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Recipients:
University of Cambridge
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Results

Mechanisms of transport of the leaderless protein galectin-3 to the cell surface 31 Jan 2017

Unconventional protein secretion is a poorly understood physiological process in which proteins without an N-terminal signal sequence exit the cell. There are currently four proposed pathways by which unconventionally secreted proteins are thought to exit the cell: by direct translocation across the membrane, via secretory lysosomes, by release from exosomes or multivesicular bodies, or through membrane blebbing. No complete mechanism has been described for any of these pathways, representing a significant gap in our knowledge of protein trafficking. Unconventionally secreted proteins play important extracellular roles physiologically, but abnormal levels are associated with several human diseases, including metabolic disease. As such, this mechanism is interesting to gain an insight into disease as well as to broaden our understanding of cell biology. I will investigate the unconventional transport of galectin-3 to the cell surface. Galectin-3 will here be used as a model to understand the mechanism of unconventional secretion. Data-driven and hypothesis-driven approaches will feed into each other to form a picture of how galectin-3 is secreted. A CRISPR-Cas9 screen has identified potential proteins that decrease cell surface galectin-3, providing the starting point for further investigation. Hypothesis-driven experiments will be used to investigate aspects of the models previously proposed.

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

Structural studies of bacterial nucleic acid transport proteins 31 Jan 2017

Horizontal gene transfer contributes to genetic plasticity in bacteria and is of great clinical relevance as it contributes to the spread of antibiotic resistance genes. One mechanism of horizontal gene transfer in bacteria is transformation. While the phenomenon of transformation has been known for many decades, little is known about the mechanistic steps of exogenous DNA uptake into bacterial cells. The most obvious problem is how the DNA gets past the cell envelopes. ComEC is believed to be the protein that forms an aqueous pore that allows transport of DNA into the cytoplasm through the bacterial plasma membrane. The protein represents a novel transport protein, and no structural and very little functional information is available. The aim of the project is to structurally and functionally characterize ComEC proteins using modern protein expression and screening techniques, advanced structural approaches (X-ray crystallography, cryo-electron microscopy) and functional studies (fluorescence microscopy, biophysics), in order to build a model for DNA transport across the plasma membrane into the cytoplasm.

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

KDEL Receptor Stuctural Determination & Biochemical Characterisation 31 Jan 2017

This project aims to characterise the KDEL receptor (KDELR) structurally, biochemically and biophysically. The KDELR is a membrane protein resident in the cis-golgi, where it binds the K-D-E-L amino-acid motif present on resident ER proteins, which have been transported to the Golgi via bulk flow. Once KDELR binds cargo, it initiates transport back to the ER via COPI mediated vesicles, where it releases its cargo, ostensibly because of the differing pH. The molecular mechanisms concerning the actions of the KDELR are largely elusive and would be greatly aided by the structural determination of the KDELR, as well as the structures and characterisations of its interactions with native cargos. Furthermore, KDELR has been predicted to be a GPCR, but does not appear to share homology with proteins in the family. However, distant homology to the SWEET family of sugar transporters has been found, suggesting these ‘receptors’ are in fact transporter like proteins. Furthermore, this project has the potential to involve live cell imaging experiments (in collaboration with Prof. Francis Barr) to test hypothesis borne from the structural and biochemical data.

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

Dissecting Polo kinase recruitment to the centrosome 31 Jan 2017

Polo kinase is an important cell cycle regulator and it is essential for the correct assembly of centrosomes, major cell organisers. Centrosomes are formed by a pair of cylindrical centrioles surrounded by pericentriolar material (PCM). Polo controls PCM assembly (at least in part through Cnn phosphorylation) and also centriole disengagement and assembly. How Polo is recruited to centrioles and centrosomes is mysterious. During my rotation I have obtained evidence that the PCM protein Spd-2 is necessary for Polo recruitment to centrosomes. During my project I aim to characterise if Polo binding to Spd-2 is necessary for Cnn phosphorylation and correct PCM organisation, what happens when Spd-2 cannot bind Polo and what upstream regulators facilitate this interaction. Furthermore, I aim to identify the other centriole/centrosome proteins involved in Polo recruitment. To do this, I will make use of biochemical assays and advanced microscopy techniques, coupled with fly genetics and a powerful mRNA injection assay to rapidly test the effects of different mutants in fly embryos. Ultimately, I hope to be able to describe in molecular detail which proteins are phosphorylated by which kinases to allow Polo to be recruited to fulfil its many functions at the centrioles and centrosomes.

