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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.

Research Staff

PhD Students

Projects

Impact of regular physical activity on the age-related changes in contractile properties of single muscle fibres

Impact of regular physical activity on the age-related changes in contractile properties of single muscle fibres

Rationale

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.

Aims/Objectives

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.

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Contributing IRM PhD students (present and past)

  • Dr. Hans Degens
  • Dr. Jamie McPhee
  • Jamie Metcalfe

Funding Source

  • HEFCE

The Nanotoxicological Influence of Nanoparticles on Vascular Contractility and Function

The Nanotoxicological Influence of Nanoparticles on Vascular Contractility and Function

Rationale

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.

Aims/Objectives

  • To investigate the direct influence of nanoparticle (Silica, gold, quantum dot) uptake on the vascular function of conduit arteries and the microvasculature, in vitro, and in vivo.
  • To determine the influence of nanoparticles of defined size, charge, shape and material composition (fabricated and fully characterised) on cellular and vascular function, using state of the art apparatus.
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Contributing IRM staff (including PhD Students)

  • May Azzawi
  • Teba Mohamed
  • Ali Shukur
  • Asima Farooq

Collaborators

  • Dr. Debra Whitehead (DRI)
  • Professor Seifalian (University College London, UK)
  • Ali Shukur, BSc, MSc (Current PhD student)
  • Asima Farooq, BSc (Current PhD student, DRI)

Funding Source

EPSRC-funded Bridging the Gaps: Nano-Info-Bio project, grant reference EP/H000291/1.

Molecular Regulators of Skeletal Muscle Degeneration and Regeneration

Molecular Regulators of Skeletal Muscle Degeneration and Regeneration

Summary of project

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.

Rationale

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.

Aims/Objectives

My general aim is to intimately improve muscle mass and functions in skeleton-muscular diseases and ageing. In our laboratories, we investigate:

  • the potential cross-talk between IGF-I/TNF and IGF-I/IL-6 and their effects on cell proliferation and differentiation using murine C2 and C2C12 myoblast cell line,
  • Interactions between skeletal muscle and the immune system during muscle regeneration and degeneration,
  • molecular mechanism underlying skeletal muscle migration and fusion.

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.

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Contributing IRM staff

  • Prof. Claire Stewart
  • Dr. Nasser Al-Shanti
  • Dr Adam Sharples (Uni of Bedford),
  • Dr. Amarjit Saini (Loughborough University)
  • Dr. Hans Degens
  • Dr. Jamie McPhee
  • Dr. Rob Erskine
  • Professor Marco Narici (University of Nottingham)

Contributing IRM PhD students (present and past)

  • Mr. Georgi Dimchev
  • Mr. Peter Durcan
  • Mrs Aisha Elbouzidi
  • Mr Ali Shukur
  • Mrs Mayada Alqaisi
  • Mr Adel Hamdi

Collaborators

Role of the cyclin-kinase Cdk5 in microvascular cellular adaptation during acute ischemic stroke

Role of the cyclin-kinase Cdk5 in microvascular cellular adaptation during acute ischemic stroke

Rationale

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).

Aims/Objectives

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).

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Contributing IRM staff (including PhD Students)

  • Dr. Alessandra Bosutti
  • Prof. Mark Slevin
  • Dr. Jie Qi
  • Kamela Ali

Collaborators

Funding Source

  • Wellcome Trust to Prof. Mark Slevin, director of the study and group co-ordinator for vascular research.

Genetic control of cell cycle arrest induced by methylglyoxal in Saccharomyces cerevisiae

Genetic control of cell cycle arrest induced by methylglyoxal in Saccharomyces cerevisiae

Summary of project

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.

