The homeostasis of skin epidermis is ensured by stem cells (SCs) located into specialized microenvironments defined as niches. The basal layer of interfollicular epidermis (IFE) contains SCs and progenitors that generate the stratified layers of the skin. Through lineage tracing experiments using K14CREER and InvCREER, I will study the hierarchical organizations of cell lineages within the IFE during mechanical force mediated tissue expansion. Mechanical force mediated tissue expansion is commonly used to grow extra skin for reconstructive surgery to fix birth defects, severe skin burns or traumas, or for breast reconstruction after breast cancer surgery. Clonal analysis and proliferation kinetic experiments will be performed following the expansion of miniaturized prosthesis placed subcutaneously. Statistical analysis of the clonal behaviour will be used to develop a quantitative model of the cell fate during skin expansion.
Through live imaging microscopy on the LifeAct-EGFP mouse strain I will follow cytoskeletal dynamics during skin expansion in order to verify the role of the cytoskeleton in SCs activation. The relative cytoskeletal tension during tissue expansion will by measured by laser cutting of cell junctions on mT/mG mouse strain. Microarray analysis and single cell gene profiling will be performed in expanded clones to define the molecular mechanisms involved in sensing and mediating tissue expansion. The activity of new candidate genes will be assessed to decipher how these signalling molecules interact with each other to activate SCs and progenitors, maintaining the architecture of the skin and orchestrating tissue expansion in vivo.

Peripheral T cells fall into two major classes, based on the mutually exclusive surface expression of CD4 or CD8 co-receptor molecules. Both lineages derive from common CD4+CD8+ double positive (DP) thymocytes progenitors in a process known as positive selection. The quality of the signals derived by T cell receptor (TCR) triggering plays a role in linage commitment: strong/persistent TCR signals drive CD4+ T cell lineage choice, whereas weak/intermittent TCR signals promote CD8+ lineage choice. During positive selection, the transcription factor (TF) Th-POK maintains Cd4 gene transcription while repressing Cd8 gene expression. Conversely, RUNX3 helps silencing Cd4 gene transcription, while it maintains and reinforces Cd8 gene transcription. It is not well understood how TCR signalling and transcription factor expression are linked. Positive selection requires 3-4 days for completion, and we do not know when, during this process, DP thymocytes commit to a certain fate. What are the TCR-signalling derived events that determine the expression of one transcription factor rather than the other? When does lineage choice initiates, and when does it become irreversible? The purpose of this research is to identify the link between TCR signalling and thymocyte development and to define the kinetics of transcription factor expression that ultimately lead to lineage choice. To achieve this goal, I will first investigate how and when ThPOK and RUNX3 are expressed in thymocytes at various steps of the lineage choice process, and then I will make use of TCR signalling inhibitors or enhancers to interfere with TF networks establishment and lineage fate choice decisions.

Growing evidence points to a disruption of synaptic circuits as the underlying mechanism of many neurodevelopmental disorders. For example, Rett Syndrome (RTT), caused by de-novo mutations in the transcriptional regulator, MECP2, is believed to be secondary to aberrant experience-dependent circuit refinement and function. A key feature of RTT is apparent typical initial development before stagnation occurs. Subsequent regression, characterized by loss of language and cognition, is also accompanied by motor symptoms and onset of epilepsy. RTT mouse models recapitulate many features of the regression phenotype and point to a misregulation of excitatory/inhibitory circuit balance as a key factor in the onset of regression. Understanding which classes of neurons are primarily affected by MECP2 deletion and are responsible of the regression is an open problem and a big challenge. The visual system is an ideal biomarker of the regression, due to the possibility to study in vivo cortical processing using 2-photon microscopy. MECP2 deficient mice are excellent models for the study of the regression and recovery of the disease. By combing advanced imaging techniques and non-invasive optical electrophysiology, we will analyze longitudinally how visual cortical circuits regress and recover after de-silencing MECP2 globally. We will then target parvalbuminic interneurons (PV) that have been shown to be upregulated in MECP2 deficient mice. Specifically we will acutely downregulate PV activity by using light-gated ion channel Halo-rhodopsin (HR) or selectively desilence MECP2 in these cells and study visual circuit response.

