- Total grants
- Total funders
- Total recipients
- Earliest award date
- 09 Jun 1998
- Latest award date
- 29 May 2020
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
We hypothesize that the visual acuity is limited in patients with infantile nystagmus due to the arrested retinal development and nystagmus characteristics. The main research question is to investigate the combined effects of retinal development and nystagmus characteristics on the visual acuity of patients with infantile nystagmus. Specifically we will quantify retinal development by grading optical coherence tomograms using a foveal hypoplasia grading system developed at Leicester. Additionally, we will segment the intra-retinal layers at the fovea to derive thicknesses measurements including the cone outer segment thickness which is a surrogate marker for cone photoreceptor spacing. Nystagmus characteristics such as intensity, eye velocity and eXpanded Nystagmus Acuity Function (NAFX) will be calculated from eye movement recordings. A mixed model will be used to determine the level of contribution of each of these factors to the visual acuity of patients with infantile nystagmus. Understanding the contributory factors to reduced visual acuity in nystagmus will help determine therapeutic strategies to improve vision and provide prognosis for patients.
DNA replication through regions of damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by the interplay of high-fidelity and error-prone DNA polymerases, that latter of which can accommodate distorted templates in their active sites. Despite its major role in the maintenance of genome stability and implication in human cancer, TLS is still poorly understood at the molecular level. In this proposal, we will set out to unravel the molecular mechanisms of TLS. We will reconstitute, in vitro, two minimal replisomes including the human high-fidelity polymerase pol delta or the TLS polymerase pol eta. For the first time, we will employ cryogenic electron microscopy (cryo-EM) to determine these replisomes' structure. This work will constitute the critical building block for a far-reaching mechanistic investigation, which will combine cryo-EM and single-molecule fluorescence microscopy to resolve the architecture and choreography of the DNA lesion bypass machinery.
Histone deacetylase 1 (HDAC1) has been implicated in almost all cellular process from cell cycle, DNA synthesis, DNA repair to gene expression. However, the key interactions required for these activities remain poorly defined because studies have largely been performed by large-scale co-IPs (which may not identify weak or transient associations) in asynchronous and untreated cells. Novel methodologies have been developed which monitor the proximity of protein associations using biotin ligation e.g. Bio-ID. This has the advantage of using intact cells, requires no cross-linking and allows the identification of both binding partners and transiently associated proteins such as substrates. In this application we intend to contribute to the identification of novel HDAC1 associated proteins and substrates using a novel ‘APEX2’ biotin-ligation approach. The power of the APEX2 approach lies in its 1-minute labelling time. This will enable us to assess HDAC1 proximate proteins during different phases of cell cycle (e.g. G1/S/G2/M) and +/- DNA damaging agents (e.g. Doxorubicin, UV-light). These experiments should provide a comprehensive view of HDAC1 associated proteins at an unprecedented level of precision in terms of cell cycle and response to signalling pathways.
Metabotropic glutamate receptor modulation of mesolimbic dopamine release: effect of phencyclidine pretreatment modelling schizophrenia 31 May 2018
Schizophrenia is a severely debilitating mental condition, affecting ~0.5% of the population. Current theories suggest a core deficit in cortical glutamate which causes dysregulation of glutamatergic control of dopamine release in nucleus accumbens. In particular phasic (high-frequency) activity, related to attentional processes, rather than tonic (low-frequency) activity, is vulnerable. However, remarkably little is known about mechanisms involved. Phencyclidine (glutamate antagonist), causes changes in people, resembling schizophrenia. In animals, short-term chronic treatment causes behavioural deficits mimicking changes in schizophrenia, providing an animal model for studying causative mechanisms. In clinical trials, type-2 metabotropic glutamate receptors (mGluR2) agonists alleviated psychotic symptoms. In rats they reverse psychomimetic effects of phencyclidine and modulate accumbal dopamine release. We suggest that these processes are dysfunctional in schizophrenia, which accounts, at least in part, for glutamate-dopamine dysregulation. The aim of this study is to use fast cyclic voltammetry in vitro, to characterise the effects of activation of mGluR2 on stimulated accumbal dopamine release, particularly focussing on differences between phasic and tonic stimuli, and assess whether this modulation is disrupted by subchronic phencyclidine pretreatment, modelling schizophrenia. Identifying fundamental mechanism determining glutamatergic modulation of accumbal dopamine function, and dysfunction in model schizophrenia, underpins further research developing novel antipsychotic treatment strategies.
