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Funders:
Paul Hamlyn Foundation
The Wellcome Trust
BBC Children in Need
Recipients:
University of Cambridge

Results

An Extended Pilot for the Human Cell Atlas: Adult tissues, human development and inflammation-mediated pathologies 30 Sep 2018

The Human Cell Atlas (HCA) is an international, collaborative effort that "...aims to define all human cell types in terms of their distinctive patterns of gene expression, physiological states, developmental trajectories, and location". Here, we will contribute directly to the first phase of the HCA by forming an ‘extended pilot’ to implement UK infrastructure for large-scale, high quality human cell atlas experiments. We will generate a high-level atlas, with spatial resolution, for multiple adult human tissues along with matched data from human fetal material. We will then illustrate the power of a deep and focused investigation of a single tissue (skin) to produce highly-detailed data describing its cellular composition and spatial organisation. Finally, for selected tissues that have been profiled in adults and fetal material, we will analyse samples from immune-mediated disorders as a comparison with our reference data to gain deeper understanding of the pathological mechanisms. This will demonstrate the utility of the HCA as a ‘healthy reference’ for comparison with disease. Throughout, we will generate profound biological insight from primary human cells and lay a foundation of technology development and optimisation with a set of hardened and scalable methods for single-cell RNA-sequencing, spatially-resolved gene expression, and tissue imaging.

Amount: £765,117
Funder: The Wellcome Trust
Recipient: University of Cambridge

Neural circuits underlying fertility 17 Jul 2018

This programme aims to establish in detail the characteristics and in vivo significance of the arcuate (ARNKISS) and rostral periventricular area of the third ventricle (RP3VKISS) kisspeptin neurons in driving the pulsatile and surge patterns of luteinising hormone (LH), respectively. Genetic cFOS-dependent activation and rabies trans-synaptic strategies will enable permanent GFP/mCherry tagging of RP3VKISS neurons activated at the time of the surge, or projecting directly to GnRH neurons, for subsequent electrophysiological and RNAseq analyses. GCaMP-based fiber photometry and GRIN lens miniscopes will be used to evaluate RP3VKISS neuron and population activity. This will be combined with microfluidics delivering pharmacological agents or adeno-associated viruses bearing CRISPR components to interrogate the key factors regulating the activity of RP3VKISS neurons in vivo. Studies aimed at understanding how the ARNKISS neurons synchronise to generate LH pulses will use the same strategies in addition to expansion microscopy and brain slice dual calcium and electrophysiological recordings. Novel strategies for slowing LH pulses in an animal model of polycystic ovary syndrome will be explored. It is expected that the in-depth understanding of these two kisspeptin populations will provide opportunities for developing new therapeutics aimed at the beneficial regulation of fertility in the clinic.

Amount: £100,000
Funder: The Wellcome Trust
Recipient: University of Cambridge

Clinical Characterisation of a Broad Spectrum of Genetic Variation in the General Population 30 Sep 2018

Inborn errors of metabolism (IEM) are severe and extreme changes in metabolism caused by mutations in a single gene. Recent large-scale human studies have shown that genes causal for IEM are associated with nutrients, or ‘metabolites’, in the blood. However, whether these associations cause disease or adverse health outcomes is unknown. In this project, I will use IEM genes identified in these studies to link genetic variation to clinical features in a large human population. To do this, I will assemble a list of IEM genes of interest that were identified in the literature and in large population datasets. I will then test for associations between the variants I find in these genes and a wide range of clinical features found in open-access population datasets. As the IEM genes used in this study have been associated with blood metabolites previously, linking variants in these genes to clinical features will shed light on the molecular mechanisms underlying genes and disease in the general population. Understanding how genetic variation affects disease will help identify novel therapeutic targets and enable health professionals to better manage disease risk.

