- Total grants
- Total funders
- Total recipients
- Earliest award date
- 10 Apr 2001
- Latest award date
- 30 Sep 2018
- Total GBP grants
- Total GBP awarded
- Largest GBP award
- Smallest GBP award
- Total Non-GBP grants
We have recently shown that fibrin, but not its precursor fibrinogen, activates platelets through a receptor complex, GPVI-FcRg-chain, which was identified by one of us (SPW) in the 1990s, and is recognised as the primary signalling receptor for collagen. The paradigm-changing observation that GPVI is a receptor for fibrin establishes a role for GPVI not only in initiation (via collagen) but also in propagation (via fibrin) of thrombus growth and may explain the increase in embolisation in thrombosis models in GPVI-deficient mice. We propose that the interaction of fibrin with GPVI represents a target for a new class of antiplatelet agent that may have benefits over current antiplatelet drugs. To investigate this we will map the site of interaction of fibrin with GPVI and develop agents (inhibitors and mouse models) that block this interaction but preserve the activation of GPVI by collagen. We will use these to determine the importance of GPVI-fibrin in haemostasis and thrombosis, and in other vascular pathways where GPVI is known to play a role including maintenance of vascular integrity.
Harnessing the molecular-scale resolution of DNA-PAINT to study the structural basis of electrical signals of the healthy and arrhythmic hearts 12 May 2017
Single molecule localisation microscopy (SMLM) has revolutionised our understanding of signalling microdomains in many biological systems. Among such molecular systems, are arrays of gap junction (GJ) channels paving the fundamental electrical signalling pathways of the heart. Conventional SMLMs are yet, limited by resolution, unable to visualise individual GJ channels within their native tissue environment. One of the newest SMLM techniques, called DNA-PAINT, promises resolution of ~ 5 nm, sufficient to resolve single proteins within tightly packed arrays or clusters and to visually observe their interactions with other molecules. However, by design, this technique is not suitable for imaging complex tissues like the myocardium. This project aims to implement three possible modifications to the DNA-PAINT protocol to enable in situ optical mapping of GJ isoforms at an unprecedented single-channel-level resolution. DNA-PAINT will be further harnessed to quantify the changes in the phosphorylation states of GJ channels within the ventricular cardiac tissue exhibiting pathological electrical disturbances known as arrhythmias. Both the improved DNA-PAINT protocol and the pilot measurements on GJ remodelling will seed a larger investigation pioneering a direct, in situ correlation of the GJ nanostructure and the disturbed electrical patterns within hearts undergoing life-threatening arrhythmias.
The proposed research uses standard molecular biology, protein purification and biophysical structural analysis methods in a focused series of experiments that comprise a complete 6-week project. This builds on existing molecular genetics studies that have identified novel missense mutations in KMT2D (also known as MLL2) as the cause of a unique phenotype (renal tubular dysgenesis, choanal atresia and athelia). Previous studies have identified KMT2D mutations as a major cause of Kabuki syndrome, a comparatively common autosomal dominant congenital mental retardation syndrome. The missense mutations occur in a central region of the KMT2D protein (2841-3876) that does not have variants associated with Kabuki syndrome. This central region contains a series of coiled-coil domains that are likely to mediate protein-protein interactions. However, the effect of the missense mutations on KMT2D structure and interactions is completely unknown. This project will determine the structure-function relationships between KMT2D and a unique phenotype that are likely to be caused by altered protein-protein interactions, as well as describing the broader genotype-phenotype correlations in this important gene. The approach described in the proposal is the only tractable way to understand possible structure-function relationships, given the large size of the gene and encoded protein.
We will be investigating the viability of using cyanobacteria as a model for our own by exploring the evolutionary links as well as the similarities between human cells and cyanobacteria cells in terms of the communication and cell differentiation. This will allow us to use the cyanobacteria as a model for human stem cells. There are 3 cases which will be investigated: metabolism of retinoic acid, nitrogen-fixing cells and prostaglandin cell signalling. In each case, we will be blocking the signal, modifying the bacteria and studying how this affects the bacteria. The production of proteins and the chemical signalling are amongst the several responses we will be monitoring. Using information gained from this we will be able to see if there is a viable link that can be used to monitor cyanobacteria that have human orthologues spliced into it.
