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

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

Epigenetics is a complex, dynamic and largely mysterious layer of gene-expression regulation. Recent studies have shown that a large part of the breakdown in cellular control that is dysregulated in cancer is in vast changes in epigenetic regulation. In normal cells, each epigenetic marks define states of gene activation, transcription/RNA processing[1, 2], which in turn drive protein production to establish appropriate cellular functionality. Not only cancer cells use epigenetics as an engine of diversification, but part of my proposal’s driving assumption is that they become addicted to transient substates enabled by dysregulation. Cancers use dysregulation as a “search & evade” mechanism. They can search for states not normally accessible to healthy cells to “solve” an environmental insult or—as a corollary—they might rapidly be able to transition out of states that are susceptible to drug action, for instance. But this might come at a price. However, rather than viewing this as a robust trait that enables rapid evolution in the face of chemotherapy or immune detection, we see a fragile mechanism that –-at his heart-- can be the target of intervention.
The aim of this proposal is to apply CyTOF mass-cytometery and state-of-art RNA-sequencing protocols to (i) map the network of co-regulated epigenetic substates for specific cancer-cell populations on a single-cell level; (ii) correlate the transcriptomes of such populations to link epigenetic states to transcription modules to derive the control points that epigenetic dysregulation enables in cancer. A critical goal of this project will be the discovery of target networks against which drugs can be screened.

Polycomb group (PcG) proteins are important for silencing of key developmental genes in higher eukaryotes. Transcriptional polycomb repression is believed to involve changes in chromatin structure. Yet it remains controversial how PcG-dependent chromatin modifications serve as signal for chromatin compaction and epigenetic inheritance of gene silencing. To gain insight to the molecular mechanisms of polycomb gene silencing, I propose to investigate the temporal order and epigenetic inheritance of PcG-induced chromatin modifications. Traditional genetic and biochemical analyses have mostly provided a static picture of PcG activity and failed to generate an understanding of the functional consequences of chromatin modifications. I will employ conditional control of PcG activity using synthetic small molecule ligands to induce H3K27me3 and determine the sequence of events, kinetics and epigenetic memory of this histone mark. Rapid and reversible recruitment of PRC2 will enable kinetic measurement of H3K27me3 at high resolution and form the basis for mathematical modeling of complex histone modification dynamics and patterns. This will lead to a better understanding of the mechanisms involved in establishment and maintenance PcG-dependent gene repression. Furthermore, tight temporal control of PCR2 activity provides a powerful tool to identify and discern novel modulators of initiation, spreading and memory of PcG-dependent gene repression. These studies aim to unravel the crosstalk between polycomb function and cell plasticity and ultimately help to incorporate epigenetic regulation into current transcriptional regulatory models.

It is well known that the context modifies the perception of a stimulus but the underlying neural mechanisms are unknown. My proposal aims to elucidate the neuronal circuits for the contextual modulation of visually evoked activity in the neocortex. Surround suppression is a classical example of how visually evoked responses in the cortex are modulated by the context within which the stimulus is presented. I aim to understand the physiological role of a large class of inhibitory neurons, the Vasoactive-Intestinal-Peptide expressing interneurons (VIP-INs), on surround suppression.
By combining two-photon imaging with specific Cre mouse lines, I will establish the visual responses of three genetically defined neuronal populations in visual cortex: pyramidal cells (PCs), that experience surround suppression, somatostatin expressing interneurons (SOM-INs), which inhibit PCs causing surround suppression, and VIP-INs that selectively inhibit SOM-INs.
I will then perturb the activity of VIP-INs through pharmacogenetic or optogenetic approaches, while monitoring the activity of SOM-INs and PCs through optical imaging and targeted electrophysiological recordings. These experiments will reveal whether, by modulating the activity of SOM-INs, VIP-INs affect surround suppression.
Finally, I will develop a behavioral task in awake head-fixed mice to determine whether the modulation of surround suppression by VIP-INs affects perception.
In conclusion, by integrating these electrophysiological, imaging and behavioral strategies, I will reveal the impact of a large group of inhibitory neurons, the VIP-INs, in the contextual modulation of visually evoked activity in the cortex.