Contributing IRM staff

  • Syed F.H. Naqvi
  • Nessar Ahmed
  • Mikhajlo K. Zubko

Publications

Impact of regular physical activity on the age-related changes in contractile properties of single muscle fibres

Associated Publications

  1. Larsson L, Li X, Yu F & Degens H. INVITED MINIREVIEW. Age-related changes in contractile properties and expression of myosin isoforms in single skeletal muscle cells. Muscle & Nerve, S5, S74-S78, 1997.
  2. Degens H, Yu F, Li X & Larsson L. Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres. Acta Physiol. Scand., 163, 33-40, 1998.
  3. Degens H, Alway SE. Control of muscle size during disuse, disease and aging. Int. J. Sports Med. 27, 94-99, 2006
  4. Degens H, Larsson L. Application of skinned single muscle fibres to determine myofilament function in ageing and disease. J Musculoskel Neuron Int 7, 56-61, 2007.
  5. Degens H. Age-related skeletal muscle dysfunction; causes and mechanisms. J Musculoskel Neuron Int 7, 246-252, 2007.
  6. Degens H. The role of systemic inflammation in age-related muscle weakness and wasting. Scand J Med Sci Sports 20, 28-38, 2010
  7. Gilliver SF, Jones DA, Rittweger J, Degens H. Effects of oxidation on the power of chemically skinned rat soleus fibres. J Musculoskel Neuron Int, 10, 267-273, 2010.
  8. Arampatzis A, Degens H, Baltzopoulos V, Rittweger J. Why do older sprinters reach the finish line later? Exerc Sport Sci Rev. 39, 18-22, 2011.
  9. Gilliver SF, Jones DA, Rittweger J, Degens H. Variation in the determinants of power of chemically skinned type I rat soleus muscle fibres. J Comp Physiol A, 197, 311-319, 2011
  10. Ireland A, Korhonen M, Heinonen A, Suominen H, Baur C, Stevens S, Degens H, Rittweger J. Side-to- Side Differences in Bone Strength in Master Jumpers and Sprinters. J Musculoskel Neuron Int, 11, 298-305, 2011.

The Nanotoxicological Influence of Nanoparticles on Vascular Contractility and Function

Associated Publications

  1. Naveed AKBAR, Teba MOHAMED, Debra WHITEHEAD, May AZZAWI. Biocompatibility of amorphous silica nanoparticles: size and charge effect on vascular function, in vitro. Biotechnology and Applied Biochemistry 2011;58:353-362. Doi:10.1002/bab.46
  2. Azzawi M. The nanotoxicological influence of nanoparticles,with special reference to the vasculature. In:Current advances in the medical application of nanotechnology, Ed.: M Slevin. Bentham Publications.

Molecular Regulators of Skeletal Muscle Degeneration and Regeneration

Associated Publications (last 4 years)

  1. Sirt1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol. Saini A, Al-Shanti N, Sharples A, and Stewart C. Experimental Physiology; 2011; in press
  2. Inhibitory effects of IL-6 on IGF-1 activity in skeletal myoblasts could be mediated by the activation of SOCS-3. Al-Shanti N, Stewart CE. Journal of Cellular Biochemistry; 2011; Oct 7:10.1002/jcb.23420in.
  3. Reduction of myoblast differentiation following multiple population doublings in mouse C(2) C(12) cells: A model to investigate ageing? Sharples AP, Al-Shanti N, Lewis MP, Stewart CE. J Cell Biochem. 2011 Aug.
  4. A semi-automated programme for tracking myoblast migration following mechanical damage: Manipulation by chemical inhibitors. Al-Shanti N, Faulkner, SH, Saini, A, Loram, I & Stewart, CE. Cell Physiol Biochem. 2011;27(6):625-36.
  5. C2 skeletal myoblast survival, death, proliferation and differentiation: regulation by Adra1d. Saini A, Al-Shanti N, Stewart C. Cell Physiol Biochem. 2010; 25(2-3):253-62.
  6. C2 and C2C12 murine skeletal myoblast models of atrophic and hypertrophic potential: relevance to disease and ageing? Sharples AP, Al-Shanti N, Stewart CE. J Cell Physiol. 2010 Oct;225(1):240-50.
  7. Ca2+/calmodulin-dependent transcriptional pathways: potential mediators of skeletal muscle growth and development. Al-Shanti N, Stewart CE. Biol Rev Camb Philos Soc. 2009 Nov;84(4):637-52.
  8. Powerful signals for weak muscles. Saini A, Faulkner S, Al-Shanti N, Stewart C. Ageing Res Rev. 2009 Oct;8(4):251-67.
  9. Two-Step versus One-Step RNA-to-CT 2-Step and One-Step RNA-to-CT 1-Step: validity, sensitivity, and efficiency. Al-Shanti N, Saini A, Stewart CE. J Biomol Tech. 2009 Jul;20(3):172-9.
  10. Pro- and anti-apoptotic roles for IGF-I in TNF-alpha-induced apoptosis: a MAP kinase mediated mechanism. Saini A, Al-Shanti N, Faulkner SH, Stewart CE. Growth Factors. 2008 Oct;26(5):239-.
  11. PD98059 enhances C2 myoblast differentiation through p38 MAPK activation: a novel role for PD98059. Al-Shanti N, Stewart CE. J Endocrinol. 2008 Jul;198(1):243-52.
  12. Beneficial synergistic interactions of TNF-alpha and IL-6 in C2 skeletal myoblasts--potential cross-talk with IGF system. Al-Shanti N, Saini A, Faulkner SH, Stewart CE. Growth Factors. 2008 ;26(2):61-73.

