Our recent sensations affect our current expectations and perceptions of the environment. Neural correlates of this process exist throughout the brain, and are loosely termed adaptation. In this proposal, we will address adaptation in the cerebral cortex. Sensory adaptation potentially carries a severe price of increasing the ambiguity of neural coding, since the same stimulus can give rise to different responses depending on the context and adaptation state. However, the ubiquity of adaptation suggests that it has considerable advantages, possibly serving similar functions across sensory modalities. More than 40 years ago Barlow suggested that adaptation reduces the dynamic range required to encode natural stimuli by emphasizing changes in intensity rather than absolute values. Later studies emphasized other roles, including predictive coding, optimizing neural coding and detection of unexpected stimuli. However, these ideas were developed using different modalities and different stimulation paradigms. As a consequence, we lack a theoretical framework that affords a general understanding of sensory adaptation in cortex. The goal of this project is to develop such a unified theory. Given the similarity in circuitry across neocortex, we suggest that neural responses adapt to ongoing statistical changes in input, independent of modality. We hypothesize that the diverse adaptation properties of specific subtypes of cortical neurons serve similar functions across sensory modalities.
Toward testing these ideas, we will use stimuli with similar time course to study adaptation in the three modalities (somatosensation, audition, and vision) and successively vary the statistical complexity of the stimulus to probe the context dependence of dynamical representations across sensory cortex; using the same recording techniques and using the same analysis methods. This approach will enable us to discover the common roles that neuronal subclasses play across sensory areas in adjusting cortical responses according to statistical properties of stimulus history. This research will be conducted in tight collaboration with theory, that will inform both the construction of the stimulus sets we will use and the analysis of our neural data. We believe that this approach will allow us to build a ‘grand unified theory’ of cortical adaptation.

Insect-transmitted parasites endanger our health and our food supply and are the causal agents of e.g. malaria and yellow fever and numerous plant diseases in economically important crops. Surprisingly, only a few laboratories worldwide investigate which characteristics at the parasite molecular level are required for insect transmission and represent key drivers of parasite epidemics. We hypothesize that evolution of the triangular host-parasite-insect relationship is a battle between genes rather than individual organisms.
Aster yellows phytoplasmas (AYPs) represent parasites that excel at controlling their plant hosts, dramatically modifying their development and thereby not only affecting the plant-AYP interaction, but also the interactions between plants and AYP vector leafhopper populations, for example by increasing plant attractiveness to the leafhoppers. When studying such an extended phenotype that even changes the development of other species within its own ecological web, it becomes evident how developmental outcomes are intertwined with ecological ones. Therefore, we selected this triangular host-parasite-insect system as a paradigm to unravel how mechanistic aspects of gene function are linked to the ecological niche of the organism and the environment.
AYP express vir genes, encoding effectors, which products have a dramatic impact on plant architecture and hence, crop yield, but which facilitate AYP spread by their vector insects. The relative AYP fitness increase that can be achieved by those different modifications of plant development can only be determined by understanding their impact on the population dynamics and migratory behavior of the insect vectors, which in its turn allow for the expansion and spread of the AYPs.
Unraveling these complex interactions requires an interdisciplinary approach and multi-scale modeling framework, in which we can zoom out from the molecular up to the ecological level. We will compare and contrast a leafhopper migration-driven context found in Wisconsin (USA) with a residential-population driven context found in Poland (Europe), allowing us to separate the relative importance of the plant and the insect dynamics. These distinct ecological settings also allow for testing hypotheses regarding how the ecological embedding is determining which effector genes are selected for, both regarding type and severity.
This study will result in a better understanding of general principles of any triangular host-parasite-vector interaction web in their full multi-level context. Moreover, it will set up a modeling framework that can be straightforwardly applied to a broader class of disease studies, including human diseases such as malaria.

