- Total grants
- Total funders
- Total recipients
- Earliest award date
- 11 Jan 2016
- Latest award date
- 07 Dec 2016
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
Connectomics, establishing comprehensive neuronal wiring diagrams at the resolution of single synaptic connections, is still in its infancy. Although most neuroscientists are confident that connectomics will eventually have a major impact, doubts remain about when this will happen. We propose a project that within 4 years could transform an important field of neuroscience – the circuit basis of learning and memory – by reconstructing the olfactory memory circuits of Drosophila. A consortium of laboratories at HHMI Janelia has generated a complete serial section transmission EM volume of an adult female Drosophila brain. This 106 TB volume (100x larger than any previously imaged whole brain) will have a major impact on over 200 laboratories working in Drosophila neurobiology. We will reconstruct input and output neurons of the primary associative learning centre, the mushroom body, along with selected upstream layers bringing teaching signals and downstream layers mediating descending control of behaviour. This will reveal the complete network and synaptic organisation of a memory centre, whose logical principles, including sparse coding, dopamine-dependent plasticity, valence segregated by neuronal population, and network recurrence, are all relevant to mammalian brains. This will enable a wealth of experimental circuit studies as well as piloting large-scale, geographically-distributed connectomics.
Regulatory potential of repeat elements in the evolution of tissue-specific transcription 05 Jul 2016
The human genome, like all mammalian genomes, is in large part composed of decayed--but once active--repeat elements, many of which carry tissue-specific regulatory information. We hypothesise that repurposing of repeats has been critical for creating tissue-specific transcriptional regulation. Our research plan is an integrated experimental and computational strategy to systematically explore how these repeat elements have shaped the regulatory genome across the recent placental mammalian radiation.
Centrioles, at the core of centrosomes, orchestrate structure and function in the interface and mitotic cell. Here we aim to understand how centriole duplication is controlled. This requires Plk4 kinase to phosphorylate Ana2 in part so it can bind Sas6. Here we address how phospho-Ana2 interacts with other core procentriole components, Dragon, Ana3 and Rcd4 and how these events are spatially regulated to achieve duplication. Second, we aim to characterize how centriole to centrosome conversion is regulated giving newly formed centrioles competence to duplicate and nucleate cellular microtubules. We will determine how Polo kinase regulates formation of the Cep135, Ana1, Asl network essential for centriole conversion. We will also assess roles of Polo and Plk4 in anchoring peri-centriolar material (PCM) to the centriole and Plk4’s role at the peri-centriolar satellites to mobilise centrosomal molecules. Thirdly, we address how centrosomes can organise membranous vesicles. We focus upon Dragon, a molecule present in the centriole and the Golgi apparatus, and Rosario, counterpart of lysozyme-like vesicle protein LYST, required to evenly distribute centrosomes in the syncytial embryo. We will characterize the process whereby primordial germ cells form in the syncytium, an event triggered by interactions of centrosomes with the embryo’s polar cytoplasm.
In response to stress conditions and environmental changes, bacteria generate scores of small RNAs that play key roles in reshaping the dynamic landscape of gene expression. This process involves chaperone proteins that facilitate the actions of such regulatory RNAs and enzymes that affect transcript lifetimes. We aim to understand the molecular basis of these processes. Trapped intermediates of the degradative machinery with bound regulatory RNAs and targeted substrates will be structurally characterised to visualize how transcripts are captured and channelled to active sites, where they meet a fate of rapid degradation or processing into matured forms. We will identify RNA targets of chaperones and the degradative machinery and explore whether the patterns change with physiological state or during the cell cycle, and why. We want to understand why the degradative machinery has a sub-cellular localization and the origins of its dynamic and cooperative interactions with substrates and the translational machinery. Our studies will help to explain how the use of RNA enables speed and accuracy to be attained in genetic regulation and enriches the capacity of even the simplest organisms to exhibit complex behaviour in homeostasis, development and pathogenesis. This knowledge could be exploited to treat threatening bacterial infections.
