- Total grants
- Total funders
- Total recipients
- Earliest award date
- 17 Jan 2014
- Latest award date
- 15 Dec 2014
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
Trypanosoma brucei, the causative agent of Human African Trypanosomiasis, constantly changes its variant surface glycoprotein (VSG) coat to avoid elimination by the immune system of the mammalian host. It is known that VSG synthesis is essential: RNAi knockdown of VSG results in precytokinesis arrest. However, it is not yet known how the cell senses that the VSG coat is intact before cell division occurs. This project seeks to identify the molecular mechanism of the sensor of VSG coat integrity. I will replace wild-type VSG with a number of different VSG mutants. These include a truncated form without a GPI anchor and a VSG with a trans membrane anchor. I will assess whether these cells are capable of division and compare the growth rates to cell lines expressing wild-type VSG. I will also investigate the requirements for VSG recycling by determining whether a VSG-GFP fusion mutant is recycled on the cell surface. To identify the genes responsible for controlling surface VSG density I will carry out a forward genetic screen using RITSeq. The identification of the VSG density sensing signal pathway will be novel biology and inform future drug design.
Neutrophils are key effectors of antibacterial immunity and inflammation. These cells often migrate in a highly co-ordinated and directed manner in order to reach sites of infection. This so-called ‘swarming’ response was shown to depend on self-production of the neutrophil attractant, LTB4. However, the cellular dynamics underlying transition from exploratory, single cell migration to collective and highly directional migration remain unclear. To address this knowledge gap I will use in vivo imaging and genetic manipulations in a zebrafish model. I will first determine the cellular triggers for LTB 4 production by neutrophils in situ. For this I will make transgenic fish expressing a reporter probe for LTB 4 production. Then I will investigate how LTB4 autocrine/paracrine signalling directs neutrophil polarity and migration. For this I will directly monitor autocrine/paracrine signalling using an additional probe for LTB 4 sensing. Finally I aim to spatiotemporally manipulate LTB 4 production and neutrophil swarming in vivo. To this end, I will develop an optogenetic tool to control the production of LTB 4 by light and use this to establish the implications of neutrophil swarming in microbial defence and tissue integrity. Thus, this study will provide a better understanding of how neutrophils self-organise their migration to sites of infection
Investigating!the!influence!and!therapeutic!potential!of!RNA!G^quadruplex!structures!on!positive! 14 Jul 2014
An!increasing!body!of!evidence!suggests!that!RNA!secondary!structures,!such!as!G] quadruplexes!and!stem]loops,!play!crucial!roles!in!the!regulation!of!translation!and!RNA! replication!during!viral!infection.!Our!ability!to!exploit!such!RNA!structures!therapeutically!is! dependent!upon!our!knowledge!of!how!they!act,!which!remains!unclear.!!One!goal!of!this! research!is!to!systematically!identify!and!examine!the!influence!of!RNA!G]quadruplexes! present!in!the!coding!regions!of!two!widely!used!model!calciviruses.!Using!the!established! reverse!genetic!systems!for!these!viruses,!the!effects!of!mutations!that!alter!the!presence!and! distribution!of!G]quadruplex!structures!can!be!studied,!both!in!terms!of!virus!efficiency!and!rate! of!RNA!synthesis!in!cell!culture.!If!suitable!mutated!viruses!are!obtained,!the!persistence!of! these!viruses!within!a!small!animal!model!will!be!examined!which!will!give!insights!into!the! suitability!of!such!modified!viruses!for!use!as!vaccines.!The!second!goal!of!this!research!is!to! investigate!the!mechanisms!by!which!helicases!control!the!regulation!imposed!by!such!RNA! structures.!Using!a!reconstitution!system!for!translation!initiation!in&vitro,!the!hierarchy!between! different!RNA!secondary!structures!and!the!helicases!that!mediate!their!unwinding!can!be! examined.
