Biomedical, Cellular and Molecular Biology
Much of the work in this research group, which extends from yeast through rodent to human cells and tissues, is concerned with understanding exercise/hormonal/immune/nutritional interactions at the cell and tissue level and their regulation of fundamental intracellular biochemical and molecular pathways in health and disease. Budding yeast presents the simplest eukaryotic model that is used to study different aspects of the cell cycle, genetic control of DNA damage, response to stress factors, senescence and ageing of eukaryotic cells. These studies complement in vitro analyses using human or rodent cell/tissue models investigating inappropriate cell cycle progression (hyperplastic growth and hypertrophy) or apoptosis (atrophy), ageing, impaired wound healing, inflammation and disease.
Under investigation are the roles of advanced glycation endproduct (AGE) and free radical-induced damage to proteins, growth factors, nucleic acids and cells and the complementary protective effects of anti-glycation products or antioxidants on the pathogenesis of e.g. diabetic complications. Of particular interest are the roles of natural products with combined anti-glycation and antioxidant properties.
Complementing these studies, questions are being addressed in order that the mechanisms underpinning hypertrophy and atrophy during disease processes and across the lifespan are understood. There is a common interest in factors that regulate tissue growth, function, ageing and repair, of: muscle fibres responding to changing workloads; muscle, fat and peripheral blood/mesenchymal stem cells responding to changes in their hormonal and cytokine milieu (relevant to childhood obesity); skin in the context of chronic leg ulcers and percutaneous wound repair; oncogenes activated in muscle tumours (Rhabdomyosarcomas) and the modulation of vascular responses.
To this end, major advances have been made in the isolation and characterisation of fat and muscle stem cells from children and adults as well as the mobilisation and characterisation of peripheral blood stem cells following damage, exercise or nutritional interventions. These developments have facilitated studies investigating the interactions of omega-3 fatty acids, amino acids, growth factors, hormones and cytokines in regulating stem cell mobilisation, proliferation and differentiation. The application of nano-technological developments in vascular biology and wound healing applications also enable the study of nanotoxicity, endothelial and smooth muscle cell responses, as well as vessel contractility and inflammatory cell function in health and disease. The development of muscle-targeted somatic gene therapy and custom microarrays has facilitated the manipulation and the study of the molecular regulators of striated muscle in vivo. Haematological investigations during pregnancy informed policy on folate supplementation aimed at preventing developmental complications and studies on haematopoietic cells demonstrated a role for Annexin II in the treatment of acute leukaemias.
The researchers in this line are therefore applying several current technologies including: human and rodent cell and tissue culture, transfection, RT-PCR, PCR arrays, SDS-PAGE & Western blotting, ELISA, CBA-based flow cytometry, proteomics, metabolomics real time live cell imaging with applied tracking programmes and confocal/fluorescence microscopy to enable the implementation of systems-based approaches for addressing questions concerned with the regulation of gene expression in relation to nutritional, hormonal, metabolic, pharmacological and physical stimuli which are relevant to exercise, ageing, nutrition, obesity, chronic and acute diseases, wound healing and rehabilitation.
Ageing is associated with muscle weakness, which is not only attributable to a loss of muscle mass, but also to changes in the contractile properties of the remaining muscle tissue. This change in contractile properties is to a large extent attributable to changes in the contractile properties of individual muscle fibres. It is known that disuse leads to similar changes in muscle contractile properties as occurs during ageing and it is thus thought that the muscle weakness at old age is largely attributable to disuses.
Therefore, we hypothesise that the age-related muscle weakness and changes in contractile properties is prevented, or at least attenuated, in people that maintain high activity levels. To investigate this we will explore the contractile properties of single fibres obtained from master athletes.
In the last decade, there has been a dramatic increase in the synthesis of engineered nanomaterials for a wide variety of applications. Despite the clear advantages of these applications, especially in medical intervention and therapeutics, the influence of nanoparticle exposure on cellular and organ function remains poorly understood. The nanoscale size of nanoparticles (<100 nm) allows their penetration into cells and, due to their large surface area per unit mass, they are more reactive than larger scale particles. This has led government and scientific organisations to call for a need to assess the safety of engineered nanomaterials and determine the mechanism of their interaction with cells and tissues, including the vasculature, before their use in nanotoxicity studies. Our research focuses on the influence of nanoparticles, of different material composition, size and charge, on the function and contractility of conduit arteries and the microvasculaure, both in vitro and in vivo. Findings will help define and optimise the use of nanoparticles in healthcare, especially in imaging diagnostics and therapeutics.
EPSRC-funded Bridging the Gaps: Nano-Info-Bio project, grant reference EP/H000291/1.
