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Recipients:
University of Cambridge
University College London

Results

Gene Expression Heterogeneity in the Maintenance and Coordinated Differentiation of Neuromesodermal Progenitors in vivo 08 Aug 2018

Modern imaging data in biology is essentially multi-scalar in that raw image data undergoes a series of processing steps until it is at a manageable size to perform quantification and analysis. While this processing pipeline might be beneficial to one set of scientific questions, it may be inappropriate to others. Computational biologists may be interested in improving the pipeline itself, while other researchers may be interested in accessing the already processed data. Thus, a central road-block in the open sharing of large-scale imaging data is the fact that there is no one size fits all solution. This project aims to generate a web-based database that stores experimental and data descriptors together with links to the raw and processed data files. This will greatly enhance our ability to upload this data to repositories that are based placed to share the datasets in question, from the raw unprocessed data files down to feature extracted and processed data. Upon submission, the website will link to the deposited data and thereby act as an integrated platform for other researchers to access and explore the data that is available to them. Thus, researchers will be able to access our data at all levels. In built in the project is a second evaluation phase, whereby will we reach out to collaborating laboratories to assess the effectiveness of our open research platform. These will include computational biologists interested in accessing raw data files and processing pipelines, and other developmental biologists who will interact with processed datasets.

Amount: £48,563
Funder: The Wellcome Trust
Recipient: University of Cambridge

Molecular mechanisms of alternative splicing regulation 28 Nov 2017

Alternative pre-mRNA splicing (AS) is a widespread regulatory mechanism enabling individual genes to generate multiple protein isoforms. We have investigated the mechanisms controlling AS events that are regulated during the transition of smooth muscle cells (SMCs) between contractile and proliferative phenotypes. We have shown how the widely-expressed RNA binding proteins (RBPs) PTBP1 and MBNL1 regulate SMC splicing events. Recently, we identified RBPMS as a potential "master" regulator of SMC AS. RBPMS is sufficient to switch AS events to the SMC pattern and its activity is strongly modulated by its own AS and by phosphorylation. Critically, RBPMS is sufficient to switch AS to the SMC pattern in vitro. This offers a unique opportunity to determine the molecular anatomy of regulated splicing complexes. We will carry out detailed mechanistic analyses of RBPMS-regulated splicing using a combination of biochemical, proteomic, single-molecule, and structural approaches including Cryo-EM. We will identify critical regulatory interactions between regulatory RBPs and core splicing factors, and test their importance by genome editing and mRNA-Seq. In a complementary aim, we will investigate how peptide-ligand interactions equip PTBP1 to regulate AS and a range of other post-transcriptional processes, and whether a family of such peptide-mediated interactions extends to related RBPs.

Amount: £1,503,005
Funder: The Wellcome Trust
Recipient: University of Cambridge

Inhibition in the Periaqueductal Gray 30 Sep 2018

Deciding which action to take, such as whether to cross a busy road, is a critical survival skill. Making decisions requires integrating complex information and identifying the cellular mechanisms of this process is critical for understanding how the brain computes decisions. In this project will investigate neurons that control defensive decisions in mice and focus on inhibitory neurons in the midbrain Periaqueductal Grey (PAG), which have the ability to veto defensive behaviours.The first main goal of the project is to use electrophysiological and advanced molecular techniques, such as RNA sequencing and gene knock-down, to identify the genes and ion channels that control the firing of PAG inhibitory neurons. The second goal is to determine key regulators of the activity of these neurons, in particular neuromodulators and long-range synaptic connections from other brain areas, using techniques such as optogenetics in combination with behavioural assays that exploit the innate defensive behaviours of mice. The results of this work will reveal new the biophysical principles that drive firing in a key population controlling a critical behavioural decision, and provide an entry point for understanding how pathological states such as anxiety lead to maladaptive decisions.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College London