Human cancers are susceptible to genomic instability, resulting in chromosomal abnormalities, such as duplications, deletions and translocations. Different types of tumors typically exhibit distinct chromosomal aberrations, but how these tumor-specific abnormalities affect the cellular phenotype and responsiveness to drugs remains largely unknown. In my research project, I aim to characterize the effect of tumor-specific chromosomal aberrations on the cellular response to anticancer drugs. First, I intend to screen human cancer cell lines against a large library of anticancer drugs and analyze the interactions between the tumor type, its recurrent chromosomal aberrations and its cellular response to the drugs. Second, I intend to introduce tumor-specific aberrations into human embryonic stem cells using the CRISPR/Cas genome engineering system, differentiate the cells into the relevant cell types and screen them against the drug library. By applying cutting-edge genome-editing, high-throughput screening and gene expression profiling technologies to these two in-vitro model systems, I hope to identify unique vulnerabilities of characteristic chromosomal aberrations in various tumor types. I will then study the genes and the pathways behind the selective drug responses, and will analyze clinical data to correlate the selective sensitivities with differential clinical outcome in human patients. This work may have profound biological and clinical implications: it will shed light on the cellular and molecular mechanisms underlying common aberrations in human cancer; and it will allow the stratification of cancer patients based on the cytogenetic status of their tumors.

Macromolecular protein assemblies exhibit phenomenal functional versatility, and have recently been suggested to conduct long-range (micrometer scale and above) electrical signals that drive biological functions. To understand how electrical conductance emerges from structure it is crucial to distinguish between different transport mechanisms. Shared structural properties with conducting polymers motivate me to explore long range conductance by phonon-assisted hopping mechanism. This mechanism is valid in elongated aromatic, pi-conjugated, regions where electrons are delocalized. However, in analyzing natural protein assemblies it is difficult to eliminate interfering influences due to the proteins’ and protein assemblies’ complexity and susceptibility to environment influence, e.g. structure integrity in measurement setups.
Instead of analyzing native complexes, we will construct artificial “de novo” ones as a model system. We propose to establish this platform using novel computational design tools for proteins and protein assemblies, based on the Rosetta modeling software, and realize an electron delocalized region using natural, aromatic amino acids. Through a design of an “ideal” assembly, we expect structure-conductance relation to be less susceptible to external environmental influences, allowing us to gain clearer insights on the structural elements that underpin conductance in protein assemblies.
The results of this research will give new perspective on electrical conductance in protein assemblies and possibly lead to fresh reevaluation of known natural protein assemblies to uncover hitherto-unknown basic biological functionalities.

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

Oligomeric lectins from bacterial toxins and animal viruses induce glycosphingolipid (GSL) reorganization on target cells, leading to the clathrin-independent construction of endocytic pits. These lectins have no sequence similarity, but nevertheless bind GSLs with similar geometries. I will bioengineer innovative 3D DNA objects to address the structural code underlying this apparently convergent evolution for a shared membrane mechanical function. I will systematically vary GSL binding geometry, and then submit the corresponding synthetic lectins to functional testing on cell and model membranes. My experimental data will be scrutinized by theoretical and computer modeling, which should in return suggest new experiments. I expect to uncover novel clustering mechanisms by which a previously unrecognized highly organized exomembrane structure is obtained, made from regularly spaced infectious disease particles in which specific geometries are optimally exploited for membrane bending.

Chronic inflammation of the gastrointestinal tract results in pathological conditions known collectively as Inflammatory Bowel Disease (IBD), affecting ~0.5% of the world population. However, only ~20% of IBD incidences are currently accounted for by genetic studies. Genome wide association studies suggest that genetic variants in many loci may account for the risk of initiation of IBD. The gut has many lines of defense against invasion of luminal microbes, which are thought to be the major cause for IBD, including the epithelial barrier, and the innate and adaptive immune responses. The traditional view of gut function suggests that intestinal epithelial cells (IEC) serve simply as a physical barrier, while immune cells, residing in the lamina propria, orchestrate mucosal immune homeostasis, and thus play a major role in IBD. However, I hypothesize that IEC may also have innate immune properties that affect the response to or tolerance towards different insults. Dissecting the relative contribution of different epithelial cells, immune cells and microflora in gut homeostasis is challenging; therefore it is necessary to study each component independently. Here, I will use a systems biology approach – combining genomic profiling, computational modeling and biological experiments – to dissect the innate immune role of IEC in mouse models and human clinical samples. I will identify a gene expression signature of IEC immune-effectors, and will test their function in mucosal immunity in vitro and then in vivo in mouse models, as well as in human IBD samples. This work will help identify novel immune effectors in IEC, broaden our understanding of mucosal immunity and IBD, and will provide a paradigm for determining the circuits underlying related pathological conditions in complex tissues.