Human influenza viruses are transmitted by inhalation of virus-laden aerosol, whereas avian viruses are disseminated by the fecal-oral route. Thus, in order to become transmissible between humans, an avian virus must adapt to a different mode of transmission. It is well established that the hemagglutinins in the envelope of avian viruses recognize differently glycosylated receptors on host cells. However, we propose that host adaptation also requires changes in the structure, fluidity, and lipid selection of the viral envelope glycoproteins hemagglutinin and neuraminidase, the proton channel, and the matrix protein.
There are several requirements on the envelope’s biophysical properties for optimal transmissibility: The membrane must be fluid at body temperature (42 °C in birds versus 33 °C in the human respiratory tract) to allow virus budding, but it should solidify to protect virions against damage during transmission between organisms. After internalization by a new host the membrane must liquefy again to allow infection. The phase state of the membrane depends on temperature, but lipid compositions determine at which temperature phase changes occur. In turn, this will have an effect on the structure and organization of the viral envelope proteins and their interaction with the viral membrane, particularly regarding their ability to laterally interact, cluster and adopt certain conformations. Hitherto, these aspects have been widely neglected when studying the adaptation of influenza virus to new hosts.
We will take a multi-pronged approach to address this problem. Avian and human viruses will be grown at different temperatures and in different types of host cells. The lipidomes of these viruses will be determined. The phase and mechanical properties of the viral envelopes under changing environmental conditions will be examined. Structural changes of the viral envelope proteins will be determined at high resolution and the reasons for their altered behaviors will be sought by determining differing interactions with specific lipids in each case. Hypotheses about the molecular reasons for envelope adaptation to different host conditions will be formulated based on these results and ultimately tested for their relevance in viral envelope lipid selection and virus morphology by generation of recombinant viruses lacking lipid binding sites.

Animals as diverse as mammals, birds and insects use odors to find mates, hosts and food sources. This is a difficult task, because natural odors occur in complex turbulent air plumes, and the relevant target odors intermingle with plumes of multiple background odors that stem from a variety of natural and anthropogenic sources (vegetation, exhaust fumes, etc.). It is essential for animals to be able to separate target from background odors. We hypothesize that animals may use temporal information from natural odor fluctuations to achieve odor-background segregation, as odorants from the same source fluctuate together in synchrony, while odorants from different sources do not. Behavioral studies have shown that insects can indeed use fast temporal stimulus cues. However, it is not yet known how the brain accomplishes the difficult task of odor-background segregation.
In this project we will investigate the neural mechanisms of odor-background segregation using the honey bee as model organism. Honey bees visit many different flower species for nectar and pollen. However, over a series of trips an individual forager bee only visits a single flower species on which it has found nectar previously, a phenomenon called floral constancy. To localize the target flowers in a natural environment bees use several cues, including odors. As the bee is challenged by multiple backgrounds in every flower patch, bees must be equipped with efficient mechanisms of odor-background segregation. We will use behavioral experiments to identify naturally evolved strategies of odor-background segregation (Brian H Smith, USA). Physiological recordings will inform us about neuronal processing and the receptor neurons’ and the brain’s different roles (Paul Szyszka, Germany). We will use computational models (Thomas Nowotny, UK) to formulate mechanistic explanations of the process of odor-background segregation and generate predictions to test in experiments. To overcome the limitations of computer simulations, we will finally transfer successful models to the embodied context of odor tracking robots (Ryohei Kanzaki, Japan). Understanding how small and fast fluctuations in relative concentrations of odor mixtures are used by animals to reliably identify odor sources will help us identify common principles of how animals’ brains represent and process their environment.

The spontaneous generation of living organisms without descents from similar organisms was decisively put to rest with the experiments of Pasteur during the 19th century. As a consequence, the logic of self-reproduction, one of the most fundamental features of biological systems, is very difficult to break down. An approach to this problem consists of synthesizing cell analogs in the laboratory from its basic natural molecular components.
The chemical synthesis of living entities is a standing challenge not achieved yet. Various approaches have been taken to construct synthetic reduced cells in the laboratory, a goal that became experimentally imaginable over the past two decades. The construction of protocells, which explores scenarios of the origin of life, has been the original motivation. With the emergence of the synthetic biology era, bottom-up engineering approaches to synthetic cells are now conceivable. The modular design emerges as the most robust method to construct a minimal cell from natural molecular components. Although significant advances have been made for each piece making this complex puzzle, the integration of the three fundamental parts, information-metabolism-self-organization, into cell-sized liposomes capable of sustained reproduction has failed so far. Our inability to connect these three elements is also a major limitation. New experimental platforms and methods should be taken to accelerate the cell-free synthesis of complex biochemical systems.
Our scientific collaboration, composed of four team members, will address the problem of biological self-reproduction by using an engineering versus evolution approach to cell analogs. While “information-less” protocells will be generated by driving Miller-Urey experiments, a prototype of minimal cell will be constructed by integrating the three indispensable molecular parts making living cells: information, metabolism and self-organization (compartment). Unique experimental platforms developed by the team members will be used to achieve major advances in the synthesis of minimal living systems in the theoretical context pioneered by von Neumann in the 50s. The societal impact of our work includes a deeper comprehension of the emergence of self-reproduction with and without genetic information and the development of powerful platforms for the construction of living entities in vitro.