Olfactory detection is essential for recognition of food sources, predators and mating partners; thus, adaptation of olfactory perception and preferences plays a fundamental role in colonization of new ecological niches and species evolution. Within drosophilid flies many olfactory adaptations to highly specialized environments have been described. Nevertheless, very little is known about the genetic changes that underlie these evolutionary adaptations. To gain molecular insights into genetic modifications that distinguish diverse odor preferences of closely-related Drosophila species, I will take a comparative functional approach. By generating novel genetic tools in non-traditional model drosophilid species, I will first characterize similarities and differences between the peripheral molecular, physiological and neuronal olfactory organization of three Drosophila species within the D. simulans clade. Second, I will compare orthologous olfactory receptors with different response profiles to identify residues that are responsible for shifts in receptor-ligand specificity. This will provide insights into general principles of olfactory receptor-ligand interactions and evolution of novel specificities. Third, I will perform interspecies hybridization and quantitative trait locus (QTL) analysis to identify developmental genes underlying changes in receptor expression and wiring of these olfactory pathways. Together, this work will provide fundamental insights into the genetic basis of structural and functional evolution of neural circuits.

Like all living cells, bacteria need an adequate supply of energy for vital processes. When under stress, for example when poisoned or exposed to light, bacteria face a difficult juggling act – use energy to respond to the stress or keep vital functions going. In fact, they do both but we do not understand how bacteria manage this difficult balance. We propose to investigate the strategies bacteria adopt to coordinate their free energy by directly measuring the flow of energy under stress.
To do this we will develop techniques that can measure in real time and in a single cell the distribution of the two main sources of free energy: the proton motive force (PMF) and the internal ATP levels. We will then monitor how this changes when the bacteria is stressed. With this information we can start to build a mathematical model that will allow us to understand just how bacteria manage their free energy supplies when stressed. Put simply, we hope to uncover the very fundamental principles of how bacteria manage energy flow and offer a very scientific definition of ‘dead’ and ‘alive.’

Excitation-contraction coupling is the process by which an action potential is converted to muscle contraction. In the cardiovascular system, this process is regulated by neurotransmitters and hormones, released - via excitation-secretion coupling - from the autonomic nervous system. Secretion of neurotransmitters and hormones is mediated by SNARE proteins, which are tightly controlled by myriad accessory proteins (e.g. synaptotagmin, complexin, munc13, munc 18). The importance of this regulation is underscored by the fact that alterations in secretion are linked to chronic heart failure, stroke and neurodegenerative disease. It is well established that increases in Ca2+ trigger the fusion of secretory vesicles with the plasma membrane, thus releasing hormones and transmitters onto target cells. Despite extensive studies, how SNAREs form the fusion pore, and how the fusion pore opens and closes, remain unresolved questions. I hypothesize that SNAREs transiently line the fusion pore in its initial open state, and that interactions between the SNAREs and accessary proteins dictate the opening, expansion, or closure, of the fusion pore. To test these ideas, I will develop novel methods based on nanodisc to carry out biochemical and functional analysis of complexin (Aim 1), characterization of the transmembrane domains of SNAREs (Aim 2) and measurements of fusion-pore dynamics with single pore resolution (Aim 3).

Humans and animals perceive their environment through different senses which together contribute to recognition of surrounding objects and situations. However, the brain mechanisms underlying this process remain elusive. The goal of this project is to use the power of the mouse model for dissection of neuronal circuits to start understanding the functional role and the mechanistic underpinning of multisensory interactions in cortical circuits. Current evidence shows that coordinated olfactory sampling and whisker contacts are routinely used by mice to discover objects and conspecifics, suggesting that the neuronal representations of olfactory and tactile scenes strongly interact. To address this question, we will measure cortical population activity using high density recordings techniques including two-photon imaging and multielectrode recordings in sensory and associative cortical areas during naive and goal-directed olfactory-tactile exploration of parametrized olfactory-tactile objects. In particular, head-fixed mice will be trained to associate different responses to two categories of multisensory stimuli with unisensory ambiguities. This design will allow testing if, due to associative learning, information from one sensory channel can bias the representation of a stimulus from another channel towards some expectation, as observed in many multisensory illusions in humans. The areas and connections underlying multisensory interactions will be tested using targeted optogenetic silencing, giving us the opportunity to quantitatively test functional models of multisensory integration, that can be extended for comparison to other modalities.