A vast number of intracellular processes have been shown to involve oscillating gene expression levels, including yeast metabolism, immune response mediated by NF-kappaB, tumor suppression by protein p53 and growth factor response via ERK. Synthetic biology has offered an efficient way of studying the design principles of the regulatory networks maintaining these rhythms, also known as biological clocks. However, investigating such functional units in isolation neglects mutual interactions with other components of the network. The proposed research aims at discovering the mechanisms that determine the emergent dynamics resulting from this interaction. Our first step is to experimentally explore the sensitivity of an already existing biological clock (a transformed Escherichia coli strain) to periodic input stimuli of different amplitudes and frequencies, delivered using micro-fluidic tools. By systematically varying experimental parameters, the free-running system can be tuned to show different dynamics with or without spontaneous oscillations, resulting in various possible responses to the stimulation, e.g., induction of oscillatory dynamics, entrainment of an existing oscillation or failure to do so. In a next step, a quantitative model constructed in prior work for the oscillatory regime of this gene network will be adapted to accurately capture the experimental results and thus will allow us to dissect the essential mechanisms that lead to tight coupling between stimulus and response and represent an efficient way of intracellular signal transmission. This information will suggest modifications of the genetic circuitry that will finally be experimentally tested.

The thyroid is a critical regulator of growth, development and metabolism in most tissues of the vertebrate body. While recent studies have identified some of the factors that regulate the migration, differentiation and growth of the thyroid gland, little is known about its regenerative capacity.
I propose to use genetic and chemical tools as complementary approaches in order to study the cellular and molecular mechanisms regulating thyroid regeneration. First, I intend to generate a zebrafish line in which thyroid follicle cells are conditionally targeted for ablation. To do so I will activate thyroid-specific expression of nitroreductase (NTR), an enzyme that converts metronidazole (MTZ) into a cytotoxic product. To examine thyroid cell survival and regeneration, I will use the photoconvertible property of the fluorescent protein Kaede to label thyroid cells before and after ablation. Upon successful setup of the ablation system I will conduct in-vivo chemical screens on the thyroid-ablated zebrafish to identify molecular pathways involved in thyroid regeneration. Simultaneously, I will investigate the possibility that cells from adjacent tissues contribute to thyroid regeneration using cell lineage tracing approaches, including those enabled by the Cre-ER system. Complementary to the chemical screens I will perform gene expression profiling of the suspected cells during thyroid regeneration. Combining these various genetic and chemical tools, I aim to identify molecular pathways and progenitor cells which contribute to thyroid regeneration.

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

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

The proposed research takes an important step in making artificial life forms from scratch, creating self-assembling cell membranes, the structural envelopes that contain and support the reactions required for life. One of our long term, very ambitious goals is to construct an artificial cell – a synthetic living unit from the bottom up – to make a living organism from non-living molecules that have never been through or touched a living organism. Self-assembling artificial cellular systems ("SACS") will be extremely useful for analyzing the interaction between a lipid membrane and encapsulating biomacromolecules. On this point, we will initially evaluate the confinement effect of encapsulating desired amounts of amino acids, sugars, peptides, proteins, nucleic acids (DNA, RNA), drugs and small substrates within our non-natural cells (size-controlled confinement space). Moreover, we will use molecular imaging for monitoring the encapsulation of a wide collection of biomacromolecules by tetrazine/cyclopropene fluorogenic bioortogonal approaches. Additionally, biomimetic vesicles will incorporate functional enzyme systems in order to host (bio)chemical reactions. Finally, de novo phospholipid membranes could be also used to control the movement of substances in and out of the cell and for the development of biosensors for small ions, proteins and large molecules, including DNA.

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

During embryogenesis a single cell gives rise to a whole organism by successive rounds of cell division. In every division, cells duplicate and segregate their DNA into the daughter cells by means of a microtubule-based structure called the mitotic spindle. During development cells change in size and shape, and accordingly, the spindle adapts by changing its microtubule mass and size to accurately segregate chromosomes over a wide range of cellular length scales. Although these changes in size and length are directly related to chromosome position and define the cleavage plane, the mechanisms underlying microtubule nucleation, spindle scaling and their relation to the cell boundary remain elusive. To address these questions, I will combine mathematical modeling, biophysics and quantitative measurements of spindles assembled in both in-vitro Xenopus laevis egg extracts, and in-vivo during the early development of zebrafish. First, I will take advantage of the ease of imaging and perturbations in spindles from extracts to develop a method to measure microtubule nucleation in the spindle based on single molecule microscopy and a laser ablation technique I recently developed. Second, I will systematically characterize the microtubule architecture and nucleation during the scaling of spindles in zebrafish early embryogenesis. Finally, I will combine the in-vivo and in-vitro measurements with biochemical perturbations of proteins that nucleate, stabilize, and destabilize microtubules to propose and test models for microtubule nucleation and spindle scaling. My studies will advance towards the understanding of spindle morphology and its role during embryo development.

