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Recipients:
University of Cambridge

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

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

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

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

Salary support 2018 - 2023 30 Sep 2018

Not available

Amount: £1,494,699
Funder: The Wellcome Trust
Recipient: University of Cambridge

Proteomic characterisation of secreted antiviral factors in cell-mediated immunity to human cytomegalovirus 30 Sep 2018

Human cytomegalovirus (HCMV) is a widespread human pathogen, infecting 60-80% of the population. Infection is asymptomatic in immunocompetent individuals but causes disease in immunocompromised patients, such as transplant recipients. Current therapeutic tools are limited, with no available vaccine and a limited array of antivirals. HCMV triggers a broad and robust immune response involving both the innate and adaptive immune systems. Antiviral immunity is mediated in part by proteins secreted by immune cells and infected cells. In order to counteract this immunity, HCMV encodes numerous evasion factors that modulate the function of immune cells and the array of proteins they secrete (‘secretomes’). In this project, I will apply mass-spectrometry to generate comprehensive profiles of the secretomes produced by different immune cells when exposed to HCMV-infected cells. Using this technique, it will be possible to identify important and potentially novel secreted antiviral factors that can subsequently be validated and investigated to determine their mechanism of action. This will contribute to a better understanding of HCMV immunity and may facilitate the design of novel effective vaccine candidates and therapies.

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

The Genetic Basis of Congenital Hypothyroidism 30 Sep 2018

I have already identified 2 novel genetic causal variants for congenital hypothyroidism (CH) by whole exome sequencing (WES); IGSF1 defects in central hypothyroidism and SLC26A7 in dyshormonogenetic CH. I will therefore continue this strategy to identify further genetic causes of CH. I will expand my CH cohort, enriched for probability of genetic mutations. After excluding candidate gene defects, cases will undergo WES. I will then undertake functional characterization of specific novel variant s using in vitro techniques and a zebrafish model of thyroid development. Human SLC26A7 mutations are a novel cause of dyshormonogenetic CH and the disorder or its pathogenesis has not been characterized; I will phenotype cases to define this syndrome in more detail. I will characterize the biological function of SLC26A7 (a key transport protein), by performing electrophysiological studies to define its role as a putative anion transporter in the thyroid. Structure-function relationships in S LC26A7 are poorly understood. I will therefore characterize the properties of naturally-occurring and artificial SLC26A7 mutants to define functional domains in this protein.

Amount: £214,297
Funder: The Wellcome Trust
Recipient: University of Cambridge

Epigenetic transgenerational inheritance of metabolic, reproductive, and endocrine phenotypes through the male germline: effects of developmental bisphenol A and dexamethasone exposure 30 Sep 2018

The majority of heredity is accounted for by transmission of genetic material from one generation to another. However, in recent years evidence has accrued that some environmental factors can cause variations in phenotype that are inherited through the germline without changes in DNA sequence – so-called environmental epigenetic transgenerational inheritance. We are interested in how metabolic/reproductive/endocrine effects of developmental exposure to two exogenous endocrine insults – bisphenol A, an endocrine disrupting chemical that leaches from plastics and thermal paper, and dexamethasone, a synthetic glucocorticoid administered to pregnant women at risk of preterm delivery – may be transmitted inter/transgenerationally through the male germline. We will expose mice to human-equivalent doses of these chemicals and breed for three generations to obtain both phenotypic data and spermatozoa for epigenetic analyses (using RNA-seq, RRBS, and ATAC-seq). We will investigate the functional significance of any spermatozoal epigenetic changes detected; for example, using zygote pronuclear microinjection to determine the role of spermatozoal non-coding RNAs. The ubiquity of human exposure to these chemicals means that even small inter/transgenerational epigenetic effects would have significant implications at the level of public health; we therefore expect this work to be of interest to the wider scientific and medical community.

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

Statistical Methods for the Analysis of Deep Sequenced Pneumococcal Data 30 Sep 2018

Streptococcus pneumoniae (the pneumococcus) is a major disease causing pathogen and can cause sepsis, meningitis and pneumonia especially in at risk populations such as young children and the elderly. Understanding genetic factors in disease virulence, transmissibility, and drug resistance informs the management and treatment of infectious disease. By using deep sequenced patient samples of S. pneumoniae it is possible to build a clearer picture of its within host diversity. I aim to develop statistical and computational methods for the analysis of deep sequenced pathogen data that are also able to deal with large datasets, of the order of thousands of samples. I aim to apply these methods to the analysis of deep sequencing data derived from nearly 4000 S. pneumoniae samples taken from patients in the Maela refugee camp, Thailand. The methods I develop will help to identify significant genetic factors for disease dynamics and antimicrobial resistance. The project will contribute to the understanding of S. pneumoniae and will also provide tools of more general applicability to the investigation of deep sequenced pathogen data.

