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.

I developed an approach to quantifying the orientation of DNA polymerases by observing the mutational patterns of strand replication in sequenced genomes. The replication terminus is clearly identified by these methods but the replication origin is not. Contrary to 50 years of biochemical characterization, a range of overlapping replication pattern indicates that initiation of replication at the origin can occur at multiple locations. I therefore hypothesize that subpopulations of bacteria exist that initiate DNA replication outside of the standard origin initiation region. Sub-population dynamics in bacteria are an important subject our ability to effectively design antibiotics to prevent bacterial growth depends not only on the bulk behavior of the population but also on individual susceptibility. The origin has been used as a target for drug design. Incomplete population control, a possible result of failing to recognize variability, can lead to the spread of antibiotic resistance and this variability should be investigated. I will first molecularly characterize the size of the sub-population of cells that initiate replication outside of the terminus region in Escherichia coli in liquid growth experiments and through fluorescent microscopy. Second, I will molecularly determine the specificity and range of potential replication start locations using ectopic insertion of a replication termination system. In addition to this molecular work I will undertake a careful bioinformatic analysis to estimate the proportion of replication starts at different locations and compare this with my molecular findings, thereby fusing molecular and bioinformatic approaches.

B cells constitute an essential part of the adaptive immune system by producing antibodies, which neutralize toxins and infected or malignant cells. B cells circulate in peripheral blood and concentrate in secondary lymphoid organs, where they migrate and search for antigens. Upon antigen recognition the cells rapidly stop and lose their ameboid shape, quickly spreading and then slowly contracting over the surface of antigen presenting cells. Concomitantly with this response, B cells completely reorganize their cell surface receptors giving rise to the immunological synapse (IS), which is a central step in B cell activation. Thus, understanding B cell migration and IS formation at the molecular level is essential to comprehend the adaptive immune response, of which very little is currently known. Here, we propose to identify novel genes that are critical for the regulation of B cell morphology. We will address this with a genome wide RNAi screen. The key candidates will be subsequently functionally characterized by cutting edge imaging techniques, such as TIRF and confocal microscopy, both in vitro and in vivo (intravital two-photon imaging). Antigen stimulation will be assessed in a highly controlled manner using synthetic lipid bilayers which, together with the imaging techniques, are readily available in the host laboratory. Furthermore, our studies will be boosted by the use of genetics to create B cells deficient in key candidates. Understanding the basic mechanisms of B cell migration and membrane antigen recognition is of great importance, not only for understanding the protective immunity of our bodies, but also for designing better antibodies for therapeutic use.

The innate immune system detects ”molecular patterns” on microbes or viruses and initiates fast responses to contain the infection. In addition, innate recognition stimulates adaptive immune responses. Therefore, the efficiency of innate detection largely determines immunity to infection. Pattern recognition receptors are integral components of the innate immune system and recognize molecules specific to pathogens. RIG-I is a DExD/H-box RNA helicase sensing viral RNA in the cytosol of mammalian cells. Activation of RIG-I induces the expression of antiviral cytokines. Previous work from the host laboratory and others shows that RIG-I agonists are RNA molecules phosphorylated at their 5’-end. During my postdoctoral training, I would like to further characterize the molecular determinants that allow RIG-I to distinguish self from viral RNA. Two lines of investigation will answer this question: (i) I would like to start with in vitro experiments to identify optimal RIG-I ligands, and I want to confirm these results by testing their agonistic activity in vivo. (ii) Next, I am planning to identify physiological RIG-I agonists by purifying, and subsequently cloning, RIG-I-associated RNAs from cells infected with different viruses. Notably, the influenza A virus NS1 protein interacts with RIG-I and inhibits RIG-I-mediated responses. Therefore, I will also analyze the mechanism of inhibition by testing (i) the role of RNA ligands in the association of NS1 with RIG-I and (ii) possible effects on downstream signaling components. Collectively, these steps will significantly extend our knowledge of RIG-I function and innate recognition, and may suggest novel anti-viral strategies.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control gene expression by targeting mRNAs for translational inhibition or degradation. They exert essential functions during embryogenesis, developmental timing, differentiation, cell growth and apoptosis. MicroRNA genes are under control of RNA polymerase II promoters enabling tightly regulated stage- and tissue-specific miRNA expression. Deregulated expression of miRNAs has been implicated in human cancer. This study aims to unravel the transcriptional network regulating miRNA gene expression in Caenorhabditis elegans. The specific aims within this research project are: (1) Identification of transcription factors regulating miRNA gene expression. Transgenic animals will be generated carrying miRNA promoter-GFP transgenes that mimic endogenous miRNA expression. I will use these strains to screen an RNAi library comprising known and predicted C. elegans transcription factors in vivo. Candidate transcriptional regulators of miRNA expression will be verified on endogenous miRNA genes. (2) Identification of transcription factor binding sites in the C. elegans genome. For this, I will perform Chromatin Immunoprecipitations of FLAG-tagged transcription factors derived from (1) and identify their promoter binding sites using commercial C. elegans genome-wide tiling arrays and a direct qPCR approach. (3) Analyses of genetic interactions between transcription factors and miRNAs. Epistasis experiments will be performed using C. elegans miRNA mutant strains along with an RNAi approach. This study will constitute the first systematic genetic screen to identify miRNA transcriptional regulation in any organism.

Nuclear reprogramming is the functional conversion of the genome contained within a differentiated somatic cell to a state of developmental pluripotency, through factors that reside in the pluripotent stem cells, such as embryonic stem cells (ESCs). The aim of this research is to decipher the molecular mechanism of reprogramming, through understanding the roles of two factors recently discovered to play essential roles in reprogramming: Polycomb repressive complex 2 (PRC2) and Oct4. Nuclear reprogramming will be studied using cell fusion between mouse ES and human B cells. PRC2 methylates histone H3 and represses many differentiation-specific genes, but also appears to upregulate a set of genes in ESCs. I will modulate expression of target genes from both categories to test their roles in reprogramming. I will also test PRC2’s requirement in reprogramming cell types at different stages of differentiation, in an attempt to decipher the nature of PRC2’s role in reprogramming. Oct4 is a transcription factor that plays a central role in maintaining ESC identity. To identify Oct4 targets that play roles at different stages of reprogramming, I will immunoprecipitate Oct4 at different times after cell fusion. Identified targets will be tested for their essentiality in reprogramming by RNAi. The significance of this proposal is two fold. First, by advancing our understandings in the reprogramming process, we will further our understandings in ESC identity and the nature of each differentiation processes. Second, this work provides essential knowledge for generating patient-specific stem cells used for cell replacement therapy.