Critical periods (CPs) are windows of heightened experience-dependent cortical plasticity during brain circuitry development. There are different CPs across cortical modalities, each with its own time-trajectory correlated with the different maturation rate of parvalbumin (PV) expressing cells across brain regions. Alterations in the timing of CPs could underlie neurodevelopmental disorders. Brain-derived neurothrophic factor (BDNF) powerfully regulates visual CP timing. The BDNF Val66Met single-nucleotide polymorphism (SNP), very common in humans and associated with reduced neuronal BDNF secretion, is attracting growing interest as a susceptibility factor for several neuropsychiatric disorders, including depression and schizophrenia.
Here, we hypothesize that this SNP may broadly alter the timing of cortical critical periods. Taking advantage of the BDNFMet/Met knock-in mouse, reproducing the phenotypic hallmarks of humans with this SNP, we will explore CP timing across different cortical modalities. Electrophysiological recordings of visual acuity in vivo and auditory topography in vitro will establish CP time-course in V1 and A1, respectively. The study of PFC plasticity, instead, will be addressed behaviorally using an acoustic preference paradigm.
Alterations in synaptic transmission and network dynamics of PV circuits will be investigated in acute brain slices from V1, A1 and PFC of BDNFMet/Met mice. Finally, using state-of-the-art brainbow technique we will explore hypothetical alterations in PV connectome.
Together, we aim to reveal exciting new insights into the hypothetical link between alterations in CPs timing and the pathogenesis of neuropsychiatric diseases.

Learning alters the information processing, by which sensory input is transformed into appropriate behavioral output. However, it is unknown how socially relevant information is associated with sensory input in the brain. I propose to investigate how olfactory sensory input acquires social meaning by virtue of its linkage to a socially-salient stimulus, and how this process is modulated by oxytocin (OT). First, I will identify the neural substrates mediating OT-dependent social learning by carrying out a comprehensive set of experiments comprised of both gain-of-function and loss-of-function experiments for oxytocin receptor (OXTR) as well as histological analysis on OT-OXTR pathway. Second, I will elucidate the mechanism for OT-mediated social learning based on a set of experiments assessing association between odor and optogenetic activation of OT-neurons. Finally, I will elucidate the neural correlate of OT-mediated social learning by comparing olfactory response of neurons in naïve and trained animal by employing in vivo calcium imaging. Identification of neural substrate during OT-mediated social learning and elucidation of its underlying mechanism will provide better understanding of neuromodulation of neural circuit during experience-dependent behavioral adaptation and neural encoding of social significance.

In our 2009 application, we proposed to develop an integrated view of transcription in bacteria that takes into account the state of the nucleoid and the hierarchy of gene regulation. In order to obtain the quantitative in vivo measurements and test the predictions from the model, we developed new optical/microfluidic technologies, a comprehensive bioinformatic approach, and characterized the biophysics of the nucleoid as a "complex polymer". We reported three major advances: (i) the largest data set (now > 200k measurements) on loci mobility, revealing a remarkable role of chromosomal coordinate, (ii) a new microfluidic chemostat that led to the first gene expression experiments in changing conditions, and (iii) bioinformatics work (www.lgm.upmc.fr/NuST) showing that nucleoid organization produces contiguous gene clusters with similar biological properties. Our renewal will leave behind the old frontier of nucleoid “statics” and move onto the new frontier of nucleoid dynamics, adding cell-cycle resolution, and, most importantly, environmental transitions to our high-throughput measurements. Our renewal focuses on a NEXT biological question, the role of the nucleoid in global transcriptional changes causing physiological adaptation and response to nutrient transitions and perturbations. Our methods will attack this long-standing question with a novel high-throughput and fully quantitative perspective. In the field of nucleoid organization, our team has one of the widest interdisciplinary spectra, with, at the same time, a proven record of tight and successful collaboration between its members.