The intestinal epithelium has the complex task of transporting water, ions and nutrients, while providing an efficient barrier to microbes and other potentially harmful luminal constituents. This delicate balance is notably altered in chronic inflammatory conditions of the gut, such as Crohn’s disease.
Luminal nutrients such as glucose have long been recognized as regulators of epithelial permeability. Recently, a role of the enteric nervous system in the control of barrier function has been proposed. Indeed, alterations to either enteric neurons or enteric glial cells leads to dysregulation of epithelial permeability. The structural basis for an interaction between the epithelium, glial cells and nerves has recently been provided, but neither the detailed nature nor the integration of this communication have been examined.
My project aims to investigate how nutrient-sensitive epithelial cells called enteroendocrine cells communicate with enteric glia and nerves in the mucosa to regulate epithelial permeability.
My approach will be to evaluate the signal integration, from the luminal stimulus to the epithelial response. We will use a combination of live cell imaging techniques and fluorescent reporters to visualize the propagation of the signals in living tissues. We will selectively block or release enteroendocrine cell mediators, inhibit enteric glial cells and/or block or stimulate enteric nerves to dissect the various components of this crosstalk.
My project will lead to a comprehensive understanding of signal integration in the intestine between the epithelium and the enteric nervous system, extending the concept of neural control of intestinal barrier function.

Extracellular interactome networks consisting of secreted and cell surface proteins coordinate complex biological processes, including embryonic development, homeostatic regulation and cancer progression. Identification of extracellular protein-protein interactions (PPIs) is central to biomedical science and therapeutics. For example, multiple cell surface and secreted proteins are targets for FDA-approved drugs. However, a description of extracellular PPIs for many of these proteins remains either unknown or incompletely known. We are particularly interested in Wnt signaling that controls the central nervous system (CNS) vascular development and blood-brain-barrier (BBB) formation and maintenance. The CNS vascular dysfunction including BBB breakdown is associated with brain trauma, stroke, multiple sclerosis and neurodegenerative disorders. So far information of extracellular PPIs of Wnt signaling for the development and regulation of CNS vasculature is limited. Here, we aim to utilize and improve an innovative technology for systematic and unbiased methods to identify novel extracellular PPIs related to Wnt signaling for the CNS vascularization and the BBB integrity. We expect to validate novel candidate PPIs using biochemical and biophysical assays. Additional experiments will assess these PPIs and their significance for CNS vascularization using cell culture systems and mouse genetic models. The information obtained from these studies will expand our knowledge of CNS vascular biology and bring a new approach to the study of extracellular interactomes.

The X-ray free-electron laser (XFEL) promises the study of systems that cannot be crystallized and the ability to follow the evolution of structures undergoing reactions or other dynamic processes, overcoming limitations of crystallography (which requires crystals) and cryo-electron microscopy (which requires cooled samples). Both of those methods are fundamentally constrained by the problem of radiation damage, which sets a limit to the exposure that can be tolerated by the sample. The XFEL breaks this limit with very intense and brief X-ray pulses that are shorter than the time atoms can move on the atomic scale, even though the sample is ultimately vaporized. This enhanced dose tolerance has been well demonstrated by high-intensity experiments using protein nanocrystals, where diffraction patterns are collected at many thousands of times higher exposures than is possible otherwise. However, even at these extreme intensities, the diffraction signal of non-crystalline objects is low, comparable to the achievable signals of biological molecules in cryo-electron microscopy (cryo-EM).
We propose to develop radically new methods to image single uncrystallized biological molecules at atomic resolution by XFEL diffraction of nano-engineered samples. By attaching DNA origami structures to the sample we obtain stronger signal than from the molecule alone. The structural information of the sample is built up from millions of diffraction patterns from such samples, collected one at a time at the repetition rate of the XFEL. These patterns can only be fully interpreted to give a three-dimensional (3D) structure of the molecule if they are individually registered to each other in all three orientations (similar to cryo-EM). The flexibility of DNA nanotechnology will be exploited to build structures that align in a flowing jet or orient on a membrane substrate such as graphene. The unknown rotation of the object about the alignment axis will be obtained from signatures based on the designed DNA structure. In addition to boosting the diffraction signal and orienting the molecule, the known DNA origami structure provides a holographic reference to phase the aggregated diffraction intensities and give the 3D electron density map. Our general-purpose technique will be applied to obtain atomic resolution imaging of biological molecules that do not readily crystallize.