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

Machine Learning methods for microscopy image data in high-content screening 31 Jan 2017

An important area in drug development is understanding low-level molecular processes and pathways that cause diseases. These cellular phenotypes are high-dimensional and are increasingly being captured using single-cell assays and high-content imaging. In understanding natural cell trait variation and engineered variants, we can elucidate the cellular consequences of disease mutations. In my project, I will exploit cellular images in a range of contexts to investigate the link between genetic variation and cell trait variability using both natural genetic variation and engineered variants. To do so, I will develop machine learning methods to extract features from high throughput microscopy data, and to accurately account for genetic, environmental, and experimental sources of variability in them. Furthermore, I will work on integrative approaches using public genomic data to bring in other omics modalities, thereby tackling key challenges in the larger aim of deciphering disease and fostering drug development. I will use existing data from the HipSci project, high throughput drug screens from AstraZeneca, and, in addition, will design and oversee the generation of datasets through high-throughput CRISPR knockouts as part of Leopold Parts’ group at the Wellcome Trust Sanger Institute and Oliver Stegle’s group at the European Bioinformatics Institute.

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

Systematic Identification of Lineage Specification in Murine Gastrulation 31 Jan 2017

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.

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

Functional proteomic analysis of novel antiviral restriction factors in primary leukocytes 31 Jan 2017

This project aims to identify and characterise novel antiviral restriction factors (ARFs) that play key roles in preventing infection of primary leukocytes. ARFs may function by preventing viral entry or exit at the cell surface, or replication at various intracellular stages. I will focus on the subset of plasma membrane (PM) ARFs, which will be identified by two properties: interferon (IFN) induction and virally-induced downregulation. For this I will employ tandem mass tag-based MS3 mass spectrometry, enabling quantitation of PM proteins in primary leukocytes. Key Goals: 1. Use IFNs and infection with two important human pathogens, human cytomegalovirus and HIV as a functional screen to identify novel cell surface ARFs 2. Investigate how these ARFs inhibit viral infection, and how are they targeted for destruction by viruses. The use of IFN as part of the functional screen will additionally enable exploration of the difference in effects between IFNalpha, beta and lambda at the PM, a subject which is currently surprisingly poorly understood. This will provide important insights into human immunity in its own right. Understanding how viruses interacts with and targets ARFs for destruction will have important implications for therapy.

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

The role of regulatory T cells in immunity to malaria 31 Jan 2017

In malaria vaccine trials conducted in the target population of semi-immune people from endemic African countries, vaccine immunogenicity is sometimes substantially reduced compared to European malaria-naïve participants. This could result from the suppression of vaccine-induced immune responses by regulatory T cells (Tregs) acquired through prior malaria infections, however there are few studies in man which have previously explored this. Using samples from controlled human malaria infection studies in semi-immune Kenyan individuals, we will investigate how Tregs affect natural immunity to infection and see if increased Treg responses correlate with vaccine efficacy following malaria challenge in participants with varying prior exposures to malaria. We will also directly compare the effect and induction of Tregs in different pre-erythrocytic candidate vaccines and adjuvants to understand how vaccine-specific effects might affect Treg responses. Additionally, we will investigate if malaria-induced Tregs affect responses to other childhood vaccines. Single cell transcriptomic analysis using the Fluidigm platform will be employed to explore the phenotype and functional heterogeneity of Tregs. This will provide insight into the mechanisms by which Tregs are involved in immunity to malaria. This work will have important implications for the design and evaluation of malaria vaccines for use in endemic populations.

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

Not just a virulence factor: capsule biology across the Neisseria genus 31 Jan 2017

This project seeks to address the relatively unexplored topic of the genetics, function and evolutionary history of the Neisseria polysaccharide capsule, beyond its established role as a virulence factor in Neisseria meningitidis (Nme). This will be achieved by examining capsular types not associated with disease, both from Nme, and capsules recently discovered in the commensal Neisseria species. The first goal is to complete genetic and phenotypic characterisation of the novel commensal capsular types. Once this is established, a key goal is to seek comparisons between these novel capsules and those of Nme within the coding sequences and regulatory regions, and at the structural level. I also plan to address the question of what the role of capsule is in colonisation and transmission, given that it most likely was not selected for its virulence properties. Finally, I seek to build a clearer history of the acquisition and evolution of capsule in Neisseria. This will bring forward new insights into the roles of capsule in normal, healthy colonisation of the nasopharynx, both by Nme and the strictly commensal Neisseria species. This work may also have implications for our interpretation of Nme dynamics and the rare transition to a state of disease.