Non-local computations in hippocampal circuits: neural mechanisms 17 Jul 2018

The overall goal of this proposal is to build a neural-level understanding of how non-local cortico-hippocampal communication mediates memory consolidation and spatial computations. The well-studied network of spatially modulated neurons in the hippocampus and associated regions provides the pre-eminent cellular-model of memory for events and places. However, the activity of these neurons mainly encodes local information, that is, the current configuration of an animal in its environment. Work conducted by us, and others, have identified transient reactivations of hippocampal neurons and cortical counterparts as a key mechanism supporting systems consolidation and spatial planning. These brief ‘non-local’ events provide a means by which remembered experiences can gradually update memory networks, equally they are theorised to support the calculations necessary for route planning. Our aims are: 1) to understand how hippocampus and cortex interacts during reactivations; 2) determine how reactivated information affects existing representations; 3) precisely define the spatial computations that guide navigation; 4) investigate how neuromodulation controls the occurrence of reactivations. To this end, our approach is to combine computational modelling, machine learning, and state-of-the-art experimental techniques. Developing a basic understanding of these processes opens the way to understand how they fail in disease and may ultimately deliver tremendous therapeutic benefits.

Amount: £1,670,136
Funder: The Wellcome Trust
Recipient: University College London

Investigating the energy homeostasis of cellular calcium signalling 31 May 2018

Cellular calcium signalling is a ubiquitous and fundamental mechanism driving many processes in cell physiology. However it carries a significant energetic cost: calcium that enters the cytosol must be removed or sequestered by ATPases. In this project we propose to explore the mechanisms involved in maintaining energetic homeostasis in the face of this energy demand. The transfer of calcium signals to mitochondria is thought to support energy production, as it upregulates the rate limiting enzymes of the TCA cycle, increasing the rate of ATP production, although extrusion of calcium from the mitochondria also carries an energetic cost. The recent development of new targeted fluorescent reporters allows detailed exploration of compartmental ATP generation, making these questions accessible. We will therefore use fluorescence microscopy and imaging of a novel mitochondrial targeted ATP reporter to measure changes in cytosolic and mitochondrial ATP in response to changes in cytosolic and/or mitochondrial calcium signals to address these fundamental questions in cell biology. It is important to understand the fundamental mechanisms of cellular energy homeostasis so that we can better understand how mitochondrial dysfunction, associated with many disease states, undermines the ability to match energy demand with energy production.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College London

New methods to predict gene function in fission yeast 30 Sep 2018

Computational biology aims to answer some of biology’s most complex questions using computational and statistical methods. The field has successfully identified genes involved in disease and has helped to discover drugs for their treatment. My PhD research takes this approach to understand the processes by which we age. Aging is a complex disease — characterised by the progressive loss of function in an organism over time — with huge social and financial cost. Faced with an aging population, breakthroughs in this area are desperately needed. I am attempting to do this using data collected from experiments that measure the lifespan of yeast in different environmental conditions. Whilst we are only distantly related to yeast, it is a useful model of human aging as it shares many cellular processes, but lives for a fraction of the time. Ultimately, I aim to use these data to predict the genes that cause yeast to age. Whilst a handful of aging genes have been identified, more genes are likely to contribute. I use networks and machine learning approaches to make my predictions. Going forward, these will help to deepen our understanding of aging and aid the development of treatments to eventually cure this disease.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College London