The Hsp90 molecular chaperone helps fold and activate 10% of the proteome and 60% of the human kinome. Together with the kinase specific co-chaperone Cdc37 it facilitates the folding and regulation of its client-kinases. This is accompanied by transitions between open and closed Hsp90 conformations triggered by ATP hydrolysis. Recently the closed Hsp90ß:Cdc37:Cdk4 complex was solved by the Agard lab using cryoEM. It reveals that Hsp90ß inactivates Cdk4, through clamping an unfolded ß-sheet and thereby separating the two kinase lobes. I aim to elucidate the biochemical and structural basis for kinase capture and refolding by the chaperone machinery. This might either occur directly by kinase binding to a very different “open” Hsp90ß:Cdc37 complex or involve unfolding by Hsp70 and subsequent transfer to Hsp90ß via a “transfer complex” of Hsp70, Hop, Hsp90ß, Cdc37 and the kinase. I will reconstitute the chaperone system in-vitro to identify key steps and necessary protein components. The kinase folding state will be monitored using ligand/inhibitor binding as well as FRET between the lobes. Using this system, I will also study the role of several co-chaperones including FKBP51, Aha1 and PP5. To allow structural characterization of the important dynamic intermediates by cryoEM, I will trap these states using a variety of strategies and determine their structures to atomic resolution by state-of-the-art cryoEM. In vivo relevance will be assessed by disrupting the underlying interactions in cancer cell lines. This will offer the future perspective to pharmacologically target the identified structural states relevant in many human cancers.

Cardiac hypertrophy from different causes represents a major culprit for premature sudden cardiac death in humans. The infrequently feeding Burmese python exhibits a dramatic change in metabolism after a large meal that requires organs to grow, including physiologic cardiac hypertrophy much as is seen in athletes after exercise. Most interestingly, such growth is reversible and cyclically occurs at every meal. This could be clinically important since regression is not seen in most pathological hypertrophy. With this project, we aim to define the mechanisms that allow the python heart to revert hypertrophy, in the attempt to provide novel insights for regression of cardiac growth in mammals. We hypothesize the presence of one or more factors promoting regression in the python plasma after the peak of digestion. We will test this hypothesis by treating hypertrophied neonatal rat myocytes with python plasma and evaluate cellular dimension as a marker for regression of growth. The sponsor’s laboratory has established this protocol. Once we have defined the trigger responsible for regression of hypertrophy, we will study the mechanisms employed by the python to reduce cardiac mass. We envision the involvement of complex signalling cascades leading to apoptosis and autophagy that are needed to reduce cardiac mass in a controlled manner. Moreover, we will extend our research on established mammalian pathological models of cardiac hypertrophy. We will attempt to activate the same signalling pathways in mammalian models in order to reduce cardiac mass. The results of this project are expected to unveil novel pathways that may lead to therapeutics for cardiovascular diseases.

When plants and animals grow they often develop patterns, such as the spiral arrangement of leaves around a stem or the overlapping pattern of scales on a snake. Some of these patterns are controlled by genes acting to shape the cells, and these patterns have been well studied. However, many biological patterns arise simply from physical forces. These patterns depend on the chemistry of the materials that plants and animals are made of, and on the forces that arise as these materials grow. We hypothesise that a single set of rules governs this mechanical pattern formation, and that these rules will relate to how tissues grow, what they are built of, and how stiff they are. By defining and understanding these rules we will be able to explain a great deal of the diversity of living organisms.
We have chosen to study the formation of a particular type of pattern – buckling, or wrinkling, of layers of the skin. Our team comprises a plant biologist, who will study buckling of the petal surface of Hibiscus trionum, an animal biologist, who will study buckling of the skin of corn snakes, and a polymer engineer, who will model buckling in artificial systems and generate rules and predictions. The two biologists will test these predictions in their different models, and the team will work iteratively to refine the models. The two biologists will also share tools and techniques to enable them to measure the same properties of their different systems.
Our findings are poised to provide new understanding of the universal principles that apply to life, and specifically growth processes in both plants and animals. They will help evolutionary biologists to explain the great diversity of plant and animal form, and will underpin many future applications in which engineers use bioinspiration to generate new materials and structures.