The ability of the brain to integrate sensory information and produce goal-directed behaviour in higher mammals is a result of complex information processing within cortical columns. These columns span all 6 layers of the neocortex and are highly interconnected with other cortical and subcortical structures. Because of the limited penetration depth of two-photon microscopy functional imaging of neurons in deeper cortex layers (L5/6) has remained challenging. Here, I propose to adapt and utilize a novel multi-photon multi-area microscope for simultaneous calcium imaging of two or more layers in mouse barrel cortex during a whisker-dependent texture-discrimination task. Application of newest red genetically-encoded fluorescent calcium indicators will facilitate deep imaging. Simultaneous recording of layer 2/3 and subcortical-projecting layer 5/6 cells will allow me to test the hypothesis that a cross-laminar mismatch operation is performed, with deep-layer neurons expected to convey information about deviations of the sensory input from the expectations represented in superficial layers 2/3. I will aim to identify the sublaminae and cellular subtypes that most saliently carry predictive and mismatch information. Chronic imaging throughout task-learning, followed by reversal learning, will be particularly informative in order to understand how the computations across cortical layers are established and how they depend on the behavioural context. The results are expected to help identify the functional roles of distinct sublaminae of sensory cortex during behaviour-relevant conditions and fundamental principles of the translaminar information processing.

Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites, which represent a general feature of all eukaryotic cells, play important roles in Ca2+ signaling and lipid metabolism. How are these junctions generated and maintained? The De Camilli lab has recently found that the three extended synaptotagmins (E-Syts) localize to the ER and mediate ER-PM tethering via C2 domain-dependent interactions with the PM in a Ca2+- and PI(4,5)P2-dependent manner in mammalian cells. Unlike E-Syt2 or E-Syt3, which strongly binds to the PM at resting Ca2+ level, the binding of E-Syt1 to the PM requires elevation of cytosolic Ca2+. This suggests that E-Syt1 may contribute to increasing ER-PM contacts in response to Ca2+ rise. Give the fact that cytosolic Ca2+ displays a rear-to-front gradient in a migrating cell, with the highest concentration at the rear end, I postulate that E-Syt1 is recruited to the cell cortex to increase ER-PM contacts at the trailing edge. To test this hypothesis, I will use total internal reflection fluorescence (TIRF) microscopy to test whether E-Syt1 is recruited to the trailing-edge PM. Increased E-Syt1-mediated ER-PM contacts may in turn hold the ER and facilitate Ca2+ release within the rear end of migrating cells to maintain Ca2+ gradients and cell polarity. Additionally, E-Syt1 may facilitate endocytosis of FGF receptors and EGF receptors to activate ERK signaling pathway, which leads to cell adhesion disassembly. I will use a combination of wound healing assay and biochemical analyses to examine these potential roles of E-Syt1 in cell migration. Together, these results will give insight into the physiological roles of E-Syt1-mediated ER-PM contacts.