Protein level knockdown of transmembrane proteins 31 May 2018

TrimAway is a newly described protein-level depletion method for degrading specific endogenous proteins. The technique relies on the experimental introduction of antibodies to the cell, which elicits rapid degradation of target antigens via the cytoplasmic Fc receptor and E3 ubiquitin ligase TRIM21. This enables acute depletion of proteins, enabling the functional characterisation of previously intractable proteins. TrimAway has been shown to act against diverse cytosolic substrates including membrane-anchored GFP. However, it is currently unknown whether TrimAway is capable of targeting transmembrane proteins. To address this, we will attempt to degrade three representative transmembrane proteins using TrimAway. With one, seven and twelve transmembrane passes respectively, the impact of topology on degradation rates will be determined. Targets, and control GFP, will be expressed as C-terminal myc-tagged constructs and expressed in human cells competent for TrimAway. Anti-myc antibody will be electroporated into the cells according to established protocols and the fate of target proteins will be monitored by western blot. Our targets have been selected as well-studied, disease-relevant proteins whose function may be illuminated by acute depletion. The results will help define the limits of the TrimAway technique and shed light on the cell's ability to degrade membrane proteins via the ubiquitin-proteasome system.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating the Contribution of SOX17 Mutations to the Pathogenesis of Pulmonary Arterial Hypertension 30 Sep 2018

Pulmonary arterial hypertension (PAH) is a rare fatal disease characterised by increased medial muscularisation of larger pulmonary arteries and neomuscularisation of small non-muscular arterioles, leading to obliteration of the vessel lumens. This increases the pulmonary arterial pressure and thus, the workload placed on the right ventricle, ultimately causing death by right ventricular failure. PAH is associated with endothelial dysfunction, namely increased permeability and apoptosis. Mutations in the gene encoding the Bone Morphogenetic Protein type II receptor, BMPR2, cause the majority of familial PAH cases and approximately 25% of apparently sporadic cases. Recently, we have co-ordinated a national DNA sequencing study of patients with idiopathic PAH to identify causal genetic mutations. Our study has identified that mutations in the gene, SOX17, are significantly associated with PAH in some IPAH patients. SOX17 is a transcription factor that is reported to control endothelial function during developmental angiogenesis, integrating with vascular endothelial growth factor signalling. We have identified a potential link with BMP signalling, whereby circulating BMP ligands that regulate endothelial stability induce the expression of SOX17. My project aims to explore the role of SOX17 in the pulmonary circulation and how SOX17 deficiency may cause dysregulated pulmonary vascular function.

Amount: £0
Funder: The Wellcome Trust
Recipient: University of Cambridge

Predicting the Strength of Zinc Metal Binding Sites in Proteins 30 Sep 2018

Many proteins bind to metal ions, and rely on these interactions in order to properly carry out their function - indeed failure to do so is responsible for a number of diseases. Understanding the details of how these metal-protein interactions work and developing tools to predict their occurence in newly discovered proteins, as well as predicting sites that are created when proteins interact with one another, will aid our understanting of these diseases. Such knowledge would also allow us to engineer metal binding sites into synthetic proteins, which would have enormous benefits in biosensing, novel pharmaceuticals, and developing synthetic biological circuits. This project will focus specifically on zinc-protein interactions - creating a publicly accessible database resource of known zinc binding sites, and developing sophisticated tools for predicting the location and strength of zinc binding sites in a given protein structure, including sites that occur when two protein chains come together. Ultimately the project seeks to allow a researcher to modify a protein without zinc binding ability, to enable it to bind zinc and to reveal sites for drug design to modify zinc binding.

Amount: £0
Funder: The Wellcome Trust
Recipient: University College London

Deciphering Notch signalling dynamics in vivo 17 Jul 2018

To make and organize different tissues, cells must decipher information from developmental signalling pathways. Transmitting this information accurately, so that cell-surface signals are translated into correct transcriptional responses, is of critical importance but how this is achieved mechanistically remains a major question. We propose to answer this focussing on the Notch pathway. Dosage and dynamics of Notch activity are fundamentally important for developmental decisions and tissue homeostasis and their mis-regulation underlies many diseases including cancers. Our ability to image events in real time within living fly embryos gives us a powerful system to investigate dynamic properties of Notch signalling in physiological conditions. Our overall goal is to decipher temporal, quantitative and mechanistic principles that govern how Notch activity is read by target enhancers in the living animal. Using cutting-edge strategies for measuring transcription responses and complexes we will address: (i) how Notch signals are decoded in real time in vivo, (ii) what mechanisms/partners are required to accurately transduce Notch signals, (iii) what roles tissue geometry and forces play in shaping signalling dynamics. Answers will provide new insights for manipulating Notch in a controlled way, enabling strategies for altering cell fates or for treating diseases driven by aberrant Notch signalling.

