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Recipients:
University of Cambridge

Results

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.

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

Dynamics of KLF17 expression in human naive pluripotent stem cells 27 Apr 2017

Conventional hPSCs represent a relatively late stage of embryonic development, termed the primed phase of pluripotency. Their use in research and medical applications is problematic because they display a differentiation bias and do not generate all cell lineages efficiently. The Smith laboratory has recently defined culture conditions that capture cells in an earlier phase, termed naïve pluripotency. Naive hPSCs have potential implications for more effective stem cell therapies because they don't display a differentiation bias. Very little is known about the genes that govern human naïve pluripotency in culture. The transcription factor KLF17, which is present in naïve hPSCs but absent in primed hPSCs, is of particular interest because it is specific to primates and not well studied. The key goal of this project is to generate a fluorescent reporter for KLF17. Alternative reporter designs will be trialled by fusing fluorescent proteins to either the start or the end of the endogenous KLF17 protein, in order to achieve optimal fidelity and sensitivity. The reporter(s) will then be exploited to monitor the dynamics of KLF17 expression in live cells both following withdrawal of factors to initiate differentiation, and also during the process of generating naïve cells by resetting.

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

Role of neuronal nitric oxide in the vasodilatory response to mental stress 27 Apr 2017

Nitric oxide (NO) is a potent regulator of vascular tone. Until relatively recently, it was assumed that the isoform of NO synthase responsible for tonic NO release was endothelial nitric oxide synthase (eNOS). However, we now know that in humans, neuronal NOS (nNOS) is the primary NOS isoform responsible for regulating vascular tone in vivo. Neuronal NOS is also activated by mental stress and contributes directly to resistance vessel vasodilatation. However, our preliminary data indicate that this response is biphasic, suggesting a second mechanism underlying the vasodilatory response to stress. We hypothesise that this additional mechanism may be mediated through agonism of beta2 adrenoceptors. This hypothesis will be tested in healthy volunteers exposed to mental stress (Stroop test), using the gold-standard technique of venous occlusion plethysmography to measure forearm blood flow, coupled with intra-arterial infusions of selective inhibitors of nNOS and beta2 adrenoceptors. The key goals of this research are (i) to better define the regulation of vascular tone in healthy humans with a view to understanding potential mechanisms underlying vascular dysfunction in disease states; and (ii) gain a broader understanding of early experimental medicine approaches in the clinical setting.

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

Rate of degradation of Aurora kinases 27 Apr 2017

Aurora kinases regulate the segregation of chromatids and are key enzymes in mitosis. AurA assembles the spindle poles; AurB faciliates cytokinesis of the daughter cells. Their ubiquitin-mediated degradation regulates the transition from mitosis back to interphase and show different kinetic profiles: AurA degrades 5-fold faster than AurB. Previous unpublished data from the Lindon lab showed that a AurA1-133-AurB78-345 chimera tagged with GFP degraded with similar kinetics to full-length AurA. Therefore all of the information required for rapid degradation of AurA resides in the 1-133 region. We plan to construct various AurA-AurB chimeras and express them in dividing cells. We will carry out a quantitative analysis of degradation of these chimeras using single-cell fluorescence timelapse assays. We aim to identify the the minimal sequence within AurA1-133 required to specify accelerated degradation kinetics. We plan to compare this with other known regulatory sequences for ubiquitin-mediated degradation ('degrons') and to gain a better understanding of how AurA engages the destruction machinery to affect its degradation kinetics. This information can assist the design of new therapeutic tools, such as PROTACs, that harness ubiquitin-mediated degradation to destroy targets not druggable by conventional means.

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

Mechanisms of endogenous analgesia. 30 Sep 2016

The programme will address how two distinct mechanisms of endogenous analgesia control activity in the brainstem periaqueductal grey. I aim to provide an account in computational terms, in order to specify the precise decision algorithms (rules) by which they operate. First I address the mechanism by which predictions reduce pain, by exploring precisely what information within a prediction controls pain perception. Specifically, I will test Bayesian models against competing accounts. Brain i maging will be used to determine how this is implemented in forebrain structures, and if/how they modulate activity in the periaqueductal grey. Second, I address the mechanism by which escape under threat reduces pain. Here I propose a theoretical analysis of controllability in escape/avoidance learning, building on my existing Reinforcement Learning framework. I will implement evolutionary simulations designed to show whether pain modulation emerges as an intrinsic reward to guide escape. On the basis of this, I will test model predictions (behaviourally) and their implementation (using fMRI), using an active versus passive escape learning paradigm. Finally, I will use deep brain stimulation to provide convergent evidence of how these mechanisms depend on the periaqueductal grey, looking for a statistical interaction between stimulation and endogenous analgesia.

