Type-1 interferons (IFNs) play critical roles in antiproliferative, antiviral and immunomodulatory responses. The phenotypic outcome for a cell depends on a complex and largely undetermined interplay between the dose and subtype of IFN it has experienced, the cell type, and any previous signalling events. One hindrance to study has always been an inability to probe the combined long-term response and cellular signalling history of an individual cell. I will design and construct synthetic memory circuits that record multiple aspects of signalling and allow subsequent isolation and profiling. In particular, the pathways activated and degree of activity will be assessed. Using these tools in combination with molecular techniques to quantify and manipulate cell state at the time of signalling, I aim to dissect the factors that direct cell response. A further aspect of signalling modulation occurs through variations in cytokine binding properties. Fundamentally understanding these factors is important for both native and therapeutic cytokines. I therefore aim to use the devices generated to investigate the influence of binding properties on signalling, particularly in the context of targeted IFNs. Together, these systems will provide an unprecedented ability to probe IFN signalling heterogeneity. The project represents a unique approach to understanding cellular outcomes in the context of complex cellular history and includes major advances to mammalian cell synthetic biology. A better fundamental understanding of these type-1 IFN signalling will also have important implications for their use in treating a spectrum of diseases that affect billions worldwide.

Two conditions in humans require allorecognition (the distinction between ‘self’ and ‘non-self’ tissue): pregnancy and transplantation. We will study the role of innate histocompatibility in a model system in Botryllus schlosseri, and in pregnancy immunology and stem cell transplantation in mammals. B. schlosseri is an organism with unusual characteristics that render it a valuable model of naturally accruing allorecognition:(i) a naturally acquired allorecognition where a single highly polymorphic gene determines acceptance or rejection of colonies; this gene (BHF) was recently identified and characterized by our laboratory (ii) genetic/ predicted hierarchies that determine competitive potential among germ line and somatic stem cells respectively;(iii) tissue regeneration with isolatable stem cells mediating formation of body organs de novo every week (iv) known adult stem cell niches;(v) a sequenced genome. The experimental design will take advantage of these natural traits to identify and characterize genes that determine successful chimerism through allorecognition. We will: (a) investigate the B. schlosseri allorecognition pathway and pathways that regulate stem cell transplantation engraftment in B. schlosseri chimeras; (b) identify genes that are associated with human pregnancy disorders and stem cell transplantation using transcriptome analysis; and (c) investigate the roles of homologues genes involved in B. schlosseri and human histocompatibility, in mouse pregnancy disorders models, and (d) stem cell transplantation immunity models.

Although it is clear that during metabolic adaptation and dysfunction information is exchanged between the major organs involved in glucose and lipid homeostasis, the mechanisms that mediate this communication remain in great part elusive. There are inherent difficulties in establishing causality in in vivo systems, and the search for circulating secreted factors is hindered by the challenge of unbiased screening in the complex blood compartment. Our aim is to model the information flow between skeletal muscle, liver, adipose tissue, and mesenchymal stem/stromal cells in a mini-tissue microfluidics system allowing the identification and manipulation of circulating factors affecting tissue function and metabolism. We will develop a system for compartmentalized culture of tissue-specific primary cells with the capability of temporal and spatial control of communication between compartments, allowing us to establish compartment-restricted perturbations (mimicking different physiological and pathophysiological states) and follow their proximal and distal consequences. Tissue-specific function will be monitored by sensors including temperature probes and contractility measurements, and the defined nature of this system will facilitate identification of circulating factors by mass spectrometry.

