Sophisticated models exist for the evolutionary pathways by which proteins have evolved in Nature over billions of years to form an impressive diversity of structure and to carry out many functions with unrivalled efficiency. Directed protein evolution in the test tube can emulate natural evolution, but is often limited by low hit rates and small improvements during evolution cycles. Burdened by their evolutionary history, proteins often show low evolvability, due to loss-of-function and/or structure or epistatic ratchets (ie deleterious or permissive mutations that only become beneficial upon occurrence of further mutations). Given the danger of getting lost or stuck in sequence space, the question arises how natural and forced evolution avoids dead ends. We will create a mathematical model to simulate the effects of mutational robustness on evolvability using parameters that can be adjusted to experimentally determined values for neutrality and accessibility of novel phenotypes. Based on experimental measurements of the dynamics of fitness landscapes of a protein population during evolution cycles we aim at insight into the fundamental as well as an effective basis for protein engineering by evolution. Investigations of duplicated specialized proteins (paralogs) and their ancestral precursors showed the latter often to be promiscuous (i.e. to have multiple functions) and more highly evolvable, suggesting that evolution may proceed by shifting the relative balance of two functions along paths of successive point mutations. We probe the evolution dynamics of promiscuous members of the metallo-ß-lactamase and alkaline phosphatase superfamilies. Ancestral sequences will be computed from alignments and databases as starting points for evolution to give a dynamic experimental picture of fitness of a protein population in response to the ancestor sequence and to selective pressure. Such distribution functions will be gathered by ultrahigh-throughput screening of >10^6 clones in picolitre droplet compartments during in vitro and in vivo evolution cycles, followed by systematic characterization of multiple fitness traits such as expression level, stability or reaction kinetics for multiple activities. These approaches will enable a dynamic picture of protein evolution at various levels from transition state recognition, protein biophysics to population genetics.

Our goal is to study physiologically important components within the voltage-gated sodium (Nav) channel signaling complex that may cause disorders when mutated, and to resolve their mechanisms of action at the molecular level. In particular, we will focus on vital members of the Nav channel signaling complex that do not contribute to the ion-conducting pore. Reflecting their medical importance, mutations found in several of these proteins have been implicated in various epilepsy syndromes, long-QT syndrome (LQTS), LQTS-associated Sudden Infant Death Syndrome, and Brugada syndrome, possibly through dysregulation of the Nav channel signaling complex in the brain and the heart, respectively. Little is known about the underlying machinery that governs the influence of these elements and their mutants on the functional properties of Nav channels. To elucidate the mechanisms by which these proteins influence Nav channel function, we will combine innovative biophysical techniques with molecular biology and animal toxin-based pharmacology, state-of-the-art biochemical approaches, mouse genetics, and X-ray crystallography. Successful completion of our aims will help define the function of the larger Nav channel signaling complex as found in humans, which is essential for developing new strategies that can correct for abnormal behaviors.

We address a key question in developmental biology: how does an organism reach its final size and shape, in the face of stochastic variation at the cellular level? Previous research has focused on mutants and conditions that affect the global size and shape of organs, enabling the discovery of a large number of regulators. However, most analyses have considered only average cell behaviors, overlooking local heterogeneity and stochastic variation. In this context, how the final size and shape of an organism are determined remains poorly understood.
Here we adopt an orthogonal strategy by taking advantage of the observation that many organisms have a remarkably consistent shape, yet at the cellular level, cell growth and shape can be highly variable. Our goal is to resolve this apparent contradiction between the cellular and organismal levels. We propose that cell-cell communication mechanisms coordinate cell stochastic variability so as to yield consistent organs. Since growth remains locally heterogeneous, these mechanisms may, counter-intuitively, maintain or even enhance local heterogeneity.
We will test this hypothesis in Arabidopsis thaliana, as it produces a large number of almost identical flowers with stereotyped floral organs. We choose to work on the sepal, which is accessible for live imaging and mechanical measurements. Using a combination of experimental and theoretical approaches, we will identify mutants with variable sepal size and shape. Because of their role in coordination, the corresponding genes are likely to include the hormonal/nutrient pathways and mechanical sensing and response. As plant growth is driven by the internal turgor pressure and restrained by cell walls, we will assess the activity of these growth regulators by quantifying the mechanics of cells in these mutants and in wild type to causally link gene activity and shape. We will use data-driven approach and information theory to extract the underlying patterns of correlations between cells, notably to qualify the most plausible coordinating mechanisms that regulate cell stochasticity. These hypotheses will be tested by combining a mechanical model of sepal growth that incorporates identified signals and local experimental perturbations, such as cell ablation, force application, and genetic mosaics.