Amount: £1,270,600
Funder: The Wellcome Trust
Recipient: University of Cambridge

The chemical biology and function of natural modified DNA bases in genomes 28 Nov 2017

I aim to elucidate the function of natural, chemically-modified DNA bases in the genomes of model organisms, using chemical biology and physical science approaches on genomic DNA. Modified bases are of fundamental importance to transcriptional programming and cell identity during and after development. The role of the cytosine derivative 5-formylcytosine and its influence on nucleosome formation, active enhancers, transcription and cell identity will be one area of focus to build mechanistic understanding, following on from hypotheses derived from our prior work. There will also be an investigation of 5-carboxycytosine and 5-hydroxymethyluridine and their potential links with transcription regulation. For other modified bases, such as N6-methyladenine, we will develop and use new chemical mapping/sequencing methods to elucidate their function in mammalian systems. The programme will include a systematic discovery of other natural DNA base modifications, building on and augmenting chemical methodologies I have developed to discover and profile modified bases in RNA. The function of newly identified base modifications will be investigated during the programme. The insights provided from these fundamental studies may have far-reaching consequences for normal biology and disease states. Keywords: chemical biology, nucleic acids, DNA, modified bases, epigenetics, sequencing

Amount: £2,198,391
Funder: The Wellcome Trust
Recipient: University of Cambridge

Building and breaking epithelial integrity in the neural tube: an optogenetic approach 18 Oct 2017

Using an innovative optogenetic approach within the zebrafish neural tube, I will directly explore how the polarity of individual cells drives the tissue organisation of a whole organ. In combination with 4D live imaging and functional abrogation, I will use light to specifically and reversibly manipulate apicobasal polarity, cleavage furrow formation and PI3K pathway signalling on a subcellular level. I will assess how apicobasal polarity and division are interrelated during morphogenesis of vertebrate epithelial tubes and how this relationship contributes to tissue integrity. Early zebrafish neuroepithelial divisions are highly predictable and coincident with de novo apicobasal polarisation. This provides a tractable model to assess a potential feedback loop between apical protein localisation and cleavage furrow positioning during epithelial establishment. The PI3K pathway is likely key to integrating apicobasal polarity with division. Within established epithelia, PI3K pathway defects are prevalent in cancers. I will manipulate PI3K pathway signalling within individual cells or groups of cells within an otherwise normal zebrafish neural tube. This in vivo method for manipulating cancer-linked signalling will allow me to test whether apicobasal polarity dysregulation is a cause or consequence of tissue disruption, providing clues to the cellular mechanisms of disease initiation.

Amount: £977,448
Funder: The Wellcome Trust
Recipient: University of Cambridge

A multi-disciplinary approach to understanding and improving hearing by cochlear implant users 28 Nov 2017

Cochlear implants (CIs) restore hearing by electrically stimulating the auditory nerve. This allows many CI users to understand speech well in quiet, but even the most successful have poor pitch perception and struggle in noisy situations. We believe there are two main reasons for these limitations.(i) Although it is possible to elicit different pitches by stimulating different electrodes, the selectivity of this place-of-excitation cue is much worse than in normal hearing (NH). (ii) It is also possible to increase pitch by increasing the pulse rate applied to each electrode, but use of this temporal cue is also much worse than in NH. We will study both of these limitations by performing analogous experiments in cats and humans, using some of the same measures in the two species. This will allow us, for the first time, to link the limitations that occur perceptually to their underlying physiological bases, and to do so even for novel stimulation methods that are not possible with existing clinical CIs. The knowledge gained wiill allow us to propose and test modifications both to implant design and audiological practice.