Rescue of secretion of disease-associated misfolded glycoproteins in UGGT1 knock-out cells 06 Sep 2018
UGGT1 is the checkpoint enzyme of the endoplasmic reticulum (ER) glycoprotein folding quality control (ERQC) machinery. Many a devastating human disease is caused by a mutation causing a slightly misfolded, but nevertheless functional protein that is flagged for ER retention by UGGT1 and ultimately degraded. The Seed Award in Science will enable me to test the hypothesis that modulation of UGGT1 can rescue the secretion of the mutant, but functional, misfolded glycoprotein, and alleviate the pathogenic consequences of the mutation. Rescue of secretion by UGGT1 deletion was tested successfully once in a plant but never in animals or mammalian cells. To do this, I will follow fluorescently tagged misfolded glycoprotein mutants in UGGT1 CRISPR/Cas9 knockout and wild-type (WT) mammalian cells. I will also use mass spectrometry to compare UGGT1 knockout cells to WT cells, to determine what impact UGGT1 deletion has on the overall secretome. These experiments if successful will make the case for the development of ERQC small-molecule modulators and the establishment of a personalised medicine strategy for the therapy of glycoprotein misfolding congenital syndromes. Unlike mutant-specific pharmacological chaperone therapies, hitting UGGT1, the one and only ERQC checkpoint, would benefit patients suffering from a broad range of rare disease.
Molecular changes occurring before cancer can be detected by commonly used methods provide potential biomarkers for early detection if routine screening were in place. To date, research has focused mainly on unique DNA largely disregarding the 55% composed of repetitive sequences like LINE-1s. LINE-1s are usually silenced in normal tissues but active in cancer and are best known for their ability to move to other locations in the genome with obvious deleterious implications. Mobilisation first requires activation of LINE-1’s promoters, but, intriguingly, those of both mobile and immobile LINE-1s become active in cancer. To fully understand the functional consequences of this transcriptional activity requires overcoming the challenges of studying individual LINE-1s. Using innovative approaches, we identified a LINE-1 whose activation is linked to silencing of a tumour suppressor gene and whose product can be detected in the blood of colorectal cancer patients. I will build on this knowledge to determine to determine why and which LINE-1’s promoters are active in colorectal cancer and what are the effects of this activation on neighbouring genes. I will examine in liquid biopsies the biomarker potential of the novel, aberrantly expressed genes so identified, with a long-term aim to help refine current tests.
Urinary incontinence (UI) and pelvic organ prolapse (POP) commonly affect women after childbirth. In the developed world initial treatment is non-surgical with pelvic floor exercises for UI and vaginal support pessaries or pelvic floor exercises for POP. Working with colleagues in Ethiopia we know that UI and POP affect much younger women, with greater severity. Conservative treatments are not offered because of lack of trained personnel, and significant difficulties with transport and access to care. Pelvic floor disorders are a modern epidemic and the introduction of sustainable treatment models is a public health priority. Our longterm goal is to design acceptable, sustainable and deliverable fully developed conservative treatment interventions for the treatment of pelvic organ prolapse and urinary incontinence to be disseminated, delivered and evaluated in a large prospective study. This application will fund three workpackages which bring key disciplinary perspectives together to lay the groundwork for this goal: a priority setting partnership in Gondar, Ethiopia; formative research with qualitative interviews to understand views and experiences of women and stakeholders to inform the design of the intervention, and an exploration of individual and community stigma associated with the condition to develop educational materials using concepts of communication for social change.
We request funds to purchase a BD FACSAria Fusion cell sorter enclosed within a Class-II microbiological safety cabinet (MSC) to establish a multi user cell sorting facility. The FACSAria Fusion is a highly flexible and advanced flow cell based flow cytometer that can perform multi-parameter four-way sorting. The Aria flow cell-based detection method provides greater fluorescence sensitivity than alternative stream-in air based cell sorters, which is critical for many of the proposed studies. We have requested funds for a four laser (blue/red/violet/yellow-green) 18-parameter instrument to enable high dimension analysis of the cells during sorting. The violet laser will enable use of highly sensitive brilliant violet fluorochromes and the yellow-green laser will allow greater use of multiple fluorescent proteins. The instrument can also perform single cell index sorting into 384-well plates, which will enable our research teams to undertake single cell transcriptomics projects and sort CRISPR/Cas9-mediated gene edited cells. The instrument is fully integrated into the biosafety cabinet enabling work to be undertaken with primary patient samples and hazard group 2 pathogens. Maintenance, assisting new users and staff training are essential for such a complex instrument, we have therefore requested funding for an experienced grade 7 manager for a 4-year period.