We propose to apply cell-free synthetic biology to study fundamental aspects of transcriptional regulation, gene regulatory network evolution, and network robustness. Cell-free synthetic biology emerged recently as an in vitro alternative for network engineering. Cell-free synthetic biology implements biological systems in a coupled transcription – translation reaction and therefore is a well-defined environment that is easy to control and interrogate. Since genetic networks can be implemented in vitro as linear dsDNA templates, as opposed to plasmids, it also circumvents time consuming cloning and transformation steps, enabling rapid prototyping of genetic systems. We have recently implemented cell-free systems on a microfluidic microchemostat array, which performs transcription-translation reactions at steady state for 30+hours, allowing us to implement complex genetic networks in vitro. Here we propose to apply this novel concept to study fundamental principles of genetic networks. We will interrogate two different types of oscillators (phase and relaxation) and determine how cis- and trans-changes to the network impact network function and robustness. Specifically, we plan to map the precise cis-binding energy landscape of these networks by characterizing a large number of network variants with modified transcription factor binding sites. To implement trans-element changes we will employ synthetic Zn-finger transcriptional regulators with defined specificity landscapes to determine how changes to the trans-element influence network performance and robustness. Finally, we will also attempt to rationally evolve network function through a promiscuous transcription factor intermediate. To determine how robust these networks are to external perturbations, we will change reaction temperatures, and independently modulate transcription and translation rates. Finally, we will characterize a subset of our networks on the single cell level in vivo to evaluate network robustness in a regime were stochasticity dominates. Gene regulatory networks are complex systems that are difficult to quantitatively characterize in vivo. The in vitro cell-free synthetic biology approach proposed here has the potential to contribute significantly to the mechanistic understanding of complex biological systems.

There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

The aim of this project is to understand how molecular forces ‘scale-up’ to generate biological outcomes. We focus on understanding the contractile actomyosin cell cortex, a sub-micron thick network that lies beneath the plasma membrane that supports cellular and tissue morphogenesis. Ultimately, functional outcomes and active mechanical behaviour at the larger scale must arise from the intrinsic biophysical properties of the force-generating molecules of the cortex. However, how these properties emerge is unclear. Our consortium will implement novel experimental approaches solutions to tackle this question. First, we will develop myosin mutants that allow us to modulate specific parameters of motor function (e.g. force, step-size, directionality, torque; Bryant) under control by light; we call this ‘Optomechanics’. Second, we will develop hydrodynamic active-gel theory for describing essential physical behaviors arising from molecular-scale force and torque generation in the cortex (Grill). We will apply these approaches to understand the contribution of the cortex to cellular morphogenesis during cytokinesis (Grill) and junctional tissue homeostasis (Yap). For this purpose, we bring expertise in myosin biophysics and molecular bioengineering (Bryant), cortical mechanics and theory (Grill), and cell junctions and morphogenesis (Yap). The bioengineering team will generate validated reagents that will be implemented in cells by the biological teams; results from the latter will reveal how modifications in myosin force generation at the single molecule level relate to in-vivo function. We will combine optical imaging, laser nanodissection with hydrodynamic theory to develop quantitative descriptions of cortical mechanics during cytokinesis in the C. elegans zygote. In parallel, we will define the impact of cadherin on cortical mechanics at mammalian epithelial cell-cell junctions, and how this influences mechanical coherence of populations. This combination of molecular manipulation with theory will allow us to describe how actomyosin mechanical activity at cell and tissue scales emerges from myosin motor function at the molecular scale, and how myosin molecular force drives cell- and tissue-level mechanics.