Amount: £1,727,476
Funder: The Wellcome Trust
Recipient: University of Cambridge

Disorders of Thyroid Hormone Action: Diagnosis, Pathophysiology and Therapy 10 Apr 2018

Having discovered that cystic degeneration of aortic media in human selenoprotein deficiency causes its aneurysmal dilatation, we will elucidate its pathogenesis using mice with conditional, aortic selenoprotein depletion and patients stem cell-derived vascular smooth muscle cells and determine whether antioxidants can inhibit this process. We will investigate structure-function relationships in SECISBP2, including how deficiency of this master regulator is variably rate-limiting for synthesis of different selenoproteins. Following our first identification of RTHalpha, a disorder due to TRalpha mutations with tissue-selective hypothyroidism but near-normal thyroid hormone levels, we will determine its genetic architecture and phenotypic spectrum. We will identify abnormalities in circulating metabolites and proteins to enable diagnosis of RTHalpha and guide its therapy. Aided by structural insights, we will design and test thyroid hormone analogues that disrupt mutant TRalpha-corepressor interaction, the basis of dominant negative inhibition which mediates pathogenesis of the disorder. We will develop biochemical markers to differentially diagnose RTHbeta cases from patients with TSH-secreting tumours and to guide RTHbeta therapy. In patients with deficiency of the MCT8 thyroid hormone transporter, we will trial whether treatment with triiodothyroacetic acid, a thyroid hormone analogue whose cellular transport is MCT8-independent, alters neurodevelopmental outcome.

Amount: £1,601,565
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of RNA modifications in myeloid leukaemogenesis 24 Apr 2018

Acute myeloid leukaemia (AML) is a devastating cancer with a long-term survival below 30% for which mainstream treatments remain unchanged for several decades. Advances in genomics have highlighted the importance of epigenetic corruption in both initiating and maintaining the disease, making the epigenome an important therapeutic focus. Recently, using CRISPR-Cas9 recessive genetic screens, we identified several RNA-binding/modifying proteins as essential for AML cell survival. We have since confirmed that the RNA methyltransferase METTL3 is required for AML maintenance through its role in co-transcriptional N6 adenosine methylation of target RNAs, but is dispensable for normal haematopoiesis proposing it as a novel "druggable" therapeutic target in AML (Barbieri, Tzelepis et al, Nature 2017). Here, I propose to extend the investigation of the epitranscriptome as a new therapeutic focus in AML by studying promising AML-essential RNA-binding/modifying proteins, including METTL1 and METTL16, using unique reagents and expertise as well as access to clinically relevant bespoke models and human samples.

Amount: £250,000
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating the effect and impact of binding RSV-G protein on pneumococcal growth and antibiotic sensitivity 21 May 2018

The pneumococcus (Streptococcus pneumoniae) is a global pathogen associated with high mortality rates in young children and the elderly. My research has shown that pneumococci rapidly increase their virulence by binding to the G protein encoded by human respiratory syncytial virus (RSV), the leading cause of viral pneumonia. This is thought to be mediated by the pneumococcal receptor for RSV, penicillin binding protein 1a (PBP1a), an important protein involved in bacteria cell wall synthesis and cell division. However, the mechanism behind the increase in virulence remains unknown. This proposal builds on my recent observations and brings together world-leaders in the fields of virology and bacterial cell wall biosynthesis to determine the immediate phenotypic and genotypic consequences of the pneumococcus binding to RSV-G protein. To do this I will: Determine whether binding RSV-G protein affects pneumococcal growth and increases pneumococcal cell lysis. Determine the effect of RSV-G on PBP1a gene expression and the enzymatic activity of pneumococcal PBP1a. Determine whether binding to RSV-G protein increases pneumococcal antibiotic sensitivity. These findings will help direct a larger study to determine the mechanisms of virulence enhancement during co-infection and the impact this has on inflammation, the host cell response and, importantly, antimicrobial therapy.