This scoping exercise is to explore perceived barriers to clinical innovation in medical practice. This important area has recently been brought into sharp focus by Lord Saatchi’s Medical Innovation Bill, as well as government initiatives such as the Innovation Pathway for NHS Products and the Early Access to Medicines Scheme. It became clear from a Department of Health consultation that no research had been conducted into what barriers to innovation exist, or are perceived to exist by health professionals. The award would support the running of five focus groups to ascertain what doctors themselves see as barriers to the use of responsible innovative practices, and how they might be removed without compromising patient safety. The findings will underpin a bid for a larger, more comprehensive international project which will fully explore in detail real and perceived barriers to innovation. Key goals include building an evidence base identifying barriers and constraints to innovation, and whether these differ between or are specific to certain specialties. We also aim to establish a network of potential participants for the larger project, as well as strengthening our ability to collaborate and work as a team using a smaller, more manageable data set.
Prefrontal control of hypothalamic feeding circuits: Balancing executive control of eating 05 Sep 2017
It is thought that diminished or excessive control over the drive to eat witnessed in eating disorders results from under- or over-activation of prefrontal cortical (PFC) brain regions important in decision-making. To investigate the executive control over eating this project aims to link the underlying circuitry between the PFC and feeding-promoting circuits of the hypothalamus to eating. Novel circuit-mapping strategies will be implemented to determine the functional relation between the two structures. This information will set the groundwork for relating PFC and hypothalamic activity in a rodent eating disorder model that promotes under- or over-eating. By consisting of two phases, one where animals restrict their food intake, the other where they over-consume food, we will monitor and relate changes in PFC and hypothalamic activity across phases where animals exhibit distinct feeding patterns. Finally, we will attempt to normalise this under-/over-eating by manipulating prefrontal inputs to the hypothalamus, thus determining a causal role for this circuit in influencing eating. In addition to linking executive circuits with feeding circuits this project aims to provide insight into the neural mechanisms underlying maladaptive eating behaviour.
Patients with T2D are at significant risk of developing heart failure and related complications. Altered myocardial fuel selection may play a central role in cardiac disease risk in patients with T2D, by affecting myocardial oxygen demand and metabolic flexibility. At any given level of cardiac work, an increased dependence on fatty acids (FA) relative to carbohydrates decreases cardiac efficiency, which may adversely affect cardiac function. Overall Aim: To evaluate, in vivo, the effect of T2D on myocardial energy metabolism and metabolic flexibility. Hypothesis: In patients with T2D i) myocardium is metabolically inflexible with a fixed FA preference and inability to increase glucose utilisation even in response to acute increases in cardiac workload; ii) the fixed preference of FA utilisation significantly contributes to abnormalities in contractile function. Methods: Cross sectional, case-control study. Population: 22 T2D patients and 22 non-diabetic individuals with preserved cardiac function. Investigations: Significant coronary artery disease will be excluded in all patients by coronary angiography. Myocardial metabolism will be determined at baseline and during dobutamine stress with measurement of transmyocardial arteriovenous differences of oxygen and metabolites. Participants will undergo a comprehensive evaluation of cardiac structure, function and perfusion using rest and dobutamine stress CMR.
Vacation Scholarships 2017 - University of Leicester
Unravelling Haemophilus influenzae and bacteriophage dynamics in the human upper respiratory tract 27 Apr 2017
Haemophilus influenzae is a pathogen that infect the human upper respiratory tract to cause diseases such as pneumonia and is associated with chronic diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) (Riesbeck, 2010). DNA sequence analysis of H. influenzae revealed the acquistion of novel genes mediated by phages which have not been identified; however, six H. influenzae phages have been identified of which HP1c1 is well characterised (Williams et al., 2002). The pathogen can change phenotype to escape HP1c1 but this makes it more vulnerable to the host immune system suggesting that phages impact the interaction of the of H. influenza with the host cell. During this project, we will isolate new H. influenzae phages from infected patients to better understand the pathogen dynamics in the respiratory tracts which could direct therapeutic towards treating these respiratory tract infections.
