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Recipients:
University of Cambridge

Results

Understanding the biophysical basis of energy storage in adipocyte lipid droplets and the metabolic consequences of the failure of this process. 01 Apr 2015

My proposal hinges on the hypothesis that having evolved in a nutritionally challenging environment, humans are particularly well adapted to ensuring sufficient energy intake, but considerably less well adapted to coping with sustained access to energy dense foodstuffs. And that failure to optimally store surplus energy as neutral lipid within cytosolic lipid droplets (CLDs) in adipocytes, results in lipid accumulation in other tissues such as the liver where it plays a seminal role in causing i nsulin resistance, type 2 diabetes, fatty liver, dyslipidaemia and ultimately cardiovascular disease. The aim is therefore to deepen and broaden understanding of the molecular basis of lipid storage within CLDs as well as the physiological consequences of overload/failure of this process. The approach we have adopted is distinguished by being primarily based on studies involving patients with a rare disease characterised by a lack of body fat i.e. lipodystrophy. Paradoxically, lipodystrophy leads to severe manifestations of the same metabolic diseases typically associated with obesity. In both cases, we hypothesise that metabolic disease arises as a result of a mismatch between the need and capacity to store surplus energy in adipocytes. This mismatch is particularly extreme in lipodystrophy, providing a more tractable model in which to study the pathophysiology of this problem. Having made some important genetic discoveries recently, we are now seeking to address fundamental biol ogical questions which have emerged from our primary genetic discoveries, whilst also seeking to broaden our knowledge of the genetic basis of other rare adipose tissue diseases.

Amount: £1,487,351
Funder: The Wellcome Trust
Recipient: University of Cambridge

Regulatory signalling networks between stem and niche cells in lung regeneration. 27 May 2015

For multipotent stem cells to properly orchestrate injury repair, it is necessary for signals to instruct stem cells to produce specialised cells that replace injured epithelia. The precise signals from the stromal/niche cells that can stimulate differentiation for lung injury repair are under-investigated. In this proposal, I will directly address gaps in the understanding of the regulation of stem cell lineage differentiation by characterising novel stromal cell populations expressing Lgr6 in the murine distal lung and their functional interactions with region-specific stem cells during injury repair. Aim 1 will define dynamics of Lgr6-expressing cells by tracking them in homeostasis and during injury repair. In vitro organoid co-culture of regional epithelial stem/progenitor cells with Lgr6+ cells will address functional interactions between epithelia and Lgr6+ stromal cells. I will also determine if Lgr6+ cells are essential for stem cell lineage differentiation in vivo. Aim 2 will further dissect regulatory signalling molecules derived from Lgr6+ cells. Gene expression profiling of Lgr6+ cells with regional epithelial cells will describe key signalling pathways that will be evaluated by in vitro organoid co-culture assay and by in vivo mouse genetics. This work will enhance our understanding of regulatory networks between stem-niche interactions in lung regeneration.

Amount: £1,226,049
Funder: The Wellcome Trust
Recipient: University of Cambridge

Characterization of inflammation driven responses in human hematopoietic stem and progenitor cells. 27 May 2015

The existence of functionally distinct haematopoietic stem cell (HSC) subsets is crucial for steady-state blood production and efficient recovery from injury. However, if and how these HSC subsets differentially respond to stress signals has not been addressed. Moreover, some HSC stress responses have been shown to be species-specific, underlining the necessity of studying human cells directly. My own transcriptional profiling of highly purified human long-term (LT-) and short-term (ST-) HSC ind icates that these HSC subsets may be differentially sensitive to a range of pro-inflammatory signals. As a handful of inflammation cues are known to directly affect HSC or progenitor cell function during development and infection, my specific objectives are to: 1) identify novel pro-inflammatory signals that act directly on human HSC subsets; 2) define how they alter HSC function; 3) determine through which molecular mechanisms this happens. This candidate-identification approach carried out on cells from healthy donors will be complemented with a comprehensive characterization of the inflammatory milieu, functional responses and transcriptional profiles of HSC and progenitor cells isolated from patients with chronic inflammatory diseases. Altogether, I expect these studies to provide new insights on the impact of inflammation on HSC biology and its contribution to disease.