Our aim is to understand how the uterine immune system regulates placentation and reproductive success in humans. We described a new mechanism of maternal allogeneic recognition that depends on KIR expressed by uterine NK (uNK) cells and their ligands, HLA-C, on fetal trophoblast. KIR and HLA-C genes are highly polymorphic and we find reproducible and specific KIR/HLA-C genetic combinations associated with reproductive disorders. We will: 1) use high throughput typing to allele level of KIR and HLA-C genes to describe how this variation affects pregnancy success. 2) translate these genetic findings into how NK cells affect trophoblast functions exploiting our new techniques, mass cytometry and long term trophoblast cell culture. 3) use transgenic mouse models to mimic the KIR/HLA-C combinations with poor outcome to study placentation in vivo and to test therapeutic anti-KIR mAbs. From a translational perspective we will: 4) investigate whether disorders such as pre-eclampsia that are common in women undergoing assisted reproductive technology with oocyte or sperm donation can be prevented by genotyping donors for KIR/HLA-C and 5) use the extraordinary variability of KIR genes in sub-Saharan Africa to study differences that can explain the increased frequency of pregnancy disorders in African women.
Achieving Selectivity in Space and Time with DNA Double-Strand-Break Response and Repair: Molecular Stages and Scaffolds Come with Strings Attached 05 Apr 2016
Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair in human cells. The aim of my research is to understand how NHEJ repairs DSBs directly without a DNA template but with maximal selectivity. Biochemical, structural and functional studies of individual components and complexes involved in NHEJ, carried out in my lab and elsewhere, suggest that several mechanisms operate throughout synapsis, end processing and ligation to maintain correct co-localisation of components over time. These are: (i) a stage provided by Ku-heterodimer interacting with DSBs supporting DNA-PKcs, APLF, BRCA1, PAXX amongst others; (ii) a second stage, DNA-PKcs, which links the kinase with DNA, Ku, PARP1, BRCA1 and Artemis; (iii) a temporary scaffold, which facilitates repair operations, constructed from XRCC4-XLF filaments, assembling to bridge Ku bound at DSB ends. Lig IV bound to XRCC4 C-termini likely terminates the scaffold, bringing LigIV close to DNA broken ends; (iv) a string provided by Artemis C-terminal-extension, which is intrinsically disordered, but includes short inear "epitopes" that recognise DNA-PKcs, DNA LigIV and PTIP, and keeps components closeby. My specific objectives are to study how these different but complementary ways provide colocalisation and efficient DSB repair.
An electron cryo-microscopy resource for macromolecular structure determination in the University of Cambridge 16 Jun 2016
We seek to establish an advanced electron cryo-microscopy (cryo-EM) facility dedicated to structural studies of biological macromolecular assemblies. The facility would provide a revolutionary new tool to the large structural biology community in the University that would enable acquisition of critical data in support of a wide and diverse range of projects tackling fundamental problems in molecular biology relevant to human health. Currently, the named applicants primarily use X-ray crystallography to study large assemblies, but many of these samples cannot be readily crystallised. The recent development of a new generation of direct electron detectors, together with sophisticated data-processing software, has dramatically improved cryo-EM analysis, which now achieves routinely sub-nanometer resolution. However, we lack access to such a facility in the University. A wide range of projects will benefit from the proposed facility, including studies of multi-drug efflux membrane transporters; assemblies of RNA metabolism; complexes regulating chromatin structure, DNA replication and repair; mitochondrial complex I and its roles in human disease; ribosome assembly defects in bone marrow failure; presenilin complexes and other assemblies related to neurodegeneration; human neurotransmitter transporters and G-protein coupled receptors; transport vesicle and organelle biogenesis; molecular mechanism of ATP synthase; and methods development in EM of biological macromolecules.
University of Cambridge - Mathematical Genomics and Medicine
University of Cambridge - Metabolic and Cardiovascular Disease
University of Cambridge 4 Year PhD Programme - Developmental Mechanisms
University of Cambridge 4 year PhD Programme - Developmental Mechanisms
Elucidation of the mammary stem cell hierarchy 30 Sep 2016
The mammary gland is a dynamic organ with many cycles of proliferation and death throughout both oestrus cycling and pregnancy. This capacity of the mammary gland for rapid growth and regeneration has been attributed to mammary stem cells (MaSCs). Despite extensive efforts over the past 60 years, definitive characterization of the MaSC, its localisation, and hierarchy, has yet to be achieve and there is still much disagreement regarding the nature and identity of mammary stem cells. Here, I propose to utilise a triad of novel techniques to unequivocally elucidate the mammary stem cell hierarchy. I will utilise continuous clonal labeling as a completely neutral approach for the labelling of a single cell and its daughters. This will be combined with optical clearing techniques and advanced imaging, giving the ability to image the entire mammary epithelial tree in three dimensions, using both fluorescent reporter genes and antibody labelling. Using these techniques, I will investigate clonally marked regions throughout the development of the mammary gland, from puberty into adulthood. This proposed program of research aims to ensure that significant progress is made in the unequivocal identification of the potential of MaSCs coupled with their localization and characterisation.