Alternative splicing of pre-mRNAs allows the synthesis of multiple gene products from a single gene and therefore dramatically expands the eukaryotic proteome, especially in mammals. The task of splicing is achieved by the spliceosome, a highly complex and dynamic macromolecular machinery. The molecular mechanism of alternative splicing has been studied intensively, however a lack of information on kinetic data has hampered a comprehensive understanding of this dynamic process and its regulation.
Here I propose to study the kinetics of human spliceosome assembly and their regulation, which are the key to deepening our understanding of the molecular mechanism of alternative splicing. I specifically plan to establish colocalization single molecule spectroscopy (CoSMoS) in human nuclear extracts to follow single splicing events. CoSMoS will enable me to follow the comings and goings of components of individual spliceosomes in real time. I will also apply this technique to study how and at which steps spliceosome assembly is regulated during alternative splice site selection. For this, I will use a combination of CoSMoS and single molecule fluorescence resonance energy transfer (smFRET) to follow spliceosome assembly simultaneously on the different splice sites of an alternatively spliced pre-mRNA. Taken together, my project will yield insights into spliceosome assembly in mammals and the mechanism of alternative splicing.

The fundamental physical processes determining microscale transport of constituents to and from a coral’s ciliated surface are largely uncharted and their exploration will strongly benefit from an interdisciplinary effort, integrating quantitative modeling of transport and accurate microscale observations. I propose to focus on the origin, characterization and consequences of the ubiquitous ciliary flows recently discovered in my future host lab at MIT. I will undertake the first quantitative study of such flows, using digital holographic microscopy to acquire 3D, time-dependent data with micrometer precision. This will facilitate tracking bacterial pathogens as they target their coral host, yielding unprecedented access to investigate the physical mechanisms responsible for the emergence of the collective ciliary motion, the spatiotemporal nature of these flows, and their effect on mass transport and bacterial motility. I will integrate this experimental effort with the development of a mathematical model that incorporates ciliary flows into the prediction of mass transport and bacterial recruitment to the coral surface. Modeling and observation will be synergistic, providing an ideal approach for hypothesis testing. Specifically, I will determine the circumstances (e.g., times of day; regions on the coral surface; external flow conditions) in which the coral pathogen Vibrio coralliilyticus is most favored in locating and reaching the surface of its coral host, Pocillopora damicornis. This will provide valuable insight into the mechanistic processes underpinning coral health, and its generality will facilitate applicability to a wide range of cilia-driven flows.