The molecular mechanisms that mediate somatostatin release from gastric D-cells have been poorly characterised. We aim to unpick some of these mechanisms by studying genes highlighted in the transcriptome of somatostatin-expressing D-cells isolated from transgenic mice with fluorescently tagged somatostatin expressing cells. The functionality of selected genes will be determined in secretion and imaging experiments utilising primary tissue cultures from the stomachs of mice and humans, with some focus on candidates underlying known D- cell responses to aromatic amino acids and variations in gastric pH. Examination of gastric organoids genetically modified using CRISPR/Cas9 genomic editing techniques will enable validation of these pharmacological findings. CRISPR/Cas9 editing will facilitate the introduction of fluorescent secondary messenger indicators to human D-cells, enabling imaging of human D-cell secretory responses. Based on findings that enteroendocrine cells form basolateral extensions (termed ‘neuropods’) which interface with surrounding enteric neurons and glial cells 25, 26, another aim of the study will be to characterise gastric D-cell cytoplasmic extensions and their physiological significance. By comparing the secretory responses of both murine and human tissue we shall examine the clinical relevance of murine enteroendocrine models. Both antral and fundic tissue shall be used to examine regional differences in gastric D-cell physiology. .
The main aim of my research is to develop new techniques for investigating transmission events in influenza A virus. These techniques will be mathematical and computational in nature and revolve around the use of publicly available time-resolved within-host data. Specifically, I propose to use maximum likelihood methods to extract information on transmission bottleneck size and selection based on the time-evolution of genetic variants. As a secondary aim, I wish to extend my methods (and perhaps those of other authors) to allow for analysis of other viral species such as HIV, norovirus and picornavirus (foot-and-mouth disease). Influenza virus is particularly convenient to work with as it has a short lifetime, large population size and lacks homologous recombination, however, other RNA viruses are more complicated in these aspects. As a result of this, my initial algorithm may require important adjustments to reflect this. Key goals: ? Incorporation of genetic selection to my model developed during research rotation ? Implementation of Gaussian transmission step in maximum likelihood approach leading to a more tractable algorithm ? Extension of algorithm to allow for analysis of multiple gene segments ? Analysis of existing influenza datasets ? Extension to and analysis of other RNA virus species
This project aims to uncover the logic of neurotransmitter specification in the developing central nervous system of Drosophila melanogaster. In the insect nervous system neuroblasts constitute the fundamental developmental units, each generating a defined set of nerve cells. Although the various neuroblast lineages have been well characterised in Drosophila melanogaster in terms of cell number, morphology, and position within a segment, for most lineages it still remains unknown what neurotransmitters the cells produce and by what mechanisms different neurotransmitter phenotypes are specified. The first objective of the project is to characterise the neurotransmitter type of neuronal lineages in the ventral nerve cord, focusing initially on a few lineages that the lab has direct genetic access to. The second objective will use the information gained about the principal patterning of neurotransmitter types in lineages to investigate the mechanisms by which neurotransmitter type is specified. Targeted DamID will be performed in collaboration with Tony Southall to uncover the genetic profiles of lineages that produce different neurotransmitters. The third objective will be to test candidate genes from the Targeted DamID for involvement in neurotransmitter specification, and thus understand the mechanisms by which this important element of neuronal functionality is established during development.
How the strength ofT cell receptor signalling controls cytotoxic T cell polarisation for targeted killing 17 Jan 2014
The proposed project aims to investigate how cytotoxic T lymphocytes (CTL) respond to variations of T Cell Receptor (TCR) signal strength at an intracellular level.Altered peptide ligands (APL) have been characterised to change the TCR: peptide-major histocompatibility complex interaction and subsequently alter TCR signal strength. Using APL provided by Ed Palmer, his biophysical measurements of these interactions, particularly dwelltime, can be correlated to physiological measurements. These will aim to identify any killing defects imposed by the APL and consequently any intracellular causes of defects will be investigated. This includes investigating strength of TCR signalling modules, such as calcium flux and extracellular signal-related kinase phosphorylation. The project will progress to investigating defects in conjugation and organisation of the immune synapse by immunofluorescence techniques. Next, defects in centrosome and granule polarisation will be investigated via live cell imaging to identify variations in velocity, granule clustering dynamics and timing post signalling and synapse formation, in order to elucidate how the order of these events are impacted by dwell time. This will provide insights into the determinate stages for target killing. Further work will involve following organelles with roles in TCR signalling but poorly defined roles in CTL killing, such as recycling endosomes.