Amount: £1,245,529
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating the spatiotemporal regulation of microtubule nucleation in Drosophila. 29 Oct 2014

Microtubules are dynamic polymers that have crucial roles in many eukaryotic cell processes. They are nucleated by multi-protein gamma-TuRCs that are concentrated at various MTOCs within the cell. Although most, if not all, of the gamma-TuRC components are known, it remains unclear how gamma-TuRCs are recruited to different MTOCs at different times during development and during the cell cycle. Moreover, it is not clear how each component functions in the complex. Previous studies have concentrat ed on specific gamma-TuRC components or cell types, and have often studied only mitotic processes. I will use Drosophila as a model to conduct a comprehensive in vivo analysis of gamma-TuRC biology in various cell types, including mitotic cells, developing oocytes and terminally differentiated neurons. Mutant and fluorescently tagged lines exist for most, but not all, of the gamma-TuRC components and I intend to complete this toolset using CRISPR, a novel and rapid genome engineering technique t hat works very efficiently in flies. By studying a range of cell types and examining the role of all gamma-TuRC components, I hope to gain a better understanding of how microtubule nucleation is regulated in space and time throughout animal development.

Amount: £912,868
Funder: The Wellcome Trust
Recipient: University of Cambridge

A high resolution platform to capture the dynamic spatial proteome. 11 Jun 2015

This proposal aims to create a platform for mapping the subcellular location of a substantial proportion of the proteome in a single experiment with high resolution. It is based on preliminary work carried out by the Lilley group in collaboration with Thermo, giving tantalising insight into what this technology could deliver if developed further into a fit-for-purpose spatial proteomics platform. The key objectives are: 1. Expand the sampling of subcellular proteome to locate proteins to multi ple compartments. 2. Capture information of the effect of post-transcriptional and post-translational modification on spatial location. 3. Develop approaches to enable the mapping of the dynamic subcellular redistribution of proteins upon biological perturbation. 4. Incorporate work-flows that will deliver spatial information about targeted sub-sets of proteins and integration with whole cell maps. 5. Develop cross-linking strategies to preserve interactions of peripheral membrane proteins and between components of multi-protein complexes. 6. Develop a set of bespoke informatics tools facilitating the application of pattern recognition for robust analysis. 7. Create a GUI to facilitate community-wide interrogation of cellular maps. 8. Develop on-line protocols. 9. Apply the technology to the co-applicants and collaborators research, adding value to projects already funded by the Wellcome Trust.

Amount: £200,788
Funder: The Wellcome Trust
Recipient: University of Cambridge

New genetic, imaging and microfluidics technologies for single cell genomics 11 Jun 2015

Two major limitations of single cell genomics is (i) the loss of information about the original location within the sample of the sequenced cell and (ii) low throughput at high cost with only hundreds of cells analysed per day. Miniaturising single cell analysis to pico-litre volumes will critically facilitate higher throughput (10^4 - 10^6cells) at lower costs and sidesteps restrictions in handling. Combined with genetic technologies to record lineage history and spatially localise cells this w ill allow questions to be address at single cell resolution that are essential to understand cell diversification following tissue-contextual interactions and the impact it has on gene transcription. 1) We will develop microfluidics based devices to increase sequencing throughput by increasing the number of cells processed at a low cost, and at the same time enabling the complex handling and manipulation of small cell numbers with minimal loss. 2) We will develop genetic technology to: i) record within the genome the lineage history of each cell, for readout at any stage of interest and ii) to provide a unique fluorescent signature to cells to enable us to provide spatial and temporal context to their transcriptional profiles.