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

Investigating non-canonical programmed ribosomal frameshifting in porcine reproductive and respiratory syndrome virus 30 Sep 2018

RNA viruses are under selection pressure to maintain a small genome, however they still need to produce a variety of proteins. To overcome these conflicting pressures, many viruses use non-canonical methods of translation control. One example of this is programmed ribosomal frameshifting (PRF), in which a percentage of ribosomes, while translating a ‘slippery sequence’ slip one or two nucleotides out of frame, consequently translating the remainder of the mRNA in a different reading frame and allowing expression of more than one protein from a single gene. This is normally stimulated by a downstream secondary RNA structure, however there are two known examples of a trans-acting viral protein being used as the stimulatory element. One example is found in the genome of porcine reproductive and respiratory syndrome virus (PRRSV): an Arterivirus that infects pigs, causing an estimated annual cost to the US swine industry of $664m. I will use structural techniques such as X-ray crystallography to derive information about the RNA/protein complex, and will investigate the efficiency and mechanism of this non-canonical PRF using ribosomal profiling in parallel with RNASeq. The latter will also allow me to analyse host and viral gene expression, to examine host-virus interactions in this important pathogen.

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

Epigenetic inheritance: the influence of variable silencing of the repeat genome. 10 Apr 2018

Evidence from epidemiological studies and experiments in animal models suggests that effects of environment and lifestyle can be transmitted across generations via non-genetic mechanisms. Such mechanisms are challenging to unravel in mouse and man. In mammals, non-genetic inheritance is best exemplified by the Agouti viable yellow (Avy) mouse where phenotypic differences in genetically identical animals are caused by insertion of a retrotransposon - an endogenous retrovirus (ERV) that provides a cryptic promoter driving ectopic expression of agouti. This ERV is variably DNA methylated in different individuals causing inter-individual variation in coat colour – a non-genetic influence on phenotype. Remarkably, a memory of parental coat colour is transmitted to subsequent generations. Variable expressivity can be modulated by in utero environmental exposures. ERVs represent ~12% of the mouse genome. Inspired by Avy, we propose a research programme, supported by preliminary data, to address the following questions: Aim 1 – To what extent do mammalian ERVs exhibit variable epigenetic silencing and what is the mechanism? Aim 2 – Is this transmitted as non-genetic memory across generations? Aim 3 – Are they sensors of environmental compromise? Aim 4 – Are there implications for phenotype? Aim 5 – Does a related phenomenon occur in humans?

Amount: £2,929,851
Funder: The Wellcome Trust
Recipient: University of Cambridge

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

Lineage tracing and early embryonic dynamics using mosaic somatic mutations 30 Sep 2018

Every cell in the human body descends from the fertilised egg cell, and during the journey from zygote to adult cell, many mutations are acquired. The mutations that happen early enough during embryonic development will be present in some parts of the body, but absent in others. One of the difficulties of this project is the discovery of these early embryonic mutations, as they are hard to distinguish from mutations that happened later in life, noise from experimental procedures, and mutations that were already present at fertilisation. Here, we cut out small pieces of various tissues from multiple individuals using a laser and sequence its DNA. Mutations shared between different samples from the same individual are candidate embryonic mutations. Each mosaic mutation gives us information on the development of the human body and with enough mutations, we can construct and visualise the human body as a large evolutionary tree, with the fertilised egg cell as the single root, and all the adult cells as its leaves. This can give us many new insights on the developmental relationship between different organs, how many different early embryonic cells give rise to one tissue or organ and developmental lineages of different cell types.

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

The effect of nutrients on maximal fat oxidation rates in adult humans measured using indirect calorimetry. 31 May 2018

I aim to study the effects of nutrient availability and mitochondrial transport capacity on the variability of maximal fat oxidation (MFO) during exercise in healthy adults. Less than half of MFO can be predicted by variables such as gender, VO2max and body composition. There are two possible reasons for this. First, nutrient availability may have a large effect on MFO and current protocols may not adequately control for it. Second, VO2max - which combines two variables with opposite effects on MFO (oxygen uptake and fat mass) - may not be the optimal predictor. Here, I will test whether heart rates at a given power are a better predictor of MFO and whether short-term fluctuations in nutrient availability can explain some of the variability of MFO seen within the general population. Nutrient availability will be altered using a glucose meal and by glycogen depletion. I will also use nitrate supplementation to test whether MFO can be increased by induced-expression of fat oxidation enzymes. The key goals are to determine to what extent short-term changes in nutrients and the expression of fat oxidation enzymes can alter MFO and whether the resultant fat oxidation rates can be predicted using simple heart rate data.