Except for the nucleus, mitochondria are the only organelles in our cells that contain their own genome. Acquired genomic aberrations in mtDNA lead to mitochondrial dysfunction, a chief cause of neurological and aging diseases. mtDNA mutations, ranging from single-base substitutions to large-scale deletions, are also found in high frequency in many tumors, and recent experiments have established their role in driving metastasis. While the underlying basis for mtDNA deletions is unknown, two scenarios can explain their formation – infidelity of DNA replication machinery or errors in DNA double-strand break (DSB) repair.
The overall goal of our proposed study is to elucidate the molecular mechanism of mtDNA instability by deciphering both facets of mtDNA metabolism – replication and repair – and test if their deregulation causes deletions in the mitochondrial genome. Then, we will directly study the impact of the most common mtDNA deletion on cancer progression.
We will combine a single-molecule DNA combing approach with super-resolution microscopy to unravel the replication profile of mtDNA and highlight specific regions that might stall replication forks, causing replicative slippage and subsequent sequence loss. Biophysical analysis will be carried out to emphasize the structural nature of these deletion-prone regions. Using these molecular and biophysical tools, we will then address the genetic basis for misregulation of mtDNA replication in cancer cells.
The prevalence of short repetitive sequences at the junction of mtDNA deletions prompted us to ask if they are due the activity of alt-NHEJ, a recently identified pathway of DNA DSB repair that is predominantly driven by sequence-homology and often associated with significant sequence loss. To address this question we plan to manipulate the mitochondrial genome using TALE-technology (Transcription Activator-Like effector) both to incorporate reporter plasmids into its DNA and induce targeted DSBs. This innovative approach will allow us to investigate the DSB repair pathway(s) operating in the mitochondria and test if alt-NHEJ is the predominant pathway in deletion-bearing cells. Once the basis of the deletions is underscored, we will recapitulate the most common mtDNA deletion in human cells by coordinated cleavage with two TALE-nuclease and directly assess its contribution to tumor progression.
Despite the profound impact of aberrant mtDNA on cellular function, mitochondrial genomic instability remains largely an uncharted territory of cancer research. Our proposed study surpasses the abundance of descriptive literature to firmly establish the role of deletions in tumor progression. More importantly, it will highlight the underlying mechanism behind their formation, a critical step towards predicting and preventing their accumulation in cancer cells.

Cohesin complexes are required for holding together sister chromatids during DNA replication, repair of doubled-strand breaks, and chromosome segregation in cycling cells. More recently it has been shown that cohesin contributes to the control of gene expression in non-dividing cells. In mammalian cells, the study of the specific effects of cohesin deficiency in transcriptional control, independently of its role in the cell cycle, requires an approach which would allow temporally-controlled cohesin depletion and restoration in non-cycling cells. We propose the use of a proteolytically-degradable cohesin subunit, in combination with regulated TEV (tobacco etch virus) protease activity, Cre transgenes and conditional cohesin alleles to investigate: 1) if cohesin is required for the regulated expression of pluripotency genes in mouse embryonic stem cells, independently of its essential role in the cell cycle; 2) the developmental potential of T cells generated from non-cycling precursor thymocytes in the absence of cohesin; and 3) whether functional defects that arise in cohesin-depleted post-mitotic neurons can be reversed by restoring cohesin expression. In humans, cohesin deficiency causes Cornelia de Lange Syndrome, which is a rare multiple malformation disorder distinguished by growth and cognitive retardation, and abnormalities of several tissues and organs. Our study will help to gain insight into the molecular pathology of this disorder, which could lead to novel approaches for its management. Additionally, it should reveal unique aspects of cohesin function in gene regulation and development.

The contractile cortex is a thin actomyosin network that subtends the membrane of animal cells. The cortex is the main determinant of animal cell shape. As such, it plays a key role in processes such as cell division, where morphogenesis is driven by cortex remodelling in response to cell-cycle dependent signalling. In spite of its physiological importance, our knowledge of the cortex remains poor. In the first grant, we have established a multidisciplinary collaboration that allowed us to uncovered the proteic composition of the cortex and identify the two key proteins that nucleate actin in the cortex: the Arp2/3 complex and the formin Diaph1. This led to the proposal that the cortex is a composite network, which mechanics can be fine-tuned by regulating the relative contribution of the two main nucleators.
The aim of this project is to build on these findings to investigate the control of cortex mechanics during the cell cycle. Taking a mutiscale approach, we will explore how changes in the activity and abundance of cortical proteins during the cell cycle modulate the network architecture of the cortex, and how this, in turn, gives rise to whole cell mechanics. Building on the first grant, we propose to systematically examine how cell-cycle dependent signalling influences cortex composition and cortical protein activity, and ultimately cortex mechanical properties.
As the cortex is an intrinsically mechanical structure, its biological properties cannot be understood in isolation from its mechanics. In particular, our preliminary results indicate that the dramatic cell shape changes occurring during the cell cycle are driven by changes in cortical mechanics. Cell cycle-dependent modulation of the activity of actin nucleators provides a potential mechanism. Therefore, having identified the cortical actin nucleators, we will determine how changes in their activity affect cortex architecture and mechanics. Coupled biochemical and proteomic studies will search for signalling pathways altering nucleator activity. In vitro studies will examine how actin networks assembled by different nucleators interact and if tension can modulate nucleation activity. Electron microscopy and subdiffraction imaging techniques will enable us to characterise cortex architecture, while Atomic Force Microscopy will be used to characterise cortex mechanics. Furthermore, we will use proteomics to identify proteins whose abundance and activity vary most during the cell cycle. Using these results, we will determine key cortex regulators by systematically depleting/overactivating each in turn and measuring the resulting changes in cortex architecture and mechanics. Our integrated interdisciplinary approach will allow us to uncover how signalling regulates cell mechanics through modulating cortex architecture.