Amount: £175,527
Funder: The Wellcome Trust
Recipient: University of Cambridge

Protein trans-activation of ribosomal frameshifting: a new paradigm in gene expression. 05 Jul 2016

Programmed ribosomal frameshifting (PRF) is a translational control mechanism widely used in the regulated expression of many viral, and several cellular proteins. The mRNA signals that induce PRF comprise a slippery sequence, where the ribosome changes into an overlapping frame, and an essential, stimulatory RNA structure which promotes frameshifting by modulating the ribosomal elongation cycle. Recently, we have uncovered two novel examples of viral PRF signals where frameshifting is trans-activated through the action of viral (cardiovirus 2A, arterivirus nsp1beta) and cellular proteins (poly(C) binding protein). The discovery of such protein-stimulated PRF opens up a completely new area in this field and offers novel opportunities for the study of protein-ribosome interactions. Our aim is to characterise the trans-acting factors and the mechanism by which they induce these novel PRF events. This will be achieved through a combination of functional assays, RNA-protein and protein-protein interaction studies and structural biology. This work will provide important new insights into ribosome structure and function, gene regulation, protein-protein and protein-nucleic acid interactions, virus replication strategies and virus-host interactions.

Amount: £774,356
Funder: The Wellcome Trust
Recipient: University of Cambridge

Maternal NK cell recognition of the placenta determines reproductive outcome 05 Apr 2016

Our aim is to understand how the uterine immune system regulates placentation and reproductive success in humans. We described a new mechanism of maternal allogeneic recognition that depends on KIR expressed by uterine NK (uNK) cells and their ligands, HLA-C, on fetal trophoblast. KIR and HLA-C genes are highly polymorphic and we find reproducible and specific KIR/HLA-C genetic combinations associated with reproductive disorders. We will: 1) use high throughput typing to allele level of KIR and HLA-C genes to describe how this variation affects pregnancy success. 2) translate these genetic findings into how NK cells affect trophoblast functions exploiting our new techniques, mass cytometry and long term trophoblast cell culture. 3) use transgenic mouse models to mimic the KIR/HLA-C combinations with poor outcome to study placentation in vivo and to test therapeutic anti-KIR mAbs. From a translational perspective we will: 4) investigate whether disorders such as pre-eclampsia that are common in women undergoing assisted reproductive technology with oocyte or sperm donation can be prevented by genotyping donors for KIR/HLA-C and 5) use the extraordinary variability of KIR genes in sub-Saharan Africa to study differences that can explain the increased frequency of pregnancy disorders in African women.

