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.

Life depends on the faithful transmission of genetic information. DNA must be accurately replicated and segregated during cell division. Cancer cells evolve due to accumulation of multiple, successive mutations within a cell lineage that result in changes in cell growth, proliferation, and survival. The selection for multiple, successive mutations favors the evolution of genetic instability (defects in DNA replication, repair, and chromosome segregation), which increases the rate of mutation. However, it is not clear how genetic instability arises and evolves. Cataloging the genetic rearrangements present in cancer cells does not allow us to re-capitulate the contribution of individual mutations to the development of genetic instability. Instead, I propose to study the evolution of genetic instability over time in a yeast model of cancer. I will evolve cells to escape growth suppression, which is analogous to the selection for loss of tumor suppression by the inactivation of genes that guard against genetic instability or unintended cell growth and proliferation. The level of genetic instability will be monitored as cell lineages will undergo several rounds of selection, and instability-associated mutations will be identified by whole-genome sequencing. To quantify the direct function of these mutations in generating instability, we will re-introduce them in a non-evolved ancestor strain. With this strategy, I aim to uncover early mutations that cause genetic instability and generate genetic fingerprints that can be used to identify the causes of genetic instability. By selecting in diploids, I will be able to identify dominant instability-causing mutations, which may be more relevant to cancer than the classical loss-of-function candidates. Due to the conservation of basic cell biology, I expect that our results will be relevant to understand genetic instability in humans.

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

We will investigate host and pathogen DNA sequence coevolution and multigenerational inheritance of epigenetic changes during plant-fungus interactions. Verticillium dahliae, an economically important vascular wilt pathogen that can infect numerous plant species and exhibits differential host virulence, will be studied as it adapts to infect Arabidopsis thaliana. The project will carry out experimental coevolution and track not only phenotype evolution but also genome, methylome, transcriptome, and small RNA changes using next generation whole-genome sequencing technology. The experiments will investigate genome stability and dynamics of epigenetic marks during pathogen evolution, plant evolution, and their coevolution by comparing data gathered prior to, during, and following their serial interactions across multiple generations. The experiments will identify specific genes and mechanisms that allow a broad host-range pathogen to gain increased infection efficacy on an initially marginal host, and characterize genome-wide changes in the pathogen. Simultaneously, host genome stability and host epigenetic changes in the face of pathogen attacks will be characterized. These experiments should offer an exciting view of the role and mechanisms of genome plasticity and epigenetic dynamics during host-pathogen adaptation.

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

Blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD-fMRI) has emerged as the cornerstone of noninvasive brain mapping in recent years. This technique measures neuronal events indirectly by detecting associated changes in a mixture of cerebral blood flow (CBF), cerebral blood volume (CBV) and cerebral metabolic rate of oxygen consumption (CMRO2). Although BOLD contrast is widely used as a surrogate marker for neural activation, to date, there is no clear demonstration of how neurons and astrocytes, the two different cell populations in brain, generate this functional signal. This limitation seriously confounds MRI data interpretation for both basic research and clinical applications.
In this project, we seek to dissect the neuronal and astrocytic components of BOLD signal using pharmacogenetic and optogenetic fMRI. We propose to use two different yet complementary genetic tools based on (i) Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) and (ii) light-activated photosensitive proteins (microbial opsins) for selective modulation of neuronal/astrocytic activities. Continuous arterial spin labeling (CASL) technology will be used for simultaneous acquisition of BOLD and CBF data before, during and after optogenetic stimulation of one of the two cell populations while the other will be pharmacogenetically silenced. We hypothesize that selective optical excitation and chemical inhibition of genetically defined cell populations will provide the first direct evidence of how BOLD signal is generated in vivo, what it actually represents and finally, put the long-lasting debates on BOLD contrast mechanism to rest.

The precise adjustment of the floral transition is controlled by external and internal cues. In the temperate cereals wheat and barley, flowering is induced by long-days and vernalization. VRN2, a long-day flowering repressor with no homologs in Arabidopsis, is repressed by vernalization and plays a central role in preventing flowering during the fall. However, even nonvernalized winter wheat plants flower after long periods at room temperature. Interestingly, VRN2 is also downregulated during plant development. This suggests that plant age could induce flowering, probably through the control of VRN2. Recent studies indicated that miR156 and its targets, the SPL transcription factors, constitute a conserved mechanism to control age-dependent processes in plants. Here I propose to study the role of the SPL genes in the control of VRN2 expression and cereal flowering by age. First, I will obtain and characterize mutants for SPLs using a tetraploid wheat TILLING population, in particular looking for mutations that eliminate the regulation of the SPLs by miR156. Also, I will knock-down and overexpress miR156 through a transgenic strategy in barley. Then, I will integrate the SPLs into the known flowering network by crossing the SPL mutants with available mutants for different flowering genes, and by performing detailed studies of gene expression. Finally I will screen barley landraces and varieties for polymorphisms in the SPL family to test their potential adaptative value. Results from this study will increase our understanding of the mechanisms involved in the regulation of flowering and can contribute to engineering improved varieties adapted to changing environments.