Aminoacyl-tRNA synthetases (aaRS) are the major translators of the genetic code by assigning the correct amino acids to the corresponding tRNAs and are thus of indisputable importance for protein biosynthesis. Additionally, several aaRS moonlight as inducer of cell signaling pathways. Recently, aaRS splice variants have been identified, which display unique cytokine-like properties. These splice variants are often truncated in their N- or C-terminus, thereby creating new termini. Terminal amine isotopic labeling of substrates (TAILS) was developed to map protease cleavage patterns. In the project suggested here, I will utilize TAILS to enrich and identify splice variants of AARS, especially of Arginyl-tRNA synthetase (RARS). Surprisingly, RARS signaling has not been studied yet, despite experimental evidence that RARS is present in the nucleus, signaling elicited by a RARS-splice variant, and the regulatory role of arginine in the immune system and cancer. Establishment of a timeline for RARS splicing events upon stimulation will decipher the underlying biological function and elucidate possible signaling functions of RARS. Investigation of RARS-induced signaling, interaction partners, and cellular dynamics will corroborate these results. As RARS splice variants lack export sequences, paracrine signaling could be mediated by exosomes. Dynamic regulation of RARS variants upon stimulation may lead to the identification of upstream regulators initiating splicing. Together, I suggest a new method for precise and quantitative detection of splice variants on protein level with a direct biological application for an enzyme class of very fundamental importance.

Sophisticated alien-like systems in squids, octopuses, and other cephalopods capture the human imagination, and are of growing research interest. Some represent unique innovations, like dynamic camouflage, high pressure jet propulsion, and stretchable arms with tasting suckers that grip. Other features are convergent traits that are surprisingly similar, but molecularly different, from familiar biological systems in ourselves and our vertebrate cousins, including large brains, sophisticated eyes, and a muscular heart that drives a high pressure circulatory system. Parallel evolution of these complex systems in cephalopods and vertebrates is likely due to an ancient evolutionary competition to dominant as large, active swimming, visual predators in early oceans, causing both groups to engineer their own genetic, cellular, anatomical and physiological solutions to similar environmental challenges. This matchless competition profoundly shaped complexity in both lineages, and offers researchers today an extraordinary and uniquely powerful opportunity to distill basic principles and reveal novel solutions of how to make a brain, complex eyes, and a heart with sophisticated cardiovascular regulation. Here, through detailed comparisons between cephalopods vs vertebrates, together with state-of-the-art technologies, we will decipher mechanisms and uncover alternative solutions: how to design a circulatory system with rhythmic heartbeats?
To characterize, reverse engineer and control novel types of circulatory systems, powerful genetic tools will be developed. We will establish, for the first time, targeted genome editing and light-based genetic tools to control activity in the world’s smallest cephalopod - the pygmy squid Idiosepius – a novel revolutionary model for biomedicine. Second, using this transparent marine organism, we will produce a genomic portrait of the entire circulatory system at single-cell resolution. Finally, with real-time imaging technologies and sophisticated genetic controls, we will develop new ways to regulate, not one, but three cephalopod hearts and their pacemakers. As a result, our international team will transform the pygmy squid into a cephalopod ‘lab rat’, and discover fundamental principles that led to the origins of high-pressure circulatory systems, providing new materials and ideas for synthetic biology and bioengineering.

Touch is one of the primary sensory percepts. Although most well-studied in vertebrates, it is critical to most organism including insects which rely on touch to navigate through the world around them. Moths use a long proboscis, a specialized mouthpart to actively acquire tactile information from flowers during feeding. They unfurl their proboscis and probe the flower surface in a manner reminiscent of how rats use their whiskers to sense their environment, an extensively studied active sensing system. However unlike whiskers that have mechanosensors only the base, the proboscis has sensors along its entire length. Moreover, the mechanical properties of the proboscis (diameter and stiffness) can also be actively controlled by the moth during feeding. Thus we see a novel sensory system that can be tuned on the fly.
Moths also acquire rapid mechanosensory information about the relative position of flower from the proboscis to stabilize flight and feed even in windy conditions. They combine this tactile information with the proprioceptive information from the neck and proboscis to form a representation of flower shape and position. Despite the central role of touch in the plant-pollinator interaction, little is known about the proboscis neuromechanics. Using a combination of behavioral, biomechanical and electrophysiological and data analytic studies on the nocturnal hawkmoth, Manduca sexta, I aim to understand how proboscis mechanics govern the interaction with flowers, how the proboscis is actuated to acquire relevant information, and in turn, how tactile information is encoded by sensors to enable this essential ecosystem service.