One of the most important unresolved questions in neuroscience is how memories are encoded and stored in the brain. Memory is a multi-step process, in which the acquisition phase begins as the animal forms a new memory. The nascent memory is then converted to a stable state through a process called consolidation. Later on, when the hardwired memory is reactivated, it transiently returns to a labile state and requires a second process of reconsolidation in order to persist. Therefore, retrieving a memory not only reactivates it, but also subjects it to further modifications such as extinction or formation of new memories. These intriguing observations give rise to questions regarding how neural circuits operate during these events. First, which neurons are part of the discrete network that is involved in encoding a given memory. Secondly, when the memory is retrieved, are the same neurons involved in the original training get reactivated? To address these questions, I will perform in vivo two-photon calcium imaging, using a genetically encoded calcium indicator, GCaMP5, in head-fixed mice during the performance of an auditory fear-conditioning task. Calcium imaging of hundreds of neurons simultaneously allows identification of precise neurons that are involved in encoding the given fear memory. In addition, I will track the same population of neurons over weeks to examine whether the same ensemble of neurons is being activated during memory retrieval. This research will close the gap between behavioral studies and underlying network plasticity, providing answers to some of the most fundamental questions of systems neurobiology.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with limited treatment options and dismal prognosis, urging the need to develop novel therapeutic strategies. Recent work from our group suggests that regulation of oxidative stress is crucial for pancreatic cancer progression, with Nrf2, a master transcriptional activator of cytoprotective genes, playing a central role. We have shown in an earlier publication that Nrf2 transcription is directly induced by oncogenic Kras to decrease levels of intracellular reactive oxygen species. The goal of this project is to delineate the molecular mechanisms by which Nrf2 promotes pancreatic cancer. We propose to generate mouse models in which the pattern and the level of Nrf2 expression can be manipulated in vivo, permitting evaluation of not only cell intrinsic versus non-cell autonomous functions of Nrf2, but also the dose and duration of activation that is relevant for disease progression. Complementary to this approach, we also propose to biochemically interrogate the molecular mechanisms by which Nrf2 promotes pancreatic cancer. This will involve identifying the most prominent Nrf2 transcriptional target genes in PDAC by performing ChIP-Seq on freshly isolated murine and human tumors. In addition, we also aim to identify proteins that carry out effector functions of Nrf2 by characterizing the global cysteine proteome of PDAC tumors. The less oxidized cytoplasmic environment following Nrf2 activation will reduce thiol groups on cysteine containing proteins, thereby activating such molecules, which will be likely candidates to carry out the effector functions of Nrf2 to promote pancreatic cancer.

Arabidopsis lateral root development is an excellent system to understand spatio-temporal regulation of stem cell fate. Oscillating-auxin-dependent priming predetermines stem cell fate of pericycle cells, although these primed cells initiate cell division only when they reach elongation zone of roots. Currently, it is unknown why this early marking of pericycle cell fate is required. Here, I propose that this early marking secures the transcriptional competence of primordium-specific genes against assembly of repressive chromatin marker. To confirm this idea, specific labeling, tracing, and isolation of primed pericycle cells as they develop into lateral root primordia for comparative analysis of epigenome and transcriptome is required. Currently, there are no primed-pericycle specific marker genes. By integrating microarray data, I will isolate marker genes that are expressed at early pericycle in phase with oscillating priming signal (auxin). Activation of ubiquitous GFP expression by primed-pericycle specific inducible recombinase allows tracing of recombinant pericycle cells. Following enrichment of recombinant cells by FACS at priming, founder cell, and primordium initiation stage, integration of 1) transcriptome, 2) histone 3 lysine 27 trimethylation (H3K27me3) and H3K4me3, 3) DNA methylation, and 4) H2A.Z profile in these cells would identify early epigenetic markers for the transcriptional competence of primordium-specific genes. This project should expend our knowledge on the epigenetic regulation of transcription competence during stem cell development that aid efficient engineering of stem cells.