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

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

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

Defining the import mechanism of pyocin AP41 and klebicin G 30 Sep 2018

There is an urgent need to develop new antibiotics against multidrug resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella pneumoniae. These organisms are major causes of pneumonia and sepsis, with recent reports identifying hospital isolates of each resistant to all known antibiotics. The present research focuses on the mode of action of a family of antibiotic proteins known as nuclease bacteriocins that have not been developed as antimicrobials, but show promise in animal models of infection. Nuclease bacteriocins are species-specific toxins that are used by bacteria to compete with their neighbours. Although folded proteins these molecules are capable of penetrating the defences of Gram-negative bacteria to deliver an enzyme to the organism’s cytoplasm to degrade essential nucleic acids by an unknown mechanism. Two types of nuclease bacteriocin will be investigated, pyocin AP41 which targets Pseudomonas aeruginosa, and klebicin G which targets Klebsiella pneumoniae. Preliminary computational and experimental work on pyocin AP41 has identified potential candidate proteins involved in its import. This will be followed up with structure and function studies of AP41, a dissection of its import mechanism and new studies on klebicin G, a nuclease bacteriocin that has only recently been identified.

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

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

Analysis of Inflammatory markers in chronic subdural haematoma 31 May 2018

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.

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

Investigating the role of mechanical forces in cell exit from the Zebrafish tailbud 30 Sep 2018

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.

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

Mechanisms of lineage commitment during exit from pluripotency in vivo 30 Sep 2018

In early mammalian development, a pool of cells in the embryo can generate all cell types of the body, an ability referred to as 'pluripotency'. Specification of the cells is regulated by selective activation of genes that define tissue identities. These developmental programs are regulated by proteins known as 'transcription factors' that direct expression of other genes. However, the precise mechanisms that control cell fate specification are still poorly understood. The aim of my project is to understand the molecular mechanism of how genetically identical cells can be instructed to differentiate. I will focus on understanding the functional role of covalent 'epigenetic' DNA modifications in cell lineage priming and specification. To be able to address this fundamental question, I will use mouse embryos and stem cell culture systems, linked to imaging and single cell technologies to study the effect of perturbation of DNA modifications on cell fate specification. The results from my project will help us to understand how cells regulate their fate in early development. This is of great importance to understand developmental defects and learn how to instruct stem cells in culture for differentiation for potential use in cellular therapies in regenerative medicine.

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

Investigating the effect of the Drosophila mitochondrial genome on complex phenotypes 30 Sep 2018

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.

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

Mycobacterial Determinants of Extracellular Success 30 Sep 2018

Tuberculosis (TB) is a severe infection which affects over ten million people a year, causing 1.7 million deaths annually. Treatment takes at least six months and four different drugs, and resistance to these drugs is an increasing problem. The causative bacteria, Mycobacterium tuberculosis (Mtb), lives mainly inside human cells. However, it must ultimately escape those cells to spread to the next host, a stage associated with worsening clinical disease. Despite its importance, little is known about how Mtb survives and thrives in this extracellular stage. This research will focus on understanding how the bacteria escape immune cell uptake, and whether they are using specific tactics such as 'biofilm' formation and 'quorum sensing'. Biofilm formation and quorum sensing are forms of bacterial behaviour that allow individual bacteria to ‘talk’ to each other via chemical signals, and set up collaborative, hardy, multifunctional colonies that can resist stresses including the immune response and antibiotics. In many other chronic human infections, biofilms are commonly seen and make the disease very hard to treat. This research will seek information about the genetics, regulation, and impact of biofilm formation in TB in order to unlock new knowledge about drug treatment and onward transmission of disease.

Amount: £0
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

Investigating the GWAS-identified target NCX3 in nociception 30 Sep 2018

Evolutionary, pain is a protective sensation. However, it can persist beyond its usefulness and become debilitating for patients. Chronic pain affects up to one half of the population in the UK (Fayaz et al, 2016). Currently, the treatment options are limited and discovering new drug targets is of great importance. In a recent genetic study (genome-wide association study), we identified a gene (SLC8A3 encoding the protein NCX3), which was associated with higher pain sensitivity to experimental pain stimuli in healthy participants. My thesis will therefore focus on studying the function of NCX3 on a molecular, cellular and systems level. NCX3 is an important part of the machinery that moves ions in and out of cells. Its role in pain is poorly understood, but previous reports show that it is involved in regulating Ca2+ levels in pain-sensing neurones. Inhibition of NCX3 can cause increased Ca2+ in these cells leading to higher activation of the central nervous system and increasing pain sensation. To investigate the function of NCX3, I will use genetically modified mice lacking the gene as well as isolated pain-sensing neurones. Our genetic data, combined with published results, makes NCX3 an attractive target for future research and drug discovery.

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