Recent patient reports have identified that microdeletions encompassing the SNORD116 cluster on chromosome 15q11.2 manifest a phenotype substantially overlapping with Prader-Willi Syndrome (PWS), including characteristic hyperphagia and developmental delay. The SNORD116 cluster encodes non-coding RNAs, including multiple C/D box snoRNAs and long non-coding RNAs, which are deemed orphans' of unknown function and have no validated targets. We aim to explore the function of SNORD116 by elucidating the molecular mechanisms linking loss of SNORD116 to the PWS-like phenotype. Preliminary data from our group suggests SNORD116 is predominantly brain-expressed, and that fasting regulates Snord116's hypothalamic expression. We hypothesise thatSNORD116 may regulate a repertoire of genes impacting neuronal development andfood intake control. In this research, we will examine the functional and transcriptional consequences of perturbing Snord116 expression in primary mouse hypothalamic neurons. Secondly, we will clarify the types of RNA transcripts arising from the Snord116 cluster in mouse brain and identify their protein and RNA interaction partners. Thirdly, two mouse models with targeted Snord116 deletions will permit evaluation of Snord116's role in neural food intake control. We ultimately hope this functional investigation may reveal novel neural control pathways implicated in the hallmark hyperphagia and early-onsetobesity evident in PWS.
Poxvirus immune evasion strategies 08 Jul 2014
The objectives of this research are to understand how vaccinia virus, the prototypical poxvirus, suppresses the host innate immune response to infection, and how this impacts on virus virulence and the adaptive immune response that follows. This investigation will provide fundamental information about the host-pathogen interactions, how the innate immune response functions and influences the adaptive response, how the innate immune system can be inhibited, and how this information can be used to develop more immunogenic and safer poxvirus-based vaccines. It is also a goal to obtain co-crystal structures of the vaccinia virus proteins with their cellular binding partners to understand in molecular detail how inhibition of the innate immune system is mediated and in the long term to use this information to develop small molecule inhibitors of innate immunity that will have anti-inflammatory therapeutic value.
Public Engagement Provision 20 May 2014
The Cairo Genizah Collection at Cambridge University Library is the single largest and most important collection of medieval Jewish manuscripts in the world. Comprising approximately 200,000 leaves, the discovery of this treasuretrove 100 years ago revolutionised the study of medieval Judaism and our understanding of the broader economic, social and intellectual history of the Mediterranean world. Intended as a resting place for religious works, the Genizah ('religious storeroom') has in fact preserved an ample number of secular texts too, both literary and documentary, and conspicuous among these is a unique medical corpus of approximately 2000 manuscripts. The medical fragments of the Cairo Genizah (which date from the 10th century onwards) include a large number of Judaeo-Arabic renditions of Arabic translations of Greek medical texts and of Arabic medical works, often annotated by their medieval users, together with original works by prominent, and less well-known, Jewish physicians. Uniquely,the collection preserves numerous prescriptions, druggists' notes, and lists of materia medica. This project will build upon previous work in the Collection, and make these important items freely available through Cambridge University's digital library platform, in the form of a detailed electronic catalogue of all the items of medical content along with high quality digital images of the manuscripts themselves. These will form a unique source of research on the study, transmission and practice of medicine by Jews and Muslims in the medieval Mediterranean world, and we believe that the medical humanities community will greatly benefit from their enhanced availability.
The relative importance of human and animal sources of vancomycin-resistant Enterococcus faecium in immunocompromised patients in hospital. 18 Feb 2014
Enterococcus faecium (Efm), a human and animal gut commensal, has emerged as a leading nosocomial infection in immunocompromised patients. The rise of Efm has been driven by the global spread of a hospital-adapted lineage, characterised by mobile genetic elements coding for antimicrobial resistance and virulence determinants. Two further lineages are recognised, one associated with community carriage and the other livestock-associated. Vancomycin-resistant Efm (VREfm) has spread across all three lineages, and represents a significant healthcare problem responsible for outbreaks and infections in vulnerable hospitalised patients. Whole-genome sequencing (WGS) promises to close the gaps in our understanding of Efm epidemiology. I will conduct a study to investigate the hypothesis that WGS will determine the origin of VREfm in a cohort of hospitalised patients at Addenbrooke's Hospital, a centre which ranks top in VREfm bacteraemia numbers nationally. The project will consist of: i) a lo ngitudinal survey of Efm gut carriage in patients with haematological malignancy and first hospital admission and their ward contacts; ii) cross-sectional surveys of Efm in farm slurry and wastewater in the East of England; iii) WGS and bioinformatic analysis of recovered isolates to determine the origin, within-host evolution, transmission of VREfm between patients, and a possible zoonotic source.
