- Total grants
- Total funders
- Total recipients
- Earliest award date
- 03 Dec 2014
- Latest award date
- 30 Sep 2018
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
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.
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.
During a virus infection our cells are able to sense and respond to the invading pathogen and try to fight it off. The ability of our cells to directly detect the genomes of invading viruses is an essential part of this defence mechanism. Inside our cells, viral genomes are detected by specialised proteins called pattern recognition receptors (PRRs). These PRRs include retinoic-acid inducible gene I (RIG-I) and melanoma differentiation antigen 5 (MDA5), that can bind to the RNA of the viral genome and trigger potent anti-viral responses. We are interested to understand how activation of these PRRs can result in inflammation and cell death. We hypothesize that a particular protein modification, called linear ubiquitin, directs the immune response to RNA virus infection by determining the outcome of PRR activation. To explore this, we will study how linear ubiquitination alters the balance between cell death and gene activation resulting from of RIG-I and MDA5 activation, as well as explore the molecular mechanism(s) by which it regulates these signalling pathways. This will enable us to measure the contribution of linear ubiquitin to PRR signalling, showing a possible mechanism for regulating the immune response to drive effective immunity against viruses.
Haematopoietic stem cells (HSCs) are situated at the top of a hierarchy of blood forming cells and are ultimately responsible for the production of all mature blood cell types. HSCs must therefore execute a balance of self-renewal and differentiation divisions to maintain the stem cell population throughout adult life while providing enough cells to meet daily needs. Over the past decades, several HSC subtypes have been described, differing in mature cell type production and self-renewal durability. De-regulation of these cell fate choices in HSCs has been implicated in ageing and tumorigenesis and recent advances in single cell technologies have allowed greater insight into transcriptional changes driving these decisions. However, little is known about how well these profiles correspond to the proteome of HSCs and we therefore aim to construct a comprehensive protein network for HSC subtypes in order to identify key proteins involved in HSC self-renewal. The importance of candidate self-renewal regulators will be assessed by both in vitro and in vivo methods. A more complete understanding of HSC self-renewal would lead the way for more effective in vitro expansion of HSCs for research and future clinical applications such as gene therapy and bone marrow cell transplantation.
During the course of development, cells divide, migrate, and specialize to form major organ systems. Furthemore, among most mammals and birds, mouse cells differentiation follows a unique morphology. Understanding the molecular mechanisms underlying such process is a core issue in Biology and a curiosity in mouse, which despite differences still share fundamental properties during the process. The challenge has been addressed by leveraging current high-throughput technologies such as single cell transcriptomics. The amount and complexity of this data requires innovative mathematical frameworks that take advantage of current computational capacities. I am intersted on resolving mesodermal diversification during mouse gastrulation. Based on the premise that single cell profiles represent snapshot measurements of expression as cells traverse a differentiation process, I will use probabilistic modeling among other statistical and mathematical methodologies to reconstruct a measure of a cell’s progression through some biological process, and to model how cells undergo some fate decision and branch into two or more distinct cell types. In particular, Bayesian Inference has shown to be a useful approach to take advantage of computational resources, and to include prior knowledge into models, by providing a formal probabilistic framework that allows learning from the data in order to make predictions.
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.
Single-cell genomics is a fantastic tool for studying developmental biology: it allows unbiased and large-scale study of gene expression at the correct resolution for cell fate decision making. New fluidics systems provide the capability to study tens of thousands of cells simultaneously - as many as there are in the young embryo. For my PhD, I will analyse scRNA-seq data generated on this platform, studying mouse gastrulation between E6.5 and E8. I will be able to study this process at both an exceptional cell-level resolution (thanks to the fluidics) and at an unprecedented time resolution, at 0.1 day intervals. My focus will be on identification of lineage specification, and how cells make their fate choices. I will need to develop new methods to account for the large numbers of cells assayed, the numerous lineage decisions made, and heterogeneity of speeds of development across and between embryos. I hope to produce a map of lineage specification from epiblast (E6.5) cells through to every cell type present at E8. This work will provide a developmental atlas through gastrulation, and general inferences on cell fate decisions may provide insight for cellular reprogramming and regenerative medicine.