During neuronal differentiation, spherical progenitor cells develop into asymmetric neurons with one axon to send signals to, and multiple dendrites to receive signals from, other neurons. The actin cytoskeleton, in concert with the microtubule network, plays a crucial part in this process. However, our understanding of cytoskeletal regulation remains fragmentary.

The actin-related protein-2/3 (Arp2/3) complex is of key importance as the only known regulator of actin filament branching, enabling the formation of complex, highly connected actin networks. Previous studies provide conflicting evidence regarding the role of Arp2/3 in the nervous system and Arp2/3 function has yet to be investigated at all in a physiological context. The aim of this project is to dissect the precise role of Arp2/3 in neuronal differentiation. With a conditional Arp3 knockout mouse available for the model system, immunohistochemistry techniques will be used to investigate the effect of Arp2/3 complex ablation upon neuronal organisation and morphology. After characterising the neuronal phenotype of Arp3 knockout mice, the mechanisms of Arp2/3 action will be explored using primary neuronal cultures in combination with live-cell imaging techniques and electron microscopy. A clearer understanding of the role of Arp2/3 in neuronal differentiation will offer valuable new insights into the regulation of the actin cytoskeleton in general during vertebrate nervous system development.

A challenging issue for bacterial pathogens is the need to modulate host cells behavior to allow their own successful propagation. Intracellular pathogens secrete a large number of proteins (“effectors”) into their host cells to either trigger entry, escape cellular defense, modulate cellular responses or interact with intracellular components that are critical for infection. We hypothesize that pathogens also secrete small RNAs that interact with host cellular mRNAs and non-coding RNAs, thus modulating cellular activity for their own benefit. Discovery of such RNAs among the vast amount of cellular RNAs is highly challenging due to lack of proper techniques; however, the emergence of ultra-highthroughput sequencing and computational inference (Sorek’s expertise), in combination with single-cell fluorescence Imaging techniques (Palmer) and extensive expertise in pathogenesis and cell biology (Cossart) will allow us to address this challenge. Our aim is to identify secreted RNAs using the model intracellular pathogen Listeria monocytogenes, and understand their roles in pathogenicity. Although scientifically 'risky', if successful these studies could lead to a major breakthrough in the fields of bacterial pathogenicity and RNA biology.

Phosphorylation serves as a nearly universal regulation of protein function. However, it is paradoxical that the phosphorylation of a number of extracellular, secreted proteins has been reported while most of the known kinases are localized in the cytoplasm or nucleus. In an attempt to elucidate how extracellular phosphorylation occurs, our laboratory recently identified a novel family of atypical kinases that are mainly localized in the ER and Golgi lumen and are secreted. We found that one family member, Fam20C, is the original Golgi casein kinase that phosphorylates a number of secreted substrates and regulates biomineralization. However, little is known about the cellular function and substrates of other family members. In this proposal, I plan to study the function of another family member, Fam198B, which is highly conserved in vertebrates and the deficiency of which likely results in impaired glucose metabolism in mice. I will characterize the kinase activity, cell line- and tissue-distribution and membrane topology of Fam198B. I will also attempt to identify substrates of Fam198B using multiple approaches, including peptide array library screening, quantitative phosphoproteomics and bioinformatics. Finally, I will study the in vivo function of Fam198B using mouse models, focusing on the potential metabolic dysregulation and phenotypes related to potential Fam198B substrates. I believe my study on Fam198B will greatly advance the understanding of this secreted kinase family and extracellular phosphorylation in general.

