miRNA, the endogenous 21nt long non-coding RNA, repress gene expression by binding to the 3’ untranslated region (UTR) of a mRNA and inducing translation repression and/or degradation of the target messages in metazoa. In human cells, miRNA repressed mRNAs are localized in a specialized cytoplasmic structure named RNA processing body or P-body. P-bodies, originally discovered in yeast as the cytoplasmic sites of mRNA degradation, could also serve as storage sites for translationally repressed mRNAs. Cationic amino acid transporter 1 (CAT-1) mRNA, encoding a high affinity transporter protein for cationic amino acids, is repressed by a liver specific miRNA, miR-122, in human hepatic cells and is stored in P-bodies. Interestingly, this miRNA-repressed mRNA get relocalized from P-bodies and translated into protein in metabolically stressed cells. Stress-induced relief of miRNA mediated repression of CAT-1 mRNA requires the RNA binding protein HuR, which relocalizes from the nucleus under stress condition and binds CAT-1 mRNA in the cytoplasm. HuR binding to a specific sequence within the 3’ UTR, is essential for stress induced relocalization and derepression of the miRNA-repressed CAT-1 mRNA. The objective of our research is to understand the molecular mechanism of HuR mediated derepression of miRNAs action in human cells. We will investigate, both using in vivo and in vitro assays, whether binding of HuR to mRNA prevents repressive function of miRNPs or displaces miRNAs from the mRNA and thereby prevent miRNA-mediated repression.

The varied arrangement and interconnectivity of collagen fibers, the primary structural protein in the body, give rise to a tremendous array of macroscopic tissue behaviors from stiff tendons to compliant skin. Interactions between fibers determine how external forces are transferred to cells, which in turn regulates the cell behavior. Quantitative knowledge of structure-function relationships of these fibrous tissues is critical for elucidating complex mechanisms of cell interactions with the surrounding matrix in health and disease. Although the mechanical properties and arrangement of collagen fibers have been studied extensively, surprisingly little is known about their interconnectivity (crosslinking, electrostatic forces, friction, etc.)
The objectives of this proposal are to learn the specialized techniques to measure the mechanical properties of individual nanometer- and micron-scale collagen fibers utilizing atomic force microscopy (AFM) developed at U Twente, and to develop novel methods for investigating the interactions between electrospun collagen fibers using AFM.
Working hands-on with AFM and nanoscale biofiber composites would catalyze my research program by providing powerful tools to investigate tissues at the cell/fiber level and to measure cell-ECM interactions. These studies will establish an international collaboration in a new area of research and will aid in obtaining pilot results for national and international grants. Without the proposed international exchange, it would be unlikely that the aims of the proposed research could be achieved.

Cell polarity, asymmetry in both geometry and concentration of proteins, is ubiquitous throughout eukaryotic cells, serving a host of purposes, including contributing to cell differentiation. The core mechanisms for establishment of cell polarity are conserved, being driven largely by differentiation of regions of the cortex. The proposed research seeks to elucidate the essential physical principles governing this process in the C. elegans zygote, a well-characterized model organism. For this system, the initial establishment of polarity is largely governed by the actomyosin cortex alone, in response to a local disruption of myosin activity near the sperm-donated MTOC, making understanding the mechanisms for polarity generation a largely physical (as opposed to biochemical) problem. As such, I will develop analytical physical models of the cortex to investigate the minimal local regulation necessary for the otherwise globally-regulated actomyosin cortex (responding to a local down-regulation of myosin activity near the sperm MTOC) to establish a phase boundary between contractile and noncontractile phases. The model will capture the essential features of the cortex, including dynamics of myosin foci, formation of a phase boundary, and furrow dynamics. The model will be informed by published results in its qualitative construction and by quantitative experiments in its parametrization. I will develop automated image processing tools to quantify RNAi rundown of proteins regulating myosin activity. Thus, I will combine theoretical physical models with quantitative measurements to understand the underlying physics of cellular polarity establishment.