Integrating genomic, transcriptomic, metabolomic and behavioural data from 12 strains of C. elegans to understand gene/environment interactions under different dietary regimens 31 Jan 2017
C. elegans can grow on a range of different bacterial diets. It has already been shown that it changes its behaviour depending on the available food. Its lifespan also depends on its diet. Autophagy has been shown to mediate the increase in lifespan when the nematode is grown on certain foods. However, the mechanisms by which the environment leads to a change in beheviour and life history traits remain largely unknown. With this project, we would like to use high-throughput sequencing and metabolomics to build a quantitative and comprehensive map of the underlying molecular networks activated in a specific dietary environment. Furthermore, we would like to harness the genetic diversity of C. elegans to study the genetic basis of its phenotype as well as the interaction of its genome and environment in the determination of its behaviour and lifespan. For this purpose, we plan to extend the latest statistical techniques to integrate all layers of genomic and phenotypic data. Many of the genes involved in metabolism are conserved between humans and nematodes. Therefore, we expect that the findings will be relevant to human physiology.
Understanding the Pathogenesis of Inflammatory Bowel Disease via Whole-genome Sequencing 31 Jan 2017
We will use a new whole-genome deep-coverage IBD dataset (15x+ coverage, 20 000 cases, 50 000 controls) to conduct genetic association studies. Several analyses are currently planned. The first study will use the data from >1000 IBD patients, who are part of a deep clinical phenotyping experiment, on their response to treatment with anti-TNF medication. We are hoping to determine specific genetic variants associated with successful treatment, non-response, loss of response, and unfavourable drug reactions. Once more samples are sequenced, we will attempt to discover novel low-frequency, rare, and very rare genetic variants associated with IBD. A recent low-coverage sequencing study has identified a rare missense variant in ADCY7 that doubles the risk of ulcerative colitis. In addition, a burden of very rare, damaging missense variants in genes associated with Crohn's disease was detected. The increased coverage and the size of the dataset may confirm the significance of such variants. Discovery of novel rare variants brings important insights into IBD biology, and improves the overall understanding of the genetic landscape of complex diseases.
Developing an in vivo MT nucleation assay to investigate g-tubulin independent centrosomal MT nucleation 27 Apr 2017
Centrosomes are major microtubule organising centres (MTOCs) in animal cells. During mitosis they recruit large numbers of gamma-tubulin ring complexes (g-TuRCs), which nucleate and anchor the microtubules required for spindle formation. Recent work in the Conduit lab has surprisingly shown that centrosomes lacking g-TuRCs can still organise microtubules. Nevertheless, it remains unclear if these microtubules are generated at centrosomes, or generated in the cytoplasm and then anchored at centrosomes. I aim to establish an in vivo microtubule nucleation assay to test these alternative possibilities. Drosophila larval brains, which are highly mitotically active, will be dissected from either wild-type flies or from mutant flies where the centrosomes lack g-TuRCs. They will be cooled on ice for 40 minutes in order to depolymerise all microtubules and then transferred to 25 degrees and chemically fixed at different timepoints. The brains will be stained for microtubules, centrosomes and mitotic DNA using antibodies already available in the Conduit lab and images will be taken on a confocal microscope. The location and intensity of new microtubule growth will be assessed. If the g-TuRC negative centrosomes do nucleate microtubules, the assay will be used to test candidate proteins for their role in centrosomal non-g-TuRC mediated microtubule nucleation.
Anaplastic Large Cell Lymphoma (ALCL) is a paediatric T cell lymphoma whereby tumours have an 'activated' cell surface protein expression phenotype, defined by the presence of CD30. However, it has long been an enigma as to why these supposed ‘transformed T cells’ do not express a T cell receptor (TCR) despite having the capacity to do so (as evidenced by the presence of molecular rearrangements of VDJ genes). The presumed cell of origin is a cytotoxic T cell as the large majority of tumours produce perforin and granzyme B yet in many cases expression of the helper T cell protein CD4 is also observed. We have refined the tumour cell phenotype to show expression of RORgt and production of cytokines including IL26, IL22 and IL17 hence suggestive of an origin in an innate lymphoid 3 cell (perhaps when the TCR is 'missing') or Th17 cells (when the TCR is expressed) that develop into tumours as a consequence of an inflammatory environment. Hence, the aim of this project is to establish this cellular phenotype and define the role of the (sometimes missing) TCR.
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.
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.
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
Eros is a recently described endoplasmic-reticulum transmembrane protein that controls the phagocyte respiratory burst. Eros is essential for the generation of reactive oxygen species because it is necessary for protein (but not mRNA) expression of the gp91phox-p22phox heterodimer, which is almost absent in Eros-deficient mice. Consequently, Eros-/- animals succumb quickly following infection with Salmonella or Listeria. Eros is highly evolutionarily conserved and has a human orthologue C17ORF62 , which exhibits approximately 90% sequence similarity. Dr Thomas's group have preliminary data that the role of Eros is fully conserved in humans. However, the exact mechanism by which Eros controls gp91phox-p22phox abundance remains unclear. Using a yeast 2 hybrid screen Dr Thomas has shown that Eros's most significant interaction partner was OS9, an ER-resident lectin that regulates the degradation of misfolded transmembrane glycoproteins. Given that gp91phox is a transmembrane glcyoprotein, it is possible that Eros regulates gp91phox through a mechanism that involves OS9. While OS9 is highly expressed in the immune system, its role in the respiratory burst has not been studied. Using OS9 knockout mice and lentiviral over-expression systems, I will determine whether it regulates expression of gp91phox-p22phox or indeed, Eros itself.
