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BMS 01 Project Title: Characterisation of a novel mechanism for modulating the activity of pain-sensing nerves Primary Supervisor: Dr. Charles Kennedy Email: [email protected] Secondary Supervisor: Dr. Trevor Bushell Email: [email protected] Project Area: (one or more of the following) Pharmacology Project Description: Overview: At present the therapeutic options for treating chronic pain are limited, but new targets have recently been identified, including P2Y receptors and 2-pore potassium ion (K2P) channels. Both are expressed in the pain-sensing C and A� sensory nerve fibres, which have their cell bodies in the dorsal root ganglia (DRG) and so are potentially important targets for controlling the activity of pain-sensing nerves. Our MRes projects in previous years in this area were highly successful and produced sufficient novel data for a research paper. Now we aim to continue these studies and further develop our understanding of the interaction between P2Y receptors and K2P channels. Background: P2Y receptors are a family of G protein-coupled receptors that are activated by nucleotides, such as adenosine 5'-triphosphate (ATP) (Abbrachio et al., 2006). K2P channels are highly selective for K+ ions and make a substantial contribution to the neuronal resting membrane potential in many regions of the CNS (Kim, 2005). We have shown that native, ATP-sensitive P2Y receptors inhibit the K2P background current normally present in rat cerebellar granule neurones and that recombinant human P2Y1 and P2Y12 receptors inhibit currents carried by K2P channels co-expressed in a cell line (Parmar et al., 2008). Both P2Y receptors (Kennedy, 2008) and K2P channels (Alloui et al., 2006) are expressed in sensory neurones. Aim: The aim of this project is to characterise how P2Y receptors and K2P channels interact to modulate cellular activity. The student will use the patch clamp technique to record ion currents carried by K2P channels and characterise their modulation by P2Y receptor activation. This will be supported by the use of recombinant channels and receptors. They will also learn tissue-culture and will use immunohistochemical techniques to study native protein expression. Together, these experiments will help advance the search for new, effective analgesics. Techniques to be used: Patch-clamp electrophysiology, molecular biology (expression of recombinant receptors & ion channels), tissue-culture References: 1. Abbracchio, MP et al., (2006). Update of the P2Y G protein-coupled nucleotide receptors: from
molecular mechanisms and pathophysiology to therapy. Pharmacol. Rev., 58, 281-341. 2. Kennedy, C. (2008). P2X3 receptors and sensory transduction. In: Sensing with Ion Channels.
ed. Martinac, B. (Springer, Heidelberg), 247-266. 3. Kim, D. (2005). Physiology & pharmacology of two-pore domain potassium channels. Curr Pharm
Des. 11, 2717-36. 4. Alloui A et al., (2006). TREK-1, a K+ channel involved in polymodal pain perception. EMBO J., 25,
2368-76. 5. Shrestha, S.S., Parmar, M., Kennedy, C. and Bushell, T. (2009). 2-pore potassium ion channels
are inhibited by both Gq/11- and Gi-coupled P2Y receptors. (submitted for publication).