The ATF4/ATF3 stress pathway is strongly upregulated in both neurons and microglia during neuronal injury, ER stress and in response to neuroinflammatory stimuli. However, the role of this pathway in mediating microglia cell function and in the pathogenesis of neurodegenerative diseases such as Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) remains to be elucidated. Here, I hypothesize that the ATF4/ATF3 stress response has a crucial role in maintaining CNS homeostasis and preventing aberrant neurodegeneration by repressing damaging pro-inflammatory cytokines in microglia and promoting neuronal cell proliferation. I will first assess how the ATF4/ATF3 pathway affects microglia function and gene expression ex vivo by isolating primary microglia cells from C57Bl6 wild-type mice (WT), ATF3-deficient mice (ATF3-/-) and ATF-deficient mice (ATF4-/-) and assessing their proliferation, microglia activation markers, cytokine production, gene and microRNA profiling and phagocytic capacity. Next, I will assess the role of the ATF3 stress response in mouse models of chronic neurodegeneration by generating ATF3-/-APP/PS1 mice to model AD and ATF3-/-SOD1G93A mice to model ALS. The survival, disease severity, microglia activation and CNS pathology will be assessed in the ATF3-/-APP/PS1 and ATF3-/-SOD1G93A mice compared to their respective controls. Finally, I will determine the impact of specifically deleting ATF4 or ATF3 in microglia in vivo by crossing ATF3flox/flox and ATF4flox/flox mice to the microglia-specific CX3CR1(cre/ER-EYFP) mouse, which will generate mice deficient for ATF3 and ATF4 in CX3CR1+ cells, including all microglial cells.

A particularly well-understood model system for studying the mechanisms of morphogen-mediated patterning is the vertebrate neural tube. Here, Sonic Hedgehog (Shh) induces discrete neuronal progenitor domains through regulation of a combinatorial code of transcription factors. The observed precision and reproducibility of gene expression appears greater than can be accounted for by the precision of Shh signaling. Thus, mechanisms must exist that filter signaling noise. We hypothesize that these involve properties of the downstream transcriptional network regulated by Shh signaling and/or active cell sorting.
I propose an interdisciplinary project to quantify the amount of noise in the pattern of gene expression in the neural tube and investigate how noise is transmitted and buffered. To achieve this, I will generate fluorescent reporters for the expression of three key Shh target genes, Nkx2.2, Olig2 and Pax6, and spatiotemporally correlate their activity with the activity of the Shh pathway. In parallel, a quantitative analysis of the behavior of cells will be performed to elucidate the contribution of differential cell adhesion and active cell sorting. The combined results will be analyzed using dynamical systems, theoretical frameworks, and computational tissue level simulations. For this I will take advantage of an existing collaboration with mathematical physicists and computer scientists.
Despite the importance of precision in development, relatively little is known about the underlying cellular and molecular mechanisms, thus the mechanisms deduced from our quantitative studies are likely to be relevant in other tissues patterned by morphogens.

Epithelial cells have an apical-basal polarity, which is regulated by polarity proteins. Lgl is an important polarity protein that localizes to basolateral membrane. The fly genetic studies suggest that Lgl functions not only as a polarity protein but also as a tumor suppressor.
In the breast cancer cells, the downregulation of Lgl is frequently observed but the pathophysiological significance of Lgl is not understood. Thus, this research project aims to reveal the molecular mechanisms on how Lgl contributes to the development of mammary carcinoma. Especially, this project will focus on the effects of Lgl on cell proliferation and try to reveal the molecular mechanisms on how Lgl regulates cell proliferation in mammary epithelium. To achieve the goal of this project, I designed several experiments.
First, to examine the effects of Lgl in mammary epithelial cells, Lgl will be knockdowned in mammary cells using RNAi techniques. The effects of Lgl-downregulation on cell proliferation will be examined in the culture condition of 3D space, which enable the mammary cells to form acinus structure in vitro. In addition, the effects of Lgl-downregulation on tumorigenicity will be examined by xenograft assay. Secondly, to confirm the results obtained from cultured cells, the phenotype of Lgl null mice will be examined. Finally, the molecular mechanisms on how Lgl-downregulation contributes to carcinogenesis will be addressed. Briefly, by using global analyses such as expression analyses with microarray and mass spectrometry, the downstream molecules of Lgl, which relates to cell proliferation, will be searched to understand the Lgl-mediated mammary carcinogenesis.

Protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination are important for controlling the cell cycle and preventing genome instability. Using the budding yeast Saccharomyces cerevisiae, I will characterize the significance of PTMs by developing an approach in which I can bring an enzymatic activity that removes those PTMs into close proximity to a substrate in trans.
As my first aim, I will generate a system with which to co-localize a phosphatase or deubiquitinase (DUB) activity and potential substrates. To accomplish this, I will create fusions between phosphatases/DUBs and an IgG constant region, and this fusion will be expressed in a strain where the potential substrate is fused to the TAP tag, which associates with the IgG constant region. I will validate this system using well characterized phosphorylated/ubiquitinated proteins.
As my second aim, I will transform plasmids that express these DUB/phosphatase fusions under an inducible promoter into a pre-existing library of yeast strains in which every gene is fused to the TAP tag. I will screen for slow growth or DNA damage sensitivity after induction of the DUB/phosphatase fusions.
As my third aim, I will determine the significance of the identified PTMs. In order to do this, modified sites for each candidate protein will be mapped and mutated.