Microorganisms are the most abundant living organisms on earth and they thrive in virtually every possible habitat, including in our bodies and those of other organisms. Understanding the causes and consequences of their diversity is therefore essential for controlling microbes in health, agriculture and biotechnology. Studies of microbial diversity are also increasingly important for advancing general ecological and evolutionary theory, because microbes are powerful experimental model systems. We seek to advance our understanding of the mechanisms underlying microbial diversity by focusing on the role of extra-cellular products (EPs). EPs play an interesting but largely unknown role in the evolution of microbial diversity. Given possible effects of EPs on the fitness of cells other than the producer, a growing perception is that EP production is a form of cooperative behavior, suggesting that it has evolved due to benefits conferred on the population of EP producers rather than on individual producers. However, this view rests on models that make simplifying assumptions that need to be rigorously tested. Our international collaborative project combines the expertise of: (1) two biologists, who will use existing and newly developed millifluidic droplet technology to test for collective benefit and its evolutionary consequences in experiments with two bacterial EP systems with different fitness effects and availability to others, (2) a colloid chemist, who will develop novel millifluidic tools that provide a unique opportunity to quantitatively assess the evolutionary consequences of EP production, and (3) a theoretical biophysicist, who will develop computational models that incorporate biological details of both experimental EP systems, and will both generate specific predictions that will be tested experimentally and facilitate the results from these experiments. By generating unique quantitative data on two very different bacterial EP systems and comparing results against predictions from realistic models, we expect that our project will provide conclusive answers and set new standards for the study of microbial interactions.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a dismal prognosis and an urgent need for new treatments. Tumours generally increase glycolysis to sustain their growth, resulting in the efflux of lactic acid and extracellular acidification, which has been shown to promote cancer progression. Low intracellular pH impairs tumour growth, suggesting that pH modulation represents a potential anti-cancer strategy. The regulation of pH in PDAC has not been investigated. My aim is therefore to study the roles of pH and pH regulators in normal pancreas and PDAC. I hypothesize that key pH regulators contribute to cancer progression, and that impairing their function may result in eradication of the tumour. I will employ mouse models of PDAC alongside a newly-established system for culturing normal, pre-malignant and malignant pancreatic cells as 3D organoids. These systems faithfully recapitulate the human disease. I will measure pH changes in normal pancreas and from early through late stages of PDAC by metabolic profiling, and identify putative pH regulators using transcriptomic and proteomic analyses. I will then functionally validate these proteins in vivo using a shRNA-mediated strategy, to evaluate the effects of their depletion on tumour growth. Down-regulation of key pH regulators may trigger intracellular acidification, resulting in tumour cell death, while the concomitant increase of extracellular pH may prevent activation of proteases that have been shown to promote metastasis formation. In summary, this project aims to investigate the regulation of pH in normal pancreas and PDAC, and to understand how pH changes influence PDAC progression.

It is becoming clear that parental environmental conditions can affect phenotypes of future generations, and that such information can be transmitted via sperm. However, how the male germline senses and is able to transmit environmental cues to the developing organism is unclear. I propose here a study focused on 5’tRNA fragments (5’tRFs) as potential mediators of paternal intergenerational epigenetic inheritance. While tRNA cleavage in somatic cells has been shown to occur in response to stress conditions, 5’tRFs are highly abundant in mammalian sperm even in animals maintained under standard husbandry conditions. Although a role of 5’tRFs in regulating translation is beginning to emerge in somatic cells, their function during fertilization and development remains mysterious. Intriguingly, preliminary studies in the Rando lab indicate that several of these highly abundant RNAs change abundance in murine sperm in response to dietary conditions. In this project, I will identify the RNase responsible for tRNA cleavage during spermatogenesis, and study the effects of 5’tRF depletion on male fertility and development. Secondly, I will investigate the tissue of origin of precursor tRNAs which are cleaved to 5’tRFs, to understand the spatiotemporal dynamics of 5’tRF biogenesis. Finally, I will assess the functionality of 5’tRFs as potential epigenetic information carriers. Particular focus will be put on developmental progression, with regards to acquisition of totipotency, cell fate allocation and gene expression. The research project proposed here will greatly contribute to understanding the molecular mechanisms of paternal contribution to epigenetic information in mammals.

Successful defense against pathogens is critical for survival. Microbes have developed many ways to invade larger organisms which in turn have evolved numerous immunity mechanisms. Lipid droplets (LDs) are dynamic and complex organelles that provide all eukaryotic cells with lipidic substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules. Probably for this reason, LDs are part of the infection cycle of viral, fungal, and bacterial pathogens. Mechanistic details of LD/pathogen interactions are largely unknown, but many pathogens stimulate LD biogenesis. The current view is that LDs are “hijacked” to provide lipids and energy to pathogens for effective and rapid growth.
Our recent work has challenged this dogma and demonstrated that LDs are organelles that actively participate in the Drosophila organismal antibacterial response. This new type of innate immunity could well be at work in every infected tissue of our body since all cells accumulate LDs. Preliminary data, specially generated for this project, supports the idea that this innate immune system has been conserved during evolution.
Thus, based on solid preliminary data, we here propose a multidisciplinary research project to characterize cells, sites, and mechanisms of action of this innate immunity. Whole animal studies will determine cells of interaction, high-resolution ultra-structural analysis of infected tissues and cellular models will determine sub-cellular locations and details, single-organelle biophysical studies and in vitro LD-bacteria killing assays combined with ultra-structural studies will develop a mechanistic understanding of the process, and comparative proteomics will identify protein complexes conferring chemotaxis and antibiotic activity to LDs.
In conclusion, here we present a project designed to identify and characterize a new innate immune system that will be paradigm-shifting in Immunology, Physiology, and Cell Biology. Further, the combination of the detailed single-organelle killing assays with the super-resolution studies specially developed for these studies will allow unprecedented mechanistic details establishing an entirely new dimension in the ongoing host pathogen conflict.