Amount: £2,140,661
Funder: The Wellcome Trust
Recipient: University of Cambridge

Open Access Block Grant 2016/17 30 Sep 2016

Not available

Amount: £634,951
Funder: The Wellcome Trust
Recipient: University of Cambridge

University of Cambridge - Mathematical Genomics and Medicine 30 Sep 2016

Imperial College London - Theoretical Systems Biology and Bioinformatics

Amount: £133,023
Funder: The Wellcome Trust
Recipient: University of Cambridge

University of Cambridge - Metabolic and Cardiovascular Disease 30 Sep 2016

University of Cambridge - Metabolic and Cardiovascular Disease

Amount: £133,023
Funder: The Wellcome Trust
Recipient: University of Cambridge

University of Cambridge - Developmental Mechanisms 30 Sep 2016

University of Cambridge 4 year PhD Programme - Developmental Mechanisms

Amount: £133,023
Funder: The Wellcome Trust
Recipient: University of Cambridge

Cambridge - Infection, Immunity and Inflammation 30 Sep 2016

Cambridge - Infection, Immunity and Inflammation

Amount: £133,716
Funder: The Wellcome Trust
Recipient: University of Cambridge

Virtual Fly Brain 06 Jul 2017

Neuroscience is accelerating: the capability to generate circuit level hypotheses is now matched with the ability to visualise, manipulate and record from individual neurons, in vivo. Drosophila, with its complex adaptive behaviors, powerful genetic toolkit and small nervous system, for which we will soon have complete connectomes, is uniquely placed to contribute to this work. Virtual Fly Brain (VFB) is a unique resource for Drosophila neuroscience, integrating disparate, large-scale datasets and linking them to curated literature and other resources. VFB works with international data providers and bioinformatics resources to ensure efforts are complementary, non-redundant, and make best use of resources. VFB users browse and query curated information from many sources to understand structure, function and relationships in the brain. Critically, VFB provides the data to generate circuit hypotheses and identify research tools to test them. This proposal continues this vital service and extends it to incorporate rich new data types. We will incorporate synaptic resolution connectomic data, develop bridging registrations to make it bidirectionally queryable from light level data. We will add phenotypic and transcriptomic datasets and enhance tools that enable researchers to find reagents. We will enable users to upload, view and query their own 3D datasets.

Amount: £996,004
Funder: The Wellcome Trust
Recipient: University of Cambridge

The role of Eros in Innate and Adaptive Immunity 25 May 2017

I will investigate the role of a novel protein, Eros, in immunity. I discovered the fundamental importance of this protein by demonstrating that Eros-deficient mice die from Salmonella infection because their phagocytes cannot make reactive oxygen species. This is because Eros is essential for expression of vital components of the phagocyte NADPH oxidase. My work represents the only paper on this protein. I have found that Eros-deficiency has effects that go far beyond the generation of reactive oxygen species. In particular: Eros regulates the expression of other key macrophage proteins including P2X7, a key activator of the NLRP3 inflammasome Eros regulates the expression of numerous cytokines from CD4+ T cells. Eros -/- T cells make 10-fold more IL-4 than control cells In mouse and human systems, I will investigate the molecular mechanisms by which Eros: controls the abundance of a subset of proteins working on the hypothesis that it is a novel component of the protein quality control pathway using structural, biochemical and cell biological techniques. controls T cell cytokine secretion. I will spend time working with John O'Shea, a world leader in this field.

Amount: £1,319,075
Funder: The Wellcome Trust
Recipient: University of Cambridge

21st Century Families: Parent-child relationships and children's psychological wellbeing 25 Jul 2017

New pathways to parenthood have recently emerged that did not exist, nor had even been imagined, at the turn of the 21st century. Individuals who were previously unknown to each other have begun to meet over the internet with the purpose of having children together; transgender men and women have begun to have children through medically assisted reproduction; single heterosexual men have begun to use surrogacy to become single fathers by choice; and women have begun to use identifiable egg donors to have children. These emerging family structures raise new ethical, social and psychological concerns, particularly regarding the potentially negative consequences for children. The proposed research will provide empirical evidence from a multidisciplinary perspective on the social and psychological consequences for children of growing up in family arrangements involving non-cohabiting co-parents, transgender parents, elective single fathers and identifiable egg donors. In this emotive area of family life on which people often hold strong opinions, our aim is to challenge prejudice and assumption with evidence on the actual consequences – good, bad or neutral – for children. The ultimate goal of the proposed research is to increase understanding of diversity in family life and improve the lives of 21st century children.

Amount: £1,552,401
Funder: The Wellcome Trust
Recipient: University of Cambridge

Behaviour Change by Design: Generating and Implementing Evidence to Improve Health for All 11 Jul 2017

Reducing food, alcohol and tobacco consumption would dramatically reduce non-communicable disease and, since these behaviours cluster by deprivation, would also reduce health inequalities. However, progress in achieving such behaviour change is slow. Traditional approaches to behaviour change involve providing information with, at best, modest population-level effects and sometimes increased inequalities. Conversely, Choice Architecture interventions ("Nudges") have potentially larger, more equitable effects, involving re-designing environments e.g. reducing plate size to reduce food consumption. However, evidence of effectiveness in real-world settings and understanding of mechanisms are limited. We will bridge this knowledge gap through a novel collaboration between behavioural and cognitive sciences. In the most ambitious co-ordinated set of studies to date, we propose field studies to estimate effect sizes of promising Choice Architecture interventions to reduce food, alcohol and tobacco consumption. Enabled by unprecedented collaborations, these will be conducted in supermarkets, bars and cafeterias and interventions optimised through laboratory studies determining mechanisms. We will run international workshops, public engagement activities and a Behaviour Change Summit to facilitate implementing the evidence generated, overseen by an Implementation Advisory Panel. This will enable us to realise our vision of accelerating progress in changing behaviour by re-designing environments to improve health for all.