The crucial importance of secreted extracellular scaffolding proteins (ESPs), such as precerebellins, neuronal pentraxins, and C1q-like molecules, for the formation and function of neuronal synapses in vivo has only recently been discovered. These molecules share a modular structure, with domains that specifically interact with pre- and postsynaptic cell surface receptors to bridge neurons across the synaptic cleft. Guided by structural information of known ESPs, we aim to develop synthetic connector molecules that will expand the range and affinity of trans-synaptic interactions. These synthetic ESPs will likely become new and powerful tools to manipulate the number and properties of excitatory and inhibitory synapses, and to regulate synaptic communication and cognitive abilities in mouse models in vivo.
To achieve our goals, we will develop and implement a highly multi-disciplinary approach based on the diverse and complementary expertise and state-of-the-art techniques established in the applicants’ laboratories. Armed with structural and biophysical knowledge about physiological ESPs and their receptor interactions, we will design hybrid molecules that contain novel combinations of binding domains and thus promote a novel spectrum of molecular interactions across neuronal synapses. The synthetic ESPs will be further refined by mutagenesis or by replacing native domains with nanobodies of desired specificity. These novel neuronal connectors will be recombinantly produced, purified and delivered into subregions of the hippocampus and the cerebellum by acute or chronic injections using osmotic pumps. We will also express genes encoding the designer ESPs using viral vectors with neuron type-specific promoters. Electrophysiology, confocal microscopy, nanoscopy and behavioral analyses will be then employed to verify the targeting and functional efficacy of synthetic ESPs at the synaptic, neural network and organismic levels. Their effects on motor and spatial learning, reversal learning, pattern separation and completion will be studied in combination with recordings of neural activity oscillations and single-cell firing.
Considering the huge potential variety and versatility of these novel tools, we anticipate an immediate impact onto basic neuroscience and, in longer term, potential new avenues for the treatment of neuropsychiatric disorders.

The tiny nematode worm C.elegans has been instrumental for identifying genes that increase lifespan. Although most of the genes increasing lifespan in C.elegans are also present in humans, only very few have been associated with human longevity. This is mainly due to the fact that humans are more complex than worms. Instead of studying single genes we will investigate a multi-gene approach where we assume that lifespan in the worm can be predicted by the behaviour of all these genes together. We do this by viewing the worm genetic system as an ecological system. Similar to the fact that ecological systems display characteristic behaviors before they can collapse, we will test if the gene-regulatory network of worms displays characteristic behavior when approaching a collapse leading to death. This will provide insight in the mechanisms of aging and might help to translate the results to humans.

1. Astrocytes, classically considered as simply supportive cells for neurons, are emerging as relevant elements in brain information processing through their ability to regulate synaptic activity. Indeed, the tripartite synapse formed by pre- and post-synaptic neurons and surrounding astrocyte structures has been proposed as a functional unit of brain processes. These novel and important functions of astrocytes are under control of G protein signaling-dependent processes, which trigger astrocyte Ca2+ signals eventually leading to the release gliotransmitters and other mediators regulating synaptic functions.
2. Recent evidence indicates that the roles of mitochondria in the brain may go beyond the mere needs of energy supply for cell survival and maintenance, being possibly involved in the regulation of synaptic functions. It is conceivable that mitochondrial G protein signaling participates in these processes. Various subtypes of G protein-coupled receptors (GPCRs) and the associated signaling molecular elements are present within mitochondria, suggesting the existence of mitochondrial G (mtG) protein signaling pathways.

Thus, astroglial mtG signaling potentially plays an important role in the regulation of tripartite synapse and hence brain functions. However, no studies have addressed this issue so far.
The present project proposes to investigate the consequences of the activation of astroglial mtG signaling pathways in brain physiology, identifying the underlying mechanisms at cellular, network, behavioral and theoretical modeling levels. We propose to generate and develop novel pharmacogenetic tools (DREADDs specifically expressed by astroglial mitochondria, mtDREADDs) that will allow the specific control of astroglial mtG protein activity. We will experimentally and theoretically analyze the consequences of activation of different mtG proteins (via mtDREADDs) on neuronal, synaptic, and network activity as well as brain functions in living animals.
The expected results will reveal novel processes of cellular signaling in the CNS, and will identify new regulatory mechanisms mediated by astroglial cells in brain function.