Neuronal differentiation is a fundamental process for the formation of neuronal circuits within the developing brain. In neurons the microtubule cytoskeleton plays an essential role during the different phases of neuronal differentiation. In most animal cells, the centrosome is the major microtubule organizing center (MTOC) coordinating cell division, motility and polarity. In neurons the role of the centrosome as a MTOC is highly debated, particularly during later stages of differentiation when the centrosome is thought to become inactivated. Thus, it remains unclear what the contribution is of centrosomal vs acentrosomal microtubule nucleation during neuronal development. The proposed project is aimed to investigate the role of centrosome inactivation in neuronal polarization and differentiation. Using a combination of different biochemical, molecular and cellular biology techniques and cutting-edge live imaging microscopy in hippocampal neurons we will address the role of the centrosomal and acentrosomal nucleation during neuronal development. In this application we propose the following objectives: 1) Determine how and when the centrosome becomes inactivated in hippocampal neurons, 2) Address the consequences of increasing centrosome number and changing their localization in neuron polarization and 3) Investigate whether non-centrosomal microtubule nucleation occurs at the Golgi and how it may be related to neuron polarization.

While chromosomes fill the nuclear space as decondensed chromosome territories during interphase, they are dramatically re-structured into compact rod-shaped rigid bodies during mitosis. The aim of my work is to elucidate the molecular mechanisms underlying this cell cycle-dependent chromosome reorganization. Unlike previous studies, I plan to focus on a yet poorly characterized chromosomal domain defined as ‘chromosome periphery’. I first plan to establish a detailed map of the proteins that target to the chromosome periphery in human cells, applying both high-resolution three-dimensional microscopy and superresolution fluorescence imaging techniques as well as molecular biology techniques, including chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq) analysis. The combination of these approaches will provide me with information on different scales, first at the cell biological level and second at the DNA sequence level, to obtain a complete picture of the localization of the proteins that may organize the chromosome periphery. I will then investigate the functional relevance of chromosome periphery components in mitotic chromosome morphogenesis by RNAi screening in tissue culture cells. I expect that this work will provide insight into how a protein network sets up a chromosomal domain and how such a domain can contribute to the formation of mitotic chromosomes.

Clathrin-mediated endocytosis (CME) leads to the internalization of receptor-ligand complexes and nutrients. In a multistep pathway, modules of proteins are recruited to the plasma membrane and begin to form clathrin-coated vesicles (CCV). This process is initiated by proteins that bind to phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2). The role of PI(4,5)P2 during CME has been studied extensively. However the roles of other lipids such as phosphatidylethanolamine (PE) or sphingolipids (SL) have not been investigated at similar depth. However, indirect evidence supports an endocytic role for these lipids. Impairing SL synthesis slows transferrin internalization. Furthermore, PE enhances endocytosis in living cells and clathrin coated pit assembly on liposomes. Here, I propose to dissect the role of these lipids in CME. I will first identify the lipid requirements for CME by comparing the lipid composition of the plasma membrane to that of CCVs by mass spectrometry. I hypothesize that PE will be enriched in the CCV fraction whereas SL will be absent. This approach will be useful to decipher the role of the lipids in CCV formation. Based on the information provided by these experiments, I propose to develop an in vitro assay in collaboration with Pr. Fletcher at Berkeley to elucidate lipid requirements for CCV formation. This assay will also be used to investigate the consequence of lipid localization in the inner vs. outer leaflet on CCP formation. I propose also to test the biological relevance of these results in vivo in genome-edited cells lines developed in the host lab. The proposed research promises to make important contributions to understanding CME.