Asymmetric cell fate assignation during asymmetric division relies on the biased dispatch of fate determinants between the two daughter cells. I recently discovered a novel mechanism of asymmetric dispatch of fate determinants in Drosophila. In our system, molecular motors on Sara endosomes amplify the asymmetry of the central spindle to induce robust asymmetric segregation of endosomes. Since Sara endosomes contain Notch and its ligand Delta, this asymmetric dispatch contributes to polarized signaling and asymmetric fate in the bristle lineage. This synthetic biology proposal aims at reconstituting asymmetric cell division in non-polarized, symmetrically dividing cells. The physics of the system is such that I can do this either by controlling central spindle asymmetry (i.e. modify the writing), or by controlling the biophysical properties of the motors carrying the fate determinants (i.e. the reading). Strategically, this project capitalizes on the physical understanding of the system and on pioneering pluridisciplinary assays. In particular, I will i) use opto-nanobodies to reshape the asymmetry of the central spindle in space and time in vivo, ii) use multiprotein micropatterning on glass to cluster bivalent transmembrane nanobodies in cells and thereby create cortical polarity cues in naïve cells, and iii) engineer a motor designed to exponentially amplify the low, stochastic asymmetry of the central spindle in non-polarized cells. This synthetic biology project will challenge our current view of asymmetric cell division and pave the way towards restoring asymmetric cell fate in mammalian cells in situations where it has been lost, such as tumorigenesis or ageing.

The discovery that the cytoskeletons are not unique to eukaryotes has raised several important questions about the origin and functional role of ancient cytoskeletal proteins and their evolution into modern eukaryotic cytoskeletal networks. Toward this goal, we propose to better understand the synergies required between proteins and lipids by developing synthetic cells that combine the complexity and evolved sophistication of biological processes with the control and robustness of modern synthetic and materials chemistry.
We will develop a bottom-up approach towards the design of a fully synthetic functional cytoskeleton. Our project will implement supramolecular peptides, de novo artificial lipid synthesis, and protein channel engineering. These artificial building blocks (peptides, lipids, proteins) will be symbiotically self-assembled to enable the self-regulation of size and shape of synthetic cells.
The specific objective of this proposal will be to integrate artificial cytoskeletal peptides with bioorthogonal membranes and stimuli-responsive transport proteins to create hybrid “cells” capable of mimicking the function of natural cells but with improved control, stability, and simplicity. This strategy could dramatically improve our understanding of the physical requirements needed to couple simple early proteins with lipid membranes and help us understand the fundamental reasons for how and why cytoskeletal proteins evolved to control cell division and morphology.
The potential discoveries will allow a better understanding of the origin and development of the cytoskeletal network in cells. Furthermore, the creation of hybrid synthetic cells will enable a myriad of studies that will dramatically expand our understanding of how complex living cells evolved on our planet.