One of the main challenges of this century is to better understand the function of the brain in order to solve its devastating disorders. We have learned a great deal about the basic components of the brain: the different types of neurons, each with its unique arborization, and the connections, termed synapses that enable neurons to communicate with each other. We also have a basic understanding of how information is transmitted through electrical and chemical signals. However, we know little about how individual neurons integrate the multiplicity of inputs, both excitatory and inhibitory, to compute a single output. Nor do we understand much about how individual neurons work in concert within a specific circuit to perform complicated computations. A major limitation has been our inability to monitor and control the electrical and synaptic activities of neurons inside the brain of a living animal. Overcoming this limitation requires the combination of different technologies to enable neuronal activity to be monitored with high sensitivity and fidelity while minimally interfering with brain function.
This grant exploits advances in optical methods, molecular biology, genetics, protein engineering, and electrophysiology and brings together tools developed in three laboratories, each with distinct expertise. These include a fiber-optic-based "micro-optrode" that can be inserted into the brain of a living animal in order to simultaneously record the electrical activity and biochemical signals. In particular, this instrument can detect and decode optical signals transmitted by probes that respond to the microenvironment inside the neuron. Such molecular probes, which are introduced into neurons through genetic manipulations, are designed to reveal how the neuron decodes synaptic messages that it receives from its connected partners in the brain of an animal receiving sensory stimulation. To dissect how signal processing within the various compartments and arbors of a neuron contributes to its function, we are exploiting a novel strategy to locate the molecular probes in specific neuronal microdomains. The combination of innovative approaches that we propose in this grant is expected to produce new knowledge about the contribution of specific neuronal subtypes and circuits to the complex processing of the brain in normal and pathological conditions.

Transposons are genetic elements that can cut themselves out of the genome and hop to a new location. Remarkably, they comprise half the human genome, but their functions are still ambiguous. Because transposition can wreak havoc in the genome, causing severe diseases, many mechanisms have evolved across the tree of life to quell transposons. Astonishingly, recent research revealed that some transposons possess functions essential to their host. They contribute to regulation of gene expression, human development and neuronal diversification. These findings have led to a paradigm shift in biology: no longer are transposons mere parasites of the genome but can contribute to host function.
A class of single celled eukaryotes, called ciliates, exploit transposons in a particularly impressive way. They employ them to clean up their genomes by tossing out junk DNA, and this produces easier-to-maintain smaller chromosomes that express most genes in the cell. We have proposed that ciliates specifically recruited transposons for this ‘house-keeping’ job, but how they managed to tame these intrinsically harmful elements so that they are essential members of the cellular machinery is unknown. To test our hypothesis and to answer these questions, we will merge a unique set of state-of-the-art interdisciplinary expertise spanning chemistry, physics, computer science and biology. We aim to track the evolutionary pathway of transposon recruitment, to understand the chemistry and biophysics of these ‘good’ transposons and to investigate how they differ from canonical ‘bad’ transposons. We will visualize the structure of the molecular machines at a resolution that resolves individual atoms, and analyze how these beneficial transposons are programmed and directed to act only at the right time and place to defend the cell from their potential harmful effects. Our work will discover if and how such potentially harmful genomic parasites can be adapted for essential biological roles in ciliates and beyond, providing unmatched insight into a fundamental biological mechanism for the acquisition of novel genetic and physiologic functions.
Moreover, the elucidation of how these eukaryotes have managed to customize and tame transposons to function efficiently but safely will provide invaluable implications for engineering transposon-based tools for genetic research and gene therapy.

We propose to use DNA-based scaffolds to build lectins of pre-specified stoichiometry and precisely defined three-dimensional geometry to test mechanochemical requirements of plasma membrane invagination mediated by their membrane-resident glycosphingolipid (GSL) binding partners.
It is striking that numerous pathogenic GSL-binding lectins including the B-subunits of Shiga and cholera toxins and the VP1 protein of SV40 (i) consist of a pentameric fold (ii) bind GSLs with similar geometries (i.e., angles and distances between carbohydrate recognitions sites (CRDs)); and (iii) finally adopt a curvature-active state that enables their clathrin-independent uptake into cells despite the fact that they do not exhibit any sequence similarity. This suggests the exciting possibility that their clustering, membrane bending and invagination capacities are the result of parallel, convergent evolution.
Probing lectin functionality experimentally is infeasible using conventional approaches such as targeted mutagenesis because it is impossible to alter lectin oligomerization state and CRD geometry in a controlled manner. Therefore, we will engineer synthetic scaffolds composed of DNA to display GSL-binding CRDs in specific natural and non-natural geometries and stoichiometries. Such designer DNA-based lectins indeed offer the ability to address structural questions with unprecedented molecular precision. Innovative computational and chemical biological tools will be developed that should be of general interest to the fields of DNA devices and synthetic biology. Novel DNA-based GSL-binding scaffolds will be characterized as to their capacities to cluster on membranes, to adopt a curvature-active state (i.e. to induce or sense curvature), and to traffic into cells via defined pathways. Based on structural input, experiments will be modeled at length scales from 0.1 nm to 10 microns to provide a molecular understanding of underlying mechanistic aspects, and guide the design of new scaffolds. We expect to achieve an unprecedented understanding of GSL-based membrane mechanical processes, including the discovery of novel physical principles. Beyond the world of pathogens, our study bears relevance to the GSL-dependent functions of signaling receptors and the design of novel delivery tools for therapeutic intervention against GSL-expressing tumors.