Believe it or not, the white shark on “Shark Week” is at a disadvantage: whereas your bones house a microscopic universe of cells, working in concert to fix microscopic wear and tear we cause every day, the cartilage of shark skeletons can be added to, but not repaired. Think of it this way: with bone, you upgrade components just as they go out of style or fail, but with cartilage you’re stuck working with the same old model you started with (explaining why my knees hurt). But sharks and other cartilaginous fishes use a curious work-around: although their entire skeleton is cartilage —not unlike what you’d find on spareribs at the dinner table— it’s reinforced with a thin, outer shell. The shell itself is striking under a microscope: thousands of geometric blocks called ‘tesserae’ made of the same mineral as in our bones and fitting together like floor tiles to wrap the entire skeleton. Although tesserae were first observed more than 100 years ago, we still have no idea what they are actually doing as these spectacular fish swim, bite, and glower on our TV screens. We’re curious about the mechanical role tesserae play in skeletal performance. To get a true feel for how tesserae manage and distribute forces, we plan to make our own shark bits: imitation skeletons scaled up for easier mechanical testing. Using high-resolution engineering and materials science tools, we will characterize geometries and tissue properties that define tesserae of a model species, and then use those features and a state-of-the-art 3D, multi-material printer —typically for industrial applications and capable of printing physical models with rigid and flexible parts— to manufacture tesseral mats that can be pushed, pulled, and fractured in ways that echo biological conditions. Like any design process, when the model doesn’t work, we’ll return to the source (the fish) for a deeper understanding of the template; once the parameters are clear, we can apply our models to the wide range of tesserae shapes and sizes in different species with different ecologies. This will shine a light on the functional role of this biological tiling, but also point to generalizable features useful for manmade tiled composites. Given that sharks live long lives and have existed for millions of years with irreparable, tiled skeletons, we have much to learn: why replace the old model if it never breaks?

My research has three complementary aims:

Aim 1: Develop a high-resolution temporal map of transcription factor binding in pluripotent precursor population (the mesoderm) and three of its derived tissues.

I will use isolate 2-5 million mesodermal nuclei at 5 equally spaced time points starting prior to mesodermal invagination but before mesodermal tissue differentiation. I will isolate tissue specific nuclei from three mesoderm-derived tissues (visceral muscle, heart muscle, and somatic muscle) after these cells are specified, at 8 time points after these tissues are specified. Three replicates representing different F1 crosses between inbred lines isolated from a natural population will represent biological replicates in this aim and form the basis for Aim 3. I will measure genome-wide DNase sensitivity in each chromatin sample using DNaseI-seq and infer transcription factor binding using DNase footprinting analysis.

Aim 2: Identify natural variation that impacts transcription factor binding at 2 timepoints with association mapping.

I will isolate mesodermal chromatin samples at T1 and T13 from 50 independent fly lines from a natural population and perform DNase-seq. Analysis of these data will allow us to identify cis-associated variants that modify DNase sensitivity and TF binding using previously developed QTL mapping approaches.

Aim 3: Track the effects of functional DNA variants identified in Aim 2 over the entire developmental time course with allele-specific analysis.

I will characterize the effect of individual SNPs that change TF binding over the full developmental time course using allele-specific analysis of the DNaseI data collected in Aim I. For each dsQTL identified in Aim 2, I will measure allele specific DNase sensitivity in the cis-region surrounding the dsQTL at each of the 13 time points. I will then characterize the effects of cis-regulatory variants on regional TF binding over time.

While inflammation has generally been seen as a consequence of microbial presence in wounds, it can also occur in sterile conditions, in which case it is usually triggered by ligands released by dying cells. While these two conditions call for different responses – microbial clearance or tissue repair – it remains unclear if and how organisms discriminate between these two processes. Neutrophils are usually among the first immune cells recruited during inflammation, either microbial or sterile. Given their rapidity of action and short lifespan, few studies have focused on the regulation of neutrophil function. Yet, while neutrophils are critical for microbe elimination and produce pro-inflammatory TNFa, they also participate in inflammation resolution and produce anti-inflammatory IL-10, so that regulation of these roles is likely to affect the course of inflammation. We here report that neutrophils elicited by microbial products rapidly upregulate TNFa when restimulated. Strikingly, endogenous ligands released during sterile inflammation, like necrotic cells or uric acid, can efficiently recruit neutrophils, but do not enable TNFa production. We thus hypothesize that neutrophil activation can be altered by recruitment into distinct microenvironments. In this proposal, we will solidify the nature of this priming phenomenon and test how neutrophils interact with the microenvironment of the inflammatory site. The regulation of neutrophil functions may enable the early distinction of sterile and microbial inflammation and allow the immune system to efficiently eliminate microbes while limiting unnecessary inflammation in the absence of pathogens.