Evaluation of meningococcal disease isolates for phase variation in pilin adhesion determinants 27 Apr 2017
Neisseria meningitidis is the leading cause of bacterial meningitis. Carriage rates range from 10% for healthy populations to >30% in at risk populations. Invasive serogroup W (MenW) disease is increasing and has led to inclusion of MenACWY vaccine in the national immunisation schedule. Phase variation allows for alterations in outer membrane protein surface expression through slippage across simple sequence repeats (SSRs), altering the coding frame and truncating the protein or reducing promoter activity. Phase variation within pilC genes affects adhesion and virulence. Preliminary data demonstrates that phase variation is responsible for switching between pilC1 and pilC2 expression during carriage raising the potential for an impact on carriage to disease transitions. Key goals of this project are to analyse and compare phase variation in carriage and invasive MenW isolates. Objective 1: reconstruction of pilC loci through bioinformatics and PCR. Objective 2: analysis of variability in SSRs of pilC genes by alignments and GeneScan analysis. Objective 3: expression states of pilC1 and pilC2 will be determined through bioinformatics. Due to highly similar nature of MenW ST-11 genomes, these data will provide an excellent platform to study pilC phase variation within Neisseria and the effect upon bacterial adherence.
Histone deacetylates (HDACs) are important enzymes that play an essential role in cell development and differentiation. HDACs function as part of large protein complexes that are recruited to chromatin to regulate gene expression. The NuRD complex is one of these HDAC-containing complexes, of which the core protein components are known, but the mechanism of recruitment to chromatin is unclear. Our understanding of recruitment to the genome will be enhanced by structural and functional studies on a dimer of NuRD proteins, RBBP4 and MTA1. The MTA1:RBBP4 dimer has been shown to bind to chromatin through parts of the unstructured amino-terminus of histone H3. RBBP4 has also been shown to bind to the transcription factor FOG1 through an amino-terminal motif that is highly conserved in several other transcription factors including BCL11B and SALL1. In this project we propose to purify the MTA1:RBBP4 dimer from mammalian cells and then investigate the tightness of binding to fluorescently labelled peptides using fluorescence anisotropy. We will also set up crystallisation trials to gain structural insight into the mode of binding of these peptides to the MTA1:RBBP4 dimer.
HDAC1 and 2 (HDAC1/2) form the catalytic core of four co-repressor complexes (Sin3A, NuRD, CoREST and MiDAC) which have essential roles in DNA replication, cell cycle progression and gene expression. The goal of the Cowley lab is to define a network of accessory proteins, target genes and substrates (including non-histones), for each of the four canonical HDAC1/2 complexes. In this application we intend to contribute to this process by identifying novel associated proteins and substrates of the prototypical HDAC1/2 complex, Sin3A, using a new technique termed, Biotin-ID (Bio-ID). Bio-ID uses a promiscuous version of the biotin ligase, BirA (BirA*), fused to the protein of interest, thus allowing ‘proximate’ proteins (within approx. 10nm) to be labelled with Biotin, which can then be identified using a streptavidin pull-down and mass-spectrometry. The advantage of this approach is that it allows the identification of both binding partners and transiently associated proteins such as substrates. A parallel study with the LSD1/CoREST complex in the Cowley lab has identified known binding partners (CoREST1-3, HDAC1/2, and BHC80), substrates (p53), as well as 23 other novel chromatin associated proteins. We would therefore like to extend this approach to the study of the Sin3A/HDAC1 complex.
Single molecule analysis of DNA bending by the human mitochondrial transcription factor A (TFAM) 27 Apr 2017
Visualization of the dynamics of transcription initiation by mitochondrial RNA polymerase in real-time performed at single-molecule resolution forms the basis of this proposal. The human mitochondrial transcription factor A (TFAM) carries out DNA bending and my aim is to investigate whether this is an essential feature for transcription initiation by mitochondrial RNA polymerase. Multi-colour single-molecule super-resolution microscopy is the technique which will be used to demonstrate whether this DNA bending phenomenon is required for transcription initiation. This is a high-throughput technology capable of visualising reactions at single-molecule level, especially transcription factor binding, DNA bending and production of RNA. This state-of the-art instrument makes use of three different colours for accurate detection; green and red for smFRET and blue for RNA. I am also intrigued and have a desire to investigate for the first time whether DNA bending by TFAM is correlated with transcription by mtRNAP molecules.