For a cell to maintain protein homeostasis (proteostasis) extensive networks of components are needed to safeguard and rapidly correct unwanted proteomic changes. The performance of these proteostasis networks may be reduced under stress and during aging, which results in misfolding of proteins, their aggregation and/or loss of functionality. Such instability of the proteome manifests in age-dependent neurodegenerative diseases, like Alzheimer’s and Parkinson’s and other medical disorders.
Currently there is a shortfall of capacity to quantitatively measure how well the networks maintaining proteostasis operate. The central objective of this project is to build a new biosensor system that can quantitatively measure how efficiently proteostasis is managed. We will probe proteostasis in space (i.e. spatially inside mammalian cells) and over time in mammalian cells as well as in the whole organism context using an animal model (the nematode) that is ideal for the study of age-dependent changes in proteostasis. Success in this central objective will enable us to measure the latent buffering capacity of the quality control networks under normal and stressed conditions. In turn this will enable us to gain insight into how cells respond dynamically to stresses, how resilient cells are to such stresses, and how quality control systems become degraded or overwhelmed in disease contexts and upon ageing.
Our team bridges four disciplines needed to bring this system to light. Hatters (Australia) brings experience in biosensor design and molecular biology. Ebbinghaus (Germany) is the co-inventor of a temperature jump microscope, which will be employed to extract in-cell kinetic and thermodynamic folding information about the biosensor that will be used to understand proteostasis efficiencies. Dickson (United States) is a computational expert in protein-chaperone interactions and will build coarse-grained models of proteostasis. Nicholas (Australia) has experience in nematode biology to implement the biosensors in the nematode to study proteostasis upon ageing.
Our specific goals are to:
1. Build a biosensor and mathematical framework that can measure proteostasis buffering capacity in living cells.
2. Define the proteostasis buffering capacity within different organelles of mammalian cells.
3. Quantitate proteostasis changes upon ageing in a nematode model.

According to the World Health Organization, about 600 million people around the world are obese. Not only does obesity affect developed countries, it is also becoming a major problem for low and middle-income countries. Obesity results from an increased accumulation of lipids within adipose tissue. Triglycerides are stored in lipid droplets, ultimately leading to adipocytes (ADs) hypertrophy, altered hormonal release and adipose inflammation. Hence, obesity contributes in a major way to the burden of diabetes and cardiovascular diseases (metabolic syndrome).
Growing evidence indicates that static stretching promotes adipogenesis, possibly relevant to sedentary lifestyle, while dynamic stretching or vibrations, as occurring for instance during exercise, have the opposite effect. Altogether, these observations suggest that mechanical force has a major impact on the ability of ADs to accumulate lipids, with differential responses to specific types of mechanical stress. ADs are characterized by a unique ability for volume expansion upon triglyceride accumulation, increasing their size by more than 30-fold with a marked enhancement in effective cell stiffness. Consequently, within adipose depots, hypertrophic ADs generate a mechanical stress transmitted to resident cells. We postulate that a positive mechanical feedback loop acts in the process of adipogenesis and influences hormonal production. Using a combination of transdisciplinary approaches, including soft matter physics, cell biology, biophysics, physiology, pharmacology and clinical observations we will investigate the molecular basis of adipose cells mechanosensitivity. Important questions need to be addressed: What are the mechanical forces at play in adipose tissue? Can we measure tension generated within adipose depots in vivo? How is mechanical stress transduced at the molecular level in adipose cells? Does mechanical stress impact hormonal production and adipose inflammation? We will investigate the functional role for candidate mechanosensors, including the mechanosensitive ion channel Piezo1, the adhesion molecules integrins/talins, as well as the nuclear protein lamin-A in adipose plasticity and function. In conclusion, we will provide novel insights into the mechanobiology of adipose tissue, with expected practical perspectives for the treatment of obesity.

Small regulatory RNAs carry out diverse functions in both bacterial and eukaryotic cell physiology. Despite a known role for sRNAs in ribosome biogenesis in eukaryotes, the potential role for sRNAs in bacterial ribosome biogenesis and/or (pre-) ribosome degradation and storage under stress conditions is unknown. Based on their functional properties and versatilities I hypothesize that small RNAs are involved in bacterial ribosome biogenesis. Using a combination of bioinformatic, cell biological and biochemical techniques in vitro and in vivo, I aim to identify small RNAs with physiologically relevant functions in ribosome assembly either as assembly co-factors or as regulators of ribosome biogenesis in response to environmental changes. Moreover, I will investigate at which conditions the identified sRNAs regulate ribosome biogenesis, which interaction partners are involved, which mechanisms are employed and which consequences result from sRNA function. To determine the influence of sRNAs on folding and processing of rRNA, I will use an in-vitro co-transcriptional assembly system.
In parallel, I aim to identify artificial small RNAs that could be used as effectors of ribosome biogenesis or as probes to monitor co-transcriptional folding of ribosomal RNA and thus the kinetics of ribosome assembly in mechanistic detail. The work will provide physiological and mechanistic insight into the function and regulation by sRNAs in ribosome biogenesis as well as into co-transcriptional rRNA folding.