Role of the cyclin-kinase Cdk5 in microvascular cellular adaptation during acute ischemic stroke

Associated Publications

  1. Bosutti A, Ali K, Bolton D, Qi J, Pennucci R, Matou-Nasri S, Love S, Tsai L-H, Giese KP, Degens H, Kumar S, Krupinski, Slevin M. Modulation of p35/cyclin-dependent kinase 5 signaling rectifies defective human brain endothelial cell migration produced by in vitro simulation of ischemic stroke. Abstract in: Stroke 2012 (American Stroke Association-ASA 2012).
  2. Bosutti A, Matou-Nasri S, Qi J, Pennucci R, Tsai L-H, Giese KP, Love S, Krupinski J, Kumar S, Slevin M. Over-expression of cyclin-dependent kinase 5 stimulates cell migration but not cell differentiation in human brain microvascular endothelial cells. Abstract in: Stroke 2011 (American Stroke Association-ASA 2011).
  3. Bosutti A, Pennucci R, Matou-Nasri S, Gaffney J, Love S, Tsai L-H, Giese PK, Kumar S, Krupinski J, Slevin M . Alterations in cyclin-dependent kinase 5 activator p35 signaling are associated with angiogenesis, in brain endothelial cells. Abstract in: Stroke 2010 (American Stroke Association-ASA 2010).
  4. Bosutti A, Pennucci R, Krupinski J, Kumar S, Kumar P, Slevin M. Involvement of the Cyclin-dependent kinase 5 in microvascular endothelial cell migration in vitro. Abstract in: Stroke 2009 (American Stroke Association-ASA 2009).
  5. Krupinski J, Kaluza J, Kumar P, Kumar S, Wang JM. Role of angiogenesis in patients with cerebral ischaemic stroke. Stroke 1994
  6. Mitsios N, Pennucci R, Krupinski J, Sanfeliu C, Gaffney J, Kumar P, Kumar S, Juan-Babot O, Slevin M. Expression of CDK-5 mRNA and protein in the human brain following acute ischaemic stroke. Brain Pathol. 2007
  7. Mitsios N, Gaffney J, Krupinski J, Mathias R, Wang Q, Hayward S, Rubio F, Kumar P, Kumar S, Slevin M. Expression of signalling molecules associated with apoptosis in human ischaemic stroke tissue.Cell Biochem Biophys.2007
  8. Mitsios N, Saka M, Krupinski J, Pennucci R, Sanfeliu C, Wang Q, Rubio F, Gaffney J, Kumar P, Kumar S, Sullivan M, Slevin M. A microarray study of gene and protein regulation in human and rat brain following MCAO.BMC Neurosci 2007
  9. Slevin M, Krupinski J. Cyclin-dependent kinase-5 targeting for ischaemic stroke. Curr Opin Pharmacol. 2009
  10. Slevin M, Krupinski J, Kumar P, Gaffney j, Kumar S. Gene activation and protein expression following ischaemic stroke: strategies towards neuroprotection. J cell Mol Med. 2005

Group Photos