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

Understanding the Initiation of Viral Replication & its Role in Influenza Virus Pathogenicity 31 May 2018

The development of novel strategies against influenza viruses depends on our understanding of influenza virus replication and pathogenicity. Both are directly linked to the activity of the viral RNA polymerase, which copies and transcribes the viral genome, and generate aberrant RNA products that are non-contiguous in the viral genome and strong inducers of the interferon response. Despite recent crystal structures of the RNA polymerase, we only poorly understand how it interacts with and copies the viral genome, or how it generates aberrant RNA products. This project aims to use i) structure-guided mutagenesis, ii) in vitro and in vivo activity assays, iii) cell culture-based interferon production assays, to ask if RNA polymerase residues that bind and guide the viral genome are important for the initiation of viral replication and the formation of aberrant RNA products and thereby the pathology of influenza virus infections. The project will contribute to our understanding of the mechanics of influenza virus replication and the identification of putative targets for the development of new anti-influenza virus drugs.

Amount: £0
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

Investigation into the role of RBM8A/Y14 in the development and function of megakaryocytes and platelets using a human pluripotent stem cell model of haematopoiesis 30 Sep 2018

Platelets are small blood cells, which cause blood to clot, preventing bleeding after injury. They are produced by megakaryocytes, large cells in the bone marrow. In people with low platelet counts (thrombocytopenia), life-threatening bleeding occurs spontaneously or after injury. Studying platelet and megakaryocyte development and function is important in understanding a) diseases causing thrombocytopenia, such as genetic disorders and other conditions, particularly cancer (and chemotherapy) and b) strokes and heart attacks, where platelets are excessively activated, forming clots that block vessels. Using stem cells (special cells capable of becoming any cell type) derived from adult skin or blood samples we grow & study megakaryocytes and platelets in the laboratory. We study a rare genetic disease, Thrombocytopenia with Absent Radii (TAR) syndrome, in which babies are born with very few platelets and abnormal bone formation (particularly the radius in the forearm). Our group discovered the cause of TAR, due to abnormalities in a gene called RBM8A, which helps cells control what proteins are produced; however precisely why this causes TAR is unclear. We believe our research will uncover the mechanism of this condition, helping to treat patients with TAR and improve wider understanding of how megakaryocytes & platelets develop and function.

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

Modeling cell differentiation dynamics from single cell genomics data 30 Sep 2018

We aim to understand in detail the dynamics of how white blood cells (specifically T helper cells) react to infections by multiplying rapidly and at the same time adapting their cell state to fight the infection. In particular, we focus on a mouse model of malaria where T helper cells differentiate into two subtypes: Th1 and Tfh. By quantitatively profiling the T helper cell population at different time points during a malaria infection, we expect to improve our understanding of the mechanisms which are responsible for the cell proliferation and specialisation. We study the cell population by detecting RNA expression, surface markers and cell divisions at the single cell level. The RNA expression will provide clues as to genes which are driving this process, and we will test a subset of genes using CRISPR knock outs. In addition to a better knowledge of the immune system, we hope to develop new mathematical and computational methods that will be widely applicable to modeling cell proliferation and differentiation data in diverse biological contexts.