Obesity causes brain insulin resistance and prevents the brain from regulating metabolic responses, maintaining energy balance and controlling the nutritional status of an individual. Restoring the brain’s ability to modulate metabolic functions could be an important intervention to prevent the negative outcomes of obesity and diabetes. The Dorsal Vagal Complex (DVC) in the brainstem senses insulin to regulate glucose metabolism, food intake and body weight in rodents. Three days of high-fat diet feeding is sufficient to completely disrupt the insulin response in the DVC, thus causing an increase in blood glucose levels and excessive eating. Recently, I discovered that increased mitochondria fission and ER stress in the DVC can cause insulin resistance and affect the ability of the DVC to regulate blood glucose levels. I aim to understand whether increased mitochondria fission in the DVC can affect food intake and body weight in rats. Using in vivo and in vitro experiments, I aim to uncover the mechanism by which changes in mitochondria shape and size affect DVC insulin sensing and eating habits in rodents. This project could lead the way for the development of novel approaches that target the brain to regulate food intake and body weight in obese subjects.
The structure and mechanism of cytoplasmic dynein by X-ray crystallography and electron microscopy Dynein is a microtubule-associated motor protein involved in numerous fundamental processes in eukaryotic cells including the movement of cilia and flagella by axonemal dyneins and intracellular transport of organelles by cytoplasmic dynein. Dynein-driven mRNA translocation is critical for embryogenesis and during mitosis dynein powers spindle assembly, chromosome positioning and sister chromatid separation. In neurones, cytoplasmic dynein drives retrograde axonal transport and mutations are associated with neurodegenerative diseases. Yet despite its necessity for eukaryotic life, the structure and mechanism of dynein remain unknown. The aim of this project is to determine the structure of the cytoplasmic dynein motor. This will be achieved by the following specific objectives: 1) Expressing and purifying yeast dynein motor domain for cryo-electron microscopy and crystallographic studies. 2) Obtaining 3D cryo-EM structure of Dictyostelium discoideum dynein motor domain with a yeast-like truncation for comparison to (1). 3) Expressing and purifying AAA4-stalk-AAA5 fragment of a yeast motor domain for cryo-EM and crystallographic studies.
Towards an understanding of the mechanical stability and energy landscape of proteins: An approach based on Molecular Dynamics simulations. 22 May 2006
Towards an understanding of the mechanical stability and energy landscape of proteins: An approach based on Molecular Dynamics simulations The properties of proteins or their complexes are often characterised in terms of their thermodynamic stability and yet it is becoming clear that many biological processes are in fact mechanically mediated. Examples include protein degradation, the transport of proteins across membranes, and the roles of Titin and Filamin in muscle contraction and the cross-linking of cortical Actin networks respectively. In addition, experimental data is now emerging which suggests that certain protein mutations leading to disease are correlated to the mechanical stability of the proteins in question. For instance, certain developmental diseases have been linked to mutations in Filamin and are postulated to be due to compromised cell motility and/or membrane stability. Even where mechanical properties are not directly biologically relevant, such studies provide clean, single molecule information about the underlying energy landscape. Despite the extensive amount of work that has been performed in the last decade or so, a number of fundamental issues still remain unresolved. Thus, the over-riding aim of this project is to provide insight and clarification to some of the important issues and to make more effective use of single molecule pulling experiments where Leeds is an acknowledged world leader. Specifically, the objectives are the following: 1. To determine which atomistic models used in molecular dynamics simulations are the most adequate in reproducing mechanical unfolding experiments. 2. To elucidate the molecular determinants of mechanical strength in proteins; for example, the roles and relative importance of backbone hydrogen bonding, side-chains, and water. 3. To establish the relationship between mechanical properties and other biophysical parameters (e.g. native state fluctuations or thermal unfolding pathways). 4. To establish the relationship between the mechanical properties of proteins (with and without mechanical function) and sequence conservation data. Certainly, a better understanding of these issues will also contribute to understanding of the folding and function of proteins in general.
Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques. 22 May 2006
1. Insights into the molecular mechanisms and dynamics of translocation through SecYEG: an approach using ensemble and single molecule techniques Signal sequence-bearing secretory pre-proteins, and integral membrane proteins are translocated either through the inner membrane in bacteria or the endoplasmic reticulum in eukaryotic cells. The main secretory pathway in Escherichia coli involves the SecYEG translocon, which consists of three integral inner membrane proteins: SecY, which forms the protein channel, SecE that forms a clamp around the complex and SecG. A plethora of studies demonstrated that SecYEG undergoes large and complex sequential intramolecular conformational changes upon binding of partner proteins (e.g. SecA or ribosome) and translocation of secretory proteins. However, the nature of the open and closed states, the oligomeric organisation, the regulation of channel gating and the dynamic behaviour of the reaction remain poorly understood. The emergence of fluorescence techniques at the single molecule level has already improved our understanding of such dynamic processes. In-house developments in single molecule fluorescence spectroscopy instrumentation, combined with the expertise of the Radford group in protein folding and the Baldwin group in membrane proteins, place us in a strong position to unravel the SecYEG dynamics and conformational changes involved in protein translocation through this protein pore. The aims of the project are to: (a) Design structure-based FRET labelled mutants of SecYEG, focusing on key domains of the translocon (e.g. "plug domain", "hinge region", dimerisation interface and the two halves of SecYEG, and (b) characterise the structure/function relationship of these labelled SecYEG mutants using ensemble techniques. Measure intra- and intermolecular distances within and between SecYEG and the model substrate proOmpA during translocation using single molecule FRET (smFRET). Determine the extent to which restricting the movement of key domains by "locking" the protein in specific conformations can influence SecYEG function and derive a detailed mechanistic model for protein translocation.
The characterisation of male accessory gland products of anopheles gambiae andaedes aegypti. 23 Jan 2006
The characterisation of male accessory gland products of Anopheles gambiae and Aedes aegypti.
Role of the low-density lipoprotein receptor-related protein in prion protein metabolism. 27 Jun 2006
The prion protein (PrP) is the principal agent responsible for the transmissible spongiform encephalopathies, a group of neurodegenerative diseases including Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrPC) undergoes a conformational change to the protease-resistant, infectious form PrPSc. As the conversion of PrPC to PrPSc occurs at the cell surface or in an endocytic compartment, understanding the mechanisms and molecular partner s involved in the endocytosis of PrPC is central to both the normal cell biology of the protein and to disease pathogenesis. We have preliminary experimental data indicating that the GPI-anchored PrPC is piggy-backed onto the transmembrane low-density lipoprotein receptor-related protein (LRP1) in order to access the clathrin-coated internalisation pathway. We hypothesise that this interaction is critical for the normal cellular metabolism of PrPC and regulates its conversion to PrPSc. The ove rall aim of this proposal is to determine the role of LRP1 in PrP metabolism. The specific objectives are: (1) to characterise further the role of LRP1 in the endocytosis of PrPC; (2) to map the molecular interactions between LRP1 and PrPC; and (3) to determine the role of LRP1 in the conversion of PrPC to PrPSc.
Hepatitis C virus (HCV) is one of the most common causes of chronic liver disease. Progress in understanding viral replication has been hampered by the lack of a robust in vitro culture system. We propose to exploit recent developments in our laboratory to analyse the function of the HCV NS5A protein in viral RNA replication. NS5A is a phosphoprotein that is a key component of the viral RNA replication complex, but its precise role remained to be elucidated. Our studies will proceed in two interrelated areas: Firstly, we will use baculovirus vectors capable of delivering replication competent HCV genomes and sub-genomic replicons into hepatocytes, coupled with a mass spectrometry based approach, to unambiguously define the sites of phosphorylation. This will allow us to identify the kinases involved and use this information to assess the role of phosphorylation in RNA replication. Secondly, we have recently introduced a biotin acceptor domain into NS5A in the context of the subgenomic replicon - the resulting constructs are replication competent. We will exploit this advance to purify RNA replication complexes using avidin-affinity chromatography and use proteomic approaches to identify the cellular proteins associated with these complexes.
WATCH IT- a feasibility study of a community intervention to reduce morbidity in obese children. 01 Nov 2005
WATCH IT is a community programme for obese children that aims to influence behaviour in the long-term, and so impact on adult obesity, heart disease and diabetes. This proposal sets out a feasibility study for a multicentre randomised controlled trial of WATCH IT. This research will provide 'proof of concept' as well as answers to important methodological issues that include the feasibility of recruiting children; the acceptability of randomisation to a no-treatment arm; the acceptability of outcome measurements (particularly blood tests and a DEXA scan); the sustainability of participation over a year; an accurate sample size estimate based on the change in body fat. Children will be assigned to the WATCH IT programme or a control non-intervention group for a period of 12 months. The intervention is delivered through the NHS by health trainers in partnership with sports centres. It takes a motivational counselling, solution focused approach along with group physical activity sessions. A formal uncontrolled pilot of the intervention in Leeds shows that it has been successfully implemented in disadvantaged areas, that attendance levels are good and sustained, and that there has been some reduction in adiposity for most children. The clinical outcomes of interest include changes in adiposity, lifestyle, quality of life and metabolic risk.