Autoimmunity after alemtuzumab: a human model to study the role of regulatory T cells in lymphopenia associated autoimmunity. 13 Nov 2014
The association between lymphopenia and autoimmunity is well recognised, but poorly understood. MS patients treated with the highly effective, recently licensed, lymphocyte-depleting antibody alemtuzumab offer a rare opportunity to study this phenomenon as: 30% develop autoimmune thyroid disease, 1% ITP and 20% asymptomatic autoantibodies after treatment. We previously reported increased CD4+CD25hiFoxP3+ Tregs after alemtuzumab (similar expansions are seen in other lymphopenic settings). Her e, by immunophenotyping, TCR-sequencing, TREC analysis, FoxP3 methylation and isoform analysis, RNAseq and functional assays, I will explore the hypotheses that autoimmunity occurs because these cells are: 1. Not Tregs but activated Tconv2. 2. Defective, incapable of suppressing Th2 auto-antibody responses, due to being clonally restricted, exhausted or skewed towards a Th1 Treg phenotype. 3. Plastic - generating autoimmune effector cells via the loss of FoxP3. 4. Or, the defect may lie wit h other FoxP3- regulatory cells, e.g. the newly reported CD52hi population. This work may directly benefit MS patients; once the post-alemtuzumab Treg population is understood it may be possible to augment their function, e.g. with IL-2 or rapamycin. It also has the potential to advance our understanding of human autoimmunity, and may benefit people with other forms of lymphopenia-associated autoimmunity.
Aim 1: To examine preference for, and rewarding properties, of dietary fat. Aim 2: To examine preference for, and rewarding properties of, sucrose. Aim 3: To determine whether preference for dietary sucrose and fat is mediated melanocortin signalling. Aim 4: To examine the brain activation response to manipulation of single/multiple diet macronutrients. 1. Increased dietary fat content in food will increase the preference for and rewarding properties of the food. Increasing fat content in food will be positively correlated with brain activation of areas involved in reward. 2. Increased sucrose content in food will increase the preference for and rewarding properties of the food. Rewarding properties and preference for food containing artificial sweetener in place of sucrose will be reduced compared to matched sucrose formed contents. Increasing sucrose content in food will be positively correlated with brain activation of areas involved in reward. 3. MC4R deficient subjects will display an increased preference for high-fat food (but not high sucrose food) in comparison to equally obese subjects matched for age.
Wounds are thought to be repaired by a reserve stem cell population activated in response to injury. However, excisional wounding in mouse oesophagus has revealed a new model of tissue repair. The same progenitor population that maintains the tissue is able to switch its behaviour, tilting fate towards proliferation to generate the excess of cycling cells that will repair the tissue (Doupe, 2012). Progenitor cells are then able to regain stem cells properties to achieve a rapid and efficient hea ling response. This illustrates recent findings that show how cells committed to differentiation acquire stem cell-like behaviour upon damage in other epithelial tissues (van Es, 2012; Tata, 2013). Although this cellular plasticity is a strictly controlled and limited process, if inadequately activated in cells with pre-existing tumour initiating mutations it will have implications for cancer development. In the current proposal I set up to use my expertise in in vivo single cell lineage tracing to investigate short and long term changes in epithelial cell fate in response to injury, as well as the mesenchymal contribution to this process. Mechanistic insights will be gained by transcriptional network analysis and organotypic cultures specifically designed to recapitulate the epithelial wound response in vitro.