While the fundamental functions of sleep remain unknown, deficits in learning and memory after sleep loss are shared between humans and genetic model systems, including the fruit fly. My past research has found that sleep strongly modulates the consolidation of recently learned associations into long-term memories - sleep deprivation after learning prevents memory consolidation, and acutely inducing sleep enhances the consolidation of newly acquired memories that would typically be forgotten. While these data demonstrate an important role for sleep in memory processing, the molecular processes that occur during sleep to support memory consolidation remain unknown. To date, the complexity and parallel structures of memory-encoding and sleep control circuits in mammalian systems prevent researchers from properly investigating these mechanisms. In the fruit fly, however, memory-encoding circuits in the Mushroom Body and sleep control neurons in the dorsal Fan-shaped Body are accessible for in vivo whole cell recordings and genetically addressable at the resolution of individual neurons. The proposed project will 1) use in vivo patch clamp recordings to examine the neural circuits that promote sleep during memory consolidation, and 2) profile actively translated mRNAs in memory-encoding circuits to identify proteins that facilitate memory consolidation during sleep. Understanding and controlling these mechanisms will not only provide insight into the neural functions of sleep, but support the development of tools to offset the cognitive consequences of sleep loss and to influence whether recently acquired memories are retained or forgotten.

Viral entry into its host cell may results in either successful or abortive infection. Abortive infection of permissive cells, which are able to support viral replication, is rarely discussed but is quite abundant. What makes one cell resist to infection while its neighbor succumb to it is the subject of this research proposal. This is important as it could lead to the discovery of novel cellular anti-viral modalities and deepen our understanding of host-virus interactions.
My goal is to characterize the anti-viral response to viral infections in single cells, its variability among genetically identical cells and how this relates to infection outcome (successful or abortive).
To do so, I will learn and implement new technologies developed by the Tay lab. The success of the project relies on using a combination of live-cell imaging with in-lab fabricated microfluidics devices that allow the retrieval of single cells with known fates for further analyses, such as single-cell RNA-sequencing. By using viruses that express fluorescent markers I will be able to monitor infection in living cells in real-time, extract and molecularly profile cells in specific time points, for example in the process of aborting viral infection (when fluorescent proteins level stop raising or begin to drop). The results from this screening approach will be validated using standard cell-biology and virology techniques. I also plan to use this technology to investigate the response of the known anti-viral pathways.
I expect to reveal new molecular participants in the anti-viral response as well as to explore the variability in the response among single cells and how it affects infection outcome.

The purpose of this proposal is to recreate an ancient living system, an organism that uses RNA catalysts, ribozymes, as part of its translation apparatus. We will achieve this grand goal by progressively reintroducing ribozymes into cellular metabolism, ultimately replacing protein enzymes. To this end, Hiroaki Suga will develop novel ribozymes, Flexizymes, that can charge tRNAs with amino acids. Andy Ellington will adapt Flexizymes for use in cells, and replace cognate aminoacyl-tRNA synthetases. Michael Jewett will adapt Flexizyme charging to an orthogonal ribosome that can specifically utilize Flexizymes. Both Ellington and Jewett will hand organisms containing Flexizymes and orthogonal ribosomes to Philippe Marliere for high-throughput evolutionary adaptation and optimization. Ultimately, this will result in the creation of an organism that serves as a doppelganger for ancient living systems in transition from an RNA to a protein world. This ribo-organism will be unique in that it will have two translation apparatuses operating side-by-side, one of which has the normal complement of cellular machinery, and one of which has Flexizymes, orthogonal tRNAs, an orthogonal ribosome, and a new genetic code. We will initially carry out our goals by altering the machinery for the incorporation of histidine, but eventually expand the genetic code to unnatural amino acid analogues of histidine, 1,2,4-triazole-3-alanine (T3A) and alpha-hydroxy histidine (AHH), in order to highlight potential biotechnology applications. The incorporation of these new amino acids into the genetic code, both as 21st amino acids (via suppression) and in competition with an existing amino acid, histidine (via missense incorporation), should serve as a modern experimental surrogate for the ancient establishment of the genetic code. This project is also notable for its broad interdisciplinary flavor, in that it spans from a chemist (Suga) to a biochemist (Ellington) to a bioengineer (Jewett) to an evolutionary biologist (Marliere). Each individual field touches on the other, but the overall arc traverses a much broader swath of science and technology than would otherwise be possible. The intermingling of distinct cultures is also apparent in the scope of the work, which for the first time attempts multi-scale evolutionary optimization of an entire cellular sub-system, translation.