Amount: £99,958
Funder: The Wellcome Trust
Recipient: University College London

Personalising the pharmacological treatment of bipolar disorder 23 May 2018

Background Prescribing for bipolar disorder is a major clinical dilemma as long-term pharmacological treatment is often necessary. Lithium is the most effective mood stabiliser. However, only 30% of individuals have a good therapeutic response. Presently, there is no reliable way to predict response or adverse event risk, or if an alternative treatment would be better for that patient. Aim To personalise prescribing for people with bipolar disorder via prediction models that quantify potential benefits and risks of existing treatments based on clinical phenotypic characteristics of the individual. Objectives Identify individualised clinical predictors of lithium and second-generation antipsychotic response. Determine clinical predictors of chronic kidney disease in individuals taking lithium. Determine clinical predictors of pathological weight gain in individuals taking second-generation antipsychotics. Methodology Data sources Swedish population registers, Hong-Kong health registers, Taiwanese health insurance database, UK primary care data linked to secondary care admission records, and UK mental health care data. Analyses Traditional epidemiological and machine learning methods; drawing on the strengths of each approach. Prediction model generation I will combine predictors from different datasets; resulting in models predicting drug response, chronic kidney disease and weight gain. Application Prediction models will be presented as online and smartphone application clinician decision aids.

Amount: £460,704
Funder: The Wellcome Trust
Recipient: University College London

Defining the biological basis of Staphylococcus aureus carriage 17 Jul 2018

Staphylococcus aureus is major cause of infection worldwide. This bacterium persistently colonises the nose (its natural niche) in around 20% of the population, which increases their risk of S. aureus infection. Why some people carry S. aureus while others never do is not understood, but is likely to reflect a complex trait influenced by multiple factors. This may include the human genome, host immunity, the nasal microbiota, bacterial-nasal epithelial cell interactions and lifestyle choices. We propose that key determinants for S. aureus carriage can be defined in a powered cohort study in which these parameters are established. Our study will capitalise on existing cohorts (INTERVAL & COMPARE) of healthy volunteers who have been extensively characterised through human genome sequencing and phenotypic profiling. We will screen 25,000 INTERVAL participants for S. aureus carriage, and using sequencing methods define their nasal microbiota composition. We will use existing as well as generate additional data on lifestyle. These datasets will be mined during a series of genome-wide and phenotypic association studies to identify factors that influence the nasal microbiota and S. aureus carrier status. Selected phenotypic and genetic variants of interest will then be tested in relevant experimental systems.

Amount: £2,341,255
Funder: The Wellcome Trust
Recipient: University of Cambridge

Efficient and transparent methods for linking and analysing longitudinal population studies and administrative data 05 Jul 2018

The Wellcome Trust LPS Strategy states that research is needed to i) underpin efficient linkage of multiple datasets, ii) quantify potential biases resulting from linkage, and iii) to handle linkage error in data analyses. We propose a programme of methodological research to develop efficient and transparent methods for linkage and analysis, to help maximise the joint potential of existing longitudinal population studies and multiple administrative datasets. Our proposed work packages will focus on linkage of LPS and multiple administrative datasets: WP1: Facilitate development of innovative linkage methods for multiple datasets, by developing shareable software to generate synthetic datasets that are generalisable to a range of research settings. WP2: Develop methods for efficient linkage of multiple datasets by expanding existing probabilistic linkage methods to the setting in which multiple administrative or LPS datasets are to be linked dynamically, and where there are multiple sets of identifiers (e.g. collected at different time-points). WP3: Provide appropriate tools for analysis of linked data by extending existing imputation methods for handling linkage uncertainty and avoiding bias within analysis. WP4: Maximise the value of approaches developed in WP2-3 through evaluation using exemplar linkages and dissemination of methods to the LPS community and other key stakeholders.

Amount: £301,055
Funder: The Wellcome Trust
Recipient: University College London