The goal of the proposed work is to analyze the gut microbiota of Drosophila melanogaster, the effect of this microbiota on host immune function, and the impact of host immunity on gut microbial colonization and microbiota composition. This study will identify specific gut microbes of both lab colony and wild-caught populations of D. melanogaster and determine the stability of these associations by examining the effect of diet and source on composition. The proposed work will determine whether the host innate immune system responds to microbial colonization of the gut by comparing the local and systemic immune response of axenic Drosophila to those with a gut microbiota. To determine if the gut microbiota mediates host response to pathogens, Drosophila with and without an indigenous community will be challenged with natural pathogens that are known to affect both local and systemic host immune responses. The impact of host immunity on the persistence and composition of gut-associated microbes will be examined by comparing gut-associated microbes of Drosophila lines deficient in key innate immune functions affecting gut physiology, gut immunity, and systemic immunity. Together these results will identify interactions between Drosophila host immunity and its associated microbiota.

Cell division consists in the duplication of both the genome and the cytoplasm into two genetically identical cells. Multicellular organisms rely on the constant generation of new cells to survive. During cell division, motor proteins organize microtubules in a bipolar structure, the spindle, which serves as scaffold to transmit the necessary forces for chomorome segregation. Although the spindle is apparently static, it is highly dynamical and there are constantly fluxes of molecules. There is no satisfactory quantitative understanding of how such a highly dynamical structure results from the self-organization of individual components and maintains its shape even under external perturbations. Moreover, there are no any direct measurements of the correlation between the dynamics of the spindle components and the forces involved in its maintenance, which are ultimately responsible of chromosome segregation. I propose to experimentally examine the relationship between global properties (force, shape and length) and local properties (motor and tubulin densities, kinetics and treadmilling velocities) in the spindle. I will develop an experimental set up to simultaneously probe force production and dynamics based on magnetic tweezers and fluorescence imaging. I will also develop a quantitative model based on contiuum coarse grained descriptions taking explicitly in account the coupling between local forces and motor kinetics. This fellowship will grant me the possibility to change my research direction from biophysics to experimental cell biology and consolidate my career towards having my own research group addressing cell biology problems in an interdisciplinary approach.

Advances in genetics are giving researchers unparalleled access to the complexities of the nervous system. Key to this advance is the ability to drive the expression or removal of select genes in discrete neuronal populations. It is now possible to tag cells, track their development and potentially modify their function at any time during this process. This is particularly beneficial in terms of an evolving, high dynamic environment such as the developing cerebral cortex. The aim of the present application is to relate the embryonic specification of GABAergic interneurons to the emergence of functionality in the early postnatal cortex. We propose that interneurons acquire their mature characteristics via a cascade of genetic programs regulated by a transcriptional code akin to that determined in the spinal cord. However our knowledge of this progression has been distorted by (1) the diversity of cortical interneurons (2) the spatial and temporal distances that interneurons have to travel from the point of proliferation to integration and function. To resolve these issues we will use inducible fate-mapping technologies to track and profile discrete temporal cohorts of interneuron. A multidisciplinary approach will be used to identify when and how interneurons begin to diverge in terms of acquiring their functional identity. It will focus on (1) changes in gene expression, notably transcription factors (2) alterations in responsiveness to environmental cues e.g. neurotransmitters (3) in vivo recruitment into emergent network activity. The resultant profile will provide novel insight into the function and conversely potential malfunction of this emergent complex network.

The purpose of this fellowship is to gain experience of functional connectivity analyses in a centre that specializes in this approach for aging research, pioneering work in this field having been conducted by Professor Cheryl Grady at the Rotman Research Institute (RRI). I aim to perform such analyses on two existing fMRI data sets of normal aging, in order to learn the specific techniques developed by Professor Grady and her colleagues.
Experiment 1, uses data collected by the host supervisor (Prof Grady) investigating auditory spatial and non-spatial working memory. 1-back working memory data is available for two subject groups (16 younger and 18 older adults). Differences in the patterns of functional connectivity between the groups, and by stimulus type will be assessed.
Data for Experiment 2 was collected as part of the GENIE study at St George’s University London, where I have been investigating neural correlates of cognitive aging. Fifteen older adults (aged >70 yrs) completed a verbal working memory n-back task (with 4 conditions). Changes in functional connectivity specifically with task difficulty will be assessed and combined with diffusion tensor imaging (DTI) to explore the white matter changes and their association with loss of connectivity.
The fellowship will allow me to establish collaboration with Professor Grady, one of the leading researchers in neurocognitive aging and also visit the RRI, a première aging research institute in the world, and learn from their expertise using this technique. I will be able to apply this analysis to other data sets on my return to the UK, and develop further research combining functional connectivity and DTI analyses.