How does a nervous system adapt to the body plan of a growing animal from birth to adult? This question is fundamental to biology, yet is currently impossible to answer without turning to small model organisms, which provide an extraordinary level of access to the nervous system at the molecular, anatomical, and physiological levels. We will explore how a nematode C. elegans manages to drive undulatory locomotion at all life stages, with different motor circuit components, using powerful new tools in serial-section electron microscopy, optical neurophysiology in freely behaving animals, super resolution light microscopy, and biophysical modeling. This research program should provide a unique data set that illuminates developmental dynamics from neural activities to animal behaviors.

Ribosomes stand amongst the most complicated structures in biology. Their assembly is a question of fundamental interest, but is still poorly understood in spite of efforts of excellent groups. We propose to address this challenging question using a quantitative approach, inspired by recent results in cell-free translation and in single-molecule biophysics. By applying a force to specially designed DNA/RNA duplexes, we can repetitively release and re-anneal RNA with full control over velocity and orientation of the strands. We can thus mimic the gradual appearance and folding of rRNA in co-transcriptional assembly of bacterial ribosomes, including varying transcription rate and pauses. Combined force and fluorescence experiments will allow us to follow in real time the binding of ribosomal (r-)proteins to the gradually released rRNA. We will focus with our single molecule measurements on the initial phase of the assembly of the large ribosomal subunit of E. coli. This involves the 23S rRNA, the five r-proteins that have been identified as essential and sufficient for the first assembly stage (L4, L13, L20, L22 and L24), and a selection of assembly helper proteins. The single-molecule studies will be complemented by coupled transcription-assembly experiments, in which the assembly will be followed in fluorescence experiments using Förster Resonance Energy Transfer between fluorescent labels on r-proteins adjacent in the ribosome and by testing the ribosomal capacity of protein synthesis. Using this combination of approaches, we will provide a dynamic view of the choreography of ribosome assembly, identify essential assembly factors among the about 15 known assembly helpers and identify their roles. Our interdisciplinary team combines two biophysics groups, well known for single-molecule studies on nucleic acids and associated proteins, and two biochemistry groups, well known in the fields of ribosome assembly and cell-free translation. Close collaboration between these four groups will bring together the expertise required to successfully perform the proposed experiments and will bring our understanding of ribosome assembly to a new level.

The ENCODE project and RNA deep-sequencing have revealed that the genome is pervasively transcribed, however, a considerable fraction of the RNA that is produced does not code for protein, but instead functions as a crucial layer of regulation over the messenger RNAs that do code for proteins. A major question in biology is how the activity of these regulatory non-coding RNAs (ncRNA) are controlled. Our 4 groups have now independently reported, through different fields of study, that the seemingly unruly network of ncRNAs are regulated by programmed interactions between the ncRNA themselves.
We propose to combine our expertise in RNA biology, computation, biotechnology, and oncology, to uncover the principles governing ncRNA interactions, with particular focus on ncRNA regulation of miRNAs, and to test the hypothesis that a major function of ncRNAs is to regulate other regulatory ncRNAs, particularly miRNAs by acting as competitive antagonists through cis-encoded sequences and recruitment of RNA binding proteins, and that ncRNA regulation of miRNA activity is important for controlling cancer relevant cell functions. To test our hypotheses, we will, (1) determine which ncRNAs have ceRNA/sponge activity through large-scale loss-of-function studies, (2) determine how the target site sequence encoded by an ncRNA affects the efficiency of ncRNA-mediated sponging, and (3) determine the relevance of linear and circular ncRNAs in cell proliferation and survival using a high-throughput functional approach.
These studies will assign functions to 100s of little studied non-protein coding genes, determine the mechanisms and principles of a novel form of gene regulation, and establish a new understanding of how a major part of the transcriptome, the ncRNAs, interact with each other, and potentially identify genes with relevance to cancer cell biology.