BMS 03 Neuroprotection against oxidative and carbonyl stress Primary Supervisor:Elizabeth Ellis Email:[email protected] Secondary Supervisor: Eve Lutz Email:[email protected] Project Area: Biochemistry Molecular Biology Pharmacology Project Description: Oxidative stress has been implicated in a range of neurodegenerative diseases, as well as ischaemia-reperfusion type injury such as stroke. Neuronal cells have limited intrinsic ability to protect themselves against oxidants, and this may lead to cellular damage to lipids, proteins and DNA. Oxidation of lipids leads to the production of lipid peroxidation products, many of which are highly toxic aldehydes (1). Previously we have shown that the human neuroblastoma cell line SH-SY5Y cell line is extremely sensitive to carbonyl stress (2). We have also shown that treatment of cells with certain phytochemicals can lead to the induction of protective enzymes and that this is sufficient to cause increased protection against reactive aldehydes and oxidants (2). The SH-SY5Y cell line is neuronal-like and can be induced to differentiate (3,4). What is not known is to what extent differentiated cells have increased levels of protective enzymes and whether or not this can protect them against reactive carbonyls. In this study, SH-SY5Y cells will be differentiated, and the effect on protective enzyme expression measured using Western blots and RT-PCR. The effect on cell survival will be monitored using MTT assays. Techniques to be used: Mammalian cell culture Cytotoxicity assays Gene Expression studies References: 1. Ellis, E.M. 2007 Pharmacol Ther. 115:13-24. 2. Biedler et al., 1978. Cancer Res. 38:3751-7. 3. Monaghan T et al (2008) J. Neurochem. 104, 74-88
BMS 04 Project Title: How old are your organs? Primary Supervisor: Professor David Flint Email:[email protected] Secondary Supervisor: Dr Gordon Allan E-mail: g.j.allan @strath.ac.uk Project Area: (one or more of the following) Cell Biology/Pharmacology Project Description: What links binge drinking and growing old? Well, alcohol may actually reduce the chances of a long life but in fact the biological events that develop from excess alcohol intake and the natural ageing process have much in common. This is because liver damage from alcohol toxicity induces a form of premature ageing in the liver that leads to eventual collapse of liver function. However, this ageing process occurs in many organs of the body, as we age. For example, reduced function of the lungs, kidney, skin and cardiovascular system become evident in middle age (40-50years) and appear to be due to cycles of repetitive micro-injury and repair. The repair mechanism involve cell proliferation and eventually these cells can no longer replicate due to senescence. This project aims to identify the role of 2 major growth factors involved in the response to injury that occurs during repetitive insults to our organs and to use this knowledge to devise therapeutic strategies and identify novel drugs to treat these various diseases. Our laboratory has model systems in the lung, liver, skin and cardiovascular system and students undertaking this project would have the opportunity to choose the target system which most interests them. You will receive training in tissue culture, including cell proliferation, death and migration, in immunocytochemistry and drug screening. Techniques to be used: cell culture, immunocytochemistry, western blotting References: 1. MOHAN et al (2002) IGF-binding proteins are multifunctional and act via IGF-dependent and –independent mechanisms. Journal of Endocrinology 175: 19-31. 2.ALLAN GJ, BEATTIE J, FLINT DJ (2004). The role of IGFBP-5 in mammary gland development and involution. Domestic Animal Endocrinology 27: 257-266. 3.FLINT DJ, BOUTINAUD M, TONNER E, WILDE CJ, HURLEY W, ACCORSI PA, KOLB AF, WHITELAW CB, BEATTIE J, ALLAN GJ. (2005) Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland. Domest Anim Endocrinol. 29:274-82.
BMS 05 Project Title: Examining the effects of IGFBP-5 in epithelial-mesenchymal transition Primary Supervisor: Professor David Flint Email:[email protected] Secondary Supervisor: Dr Gordon Allan E-mail: g.j.allan @strath.ac.uk Project Area: (one or more of the following) Biochemistry/Pharmacology Epithelial cells serve a variety of functions, one of which is to act as a barrier to infection and external insults. Injury, including death of epithelial cells, provokes a wound-healing response which results in migration of cells into the wound site in an attempt to seal the injured tissue. This process also activates the underlying cells (principally fibroblasts) to secrete collagen and fibronectin, proteins which help to plug the wound. If this is not controlled, excessive deposition of these proteins can lead to scarring (fibrosis) and can lead to death in, for example, lung fibrosis or cirrhosis of the liver. It is now apparent that chronic damage to a variety of epithelial cells leads to fibrotic disease but that this is the result of a relatively minor but repetitive injury which does not activate inflammation to any great extent. Instead it appears that a factor(s) produced by injured epithelial cells stimulates the underlying collagen-producing cells, resulting in fibrosis, which progressively impairs the function of major organs such as the lungs, kidneys and liver. This repetitive stimulus to cell proliferation may also be a trigger for premature cell aging (senescence) which has recently been linked to fibrosis. Our focus will centre on a protein, IGFBP-5, secreted by the epithelium, which plays a significant role in cell death and has been proposed to induce fibrosis in the adult and is also implicated in the process of cell aging. This mechanism is consistent with the late-onset of fibrotic diseases, which typically occur from middle-age onwards. We will also examine the effect of epithelial damage on the response of the underlying tissue, which contains the collagen-secreting fibroblasts. Responses will include changes in cell proliferation, migration and aging, as well as expression of various genes and proteins implicated in these events. The use of confocal microscopy will allow us to visualise these changes in living cells in complex reconstructions of the tissues under study. This proposal thus seeks to provide evidence that IGFBP-5 is a pivotal initial response to epithelial injury and that, by understanding this process, we will be able to develop novel approaches to deal with the consequences when this repair mechanism malfunctions in processes such as fibrosis and cell aging. Techniques to be used: cell culture, immunocytochemistry, PCR, Western blotting References: Yasuoka H, Jukic DM, Zhou Z, Choi AM & Feghali-Bostwick CA 2006a Insulin-like growth factor binding protein 5 induces skin fibrosis: A novel murine model for dermal fibrosis. Arthritis Rheum 54 3001-3010. Kim KS, Seu YB, Baek SH, Kim MJ, Kim KJ, Kim JH & Kim JR 2007 Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism. Mol Biol Cell 18 4543-4552.