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

Principles of human development and germ cell program 28 Nov 2017

Specification of human primordial germ cells (hPGCs) occurs around gastrulation, a critical juncture when the specification of the primary somatic lineages also occurs. In combination with human preimplantation embryos, in vitro models and hPGCs from aborted fetuses, our objective is to elucidate the origin and properties of the early human germline. For the mechanism of the hPGC fate, we will use experimental models that simulate early human development. We aim to investigate how cells gain competence for germ cell fate, and then respond to combinatorial effects of the critical transcription factors, which induce hPGC specification. Altogether, this study will reveal the organisation of the very early human embryo, and mechanisms of hPGC and somatic outcomes, which is essential for advances in regenerative medicine. Following hPGC specification, epigenetic resetting of the early human germline leads to extensive erasure of DNA methylation and epimutations in response to the critical regulators of chromatin organisation and nuclear architecture towards the epigenetic ground state. Some conserved resistant loci ('escapees') evade reprogramming. We will explore if some escapees have been exapted to function as regulatory elements. If so, this may have a crucial influence on human development, including brain development and neuronal diseases.

Amount: £2,750,000
Funder: The Wellcome Trust
Recipient: University of Cambridge

Metabolic magnetic resonance imaging across the human heart 18 Oct 2017

During my fellowship, I proved the feasibility of measuring cardiac energetics in volunteers and patients using ultra-high field (7T) MRI scanners. The sensitivity and the separation of signals from different metabolites both improved significantly compared to standard research scanners. I recently secured £340k funding to fit a new phosphorus coil on the Oxford 7T scanner, which I am now testing in volunteers. Theory predicts that this coil will have several complementary technical advantages. These will enable mapping of cardiac energy metabolism across the whole heart, with sufficient spatial resolution to distinguish signals from healthy from diseased tissue. It will also enable quantification of cardiac energy metabolism with high precision to study single subjects rather than groups. I request funding to validate these new whole-heart methods, proving their value in three carefully-targeted groups of patients, via an extension of my fellowship. My goals are (A) to study patients in which the metabolic pattern is known by other means; (B) others where the metabolic pattern will reveal previously-inaccessible aspects of disease mechanism; and (C) to prove I can resolve metabolic changes in single patients. Success in each of these studies will give me the pilot data needed for competitive Senior Fellowship applications.

Amount: £584,259
Funder: The Wellcome Trust
Recipient: University of Cambridge

Investigating the mechanism of influenza virus translation 30 Sep 2018

When viruses infect a cell, they need to hijack host machinery to produce their own proteins from mRNA, in a process called translation. The host cell requires several factors for translation, including proteins called eukaryotic initiation factors (eIFs). EIF4F plays a central role in this process and is a complex made up of the proteins eIF4A, eIF4G, and eIF4E. Together, these proteins act along with other factors to recruit the cellular machinery required for translation to begin. Influenza can promote the translation of its own proteins whilst host protein synthesis is impaired. As viral and host mRNA are highly similar, influenza virus was thought to only use the same mechanism of translation as the host. However several findings, such as the fact that influenza can translate its proteins without eIF4E, suggest that this is not the case. My hypothesis is that influenza can employ a different mechanism of translation from the host. I will use several RNA/protein analysis approaches to identify the key components required for influenza translation, and attempt to dissect the mechanism(s) of translation used by influenza. Identifying key differences between host and viral processes is important for identifying novel therapeutic targets.

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

INVESTIGATING THE ROLE OF DDX17 IN ANTIVIRAL INNATE IMMUNE SIGNALLING DURING VIRAL INFECTION AND ITS IMPACT ON VIRUS SPREAD AND REPLICATION 31 May 2018