The aim of this proposal is to better understand the role of the intestinal mucosal barrier in metabolic diseases as obesity and type-2 diabetes, which cause more than 3 million deaths per year. While the gut microbiota has been found to be a crucial factor for obesity and insulin resistance in mice and humans, the underlying disease-causing mechanisms are still unclear.
The human intestine is colonized by a complex microbial community. While its composition is specific for each individual, a core gut microbiota is shared between humans. However, the composition of gut microbiota is altered in individuals who are obese or diabetic.
The high density of the gut microbiota is a permanent challenge for the gut epithelium. As only one single, mucus-covered cell layer separates microbes from human tissue the mucosal barrier is essential in preventing microbial translocation. One epithelial defense mechanism is the secretion of antimicrobial peptides (AMPs). These endogenous peptide antibiotics have broad-spectrum antimicrobial activity and can modulate the composition of gut microbiota.
In this proposal I will focus on the intestinal barrier between host and microbiota. I will test if and how the shifted microbial composition in metabolic diseases is associated with alterations in mucosal AMP production and function. Furthermore, I will analyze mucosa-associated microbiota in metabolic disease by using mouse models and samples from human patients. Also, I will compare the peptide repertoire in the mucus of different mouse models and human diabetes patients to identify alterations in metabolic disease.
The planned investigations will help to better understand the relationship between the mucosal host barrier and metabolic diseases.

Microbes are present within and significantly affect the dynamics of all natural habitats, playing vital roles in a plethora of medical, ecological and industrial contexts. Consequently, a better understanding of the fundamental functions of these important organisms is crucial. Here, I focus on one of the most fundamental microbial functions – the uptake of nutrients – and ask how this is affected by a universal environmental parameter – the flow of the fluid that microbes live in. It is predicted that fluid flow relative to a microbe could enhance the nutrient uptake. Yet, this ubiquitous, fundamental process has, to date, eluded experimental investigation and our current knowledge rests entirely on theoretical predictions. The archetypal problem in this context – uptake of a single compound by a single microbial cell when the relative motion of the fluid and the cell is laminar and unidirectional – remains unsolved, despite its deceiving simplicity, and will represent the core goal of this project. I propose to develop the first experimental framework and perform seminal experiments to understand flow-enhanced uptake quantitatively, based on a combination of cutting edge approaches including microfluidics, optical tweezers, advanced microscopy, Raman spectroscopy, and particle tracking. This will provide a quantitative experimental approach to this unsolved problem, ground theoretical predictions in observational data, reveal any processes not accounted for in current theoretical frameworks, and help resolve existing inconsistencies in the theory. Further afield, the approach I will develop will trigger solutions for investigating the full range of realistic, more complex scenarios of flow, nutrients, and population structure.

The main cause of mortality in human cancers is the formation of metastases, in which tumor cells exhibit resistance to established anti-cancer therapies. My postdoctoral lab (PI: CS Mitsiades) has documented that this observation may be due, at least in part, to the fact that nonmalignant cells in metastatic sites can function as "accessories" to the tumor cells, conferring to them increased threshold to treatment-induced cell death. My research will characterize the molecular basis of this crosstalk between cells of colorectal cancer (CRC), one of the leading causes of cancer-related deaths), and hepatocytes, as key "accessory" cells in the liver, a major site of CRC metastases. I will also examine how the genetic features of CRC can influence the consequences of their interaction with hepatocytes; and, conversely, how the latter cells alter the sub-clonal architecture of the CRC cell population. My studies will involve 2- and 3-dimensional in vitro co-cultures of CRC cells and hepatocytes; and "orthometastatic" in vivo models to simulate the CRC liver metastases. In addition, I will apply a novel combined marking of individual cells with sequence-based "barcoding" and "Brainbow"-based multicolor labeling, to monitor how hepatocytes alter the clonal architecture of CRC cell populations, and sort out individual clones with differential responsiveness to hepatocytes. Using these models and my prior work on molecular pathways associated with CRC, I will determine which molecular pathways are essential for the hepatocyte-induced increased in the threshold of metastatic CRC for cell death. This study will provide new insights into the biology of metastatic cancers.