BMS 06 Project Title: Determination of the effects of IGFBP-5 on fibroblast activation in fibrotic disease Primary Supervisor: Professor David Flint Email:[email protected] Secondary Supervisor: Dr Gordon Allan E-mail: g.j.allan @strath.ac.uk Project Area: (one or more of the following) Biochemistry/Pharmacology Epithelial cells serve a variety of functions, one of which is to act as a barrier to infection and external insults. Injury, including death of epithelial cells, provokes a wound-healing response which results in migration of cells into the wound site in an attempt to seal the injured tissue. This process also activates the underlying cells (principally fibroblasts) to secrete collagen and fibronectin, proteins which help to plug the wound. If this is not controlled, excessive deposition of these proteins can lead to scarring (fibrosis) and can lead to death in, for example, lung fibrosis or cirrhosis of the liver. It is now apparent that chronic damage to a variety of epithelial cells leads to fibrotic disease but that this is the result of a relatively minor but repetitive injury which does not activate inflammation to any great extent. Instead it appears that a factor(s) produced by injured epithelial cells stimulates the underlying collagen-producing cells, resulting in fibrosis, which progressively impairs the function of major organs such as the lungs, kidneys and liver. This project will examine aspects of the activation of fibroblasts during this process. Our focus will centre on a protein, IGFBP-5, secreted by the epithelium, which plays a significant role in cell death and has been proposed to induce fibrosis in the adult and is also implicated in the process of cell aging. This mechanism is consistent with the late-onset of fibrotic diseases, which typically occur from middle-age onwards. Responses will include changes in cell proliferation, transdifferentiation, migration and aging, as well as expression of various genes and proteins implicated in these events. The use of confocal microscopy will allow us to visualise these changes in living cells in complex reconstructions of the tissues under study. This proposal thus seeks to provide evidence that IGFBP-5 is an important activator of fibroblasts and that, by understanding this process, we will be able to develop novel approaches to deal with the consequences when this repair mechanism malfunctions in processes such as fibrosis and cell aging. Techniques to be used: cell culture, immunocytochemistry, PCR, Western blotting References: Yasuoka H, Jukic DM, Zhou Z, Choi AM & Feghali-Bostwick CA 2006a Insulin-like growth factor binding protein 5 induces skin fibrosis: A novel murine model for dermal fibrosis. Arthritis Rheum 54 3001-3010. Kim KS, Seu YB, Baek SH, Kim MJ, Kim KJ, Kim JH & Kim JR 2007 Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism. Mol Biol Cell 18 4543-4552.