Amount: £410,687
Funder: The Wellcome Trust
Recipient: University of Cambridge
Amount: £2,000,004
Funder: The Wellcome Trust
Recipient: University of Cambridge

Decoding how neutrophils self-organise their migration to sites of infection in vivo 14 Jul 2014

Neutrophils are key effectors of antibacterial immunity and inflammation. These cells often migrate in a highly co-ordinated and directed manner in order to reach sites of infection. This so-called ‘swarming’ response was shown to depend on self-production of the neutrophil attractant, LTB4. However, the cellular dynamics underlying transition from exploratory, single cell migration to collective and highly directional migration remain unclear. To address this knowledge gap I will use in vivo imaging and genetic manipulations in a zebrafish model. I will first determine the cellular triggers for LTB 4 production by neutrophils in situ. For this I will make transgenic fish expressing a reporter probe for LTB 4 production. Then I will investigate how LTB4 autocrine/paracrine signalling directs neutrophil polarity and migration. For this I will directly monitor autocrine/paracrine signalling using an additional probe for LTB 4 sensing. Finally I aim to spatiotemporally manipulate LTB 4 production and neutrophil swarming in vivo. To this end, I will develop an optogenetic tool to control the production of LTB 4 by light and use this to establish the implications of neutrophil swarming in microbial defence and tissue integrity. Thus, this study will provide a better understanding of how neutrophils self-organise their migration to sites of infection

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

The molecular mechanism of virulence effector protein translocation by Salmonella SipB. 14 Jul 2014

A critical step in pathogenesis of enteric bacteria such as Salmonella is hijacking the host cell. This event depends on the type three secretion system (T3SS) which enables the delivery of subversive virulence effectors into the host 3,11. A hydrophobic protein called SipB is part of the T3SS and forms the translocon element 3. While the mechanism of effector translocation across the host cell plasma membrane remains elusive, previous work in our lab revealed that SipB is essential for translocation and has liposome fusion activity 7. When fusion is inhibited with a truncated derivative of SipB (SipB428-593), Salmonella entry into host cells is prevented6. The aim of this project is to build upon this foundation to research the mechanism of effector delivery into host cells. We will use established in vitro liposome fusion assays – together with biochemical reconstitution and high-resolution structural studies – to reveal the mechanism of translocation. Following the successful work with SipB 428-593, we aspire to screen natural extracts and synthetic compound libraries in search of a T3SS inhibitor, which could have therapeutic value. Finally, we will also extend our work onto homologous T3S systems from other pathogens, such as Shigella, Yersinia and E. coli.

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating!the!influence!and!therapeutic!potential!of!RNA!G^quadruplex!structures!on!positive! 14 Jul 2014

An!increasing!body!of!evidence!suggests!that!RNA!secondary!structures,!such!as!G] quadruplexes!and!stem]loops,!play!crucial!roles!in!the!regulation!of!translation!and!RNA! replication!during!viral!infection.!Our!ability!to!exploit!such!RNA!structures!therapeutically!is! dependent!upon!our!knowledge!of!how!they!act,!which!remains!unclear.!!One!goal!of!this! research!is!to!systematically!identify!and!examine!the!influence!of!RNA!G]quadruplexes! present!in!the!coding!regions!of!two!widely!used!model!calciviruses.!Using!the!established! reverse!genetic!systems!for!these!viruses,!the!effects!of!mutations!that!alter!the!presence!and! distribution!of!G]quadruplex!structures!can!be!studied,!both!in!terms!of!virus!efficiency!and!rate! of!RNA!synthesis!in!cell!culture.!If!suitable!mutated!viruses!are!obtained,!the!persistence!of! these!viruses!within!a!small!animal!model!will!be!examined!which!will!give!insights!into!the! suitability!of!such!modified!viruses!for!use!as!vaccines.!The!second!goal!of!this!research!is!to! investigate!the!mechanisms!by!which!helicases!control!the!regulation!imposed!by!such!RNA! structures.!Using!a!reconstitution!system!for!translation!initiation!in&vitro,!the!hierarchy!between! different!RNA!secondary!structures!and!the!helicases!that!mediate!their!unwinding!can!be! examined.