Cell differentiation is a fundamental biological process that underlies all multicellular organisms. It is clear that mammalian differentiation requires DNA methylation; However, we still don't know why certain sequences are methylated and others are not. In addition, recent studies propose that not just methylation status, but also methylation variability is relevant in differentiation, because erroneous methylation variability can lead to disease. The canonical role of DNA methylation in parental imprinting and X-inactiviation is to maintain already silenced DNA, not to establish silencing. DNA methylation may have a similar role in development, namely, to maintain already silenced DNA. What, then, is the driving cellular signal that determines epigenetic status? One possibility is transcription factors, which are known to drive differentiation. To study this interaction, I propose to query simultaneously DNA methylation status, transcription factor binding, and gene expression at many time points through a cellular differentiation process. This unprecedented time resolution and combination of experiments will enable me to identify causal relationships between factor binding and DNA methylation. Furthermore, I will measure not only mean DNA methylation, but also its variability. To aid in this task, I will develop a computational model to capture important sources of variation and apply this to the experimental data. Using the model of granulocyte differentiation, combining these experiments will yield new insights into causal interactions between TFs and either mean DNA methylation status or variability of DNA methylation.

Reprogramming of somatic cells to pluripotency using ectopic expression of defined factors is both a long and inefficient process, which consists of many unknown epigenetic changes. In contrast, somatic cell nuclear transfer (SCNT) is rapid and more efficient process, which results in pluripotent stem cells that are indistinguishable from their naturally occurring embryonic stem cells counterparts. In my postdoctoral work, I intend to comprehensively study the differences between the two processes, consequently aiming at identifying factors that may, upon ectopic expression, reprogram somatic cells to pluripotency in a more rapid and deterministic fashion. I therefore intend to conduct single cell analyses in untreated mouse oocytes and SCNT embryos to identify possible facilitators of SCNT-mediated cellular reprogramming. Next, to comprehensively compare between gene activation during iPS formation and SCNT, I intend to generate mouse embryonic fibroblasts (MEFs) carrying cassettes of predictive markers for both processes, thus enabling single-cell population tracing and cell-type specific gene-expression analyses. My ultimate goal will be to test whether the newly identified factors may enhance cellular reprogramming upon ectopic expression in somatic cells. I thus intend to generate secondary reprogrammable MEFs, harboring different combinations of factors, and assess their ability to reprogram somatic cells to pluripotency.

A fundamental question in biology is why different individuals show different behaviors. Behavioral variability among individuals may arise from genetic heterogeneity, the effects of different external environments, or experience. However, phenotypic variation, including in behavior, is widely observed even between genetically identical individuals that have been raised in the same environment. This variability suggests that epigenetic or other non-genetic differences between individuals may induce different behavioral states.
My overarching goal is to study the non-genetic differences underlying behavioral variability among individuals, and to investigate how different environments, in particular stressful conditions, affect this inter-individual variability.
C. elegans is an ideal model for studying these issues due to its relatively simple nervous system, the substantial knowledge about its behavior, and the available genetic and molecular tools, as well as high-throughput methods for quantitative, simultaneous monitoring of behavior in many individual worms.
As my first specific aim, I will quantitatively characterize inter-individual behavioral variability in isogenic C. elegans populations exposed to various environments. In my second aim, I will use these behavioral paradigms to decipher neuronal circuits involved in generating variability. As my third aim, I will identify potential molecular components underlying these neuronal functions, with an initial focus on epigenetic mechanisms.
Identifying the non-genetic sources of behavioral variability among individuals may have a major impact for advancing basic research on individuality, epigenetics, and behavior.