This project plans to measure levels of tissue plasminogen activator (tPA) which is involved in fibrinolysis of blood clots within the CSDH lesions. This bleeding is an essential part of CSDH formation, followed by coagulation and fibrinolysis which is triggered by the cleavage of plasminogen by tPA to generate plasmin. tPA will be measured in these samples using the commercially available ELISA kit. I will determine whether levels of tPA are correlated with levels of other inflammatory markers in CSDH fluid or in blood, and also to examine if increased tPA levels at the site of the haematoma predicts risk of CSDH recurrence. If tPA concentrations in blood or CSDH fluid correlate with clinical outcome, this could be used clinically to decide whether surgical or pharmacological management are most appropriate for individual patients. Finally, the effects of dexamethasone treatment on levels of tPA and other cytokines will also be determined, by comparison of dexamthasone and placebo-treated patients. These patient samples are anonymised and will only be unblinded after measurement of the above analytes has been completed.
Oxygen is essential for almost all animal life on the planet, acting as a key player in the energy production in cells. Cells are able to adapt to low oxygen environments by activating a factor named hypoxia-inducible factor (HIF), which shifts the metabolism of cells away from oxygen-consuming processes while increasing alternative energy-producing pathways. In T cells, an important type of immune cell involved in the body’s defence against infections and tumours, this metabolic shift alters cell function, resulting in a more aggressive immune response. Understanding how this process is regulated may allow us to target and harness immune cells to treat a variety of diseases, such as autoimmune disease or cancer. Our group and others have shown that boosting HIF levels in T cells makes them more effective in clearing tumours and resolving viral infections. We have also studied the effect of factor inhibiting HIF 1-alpha (FIH), a protein that blocks HIF activity. Interestingly, FIH itself also appears to alter the metabolism of cells, although its effect on immune cells is currently unknown. During my PhD I will assess the role of FIH in mouse T cells, focussing on how FIH-driven metabolic changes can augment immune responses.
During the elongation of the embryonic body, groups of stem cells within the tip of the embryo continually generate progenitor cells that later make up the spinal cord and segmented vertebrae. Interestingly, differentiation of other embryonic cell types has been shown to be influenced by mechanical forces from the environment surrounding the cells in culture. Over the course of my PhD I will investigate the influence of the native mechanical environment on the differentiation of progenitor cells in the zebrafish embryo into cell types contributing to the formation of specialised tissues. This will aid in our understanding of how mechanical properties of tissues, such as their stiffness, can influence cell differentiation. Firstly, I will characterise cell movement, cell shape, and environmental stiffness coinciding with cell state transitions in the tailbud. Secondly, I will investigate the influence of mechanical forces on differentiation and epithelial to mesenchymal transitions. Finally, I will investigate the role of YAP in regulating differentiation into spinal cord and mesodermal cell types. These studies will provide important insight into the fundamental problem of how cell fate decisions and cell movements are coupled during embryonic development.
The mitochondrion, known as the powerhouse of the cell, contains its own genome (mtDNA). The multi-copy mtDNA works with the nuclear genome to control energy production and various cellular activities. To date, mtDNA mutations are among the most common genetically inherited diseases and the mitochondrial replacement therapy has been approved in the UK to make three-parent babies. However, our knowledge of mtDNA biology and how it can affect organismal traits is rather limited. This is largely due to a lack of powerful genetic tools to study mtDNA. A recent study in Ma's group shows that Drosophila mtDNA can undergo homologous recombination12. Further, they established a system to induce recombination at specific sites and select for different recombinant genomes. This work not only provides a definitive resolution to the existence of recombination in animal mitochondria, but opens up the possibility of developing a recombination system for functional mapping and manipulating animal mtDNA. In this project, I will isolate recombinant mitochondrial genomes to map/define mtDNA variations responsible for longevity and fertility to accelerate our understanding of how mtDNA impacts health. Meanwhile, I will identify key components of the recombination machinery to better understand how mtDNA is maintained during aging and evolution.
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.