Many bacterial pathogens have acquired the ability to replicate within host cells. Therefore the host cell cytosol is closely monitored by a variety of innate immune receptors, which promote inflammatory responses and can even trigger host cell death by activating inflammasomes. Given the broad spectrum of cytosolic defenses, it is not surprising that the vast majority of intracellular bacteria does not reside directly in cytosol, but replicates in specialized vacuoles.

While it was previously assumed that bacteria within such vacuoles are hidden from cytosolic recognition, mounting evidence indicates that host cells feature a yet uncharacterized mechanism to detect vacuolar bacteria. We have recently identified such a mechanism, and shown that it requires several interferon-inducible GTPases, the so-called Guanylate-binding proteins (GBPs).

GBPs target and destroy pathogen-containing vacuoles, resulting in the release of bacteria into the cytosol and subsequent detection by innate immune sensors. However, little is known about how GBPs recognize pathogenic vacuoles and about their physiological role in regulating host defense against invading microbes. Here we will define this novel mechanism by 1) determining how GBPs are selectively targeted to vacuoles, 2) identifying GBP interaction partners and 3) defining how GBPs regulate host-defense in vivo. We will use a combination of new genome engineering techniques (CRISPR/Cas9), advanced biochemical and proteomics approaches and host genetics. Understanding how the innate immune system recognizes and fights invading pathogens is a key step towards the development of novel immune modulators and anti-infectives.

There is now a large body of experimental evidence indicating that the ability of many newly synthesized proteins to reach full functionality in a cell depends strongly on the rate at which individual codons are translated by the ribosome during protein synthesis. These codon translation rates can determine whether a nascent protein will fold and function, misfold and malfunction, aggregate or efficiently translocate to a different cellular compartment. That is, changing the translation rate of a codon can change the fate of a protein in living cells. This indicates that the sequence of nucleotides in a transcript not only encodes genomic information but also kinetic information that is used by cells to coordinate co-translational processes and influence nascent protein behavior. Therefore, quantifying and predicting the influence codon translation rates on nascent protein behavior is an immediate and pressing need that if met will provide fundamental insights into the coordination of co-translational processes and nascent protein maturation. This proposal addresses this challenge by proposing a novel collaboration between the theoretical biophysics lab of Ed O’Brien and the experimental molecular biology lab of Bernd Bukau. The goals of this proposal are (1) to experimentally quantify the influence that codon translation rates have on E. coli’s nascent protein behavior at the proteome level, (2) to create an experimentally verified model that accurately predicts the consequences of a transcripts’ translation-rate profile for the behavior of the newly synthesized protein, and (3) to address fundamental biology questions that include (i) how robust are nascent protein behaviors to changes in codon translation rates? (ii) What codon positions in an open reading frame cause major changes in a nascent protein’s behavior upon a change in their translation rate? (iii) How does changing codon translation rates affect downstream cellular processes involving the nascent protein? By bringing together the complementary skill sets of the O’Brien and Bukau Labs, this research will significantly advance our quantitative understanding of the temporal coordination of translation and co-translational processes affecting nascent protein behavior.