Viruses are completely reliant on the host cell machinery and have evolved many mechanisms to hijack it for their propagation. At the same time our cells have developed defense mechanisms to cope with infections. A key way by which viruses influence host physiology is through interaction with the cellular translation machinery. Not only viruses must co-opt this machinery to translate their own mRNAs, they also have to hinder host defenses that are aimed to inactivate the translation machinery. My research aims to elucidate the complex cellular changes that occur during these virus-cell encounters. I plan to concentrate on two major pathogens; a large DNA virus, Human cytomegalovirus (HCMV), and a small RNA virus, Influenza A. Although these viruses are very different, for both of these viruses the global effect of infection on the translation of host genes is still poorly defined. I therefore propose to tackle this important issue by employing a novel deep-sequencing technique termed ribosome profiling, which allows a comprehensive view of translation events. The robustness, scale and accuracy of this analysis will enable us to determine the identity and the relative levels of translation of each host protein during the course of infection and would therefore provide valuable information regarding translational control strategies used by these viruses to reprogram the cellular host translation machinery. I then plan to develop and apply more directed experiment to confirm translational changes and to elucidate their importance to viral replication. These experiments should provide a unique opportunity to understand the underlying changes that occur during infection.

Pr. A. Ting and coworkers have recently demonstrated the conceptual and technical feasability of spatially resolved proteomic mapping in live cells using an engineered Ascorbic PEroXidase (APEX). Such a methodology is meant to circumvent the limitations of classic MS spectrometry by extracting the desired proteomic information at the level of the living cell, with its structures preserved. This tagging methodology takes not more than 1 minute, paving the way potentially for spatially AND temporally resolved biological studies. In addition, certain biological compartment of high relevance like the mitochondrial intermembrane space (IMS) or mitochondria-ER junctions cannot be purified, consequently their proteomes are largely unknown. In this postdoctoral work we aim at establishing the proteomic maps of these two highly interesting cellular localizations which will certainly prove themselves illuminating in unraveling the biological processes in which they are involved (apoptosis, regulation of mitochondrial division, calcium and phospholipid exchange, protein import into mitochondria etc...). Another aim of this postdoctoral work is to develop ultra spatially resolved proteomic mapping methodologies to enable the mapping of macromolecular protein complexes found in very specific sub cellular localizations. Such endeavour requires developing bio-chemical approaches to restrict the tagging radius of the reactive species generated by APEX or to explore light based methodologies that serve the same purpose.

My goal is to elucidate the molecular mechanism of major histocompatibility class II (MHCII) trafficking to the immunological synapse (IS) between T-cells and dendritic cells (DCs). There is substantial evidence that in DCs peptide-bound MHCII is selectively trafficked from intracellular MHCII containing compartments (MIIC) to the IS where it interacts with the T-cell receptor for activation of T-cells. Nevertheless, the IS has been almost exclusively studied from the T-cell side and not from the DC and the molecular pathways that govern delivery of MHCII at the plasma membrane of DCs are still completely unknown. Specifically, the SNARE proteins, which are key trafficking proteins that catalyze intracellular membrane fusion, need to be identified. Based on my extensive experience in membrane trafficking in neuroendocrine cells, I propose to develop a new and innovative approach to identify the SNAREs responsible for MHCII trafficking to the IS. I will first position an IS between an antigen-specific T-cell and a DC parallel to the optical plane of a microscope with a micromanipulator, thereby enabling a high spatial resolution. I will then employ a combination of Förster resonance energy transfer (FRET)-based microscopy and functional assays to unequivocally identify the SNAREs that catalyze release of MIIC at the plasma membrane of DCs. Understanding how MHCII is selectively presented to the T-cell is essential for understanding the high sensitivity of T-cell activation by DCs. My study will also result in the development of new tools and insights that enable unraveling the release mechanisms of other surface exposed molecules and secreted cytokines from immune cells.

During multicellular development, cell differentiation is often controlled by morphogen gradients. Here, cells measure the local morphogen concentration to direct their cell fate. However, such gradients form by diffusion of a finite number of morphogen molecules and hence can exhibit strong stochastic fluctuations. How such ‘noisy’ morphogen gradients can induce cell fate patterns with the extreme precision observed during development is poorly understood. To address this question, we propose to study pattern formation by morphogen gradients in the simple nematode worm C. elegans, using a quantitative approach that relies on two innovative techniques: first, we will use a new microscopy technique, Single Plane Illumination Microscopy, to quantify with high precision the dynamics of both the morphogen gradient and the cellular response it generates. Then, using an information theory approach we will establish whether the cell’s response improves the information provided by the gradient and if so, what mechanisms are used. We will study a system where morphogen gradients provide the directional cue for asymmetric cell divisions, an important mechanism for generating spatial cell fate diversity. Here, cells measure the change in morphogen concentration across the cell surface to establish from what direction the gradient emanates. Such measurements are more challenging than measuring the local morphogen level alone and it is an open question how direction is sensed robustly from noisy morphogen gradients. Our research will likely elucidate general mechanisms underlying this robustness that are important for related processes such as cell polarity and eukaryotic chemotaxis.

