenylpropanoid pathway in particular. I am going to develop genetic tools based on the CRISPS/Cas9 system to allow the fast incorporation and screening of various pathway enzymes and promoter combinations. I furthermore propose to investigate the effect of tethering Cytochrome P450 (P450) enzymes – a typical bottleneck in reconstituted pathways - to their redox partners, on in vivo P450 activity. Lastly, I propose to investigate the correlation of sequence, structure and function of crucial product tailoring O-methyltransferases from various organisms to enable the classification of such enzymes based on sequence homology.

The proposed study will not only increase the efficiency of the synthetic phenylpropanoid pathway but provide tools to further our abilities in manipulating microbial hosts for fermentative production of valuable chemicals.

The biggest challenge in glioblastoma research is to understand the inherent cellular heterogeneity. While much research has been dedicated to glioblastoma stem-like cells (GSCs), a small population of tumor cells that resides at the apex of a differentiation hierarchy and underlies therapy resistance, differentiated glioblastoma cells have not received the same attention.

I hypothesize that glioblastoma is characterized by a previously underestimated extent of cellular differentiation. Dedicated differentiated cell types provide the tumor with a selective advantage, e.g. by promoting angiogenesis and evading the immune system, and by supporting GSCs with essential maintenance and cell division cues. Therefore I propose to accurately define the cellular composition of primary human glioblastoma using microfluidics-enabled transcriptome profiling of thousands of individual cells. Using sophisticated computational methods, this dataset will reveal previously unrecognized cell types, and inform on their roles in tumor formation. Additional epigenetic characterization of tumor cell subpopulations will define cell type specific gene regulatory networks and inform on the molecular consequences of common genetic alterations.

I further hypothesize that tumors of different patients are composed of varying stem-like and differentiated cell types and that cell type frequencies differ between patients. Incorporating the new information on tumor heterogeneity will allow me to redefine glioblastoma subclassification. This will lead to more accurate patient prognoses, and ultimately to the development of subtype-specific therapies and the design of more targeted clinical trials.

In order to ensure genome stability, DNA repair is highly regulated by the cell division cycle. A major, but largely unexplored question pertains to mechanisms that promote genome stability when DNA damage occurs on segregating chromosomes. My host laboratory discovered that cells suppress the repair of DNA double-strand breaks (DSBs) in mitosis to prevent chromosome segregation errors. These occur as a consequence of repair-mediated telomere fusions. The overarching objective of my project is to address the biological significance of mitotic DSB repair inhibition in vivo by generating a transgenic mouse in which I can conditionally de-suppress DSB repair in mitosis. This system will enable me to identify the tissues and developmental processes sensitive to the genome instability caused by mitotic DSB repair and to test whether inhibition of mitotic DNA repair is a tumour suppression mechanism. In parallel I will elucidate the molecular mechanism that causes the observed telomere fusions. Based on previous results, I hypothesize that the kinase Aurora-B mediates mitotic telomere uncapping through targeting of the telomeric Shelterin complex. I will test whether active Aurora-B localizes to telomeres using a FRET-based activity sensor and identify its target among the Shelterin subunits. Since telomere fusions are enhanced upon exposure to ionizing irradiation, I will also clarify the crosstalk between Aurora-B and the DNA damage response. This project comprises phenotypic animal studies and mechanistic cell based studies and thus represents a major departure from my previous work on the genomics of yeast DNA replication.

To understand how brains integrate sensory information and generate behavioural responses is a central goal of systems neuroscience. Larval Zebrafish have emerged as a very promising vertebrate model system to address this challenge because their small size and transparency enable optical access to the majority of neurons within the brain at cellular resolution. Important insights on circuit function have already been gained by two-photon imaging of neuronal populations in restrained larvae behaving in virtual environments – however, critical limitations of virtual environments as a replacement for real environments are widely acknowledged and remain a barrier to progress. An ideal solution to this problem would be to image larvae during natural, unrestrained behaviour, and this is the goal we aim to achieve in this project. It has so far not been possible to track naturally moving zebrafish, nor any other vertebrates, while imaging at cellular resolution. Here we propose to overcome this limitation by forming a novel collaboration across disciplines and three different laboratories. We will pool our collective expertise in optical systems design, wavefront-shaping, electrical engineering, and zebrafish neuroscience to perform an experiment that has until now been impossible: imaging comprehensive neural activity in a freely moving, untethered vertebrate. We will collectively design and validate a real-time optical tracking and imaging system to measure brain activity with single neuron resolution in a freely moving vertebrate. This will allow us to monitor, with unprecedented detail, the population activity of the reticulospinal system in larval zebrafish and understand how this population of ~ 300 supraspinal neurons combinatorially codes for the full range of locomotor and postural behaviors.