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Elucidation of the mammary stem cell hierarchy 23 Jun 2014

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.

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Characterisation of fate choice regulators in single mouse haematopoietic stem cells 23 Jun 2014

Virtually nothing is known about the exact molecules responsible for orchestrating haematopoietic stem cell (HSC) self-renewal and lineage differentiation. Identifying molecules governing stem cell self-renewaland differentiation will not only provide a greater insight into stem cell biology but may also uncover novel therapeutic targets for cancer since cellular mechanisms regulating clonal expansion in cancer are likely analogous to those operating in stem cells. My PhD will focus on the identification and assessment of molecular drivers of stem cell fate choice and has two aims: 1) Identification of genes directing lineage choice in HSCs. Two approaches will be taken to identify candidates: i) Clustering of single HSC gene expression profiles and functional transplantation outcomes based on cell surface marker expression, allowing the correlation of functional outcome with a particular gene expression profile. ii) Gene expression profiling of single HSCs and their direct progeny in conditions which support either self-renewal or promote differentiation. 2) Functional validation of gene candidates in vitro and in vivo with respect to effects on self-renewal and differentiation. This will be achieved by perturbing gene function and by performing a series of single cell functional assays.

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Quantitative clonal analysis and modelling of retinogenesis in zebrafish 23 Jun 2014

In order to understand how organs achieve their specific sizes and cell compositions, it is critical to have quantitative details on the proliferation and fate choices of the progenitor cells through developmental time. The zebrafish retina is an excellent system to study this process in the central nervous system of a vertebrate, and to date we have quantitative models that do a reasonable job at predicting how the zebrafish retina is generated. There are still some uncertainties and non-fixed parameters in these models and the models are not yet fully accurate. The project here is to obtain a great deal more data so that we can generate more accurate and constrained models. Key Goal 1: To optimize a protocol for imaging the whole zebrafish retina so that it will be possible to follow the full clonogenesis and differentiation of retinal progenitor cells (RPCs), from 24 hours post fertilization (hpf) to 72 hpf when central retinal development is complete. Key Goal 2: To generate a computational image analysis based toolset to track the lineages resulting from RPCs, as well as their movement in three dimensions. Key Goal 3: To extend the current model the RPC fate decision-making process so that it more fully and accurately explains clone size and cell composition. Key Goal 4: To develop a method for in vitro clonogenesis and thereby to study the development of zebrafish RPCs plated at clonal densities

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Identifying and understanding the function of trait associated non-coding variants 14 Jul 2014

In this project, I plan to expand on existing genome wide association study analysis techniques, to empower our ability to detect trait associated variants. The techniques will aim to target the ‘missing heritability’ and the current lack of knowledge surrounding the functionality of non-coding variants. This will be achieved by calculating, in an unbiased manner, the likelihoods of non-coding variants to control downstream gene products. In order to optimally evaluate the likelihood, I will impute publically available annotations into existing sizeable cohorts, for which whole-genome (or genotype) sequence data is available. These prior probability distributions will be subsequently incorporated into existing gene set test methods, in combination with multi-trait strategies. I will apply the developed method to existing cohorts, for which quantitative glycaemic and lipid trait measurements are available in an attempt to identify trait associated loci, which have not yet been uncovered. Additionally these likelihoods can be used to generate hypotheses, which explain how a variant mediates its effect on the observed phenotype. These theories will be subsequently tested, using methods that are able to elucidate causation from consequence and account for confounding factors.