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

Cholera vaccine effectiveness in Nsanje and Chikwawa, Malawi 30 Sep 2018

Following the 2015 oral cholera vaccine (OCV) mass campaign of 160,000 people in Nsanje District, Malawi, the International Vaccine Institute (IVI) was funded to setup diarrheal disease surveillance in Nsanje and adjacent Chikwawa districts. Surveillance is ongoing at 22 and 18 health care facilitiesin Nsanje and Chikwawa, respectively. Research activities include to 1) analyse the vaccine effectiveness (VE) in Nsanje, through a 1:4 case-control study and 2) conduct a cost-of-illness study to help estimate OCV cost-effectiveness. The IVI is working in parallel in neighboring Mozambique. Diarrheal disease surveillance is ongoing in the Cuamba study area and an OCV has been conducted in 08/2018. The Mozambique study area borders the Malawian Nsanje/Chikwawa districts. We propose to continue the research in Malawi through extending the surveillance work and the case-control study, to ensure the assessment of long-term VE and cost-effectiveness. Further, the extent of herd protection through OCV needs to be assessed; the Chikwawa setting, after the 2018 OCV campaign constitutes the perfect scenario. The GFTCC is currently preparing a research agenda for "End cholera by 2030" roadmap and the Malawi/Mozambique scenario with surveillance ongoing in both countries, provides an unique opportunity to answer research questions identified through the GFTCC.

Amount: £155,986
Funder: The Wellcome Trust
Recipient: University of Cambridge

Adaptive decision templates in the human brain 30 Nov 2016

Interacting with the surrounding environment depends on our ability to extract meaningful patterns from incoming streams of sensory information. Learning and experience are known to facilitate this skill; yet, we know little about how the brain extracts structure and generalises this knowledge to novel settings. Here, I propose to test the brain mechanisms underlying structure learning using contrasting tasks that involve learning structure in space vs. time at different levels of complexity (simple vs. complex feature contingencies). I will use computational modelling to interrogate the processes involved in learning behaviourally-relevant structures (i.e. decision templates). I will relate this system-level insight to multimodal neuroimaging to provide converging evidence for brain mechanisms that mediate learning specialisation and generalisation. I will exploit high-field imaging to test fine-scale decision templates in the visual cortex. I will combine 7T imaging with human electrophysiology (MEG/EEG) and interventions (TMS) to test for local and larger-scale brain circuits that retune decision templates through feedback and inhibitory interactions. Finally, I will test whether these mechanisms support our ability to generalise previous experience to novel contexts and tasks. This integrated approach will advance our understanding of the brain’s capacity for adaptive and resilient behaviour with implications for promoting lifelong learning.

Amount: £1,344,200
Funder: The Wellcome Trust
Recipient: University of Cambridge

Circuit principles of memory-based behavioral choice 30 Nov 2016

We aim to elucidate the circuit mechanisms underlying three key computations essential for memory-based behavioral choice: 1) updating valences attached to sensory cues, when actual and expected outcomes differ; 2) computing the “value” for each action, based on multiple, conflicting cues; and 3) selecting one action and suppressing other physically incompatible competing actions. One obstacle to progress in this field has been the problem of identifying underlying circuits with synaptic resolution, and causally relating structural motifs to their proposed function. Both insects and vertebrates have evolved cerebellar-like higher-order parallel-fiber systems specialized in forming large numbers of associative memories and in guiding memory-behavioral choice. However, no synapse-resolution wiring diagram of any such system has been available to guide analysis and inspire understanding.    We have recently mapped the synaptic-resolution wiring diagram of one such system, the insect mushroom body, in Drosophila larva, which reveals multiple novel circuit motifs and provides clues about learning and decision-making models and their neuronal implementation. An exquisite genetic toolkit available in this model system allows selective manipulation of individual neuron types to establish causal relationships between their activity and behavior. We are now in a unique position to causally relate the identified structural motifs to their function.

Amount: £1,737,376
Funder: The Wellcome Trust
Recipient: University of Cambridge

Metabolic and Cardiovascular Disease. 30 Sep 2017

Not available

Amount: £2,352,000
Funder: The Wellcome Trust
Recipient: University of Cambridge