The host DEAD box RNA helicases are master regulators of pathogen RNA and DNA sensing dependent IRF3 signalling and are crucial for host survival and infection outcome in response to a multitude of both viral and bacterial pathogens. Here we report the DEAD box RNA helicase DDX17 as a novel pathogen recognition receptor essential for IRF3 driven IFNbeta expression in response to immunostimulatory DNA and dsRNA. Our current data maps DDX17 to act independent to the canonical IRF3 signalling cascade at the level of gene transcription, independent of IRF3 phosphorylation. We hypothesise that DDX17 may regulate beta-catenin nuclear shuttling, an essential IRF3 transcriptional cofactor. We aim to investigate the impact of DDX17 on beta-catenin activation and phosphorylation status as well as subcellular localisation following stimulation of WT or KO MEFs. Furthermore, we aim to investigate the biological relevance in viral infection following vaccinia virus and herpes simplex virus type 1 infection of WT and KO MEFs through quantification of viral replication and spread as well as IRF3 pathway activation. This project will contribute to understanding the role of DDX17 in innate immunity and host-pathogen interaction with implications in the immunological understanding of viral infection. Key words: DDX17, IRF3, IFNbeta, helicase

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

Epigenetic control of neurodevelopmental gene regulatory networks linked to neurodegeneration 30 Sep 2018

Dementia is predicted to affect 130 million people worldwide by 2050 according to the World Alzheimer 2015 Report30. Some familial forms of dementia inherited in autosomal-dominant fashion are linked to mutations altering gene dosage2,8,14,16,19,23,25. Patients with the mutations display a long pre-symptomatic phase during which cellular changes may take place before the onset of the disease decades later23. The cellular changes are reflected in gene regulatory networks3,4,5,12,28,31. As evidence from other neurodevelopmental conditions suggests10,13,17, changes during early neural development may lead to onset of the disease decades later. In order to study the neurodevelopmental gene regulatory networks and their links to dementia, I would like to focus on two forms of their regulation: small RNAs and demethylation escapees. Demethylation escapees are regions of the genome that escape epigenome resetting during early embryonic development27. Small RNAs have an important role in neural development and gene regulatory networks controlling them1,6,20,24. In order to address the question, I will use RNA and whole genome bisulfite sequencing methods of neurons derived from human stem-cells from familial dementia patients, combined with bioinformatics analyses. Focusing on small RNAs and demethylation escapees, the project might hint at neurodevelopmental gene regulatory pathways dysregulated in autosomal familial dementia.

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

Investigating stiffness alterations in spinal cord injury 30 Sep 2018

Spinal cord injury is a devastating condition that may lead to loss of limb movement, sensation and bladder control. Despite intense research, treatment is still very limited. Most research to date has focused on biochemical signalling. However, some more recent studies have hinted that mechanics might play an important role in spinal cord regeneration. Using atomic force microscopy (AFM), a cutting-edge technique which allows us to very precisely measure stiffness maps of biological tissues, we will investigate the stiffness of spinal cord tissue at various time points after injury and compare this to the stiffness of healthy spinal cord. We will test whether artificially modifying the stiffness of the damaged spinal cord or modifying mechanosensing in spinal cord cells improves regeneration of neurons after spinal cord injury. Our studies will be carried out in a cervical contusion model in rats which closely mimics the pathology seen in the human spinal cord after injury, even though the behavioural impairments the animals show are markedly less grave.

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

Regulation of Neural Stem Cells 30 Sep 2018

Of all the tissues and organs in the human body the nervous system is the most intricate and complex, consisting of more than 100 billion neurons. These neurons make precise connections with each other to form functional networks that can transmit information at amazing speed over considerable distances. Neurons are produced by neural stem cells, which renew themselves at each cell division while also giving rise to all of the diverse types of neurons in the brain. The Brand lab is interested in how the environment influences stem cell behaviour, in particular how nutrition regulates neural stem cell proliferation. Uncovering the molecular mechanisms that control whether a stem cell chooses to proliferate or remain dormant is crucial for understanding tissue regeneration under normal and pathological conditions and in response to ageing. It is critical to learn not only how stem cell proliferation is induced but also how stem cells can return to a dormant (‘quiescent’) state, as uncontrolled stem cell division can lead to cancer, including brain tumours like glioma. A thorough appreciation of the signals, both extrinsic and intrinsic, that control stem cell behaviour is necessary to understand how homeostasis is achieved and maintained in the brain.

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