We aim to explore the emerging paradigm that transposable elements (TEs) could be sensors of bacterial stress, and that their reactivation could contribute to a transgenerational and adaptive memory of the immune response through heritable changes, either epigenetic (heritable modulation of nearby-genes expression) or genetic (restructuration of the genome and creation of new regulatory networks via transposition). This project comprises two aims. First, we want to test if the modulation of defense gene expression, through epigenetic modifications at nearby TEs, can be inherited to the progeny. As a first step towards answering this question, we will determine whether the DNA methylation changes previously observed in the course of the immune response, and other related epigenetic marks, can be passed to and mitotically maintained in Arabidopsis stem-cells (precursors of the germ cells), then propagated in the plant to the next generation, thereby contributing to transgenerational immune priming. Second, we want to set-up a reporter system to analyze the pattern of somatic transpositions in a plant challenged with bacteria and to isolate the rare individuals that display the transgenerational memory of transposition events. In fact, transposition upon environmental challenge has not been much studied experimentally so far in plants due to the lack of appropriate tools. We propose that there is a timeframe during which transposons can be reactivated upon bacterial challenges and our system should allow us to unravel the conditions for transposition, the specific tissues/cell types and the frequency of their occurrence as well as their transgenerational inheritance.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

How genetic and epigenetic mechanisms control proper gene expression is a fundamental unresolved problem. At the sequence level, enhancers regulate transcription in a cell-type- and cell-state-specific manner. On top of enhancer sequence, dynamic DNA methylation and histone modification processes have been correlated with transcription regulation. The host lab has provided the first indication that histone modification patterns can be used for enhancer annotation. However, mechanisms of enhancer regulation by chromatin modification are not completely understood, mostly due to the inability of targeted reprogramming of epigenome. Here I will develop tools which enable us to targeted edit the epigenome in human cells(Aim 1). In hESC, I will express fusion proteins containing mutant Cas9(dCas9, lacking nuclease activity) and chromatin modifying enzymes, which can be guided to target DNA sequence by sgRNA to edit chromatin modifications. Next, I will study the role of enhancer-like chromatin modification in gene regulation and cell fate determination(Aim 2). I will create H3K4me1 modification near NKX2-5 locus in hESC, a master regulator of cardiac progenitor cells, followed by monitoring changes in NKX2-5 expression, histone modification and cell fate. I will also study if chromatin modifications play a causative role in enhancer activity and transcription(Aim 3). The chromatin modifications on OCT4 enhancers in hESC will be reprogrammed by CRISPR/Cas9 system. I will then perform a rigorous genomic analysis to study the dependency between chromatin modification, OCT4 enhancer activity and hESC pluripotency. Using these data, I will explore the role chromatin modifications in enhancer regulation. I expect this project will have a major impact on the understanding of functional human epigenome and on developmental biology. The developed CRISPR/Cas9 system will be widely applicable in epigenetic studies in other model systems.

Autophagy, the process whereby cells degrade their own components, is essential for normal cell function and mammalian development, and also has been implicated in numerous human diseases. Autophagy involves a wide array of protein-protein interactions, including a large number involving ubiquitin, ubiquitin-like proteins and various adaptor modules. The ability to target and disrupt these protein-protein interactions would enable dissection of the molecular details of autophagy, which would greatly enhance our basic understanding of the process and could lead to the identification of targets for therapeutic intervention. This project will develop and apply a toolkit of protein-based modulators to explore the roles of distinct molecular networks in autophagy. We will first identify protein interaction interfaces, and subsequently, will use computational design and combinatorial protein engineering to develop high affinity sensors or inhibitors that can function in live cells. Structural studies will be performed to determine the molecular basis of their action. These tools will then be used in vivo settings, using variety of cell and mouse models, to monitor and control physiological and pathophysiological autophagy processes.

Adaptation to environmental change is required for species to be successful, however the potential and rate at which this takes place is unclear. In addition, recent studies have highlighted the importance of ‘extended genotype’ features as factors influencing a wide variety of adaptive phenotypes. This project aims to determine the genetic basis of environmental adaptation and to investigate extended genotype factors as both a cause and consequence of the adaptation process using natural populations of the model cereal Brachypodium distachyon. First, the project will aim to determine the genetic architecture of DNA methylation variation within natural populations. As DNA methylation may lead to phenotypic changes, the genetic loci regulating methylation may be closely associated with the variability of adaptive traits such as flowering time and growth rate. Second, allelic variation in DNA methylation (epialleles) will be examined to determine if they are causal, and directly associated with adaptive traits and extended genotype variation. Methylation variation will be assessed within both inbred populations and samples selected from across globally diverse environments to determine if there are specific regions of the epigenome that vary or have become fixed within their respective populations. Beyond this, both inbred and diverse allopolyploid lines will be assessed to determine the impact of genome duplication on both DNA methylation and adaptive trait variation. These experiments will allow for genome-scale analyses of the extended genotype across natural and synthetic B. distachyon lines to determine the underlying basis of adaptation ability in harsh environments.