Skeletal muscle is an essential peripheral metabolically active tissue that not only comprising ~ 40-50% of the body mass and but also playing a central role in maintaining metabolic body health. Muscles maintain their mass and function though the balance between protein synthesis (hypertrophy) and protein degradation (sarcopenia and cachexia) associated with rates of anabolic and catabolic processes, respectively.
As a consequence, my research focuses on different intracellular molecular pathways that regulate the balance between the anabolic (IGF-I positively impacts on muscle anabolism) and catabolic signals (TNF-a system act to trigger catabolism). Accordingly, a better understanding of the cross-talk among multiple signalling pathways leading to muscle regeneration and wasting may gain new insight into highly attractive, when considering not only muscle wasting and its associated co-morbidities but also muscle regeneration and repair.
My general aim is to intimately improve muscle mass and functions in skeleton-muscular diseases and ageing. In our laboratories, we investigate:
To enable us to examine our hypothesis, several technologies are applied including: human and rodent cell and tissue culture, transfection and cloning, RT-PCR, PCR arrays, SDS-PAGE & Western blotting, ELISA, Flow cytometry, CBA-based Flow cytometry, proteomics, metabolomics real time live cell imaging with applied tracking programmes and confocal/fluorescence microscopy.
Stroke is one of the major causes of death and disability in developed countries. Hypoxic stress and the abnormal activation of multiple neuronal and blood vessel cell death pathways characterize the acute event, whereas vascular remodelling, angiogenesis and neurogenesis are vitally involved in the regenerative response (5-8). The molecular mechanisms governing tissue recovery after ischemic injury are still unclear. Modulation of cell cytoskeletal organization and the control of cell cycle- kinase activities are key requisites for cellular adaptation during angiogenesis and neurogenesis. In particular, growing observations underline the crucial role of the cyclin-dependent kinase Cdk5 in neuronal migration, neuron cytoskeletal organization and neuron survival, suggesting a possible implication of this
kinase also in microvascular cellular adaptation during angiogenesis (6,9,10).Recently, we have demonstrated the deregulation of Cdk5 and its non-cyclin activator p35 in surviving microvessels from human brain peri-infarct regions, following acute ischemic stroke (6). Our current investigations demonstrate that the inhibition of Cdk5 activity with the kinase inhibitor r-roscovitine impairs angiogenesis and cell cytoskeletal structure in human brain microvascular endothelial cells (3-4), and the modulation of p35/Cdk5 pathway can rectify defective endothelial cell migration during in vitro acute hypoxia (1,2).
The role of p35/Cdk5 signalling in revascularization following brain ischemia is still undefined. Our project is directed to investigate the impact of p35/Cdk5 pathway in the regulation of several aspects of endothelial signalling in response to hypoxic injury. Our study on cell dynamic during angiogenesis is combined with the use of Cell-IQ® Continuous Live Cell Imaging & Analysis System (Chip-Man Technologies Ltd), confocal microscopy and in vitro simulation of ischemic stroke (1-4).
Methylglyoxal (MG) is an intrinsic toxic by-product of glycolysis that cases damages to cellular macromolecules resulting in disadvantageous conditions and pathologies including diabetes. MG causes cytotoxic and cytostatic effects at different stages of mitosis. However, mechanisms and genetic pathways of cellular responses to MG exposure remain obscure. Using Saccharomyces cerevisiae as a model we have showed that in this species MG induces temperature-dependent G1/S cell cycle arrest. Cells arrested at low temperatures (18oC-23oC) recover effectively at enhanced temperatures (26oC and above) or after long exposure at low temperature. The process of the cellular arrest is under control of a checkpoint gene, RAD24, and EXO1 gene encoding the exonuclease 1. Cells of deletion mutants lacking these genes escape from the arrest. Two other checkpoint genes, RAD9 and RAD17, which are normally involved into DNA damage checkpoint control, are not responsible for the MG-induced arrest, but they play roles in the recovery of cells from the arrest. We have established that the recovery of cells from the MG-induced arrest is controlled by gene RAD52, one of the main genes functioning in repair of DNA breakes. Interaction of pUC18 DNA with MG in vitro assays resulted in the conversion of supercoiled form of the plasmid into relaxed form suggesting that MG indeed produces DNA breaks. In addition, chronic exposures to MG uncovered telomere shortening in MG treated cells that reveals another detrimental aspect contributing to genomic instability caused by MG. We suggest that further studies of this novel checkpoint pathway towards the fine control of MG associated cellular responses will facilitate understanding ways of curing and preventing conditions caused by excessive accumulation of MG.