The goal of this project is to investigate the changes in structure of the ventricular wall of the mouse heart during embryogenesis, in order to better understand the origins of the human cardiomyopathy, ventricular non-compaction. This recently recognised disease is characterised by abnormally prominent and extensive trabeculae, (finger-like projections from the muscular wall), and a relative thinning ("non-compaction") of the wall itself. Neither the aetiology of non-compaction nor its pre-symptomatic progression is understood, but the formation of trabeculae and compaction of the ventricular wall normally occur during embryonic development. The disease is likely therefore either to be developmental in origin, or result from untimely onset of embryonic genetic programme in adults. Familial isolated ventricular non-compaction (IVNC) patients, detected from childhood years, suffer from cardiac dysfunction with poor prognosis. As treatment options are limited, the utmost important challenge is to better understand the aetiology of the disease so that pre-symptomatic diagnostic tools might become available. This requires a better characterisation of ventricular wall remodelling during development and availability of animal models of non-compaction. I propose to (I) investigate ventricular trabeculation in the mouse embryo, using 3D modelling and morphometric analysis (II) identify genes regulating normal ventricular wall development through the use of transgenic mutants in which trabeculation can be induced or suppressed (III) attempt to construct a mouse model of non-compaction based on mutations known to be associated with IVNC.

T cells are involved in virtually all aspects of human disease including; viral, bacterial and anti-cancer immune responses as well as during autoimmunity and organ transplant rejection. We have developed multimeric forms of peptide-major histcompatibility complexes (pMHCs) which are currently being used to identify and grade T cells in the clinic. However, the ability of these multimers to stain T cells depends on the strength of the tripartite interaction that controls T cell activation between the T cell receptor (TCR), pMHC and T cell co-receptor. Most CD4+ T helper cells (Th cells), due to the combination of generally weak TCR/pMHC class II interactions and an extremely weak pMHC/CD4 co-receptor interaction, are beyond the limits of multimer staining. This represents a major bottleneck for the analysis of Th cells in human disease. Using our previously published methods of ‘intelligent design’, I aim to modulate the pMHCII/CD4 interaction to improve its affinity. In this way, it will be possible to develop pMHCII multimers with enhanced ability to identify Th cells. The HFSP short-term fellowship will allow me to visit Prof Lawrence Stern at UMASS medical school, a world leader in the field of molecular immunology. This collaboration is necessary for the success of this project because it will allow me to learn essential techniques and approaches and to forge new contacts with internationally renowned scientists. This proof-of-concept study will provide a novel resource that has far reaching implications for the initiation of new avenues of research concerning the role of Th cells in disease.

Maintenance of the ratio between the nuclear and cytoplasmic volumes, referred to as the nucleocytoplasmic ratio (N/C ratio), is a fundamental problem of cell biology. Its implications however are unknown. The fission yeast Schizosaccharomyces pombe shows a generally constant N/C value, and its simple geometry is a great advantage to addressing the question of N/C ratio homeostasis. We therefore propose to use S. pombe as a model organism to investigate the molecular mechanisms that control the N/C ratio, and to determine the physiological consequences of its maintenance and potential modifications. This project first relies on a detailed analysis of nuclear and cellular growth rates throughout mitosis and meiosis, and when cells exit these cycles. Second, a genome-wide reverse genetic screen will be performed in order to identify genes, whose products regulate the N/C ratio. Finally we will characterize the cellular processes that may depend on specific N/C values. Such an approach will shed light on this poorly understood property of eukaryotic cells.