We have shown that specific combinations of maternal and fetal immune genes predispose to reproductive failure. We will now identify the specific alleles that predispose to reproductive failure in humans. We will correlate, for the first time, maternal and fetal immune genotypes of large cohorts of normal and abnormal human pregnancies with blood flow dynamics at the experimentally inaccessible human maternal fetal-interface. Informed by these human genetic data we have designed experiments in mice to determine the downstream consequences of the interactions between maternal NK cell receptors (NKR) genes and fetal Major Histocompatibility Complex (MHC) genes on reproductive success. We will investigate in vivo exactly how these interactions control the mechanisms of vascular remodelling, placental development and fetal growth. A combination of cellular assays and transcriptome analysis will reveal the pathways activated in both maternal NK cells and fetal trophoblast cells upon int eractions with one another in both species. Bringing together human and mouse genetics, quantitative in vivo and in vitro assays and uterine
This program will address some of the fundamental unanswered questions about L-cell biology. L-cells produce hormones affecting glucose homeostasis and appetite, and are the target of current drug development programs for type 2 diabetes. The prediction is that stimulating GLP-1 release from L-cells will provide oral therapies that could be used alongside recently-licensed DPPIV inhibitors. Understanding L-cell physiology is critical to the success of this type of approach, but has been hindered by difficulties in identifying and culturing intestinal endocrine cells. We made transgenic mice with fluorescently labelled L-cells, and developed methodologies to characterise the cells by expression analysis, electrophysiology and imaging, providing a first opportunity to study primary L-cells at a single cell level. This technology will be used to probe the physiology of the L-cell using a range of electrophysiological and optical recording techniques, and to address key questions and hypot heses about L-cell function, such as that electrogenic transporters act as sensors of the luminal constituents, that L-cells from different gut regions are not identical and that the cells exhibit plasticity in number/function in an altered metabolic environment. In parallel, we will develop new technologies to study L-cells from humans, and downstream targets of GLP-1 in the intestine.
In the intestine and stomach, organs with extensive self-renewal, Lgr5 marks adult stem cell populations that are constantly cycling. In the liver, an organ with limited cellular turnover but huge regenerative capacity, Lgr5 is not detected under physiological conditions. However, marks a new stem/progenitor cell population that gets activated upon damage and contributes to restore the tissue and reinstall homeostasis. The main goal of this proposal is to understand the molecular mechanisms, t hat following tissue damage, regulates the transition of adult liver cells from a quiescent to an activated state, from the role of the niche during this transition to the epigenetic changes triggering this activation. The main focus of this research plan will be: Plan1) Identify the quiescent cell/s that become activated cells during damage-regeneration; Plan2) Elucidate the niche for activated stem cells; Plan3) Identify the gene regulation mechanisms (epigenetic) involved in liver stem cell activation following damage. As liver disease and liver cancer are highly associated to liver damage, understanding the mechanism regulating stem cell activation holds promise to extend our knowledge not only of liver regeneration but also of liver disease and cancer.
We propose to form a consortium of academic and industrial partners to address the therapeutic challenges of Alzheimers disease and mood disorders by a shared, innovative focus on immunological mechanisms of these key neuropsychiatric disorders and their treatment by anti-inflammatory drugs. The industrial partners (Janssen and Lundbeck) will contribute drug discovery know-how and access to anti-inflammatory drugs. The academic partners will contribute expertise in neuroimaging, clinical phen otyping, animal models and informatics. The work will be organised as two programmes, focused on Alzheimer's disease and mood disorders, and four cross-cutting technical platforms (cytometry, proteomics, PET and MRI). In phase 1 (years 1-3), we will validate peripheral immunological phenotypes, e.g., cytokine levels or macrophage transcriptomes, as markers of Alzheimer's disease and mood disorders. We will seek mechanistically to link peripheral immunophenotypes to markers of central (brain) inflammation and microglial activation; to brain function and behaviour; and to longitudinal changes in the clinical phenotypes and animal models. In phase 2 (years 3-5), we will use these biomarkers in experimental medicine studies designed to test mechanism-of-action and / or therapeutic proof-of-concept of anti-inflammatory drugs in immunologically stratified cohorts of patients with Alzheimer's disease or mood disorders.