BMS 07 Project Title: Investigation of cell transformation during tumour metastasis Primary Supervisor: Professor David Flint Email:[email protected] Secondary Supervisor: Dr Gordon Allan E-mail: g.j.allan @strath.ac.uk Project Area: (one or more of the following) Biochemistry/Pharmacology Project Description: Breast cancer and metastasis involve 3 major processes, 1) inappropriate cell survival signals, 2) de-adhesion of cells to allow migration and 3) activation of proteases which degrade the extracellular matrix (ECM) to facilitate cell migration. Recent epidemiological evidence has suggested that insulin-like growth factors (IGFs) are a risk factor for breast cancer since elevated concentrations of IGF-I in the blood are associated with increased incidence of breast cancer. The actions of IGFs are in turn inhibited by a family of IGF-binding proteins (IGFBPs). One of these, IGFBP-5 has been shown to inhibit IGF-mediated cell survival and thus can be considered as a potential tumour suppressor. In addition, IGFBP-5 has actions, independent of IGF-I. For example, we have shown it to be involved in cell attachment to the extracellular matrix (ECM) and to be able to influence the activity of proteases, such as tissue plasminogen activator, which can degrade the ECM and allow cells to escape from their normal environment and thus become metastatic. This project aims to investigate the ability of IGFBP-5 to influence cell death and migration using a mammary cell line which has the characteristics of a tumour. We also have mutant forms of IGFBP-5 which will be examined to determine if they have increased anti-tumour properties. The mechanism by which IGFBP-5 influences cell activity independently of IGF-I is unknown and a major part of this project will examine changes in intracellular signalling in cells treated with IGFBP-5. Visualisation of the changes in cell adhesion will involve immunocytochemistry to identify various transmembrane proteins involved in cell adhesion and migration. The mechanisms involved in these phenomena are likely to be shared with aspects of cardiovascular disease and wound healing and, if time permits, cell culture models involving these processes could also be explored. Techniques to be used: cell culture, immunocytochemistry, PCR, Western blotting References: 1. MOHAN et al (2002) IGF-binding proteins are multifunctional and act via IGF-dependent and –independent mechanisms. Journal of Endocrinology 175: 19-31. 2.ALLAN GJ, BEATTIE J, FLINT DJ (2004). The role of IGFBP-5 in mammary gland development and involution. Domestic Animal Endocrinology 27: 257-266. 3.FLINT DJ, BOUTINAUD M, TONNER E, WILDE CJ, HURLEY W, ACCORSI PA, KOLB AF, WHITELAW CB, BEATTIE J, ALLAN GJ. (2005) Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland. Domest Anim Endocrinol. 29:274-82.
BMS 08 Project Title: Development and characterisation of fluorescently-tagged VPAC/PAC receptors for in vivo and in vitro analysis Primary Supervisor: Dr Eve Lutz Email: [email protected] Secondary Supervisor: Dr Elizabeth Ellis Email: [email protected] Project Area: (one or more of the following) Biochemistry /Pharmacology/Molecular Biology Project Description: Research in our laboratory has focussed on defining the molecular mechanisms that determine the function of two G protein-coupled receptors (GPCRs), the PAC1 and VPAC2 receptors [1-4]. These receptors mediate the actions for two structurally related neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PAC1 and VPAC2 receptors share ~50% amino acid sequence similarity, however there are marked functional differences with respect to their distribution, ligand selectivity, levels of expression and G protein-coupling. In particular, the PAC1 receptor is selective for PACAP and VIP is approximately 1000-fold less potent at this receptor, whereas the VPAC2 receptor is a high affinity receptor for both PACAP and VIP. In addition, several amino terminal domain and intracellular loop 3 splice variants exist for the PAC1 receptor, increasing receptor diversity [4]. We are investigating the role of different receptor domains in determining their ligand selectivity and G protein-coupling [2,4].
Traditionally, assays used to determine receptor expression, localisation, trafficking and internalisation rely on radioactively-tagged ligands and/or on antibodies that bind specifically and with high affinity to the receptor protein. These assays are useful also for determining the effects of mutations on receptor functions, such as receptor expression/trafficking, ligand binding and activation of intracellular signalling pathways. However, suitable ligands and/or antibodies may not be available or can be expensive to obtain and the assays need to be stopped before measurements are taken. The use of variants of the Green Fluorescent Protein (GFP) that can be fused to the target protein by recombinant techniques and also fluorescent tags that can be covalently linked to amino acid sequences present in peptides, such as the FlAsH method [5] has enabled the development of several types of assays that capitilise on the ability to excite the fluorescent tag at a specific wavelength. Moreover, assays utilising these can be conducted in live cells in real time. Multiple labels that have different excitation/emission spectra can be utilised at the same time because they can be individually detected. In addition, fluorescent methodologies, such as fluorescence resonance energy transfer (FRET) and fluorescent polarisation (FP), have been developed so that more refined biological questions regarding ligand binding and protein:protein interactions can be answered [6]. The aim of this project is to develop and characterise fluorescently-labelled PAC1 and VPAC2 receptors for use in in vivo and in vitro functional studies. Fluorescently-tagged receptors will be used to visualise receptor localisation in whole cells using fluorescent microscopy in order to determine cell surface expression and internalisation following receptor activation. The receptors will be tagged by fusion with variants of the fluorescent protein GFP. In addition, the amino acid sequence CCPGCC will be incorporated into the 3rd intracellular loop or at the carboxyl tail terminus in order to attach the FlAsH fluorophore. Biological activity of the recombinant receptors will be determined in second messenger assays and expression monitored in whole cells and on Western blots. Techniques to be used:
The project will involve molecular biology techniques (PCR and cDNA manipulation) and biochemical techniques (cell culture and transfection, fluorescent microscopy, second messenger assays, Western blotting). References:
[1] Lutz, E.M., Sheward, W.J., West, K.M., Morrow, J. A., Fink, G. & Harmar, A.J. (1993) FEBS Lett. 334, 3-8.