The human microbiome is increasingly recognized as a vital yet poorly understood ‘organ’, central to many aspects of host health, including auto-immune disorders, obesity, cancer and even psychiatric conditions. While the avalanche of metabolic data provides correlative evidence for potential roles of the microbiome in health and disease, insights are limited by a lack of testable models of human microbiome development, organization and functioning, and tractable experimental systems.
We aim to build a science of microbiome developmental biology, to understand how microbial consortium structure and functioning emerge from metabolic and demographic feedbacks among expanding lineages of bacterial species. Specifically, we will use an experimentally- and computationally-tractable two species ‘minimal microbiome’ platform to ask: (A) how do metabolic interactions influence microbe-microbe spatial structure (species mixing) and functional relationships (mutualism, exploitation, competition)? (B) How do demographic feedbacks influence emergent structural and functional relationships? (C) How do emergent structural and functional properties shape community antibiotic resistance? (D) How can we effectively manage microbiome health via modified nutrients, drugs and mixing?
Our explicit focus is on a metabolically interactive two-species model system composed of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans (Aa) and the common commensal Streptococcus gordonii (Sg). We will interrogate the Aa-Sg system using an integrated mix of individual-based simulations, ecological theory, picolitre-scale porous confinement structures, confocal and scanning electrochemical microscopy. Our project will break new ground by establishing the molecular and demographic mechanisms shaping causal relationships between microbiome environment (nutrients, drugs, mass-transfer regimes) and its structural and functional properties, including contributions to host health/disease.

Non-apoptotic cell death (NACD) is now recognized as tightly regulated forms of cell death, although the molecular mechanisms driving the underlying processes remain to be unveiled. Compelling evidence suggests that NACD sub-routines require oxygenated lipids as critical signals, demanding many questions to be answered: What are the enzymatic mechanisms involved in the formation of oxygenated lipids? Which of these lipid species play an active role in NACD signaling and what are these roles? Are they triggers and/or executors? And what are the targets? Lipid chemistry is remarkably diversified, harboring unlimited opportunities for a refined and meaningful signaling, but, at the same time, this diversity represents a major technical challenge for experimental studies. Here, we propose an analogy between language and biology to address this challenge in an interdisciplinary framework combining experimental and computational studies. Assisted by computational tools developed originally for language applications we plan to decipher the unspoken oxi-lipidome language as follows: Step 1) Identifying the words: We hypothesize that oxygenated lipids formed during different processes of cell death are analogous to words. To define the list of words relating to different forms of NACD, we will generate oxi-lipidome profiles using distinct cell death triggers and well-defined genetic models. Their analysis will yield a modulatory profile used to provide a signature for different cell death modes; Step 2) Deriving a lipidome dictionary: Oxidized-(phospho)lipid (oxi(P)L) heat maps as generated in Step 1 will lay the basis to characterize prominent differences between normally functioning and death-destined cells. This will lead to the identification of potential NACD oxi(P)L signals that will undergo further scrutiny through the language-inspired bioinformatics “filters”. Rational synthesis of the thus defined types of informative phospholipids will be thoroughly analyzed in the context of their interactions with target proteins known to shape the oxi-lipidome. Step 3) Deciphering the meanings of lipid words: In an iterative process integrating predictions and experimentation, we will study the interaction of many lipids with specific proteins and interactions that a specific lipid can undergo with many proteins using computational classification techniques also used in language analysis. Using biophysics and biochemistry techniques we will validate predicted covalent versus non-covalent interaction of the oxi(P)L-protein interfaces. The libraries of oxi-lipids created in step 2 will be delivered to specific intracellular destinations and tested for their signaling role in NACD pathways. Conclusively, knowledge gained by this comprehensive and highly multidisciplinary approach will provide intriguing insights on how the oxi-lipidome could emerge as a tremendous reservoir of yet unrecognized information.

All living organisms must organize their DNA within the confines of the cell. This is achieved by a combination of “passive” factors (e.g. cell size and shape) and “active” processes (e.g. DNA folding proteins). While much is known about how DNA is organized in higher organisms, we still have little understanding of the DNA folding processes that drive chromosome organization and compaction in bacteria. Given the abundance of these organisms, and their impact on human activity, this represents a startling gap in our knowledge. In recent years nano-scale cell imaging, large-scale genomic techniques, and mathematical/physical modeling of biopolymers have emerged as new tools to study DNA biology. In this program we bring together these approaches to facilitate precise dissection of bacterial chromosome structure. Thus, in a collaborative effort between theory and experiment, we will test and expand models to describe bacterial genome organization.