The processing and encoding of external stimuli is essential for all life forms to react appropriately to environmental cues. When animals move, however, they activate their own sensory systems. Expectations about self-generated sensory input and its subtraction from the total input are essential for accurate sensory processing. Organisms have optimized a solution for distinguishing between self-generated and external stimulations by involving a descending (efferent) neural pathway that modulates ascending (afferent) activity. Mechanosensory hair cells, for example, are subject to feedback regulation by cholinergic efferent neurons that originate from several nuclei in the brainstem. The present research proposal aims at understanding how efferent neurons control mechanosensory processing.
Fishes and amphibians have a superficial and accessible mechanosensory system, the lateral line, which circumvents many of these problems. The lateral line serves to detect hydromechanical variations around the animal’s body and controls two characteristic behaviors: the stereotypic fast escape responses to sudden mechanical input, and complex and highly variable obstacle avoidance and rheotaxis. The simplicity of the lateral line in the zebrafish larva makes it a particularly powerful experimental model system.
Versatile recent technological developments suggest that efferent/afferent interactions in the zebrafish lateral line can be interrogated using a combination of pharmacological and genetic manipulations of neural circuits in vivo, intravital structural and functional imaging of neuronal ensembles, and fictive behavioral paradigms. Combining the knowledge and strength of each group, we will be able to clarify efferent/afferent interactions and lay the foundations to further ask how the nervous system adjusts its sensitivity to discriminate between external- and self-stimulation. This collaborative project is based on molecular studies of efferent neurons, in toto visualization and manipulation of the zebrafish lateral line, and zebrafish neurophysiology, functional imaging and behavior.

The tumor microenvironment is becoming appreciated as a determinant factor in cancer development. Pancreatic cancer is supported by its tumor-associated stroma, including protection from the effects of chemotherapy. The use of genetically modified mouse models is at the heart of pancreatic cancer research, as they mimic well most of the features of the human disease. Accordingly, pancreatic tumors developed in mouse models are highly abundant with stromal components and resist conventional chemotherapy. Connective tissue growth factor (CTGF) is a critical component of the tumor-associated stroma in pancreatic cancer, and was shown to mediate anti-apoptotic signals and oppose chemotherapy effects. This research is aimed to investigate the roles and mechanism of action of CTGF in supporting pancreatic cancer survival, and identify other stromal components that may facilitate the malignant process. To discern the contribution of the pancreatic stromal compartment to tumor progression, we will use a newly-established ex-vivo system for growing mouse pancreatic epithelial cells in 3D matrix that keeps their natural conformation and biological features. Co-culturing pancreatic epithelial organoids with pancreatic stromal cells, such as PSCs, will provide an elegant and clean system to study CTGF signaling, and other survival signals that promote pancreatic malignancy. We will further expand this system to allow the growth of human pancreatic cancer cells in order to dissect signaling pathways that are most relevant to this deadly human disease.

Endogenous heterogeneity in isogenic T-cells renders individual cell activation unreliable. Accordingly, cytokine based cell to cell communication is necessary to proofread individual cell response and achieve a reliable self/non-self discrimination at the population level. Current attempts to understand cytokine-cell networks either neglect their dynamics or address dynamics of the average cell only, neglecting the full distribution. Our aim is to understand the dynamics of cell to cell cytokine communication in heterogeneous clonal populations at the distribution level.

To properly study cytokine and T cell population dynamics it is not enough to make precise measurements of cytokine concentration and T cell activation; a mathematical model that manifests the dynamics is required. Furthermore, since genetic perturbations cannot be used to understand immune response in human patients, it is of utmost clinical relevance to create a quantitative model of cytokine communications and how different cytokine perturbations impact lymphocyte differentiation.

We conjecture that the cytokine receptor distribution affects the cell fate decision. If true, we will test whether different sub-groups within the distribution carry different cell fates, eg. short-lived effector vs. long-lived memory cell. We will test these hypotheses by designing in vitro cultures of primary cells that mimic in vivo immune distributions. We propose to build a computational model of the distribution dynamics and variability of T cell regulation. We will use this model to test and validate experimentally diverse perturbations of collective T cell regulation with the goal of optimizing immunotherapies.