Methodologies and resource generation enabling high-throughput, large-scale recombineering for high-fidelity gene expression analysis in C. elegans. 16 May 2007
Reporter fusions are used extensively to determine C. elegans gene expression patterns. The applicants have advanced this technology by establishing a straightforward recombineering technique for seamless reporter insertion into fosmid clones. This advance allows trivial manipulation of large genetic units, with minimal disruption of the genetic material such that reliable data can be generated. We plan to: further develop our recombineering technique to increase throughput by evolving i t to 96-well pates initially with manual processing and subsequently using robotics characterize expression patterns of C. elegans genes within operons to reveal the complexities of this significant mode of C. elegans gene organization examine the distribution and function of the alternative gene products for C. elegans forkhead transcription factor genes to unravel the intricacies of these key control genes determine the expression patterns of all C. elegans winged helix and bHL H transcription factor genes as an important contribution towards construction of a model of the regulatory network controlling expression of the C. elegans genome provide these reagents to the wider worm community In this way we will further develop our recombineering protocol to answer important questions concerning the co-ordination of C. elegans gene expression.
We seek funding for an integrated ultra fast Confocal/TIRF imaging station, a powerful microscopy system not currently available for our research. The quality of our spatial and kinetic data will be significantly enhanced by the system, enabling us to better visualise subcellular events, and thus ask new and more probing questions. Key goals will be to advance fundamental understanding of mechanisms of neurotransmitter release, ion channel regulation and interaction, and cell movement. Within this application we describe examples of on-going research projects which will benefit significantly from the new equipment, which offers considerable improvements in speed and dramatic insight into membrane events. A further key goal is to enhance our existing collaborative efforts and also improve expertise in imaging techniques through our Bio-imaging Core Facility. We also wish to enable novel research projects for additional users, largely based in our £5M newly-refurbished Integrative Membrane Biology laboratories, completed in September 2005.
Alzheimer's disease (AD) is the most common cause of dementia, it affects over 24.3 million people worldwide with 4.6 million new cases diagnosed each year . Since the number of people suffering with AD is expected to double every 20 years , AD represents one of the most pressing health concerns in an aging population. There are currently no effective treatments for AD which makes insight into the mechanisms behind the disease of considerable interest. Accumulation of the small (39-43 residue) peptide amyloid beta (A~) causes AD. Though the source of the peptide, the amyloid precursor protein (APP), and the enzymes responsible for its cleavage are known, the reason for its production and particularly its accumulation in an age-dependent manner remains much of an enigma. Crucial to being able to solve this problem is knowing what effect interactions between the key players in AD and other proteins have on the disease. This project aims to identify and characterise how protein interactions with APP and the ?-secretase BACE1 (which cleaves APP to generate A?) affect the production of A?. This will involve the investigation of potential interactions with the leucine rich repeat transmembrane 3 (LRRTM3) protein, F-Spondin and apolipoprotein E receptor 2 (ApoEr2). Specific objectives What is the role of LRRTM3 in the regulation of BACE1? Does LRRTM3 interact with BACE1 to alter BACE1 activity? Does LRRTM3 alter the cellular trafficking of BACE 1? Does the expression of LRRTM3 alter in AD and/or in aging? What role do ApoEr2 and F-Spondin play in the regulation of APP processing? What effect does the interaction of APP with F-Spondin and full length human ApoEr2 have on the production of AJ3? What are the molecular and cellular mechanisms underlying these effects? What is the effect of receptor activation on the ApoEr2, F-Spondin, APP complex and its ability to alter APP processing? Can any of these interactions be exploited as potential therapeutic strategies for AD? Can small molecules be used to interfere.with (or mimic) potential interaction sites on either APP or BACE1?