We aim to establish a blueprint for how organisms tell time over the course of the year. This is not so simple as merely responding to the changing seasons, requiring preparation well in advance for changes in the environment. A key adaptation involves an internal annual timing mechanism (sometimes known as a “circannual clock”), which free-runs in constant conditions but is normally synchronized by environmental signals (entrainment). Circannual clocks are a fundamental feature of life, necessary for such diverse phenomena as the timed emergence of ground squirrels or bats from their hibernacula, the autumn migration of birds, and transitions between life-cycle stages in unicells. We aim to dissect the mechanisms driving the circannual clock and propose that these will turn out to be strongly conserved (i.e. similar) in different groups of organisms. We propose that circannual clocks are driven by specialist timer cells, with the clock based on cycling between resting and active phases (reversible cellular quiescence, RCQ).
We have two objectives. First (Objective 1, Functional biology), we will test the hypothesis that the RCQ is driven by a mechanism involving chemical modification of the cell’s DNA (epigenetics). Focusing on those tissues in which we think the circannual clock resides, we will explore circannual effects on the epigenetic make up of cells. We will test the generality of these mechanisms in other vertebrate groups and in a distantly related marine micro-organism. Additionally, we will test for the action of a conserved circadian input mechanism in driving seasonal timers over wide evolutionary divides.
Second (Objective 2, Evolutionary Biology), we predict that key genes in these ancestral long-term timing pathways have been subject to selection in response to evolutionary pressures on the function of these timers. Here, we will study the evolution of breeding seasons in at different latitudes in birds and fish, and test the hypothesis that natural selection tunes the circannual clockwork and photoperiodic input pathways to match local seasonal environments. We will also explore how seasonal timing circuits have been subject to selection in domestication in birds and mammals.
Integration of results from these two Objectives will give new insights into seasonal time-keeping mechanisms.

Transmission is a key step in the infection cycle of all pathogens. Pathogens have developed sophisticated strategies to survive through this crucial phase. Use of vectors that shuttle pathogens to a new host is a common strategy employed by many infectious agents. Shuttling may require internalization of pathogens in vectors, or shuttling occurs with the pathogens attached only to external vector parts. Both cases involve intimate interactions between vectors and pathogens. The latter mode of transmission is used by many arthropod-vectored animal (including human) and plant viruses and is referred to as mechanical and non-circulative transmission, respectively.
We will interrogate the non-circulative transmission of representative plant viruses that are spread by insects in the order Hemiptera. Insects in this order are sucking and piercing feeders that imbibe nutrients from the plant host’s phloem. We will focus on molecular and biophysical aspects of three related steps in the transmission process: 1) virus uptake, 2) virus inoculation, and 3) the step in-between: virus retention in the vector; specifically targeting mechanisms that mediate virus uptake from, and inoculation to, the phloem and, importantly, the biophysical changes occurring in virion-bound vectors. Re: 1) Uptake. Evidence for an exemplary plant virus, Cauliflower mosaic virus (CaMV), indicates that non-circulative transmission is mediated by specific interactions (among the virus, vector and host plant) that boost transmission efficiency. Indeed, CaMV responds to its aphid vector’s presence on the host by instantly forming transmission forms, a phenomenon named transmission activation (TA). However, neither ubiquity nor the mechanisms that drive TA are known. Here, we will test generality of TA of tissue-acquired viruses like CaMV, and for a first time TA of phloem-restricted viruses. Re: 2) Inoculation. We will determine dietary conditions and identify components required for virus release from vectors and compare different viruses and vectors to reveal common and diverging parameters in this never before investigated step of the transmission process. Re: 3) Retention. How viruses attach to, are retained in, and are released from vectors, is a black box. Using advanced biophysical methods, we will study virus attachment and release in dissected mouthparts and eventually life insects.

Living organisms, particularly mammals, constantly shift between different behavioral states according to their external environment. These shifts are largely regulated by specific molecules, termed "neuromodulators" that convey information to a wide range of brain systems. The mechanisms by which these neuromodulators regulate distinct behavioral states remain enigmatic. One important brain neuromodulator is the neuropeptide oxytocin, which recently became well known for its positive, “pro-social” effects on human social behavior. Oxytocin is synthesized and released by a small number of hypothalamic neurons that send their projections to various forebrain regions, to regulate a plethora of social behaviors ranging from aggression to empathy.
Relying on anatomical and physiological distinction between subsets of oxytocin neurons, we hypothesize that these neurons are segregated into functional modules associated with distinct types of social behavior. To test this hypothesis we will focus on one hypothalamic region – the paraventricular nucleus, which is the main source for oxytocin projections throughout the entire brain. We propose a comprehensive multidisciplinary study aiming to identify, analyze and mathematically model functional oxytocin neuronal modules activated during various forms of social behavior.
To pursue this hypothesis we will employ a novel genetic technique named vGAIT (virus-mediated genetic activity-induced tagging), which enables tagging and manipulating oxytocin neurons that were active during a given social behavior, ranging from simple social interaction to a complex “human-like” cooperative behavior. Using this technique in freely moving rats, we will explore the activity and connectivity, as well as the intrinsic electrophysiological and molecular properties of activated oxytocin modules at the single-cell level. Building on this information we will develop a computational model of the oxytocin system and investigate the mechanistic principles underlying its function. The unique combination of cutting-edge techniques and approaches will allow an unprecedented insight into the mode of operation of a central brain neuromodulatory system that controls mammalian social behavior. Furthermore, the expected findings will pave the way for the development of interventions directed towards specific pathologies of social behavior in humans.