Mammals have the ability to generate an infinite variety of motor behaviors, from simple actions such as walking to highly complex movements like object manipulation or speech. The flexibility of the motor repertoire is essential for animal survival because it enables the generation of goal-directed movements adapted to environmental constrains. Some motor patterns are present at birth while new motor skills can be acquired through training and experience. Not surprisingly, communication among numerous brain areas is needed to ensure accurate acquisition and execution of motor programs. The classical model for motor learning proposes that only a subset of structures along the motor command pathway within forebrain and cerebellum are subjected to activity-dependent adjustments and become reorganized during acquisition of a new skill. In contrast, downstream motor regions located in the brainstem are considered to be simple executive centers for stereotyped motor behaviors. Here I challenge this model and propose that learning-induced plasticity in brainstem motor circuits is essential to adapt the executive motor signal to drive new skills. To evaluate my hypothesis I will combine cutting-edge viral and mouse genetic tools together with in vivo and ex vivo electrophysiology to characterize how learning-induced signals are encoded in the brainstem. This proposal supports a new conceptual framework in which plasticity mechanisms along the entire motor command pathway including brainstem circuits, underlie the formation of motor memories.

The segmented body pattern of vertebrates is established in the early development through the periodic formation of somites, the vertebrae precursors, from the presomitic mesoderm (PSM). The rhythmic formation of somites is under control of a molecular oscillator, the segmentation clock, which controls the cyclic transcription of a group of genes within the PSM.
Previous microarray analyses and candidate gene approaches identify many cyclic genes in the PSM, mostly genes associated to the Wnt, Notch and FGF-signaling pathways. Yet, a comprehensive, dynamic and quantitative analysis of the transcriptional dynamics and regulation of cyclic genes is still lacking, mostly due to the challenge to obtain samples with unequivocal time resolution. By using a microfluidic system that allows controlling the rhythm and, thus, synchronizing samples in relation to an external reference time, we are now able to obtain nearly unlimited PSM cells accurately staged. With a combination of high-throughput sequencing methods, we plan to identify unknown cyclic genes and regulatory elements (enhancers/promoter) with a role in somitogenesis. We aim to establish a temporal relationship between genes and their regulatory regions, and identify molecular mechanisms that control the rhythmic formation of somites. For example we may discover that cyclic expression relies on the periodic interplay of multiple enhancers. Or, that it is caused by cyclic epigenomic modification within regulatory elements. Additionally, by identifying sequence motifs within these elements we will be able to connect signaling pathways to oscillatory gene expression.

Although the basis of nucleotide binding and oligomerization domain (Nod) receptors signaling has been discussed for several years, the molecular details that underlie this process are still being uncovered. Moreover its presence on brain neurons hasn’t been described yet. The role of Nod2 receptor goes beyond simply sensing the presence of a potential pathogen and activating the immune system. Its activation has been linked to stem cells cytoprotection, analgesic effects, protection from cerebral ischemia-reperfusion damage and altered sleeping patterns. Moreover, mutations on this gene have been associated with bipolar disorder and schizophrenia in humans. The cytosolic Nod2 receptor can be activated by muramyl dipeptides a product of the degradation of peptidoglycan, a polymer present in almost all bacteria cell wall and increasing evidence demonstrate that endogenous ligands like damage-associated molecular patters, can also activate Nod receptors. Particularly, not much is known about the impact of Nod2 activation on behavior or the mechanisms and the specific effects on brain physiology. Understanding the role of Nod2 on brain neurons can lead to better understanding of many neurological disorders. Preliminary data from Dr. Lledo lab have shown, the presence of Nod2 receptors in neurons from the striatal and thalamic areas of the brain. They also observed that Nod2 knock-outs show less anxiety, more social aggressiveness and lack of discrimination of olfactory cues in social interactions and that Nod2 signaling can selectively disrupt paradoxical sleep. Therefore the purpose of this proposal is to assess the impact of Nod2 signaling on brain activity and behavior.