[2] Lutz, E.M., MacKenzie, C.J., Johnson, M., West, K., Morrow, J.A., Harmar, A.J. & Mitchell, R. (1999) Br. J. Pharmacol. 128, 934-940.
[3] MacKenzie, C.J., Lutz, E.M., Johnson, M. S., Robertson, D.N., Holland, P.J. & Mitchell, R. (2001) Endocrinology 142, 1209-1217.
[4] Lutz, E M, Ronaldson, E, Shaw, P, Johnson, M S, Holland, P J & Mitchell, R (2006) Mol. Cell Neurosci. 31, 193-209.
[5] Adams, S. R., Capmbell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y., Llopis, J. and Tsien, R. Y. (2002) J. Am. Chem. Soc. 124, 6063-6076.
[6] Miyawaki, A. (2003) Dev. Cell 4, 295-305.
BMS 09 Project Title: Control of human inflammatory and cell responses by novel and classical steroids. Primary Supervisor: Dr. Dino Rotondo Email:[email protected] Secondary Supervisor: Prof. W.H. Stimson Tertiary Supervisor: Dr. Jillian Davidson Email: [email protected] Email: [email protected]
Project Area: (one or more of the following) Immunology /Pharmacology /Biochemistry Project Description: TNFα is a major proinflammatory cytokine which is released in response to many pathogenic stimuli especially pathogenic bacteria and their products. It thought that the atherosclerosis of blood vessels involves the presence of monocytes and macrophages which have been activated in response to high levels of oxidised cholesterol (oxysterols). In this respect it would seem that oxysterols may activate inflammatory activity such as enhanced TNF production. However, recently we and other workers have shown that oxysterols can suppress TNF production much in the same manner as other classical anti-inflammatory steroids such as dexamethasone (Davidson et al., 1990). It is possible that oxysterols induce some unknown protective mechanism. In addition several new steroids have been described which may have protective properties, especially 7��� epiandrosterone (7�OH-EPIA). This steroid can selectively enhance the production of prostaglandins D2 and 15 deoxy- PGJ2, however, the mechanism by which this occurs is unclear. The aim of this project is to compare the effects of different steroids such as 7�OH-EPIA, oxysterols and dexamethasone especially those that are used as therapeutic drugs. This will be studied on the production of inflammatory cytokines, particularly TNF-α in human blood and by human monocytes stimulated by different pathogenic pathways such as bacterial and viral routes. It also intended to study the cytoprotective actions of these compounds on their ability to prevent both apoptotic and cytotoxic cell death. Techniques to be used: Cell isolation from human blood, ELISA, thin layer chromatography, flow cytometry, radiotracer labelling References: 1. Davidson, J. Milton, A.S. and Rotondo, D. (1990) The Immunostimulatory Actions of Tumour
Necrosis Factor are Suppressed by Dexamethasone. Br. J. Pharmacol., 100, 445P 2. Davidson, J., Wulfert, E. and Rotondo, D. (2008). 7 beta-hydroxy-epiandrosterone modulation of
15-deoxy-Delta(12,14)-prostaglandin J(2), prostaglandin D(2) and prostaglandin E(2) production from human mononuclear cells. J. Steroid Biochem. Mol. Biol. 112, 220-227.