How proteins aggregate into amyloid fibrils is a key question in structural biology. Although the amino acid sequence dictates the structure of native proteins, it is now clearthat polypeptide chains can adopt an alternative conformation based upon the cross-beta structure of amyloid. Despite enormous interest in this field, the structure of amyloid fibrils, particularly those from biomedically-relevant proteins, remains unknown. Here we propose to determine the 3D structure and subunit arrangement of amyloid fibrils formed from the 99-residue protein,beta-2- microglobulin (â2m), combining cryo-electron microscopy (Saibil, Birkbeck) andprotein chemical analysis (Radford, Leeds). Building on remarkable 3D density maps recently obtained of â2m fibrils formed in vitro, we propose to determine structural restraints using cross-linking and fluorescence experiments to enable model building, and to further test and refine these models using mutagenesis. In parallel, we will improve the cryo-EM maps by collectinglarger data sets, allowing finer subdivision of the image data into more uniform subsets, and will use site-specific nanogold labelling to guide docking of the chain into the electron density maps. The overall aim is to answer the fundamental question of how the same polypeptide chain can fold into two structures: a functional monomer and an aggregated polymer.
Macrophages are able to fuse and differentiate into either multinucleate giant cells (MGCs) at sites of chronic inflammation or osteoclasts within the bone. However, the molecular mechanisms that control this developmental process remain elusive. We utilize a tractable cellular system for analyzing the gene regulatory networks that underlie macrophage differentiation. By genome wide expression analysis we identify the Hic-5 nuclear co-activator as transiently induced during macrophage developm ent. Targeting of Hic-5 by shRNA diverted macrophage precursors to differentiate along the MGC cell fate. Ectopic expression of Hic-5 within bone marrow derived macrophages resulted in the loss of osteoclast development. By these loss and gain of function approaches we demonstrate Hic-5 as a novel cell fate determinant in repressing the MGCs and osteoclasts cell fates during macrophage development. As Hic-5 binds to DNA indirectly, through protein-protein interactions, we propose to use ChIP-S eq in conjunction with bio-informatics to identify this protein partner. Secondly, we shall establish the requirement of Hic-5 for the development of macrophages by the generation of mice containing a conditionally floxed Hic-5 allele. The accomplishment of these research aims will be of fundamental importance in the greater understanding in the pathology of chronic inflammation and bone disorders.
You are what you ate: food lessons from the past 07 May 2010
How did food affect our ancestors? How can we learn from the past to improve our health? This project encourages discussion of modern nutrition in the Yorkshire region by presenting archaeological, visual and textual evidence from the medieval and early-modern periods (12th-17th centuries) to initiate debate and reflection on eating behaviours. Through innovative schools and youth activities, exhibitions, festival attendance, cooking demonstrations, and bone workshops this project explores the concept of a balanced diet in history, encouraging participants to engage with issues that affect their health in the 21st century: obesity, alcohol consumption, dental care, nutritional disorders, growth, famine, the impact of food processing and preservation techniques on diet, the significance of climate change and eating in season, the cost of food, the influence of social status, feasting and fasting, the appearance of food and the concept of taste. The project brings biomedical science, bioarchaeology and medical history to a new audience, working with schools, festivals and museums within the region of Yorkshire and engaging with as much of the local community as possible, especially children, ?hard-to-reach? youth groups and members of different religions and cultures. It encourages discussion of the global context of eating (learning about foods from the New World and past European famines widens awareness of current crises). The project pioneers a new model of public engagement, and will lead to further partnerships between the Universities of Leeds and Bradford and local communities through links with Wakefield Council.
This proposal is for the development of Switchable Nanostructured Surfaces. These are molecularly well-defined surfaces, which in a highly controlled way can dynamically present biological regulatory signals and stimuli to a cell with nanometre localised resolution. We will exploit principles of self-assembly, molecular shape change and nanofabrication to engineer the tunable biological nanoscale features on macroscopic surface materials. In order to achieve its aim, this project has three centr al objectives: Objective 1 - Development of stimulus-responsive molecular systems for real-time and reversible control of biomolecule activity on large scale surfaces. Objective 2 - Development of a nanopatterning methodology that will allow immobilisation of multiple biologically differing stimuli systems at well-defined and nanoscale locations with a high degree of selectivity. Objective 3 - Test and exploit smart switchable biological nanostructured surfaces to investigate stim ulus-activated calcium concentration [Ca2+]i signalling and cell responses in sperm and how these relate to cell quality .