The formation of liver tissue during embryogenesis requires dynamic interactions with endo the lial and mesenchymal cells of the microenvironment, which can be recapitulated in vitro to direct the production of hepatocytes from pluripotent stem cells. The adult liver is characterised by low cellular turnover, yet it is endowed with a facultative Lgr5+ stem/progenitor population that drives tissue regeneration following damage and can be expanded in vitro as 3D liver organoids. Here, we aim to study adult non-parenchymal liver lineages – liver sinusoidal endothelial cells, hepatic stellate cells, portal myofibroblasts and Kupffer cells – as functional components of the microenvironment, or ‘niche’, in progenitor -mediated regeneration. We will characterise the spatiotemporal association of putative niche cells with Lgr5+ progenitors and differentiated progeny throughout regeneration, and perform a whole-genome transcriptomic analysis on selected niche lineages. Making use of organoid co-cultures that incorporate niche cells from regenerating livers, we will validate the ability of the niche to support Lgr5+ progenitor proliferation and differentiation. The molecular effectors of the pro-differentiation niche will be identified and the cells will be ablated in transgenic mice to confirm their niche role in vivo. These studies may elucidate novel mechanisms of liver healing and result in the establishment of organotypic liver cultures.
Investigating the epigenetic mechanism behind transgenerational inheritance in germ cells of mice 30 Sep 2016
Poor nutrition leads to an increased risk for disease, which may be passed on to subsequent generations with a normal diet. This non-genetic mode of inheritance is not well understood. The goal of my project is to investigate this epigenetic mechanism by focusing on the inheritance of altered DNA methylation in sperm from a genetic mouse model of abnormal folate metabolism (Mtrrgt model). Folate is part of the one-carbon metabolism necessary for DNA methylation. Previously, the Watson lab reported that when either maternal grandparent carried the Mtrrgt mutation, their wildtype grandprogeny displayed developmental phenotypes and dysregulated DNA methylation. How this epigenetic instability is inherited to disrupt development is unclear. My hypothesis is that abnormal DNA methylation patterns caused by Mtrr deficiency escape epigenetic reprogramming, are passed onto subsequent wildtype generations and disrupt developmentally-important gene expression. My specific goals are: 1. To prove that Mtrrgt mutants have not acquired additional genetic mutations. 2.To determine whether alterations in DNA methylation patterns in sperm from the Mtrrgt model are present and inherited. 3.To morphologically and molecularly analyze testes structure and spermatogenesis in Mtrr mutants. Understanding mechanisms of epigenetic inheritance will help us predict how our environment affects our descendants’ disease risk.
Investigating the endocrine and paracrine roles of the imprinted Igf2 gene in growth, development and metabolism 30 Sep 2016
Insulin-like growth factor 2 is major regulator of growth in mammals. It is produced at high levels inside cells and rapidly secreted into the extracellular milieu and circulatory system, thus acting as paracrine and endocrine signals for cellular proliferation, organ and whole body growth. The mechanisms which govern these multiple actions are poorly understood. We recently found that the pancreatic connective tissue (or mesenchyme) is a reservoir of Igf2 that regulates both exocrine and endocrine growth during early development. The major goals of my project are to elucidate how mesenchymal Igf2 promotes growth of the whole pancreas and to contribute to our understanding of Igf2 as an endocrine signal. Specifically, I will test the hypotheses that Igf2 binding to Igf1r in pancreatic mesenchyme is required for paracrine growth signalling, and that fetal mesenchyme is a major source of circulating Igf2. My project takes advantage of precision genetics facilitated by access to unique conditional mouse models of loss and gain of function for Igf2, allied to cell-type specific transcriptomic analysis and whole-body physiologicalphenotyping. Investigations into cross-talk mechanisms between cell types that drive systemic growth are likely to provide novel insights about growth regulatory pathways and metabolic diseases such as diabetes.