Amount: £160,792
Funder: The Wellcome Trust
Recipient: University of Cambridge

Hormone release from the gastrointestinal tract: mechanisms underlying bile acid-induced GLP-1 release and characterisation of somatostatin signalling. 17 Jan 2014

Gut hormones are key to the regulation of nutrient homeostasis and therefore particularly relevant to type-2 diabetes. This proposal involves determining the mechanisms underlying secretion of two gut hormones, glucagon-like peptide1 (GLP-1) and somatostatin (SST). GLP-1 is an incretin hormone released in response to components in the intestinal lumen and acts to support nutrient disposal throughout the body. A preliminary project recently identified a G-protein coupled bile acid receptor(GPBA)-independent signalling pathway of bile acid-induced GLP-1 secretion in the distal small intestine. This study proposes to characterise this pathway and determine the roles of apical sodium-dependent bile acid transporter (ASBT) and farsenoid X receptor (FXR) in vitro and in vivo using appropriate inhibitors/agonists and knockout mice. An investigation into the relatively poorly understood inhibitory gut hormone SST is also proposed. SST negatively regulates the secretion of many hormones including GLP-1 but the stimuli and signalling pathways by which SST release occurs are poorly characterised. This proposal involves in vitro assessment ofSST secretion in response to various nutrients and pharmacological tools as well as the relationship between SST and other gut hormones. These data, together with transcriptomics of SST-producing cells, will be used to identifycell-specific signalling pathways.

Amount: £18,565
Funder: The Wellcome Trust
Recipient: University of Cambridge

Targeted mRNA Trafficking in Pathfinding Axons of the Developing Visual System. 24 Jun 2013

Building properly wired neural circuits involves guidance of axons towards synaptic targets in the brain. In the growth cone of pathfinding axons, targeted mRNA transport and localised translation is important for autonomously regulating navigational responses tocertain extracellular guidance cues. Still however, axonal mRNA trafficking remains poorly understood. We know very little about which mRNAs are differentially trafficked on guidance cue stimulation or mechanisms underpinning axonal mRNA transport. Using the well-characteriseddeveloping visual system as an experimental platform, this research proposal aims to explore the functional and mechanistic basis of targeted mRNA trafficking during axon guidance. We

Amount: £159,401
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigation of Mechanisms Linking PIK3R1 Mutations to Metabolic Diseases. 24 Jun 2013

Phosphoinositide 3-kinase (PI3K) is involved in metabolic and mitogenic signalling pathways. Heterozygous autosomal dominant mutations of Class IA PI3K regulatory subunits (PIK3R1), p85alpha, p50alpha and p55alpha, have been identified in patients with SHORT syndrome. This is a rare monogenic disease characterised by a variety of abnormalities including insulin resistance (IR) and hyperglycaemia. Research within the Semple group has focussed on in vitro and in vivo studies of the SHORT mutation Y657X (Y, tyrosine; X, stop codon) of p85alpha. Previous work has not succeeded in deciphering the mechanisms causing the disease phenotype, and this project aims to develop our understanding of how PIK3R1 mutations lead to IR exhibited by patients with SHORT syndrome. It is hypothesised that insulin signalling defects may be facilitated by the shorter splice variants, p50alpha or p55alpha, and that these mutations alter regulation of p110beta-mediated pathways. Additionally, it is proposed that IR occurs in a cell-specific manner from either hepatocytes or adipocytes. Furthermore, PIK3R1 mutations are associated with

Amount: £159,305
Funder: The Wellcome Trust
Recipient: University of Cambridge

Identification of novel molecular mechanisms of HIV latency using forward genetic screens 24 Jun 2013

The latent viral reservoir remains the major block to HIV eradication and transcriptional repression is thought to be the predominant mechanism of silencing latent HIV.However, multiple mechanisms of repression are likely to be responsible and finding a cure for HIV is clearly dependent upon an improved understanding of the molecular mechanisms of latent HIV infection. Here, I propose to use forward genetic screens to identify novel genes required for HIV silencing. I will use an HIV-based GFP reporter construct to establish a model of HIV latency in the human near-haploid KBM7 cell line. The KBM7 cells will be mutagenised with a retroviral gene-trap vector, rare GFP-expressing clones will be isolated, and their mutation sites mapped using next generation sequencing. Candidate genes will then be validated in more representative HIV model systems and biochemically characterised to elucidate their mechanism of HIV silencing.

Amount: £159,305
Funder: The Wellcome Trust
Recipient: University of Cambridge