We inherit our traits from our parents through genes embedded in the DNA of the sperm and the oocyte. It was already known that besides DNA, other components of the egg are also inherited that might contribute to the offspring. For example, a mother might pass on effects of the environment to its baby either during pregnancy, or in case of externally fertilizing animals such as fish, by deposited materials in the egg. In recent years it is becoming clear that environmental effects on the father (from various forms of stress to diet changes) can also be inherited by the offspring and even passed on to the next generations and result in changes in traits. How this inheritance occurs puzzles scientists. Increasingly, scientists are considering the possibility of the inheritance of environmental effects through 'epigenetic' marks and they call this process epigenetic inheritance. Epigenetic marks include chemical tags, which influence the way DNA is packaged (termed chromatin). Chromatin in the sperm and the egg may then influence how genes get switched on and off in the offspring and can have lasting impact by changing the traits of the offspring. In this consortium, an evolutionary biologist (Immler) a biochemist (Cairns) and a developmental geneticist (Mueller) join together to address the mechanisms of epigenetic inheritance by proposing a new model to study epigenetic inheritance: the zebrafish. Zebrafish, like us is a vertebrate, which develops outside of the mother as a transparent embryo, which makes it popular for developmental biologists and geneticists. Immler has recently discovered that zebrafish embryos develop faster than their siblings if the father was competing among males for breeding with female. This observation serves as an exciting model to study epigenetic effects - and the biochemist Cairns together with the developmental geneticist Mueller will use the male competition model alongside feeding experiments and genetic manipulation tools to elucidate how and what chemical tags are responsible for the observed changes in development. Together with Immler they will study whether the observed changes in development may also have a lasting impact on adult fitness of the animal and may be inherited over several generations. The findings will contribute to answering an important question about how vertebrates (including ourselves) pass on experience through the father to their offspring.

RNA viruses represent an existing and emerging global threat to human and animal health. Vaccination is the only known approach to prevent viral infection but suffers from several limitations, one of which is restoration of the virulent phenotype as a result of recombination between the vaccine strain and a circulating, wild strain. In general, recombination is one of the most, if not the most, important molecular process driving the expansion of the host range of viruses. Recombination is also a major contributor to viral evolution, leading to the emergence of vaccine-resistant strains. Unfortunately, we lack sufficient knowledge of the molecular mechanism of RNA virus recombination to prevent these issues from thwarting vaccine development and long-term efficacy. Therefore, an understanding of recombination is of potentially broad, practical value. For example, detailed knowledge of the mechanism of recombination may establish principles that can be exploited for development of strategies to suppress recombination and/or design of recombination-deficient vaccine strains. The overarching goal of the proposed research is to create fundamental knowledge on the mechanism of RNA virus recombination. Our model system will be enterovirus 71 (EV71), a member of the Picornavirus family of viruses. EV71 outbreaks are on the rise and are driven by the emergence of strains created by recombination.
Milestones for this project are as follows:
• We will provide a complete biochemical and biophysical description of the molecular events that ensue when a viral RNA polymerase encounters a putative RNA trigger for recombination.
• We will elucidate properties of the viral RNA polymerase that contribute to recombination frequency.
• We will determine whether recombination contributes to viral fitness in vitro and in vivo and perhaps even the extent to which new recombinant viral strains have outbreak potential by exhibiting increased virulence.
To achieve these goals, we have assembled a multi-disciplinary, multi-national team of scientists that brings a broad base of intellectual and experimental resources to the problem of RNA virus recombination. These resources include single-molecule biophysics, single-cell virology, a small-animal model to assess pathogenesis and to access currently circulating viral strains.