Neural computations underlying critical brain functions, such as cognition and perception, rely on fundamental interactions between neural excitation and inhibition. However, neural function depends critically on behavioral state, such as sleep, wakefulness, arousal, and focused attention. GABAergic, inhibitory interneurons are subdivided into distinct classes with different synaptic targets, physiology, and molecular markers. Interneuron diversity may provide a mechanism for the functional flexibility of neuronal circuits, allowing dynamic sensory processing depending on computational goals (e.g. fine discrimination vs. detection of any input) and behavioral state. However, there is little data on the contribution of specific GABAergic interneurons in sensory cortex during distinct behavioral states, because until recently it was not possible to rigorously identify or manipulate these cells in the intact brain. We will perform extra- and intracellular recordings in primary visual cortex of awake mice expressing light-sensitive channels in specific interneuron classes, and will record physiological parameters to precisely quantify behavioral state with multivariate analysis. Using optogenetic tools, we will identify cells from genetically distinct classes and control their activity with millisecond precision. We will test how the temporal recruitment pattern of distinct interneuron classes differs across behavioral states (Aim 1) and controls synaptic integration in excitatory cells on a fine time-scale (Aim 2). Further, we aim to probe their causal roles in controlling dynamic transitions in cortical network activity (Aim 3).

Coordination of rhythmic behavior at the level of cell communities manifests the fundamental processing capability of eukaryotic organisms such as plants and animals. Biological rhythms often rely on genetic oscillators or clocks that operate in synchrony across time and space. One of the major challenges in modern biology is to quantitatively understand the dynamics of oscillatory behavior in plants and its role in the self-organization of multicellular patterning processes. I will address this grand challenge with the combination of computer model simulations, synthetic biology and the customized plant-tailored microfluidic platform. Initially, computer models of minimal genetic oscillatory circuits will be developed based on the integration of state-of-the-art molecular and structural information on hormonal signaling in plants. Predictions from these computer models will aid in the in vitro design of intracellular synthetic oscillator in the plant cell with the measurable hormone-responsive fluorescent output. Furthermore, the oscillator dynamics at the level of each individual plant cell as well as cell populations will be characterized with live cell imaging under variable environmental conditions in the microfluidics device. This pioneering framework for probing and quantifying coupled synthetic oscillators in plants will provide novel insights into the dynamics of hormone-mediated cellular rhythms and their role in shaping plants.

The microbiotic flora of the human gut contains a large number of Enterobacteriaceae including E. coli. While harmless in this environment, these bacteria may cause severe infections if they escape the gut. Recently, and across the globe, there have been an increasing number of outbreaks of carbapenem-resistant Enterobacteriaceae (CRE), which show resistance to the most potent of antibiotics, the carbapenems. Because resistance (and virulence) genes in bacterial cells are commonly carried on plasmids, it is vital that we understand how plasmids are segregated, partitioned and maintained over generations in bacterial cells. In this research, we propose to perform live cell imaging of the structures and inter-molecular dynamics involved in plasmid segregation using recently developed super-resolution PALM microscopy. We will focus on the type II (ParMRC) system for cells containing multiple pairs of R1 plasmids. We will investigate quantitatively the ParM spindles during plasmid segregation, observe the growth of filaments in bacteria, determine the number of filaments spawned from a single plasmid and count the number of ParR proteins on the same plasmid. In addition, we will track the dynamics of R1 plasmids within cells that contain more than a simple pair of plasmids. We will address whether the ParMRC system acts purely through pairwise interactions or if there are higher order effects. We will also probe the motion of plasmid bundles in bacterial cells. Our experiments will provides us a new (previously-inaccessible) level of quantitative information and a mechanistic understanding of the ParMRC segregation process.