Amount: £3,123,724
Funder: The Wellcome Trust
Recipient: University of Cambridge

Putting genomic surveillance at the heart of viral epidemic response. 05 Apr 2017

This proposal is to develop an end-to-end system for processing samples from viral outbreaks to generate real-time epidemiological information that is interpretable and actionable by public health bodies. Fast evolving RNA viruses (such as Ebola, MERS, SARS, influenza etc) continually accumulate changes in their genomes that can be used to reconstruct the epidemiological processes that drive the epidemic. Based around a recently developed, single-molecule portable sequencing instrument, the MinION, we will create a 'lab-in-a-suitcase' that will be deployed to remote and resource-limited locations. These will be used to sequence viral genomes from infected patients which will then be uploaded to a central database for rapid analysis. We will develop methods for a wide-range of emerging viral diseases. Novel molecular biology methods will allow us to sequence individual viruses within a patient. Bioinformatics tools will be developed simple enough for non-bioinformaticians to use, without reliance on Internet connectivity. We will develop software to integrate these data and associated epidemiological knowledge to reveal the processes of transmission, virus evolution and epidemiological linkage. Finally we will develop a web-based visualization platform where the outputs of the statistical analyses can be interrogated for epidemiological insights within days of samples being taken from patients.

Amount: £482,639
Funder: The Wellcome Trust
Recipient: University of Cambridge

Biomechanics of Ciliated Tissues 11 Jul 2017

Many of the paradigmatic events in embryonic development involve geometric or even topological rearrangements of tissues in response to mechanical forces generated within them. While these processes are familiar and much studied from genetic and biochemical perspectives, there is a striking contrast between the great depth of such biological detail and the glaring lack of quantitative mechanical understanding of the forces and responses involved. We propose to close the theory-experiment loop in specific, carefully chosen examples of these problems, to gain a quantitative understanding of the underlying biomechanics. We seek to solve three outstanding problems: (i) the link between cell shape changes and cell sheet morphology as found in gastrulation, neurulation, and related problems in embryogenesis; (ii) the mechanism of generation of cilia orientational polarity in tissues; (iii) the origin of metachronal wave formation in carpets of cilia. The research will combine state-of-the-art light-sheet microscopy, micromanipulation, high-speed imaging and microfluidics with emerging theoretical tools for understanding complex geometrical transformations of tissues and the stochastic nonlinear dynamics of eukaryotic flagella.

Amount: £1,656,325
Funder: The Wellcome Trust
Recipient: University of Cambridge

Whole genome sequence based analysis of genetic variation and genome evolution 11 Jul 2017

DNA sequencing is a core technology for modern biomedical science, and our ability to sequence genomes with ease and use that information efficiently is still unfolding. I propose first to build new bioinformatics data structures and software to map sequence data, call genetic variants, and integrate phasing and imputation, scaling to millions of samples with high accuracy and making best use of new long read sequencing technologies. These will be based on sequence variation graphs and haplotype panels over them, effectively exploiting already-discovered genetic variation in the population. Second I will develop new statistical methods to infer the evolutionary history of genome sequences to identify ancestral populations, model gene flow between them, and date and place mutations into them. I will apply these methods to modern and ancient samples to elucidate Eurasian and African human population history. Finally, I will apply these methods to new data I collect from the Lake Malawi adaptive radiation of over 500 species of cichlid fish, to infer the evolutionary relationships in the radiation, test models of speciation, and identify genes involved in cranio-facial adaptation. These studies will empower future use of sequencing data in biomedicine, and advance our understanding of genome structure and evolution.

Amount: £3,027,662
Funder: The Wellcome Trust
Recipient: University of Cambridge