BMS 10 Project Title: Control of human monocyte and T-cell immune responses by new cytokines and lipid mediators. Primary Supervisor: Dr. Dino Rotondo Email: [email protected] Secondary Supervisor: Dr. A.B. McCruden Tertiary Supervisor: Dr. Jillian Davidson Email: [email protected] Email: [email protected]
Project Area: (one or more of the following) Immunology /Pharmacology /Biochemistry Project Description: Prostaglandins are well established secondary mediators of inflammation which are derived from arachidonic acid and their synthesis can be inhibited by aspirin-like drugs. However, more recently it has been recognised that prostaglandins, particularly PGE2 can suppress the production of the primary inflammatory mediators, specifically tumour necrosis factor-alpha (TNF-a) in a negative-feedback regulation. Indeed PGE2 has been shown to be one of the most potent immunoregulatory mediators so far studied [1, 2], with the ability to completely abolish many immune cell activities such as monocyte TNF-a release, monocyte phagocytic activity and also T-cell proliferation. There are many different series of prostaglandins in addition to PGE2 such as PGD2, PGF2a and the more recently discovered PGE2-ethanolamide (thought to be derived from anandamide which is arachidonyl-ethanolamide [3]). This raises the question of whether other fatty acid acid-derived lipids that can be synthesised during immune responses, can also regulate those responses and if so the nature/ mechanisms by which they do so. Also, a new member of the IL-1 family of inflammatory cytokines, IL-33 will also be evaluated. This project will study the effects of various fatty acid metabolites especially the newly discovered resolvin-1 compared to the effect of IL-33 on TNF-a release from human monocytic cells and the proliferation of human T-cells initially and expand to include other immune cell activities by flow cytometry e.g. phagocytosis etc. It is also intended to ascertain the signalling/ receptor systems by which the lipids induce their effects. This will be achieved both pharmacologically, using drugs which interfere with the signalling systems, and also by directly measuring the levels of intracellular mediators such as cyclic AMP and cyclic GMP. Techniques to be used: Cell isolation from human blood, ELISA, thin layer chromatography, flow cytometry, radiotracer labelling References: 1. DAVIDSON, J., KERR, A., GUY, K. and ROTONDO, D. (1998) Prostaglandin and fatty acid
modulation of E. Coli O157:H7 phagocytosis by human monocytic cells. Immunology, 94, 228-234.
2. ROTONDO, D., EARL, C.R.A., LAING, K., and KAIMAKAMIS, D. (1994) Inhibiton of cytokine-stimulated thymic lymphocyte proliferation by fatty acids: The role of eicosanoids. Biochim. Biophys. Acta. 1223, 185-195.
3. Bakker A.M., Davidson J. and ROTONDO D. (1995) Arachidonyl ethanolamide (Anandamide) modulation of cytokine-stimulated thymic lymphocyte proliferation. Br. J. Pharmacol. 116, 353.
BMS 11 Project Title: Control of human brain tumour cells (glioma) by Prostanoid Receptor Pathways
Primary Supervisor: Dr. Dino Rotondo Email: [email protected] Secondary Supervisor: Prof. W. Harnett Email: [email protected] Tertiary Supervisor: Dr. Jillian Davidson Email: [email protected]
Project Area: (one or more of the following) Immunology /Pharmacology /Biochemistry Project Description: Prostaglandins are well established secondary mediators of inflammation which are derived from arachidonic acid and their synthesis can be inhibited by aspirin-like drugs. It is well established that prostaglandins, particularly PGE2, can control many cellular processes such as cell proliferation or cell death. This is also the case with human brain tumours where it has been shown that PGE2 can enhance the growth of tumour cells in vitro [1, 2]. In addition, the expression of enzymes involved in the biosynthesis of prostaglandins are also upregulated in glioma cells [1, 2] indicating that glioma cells not only respond to PGE2 but they can also produce it in an autocrine manner. The disruption of PGE2-induced proliferation, by either inhibiting PG biosynthesis or blocking PGE2 receptors, can lead to the death of these cells by apoptosis [1]. Thus, interfering with these pathways is a potential target for tumour therapy. This is supported by a study which has shown that the use of celecoxib (a cyclooxygenase inhibitor of PG production) can prevent the occurrence of colon cancer in patients over a 5 year period [3]. It is not known whether this also occurs for other tumours such as gliomas. In addition, it is unclear which PGs are involved in the control of glioma functions as there are a very wide variety of arachidonic acid-derived metabolites from cyclo-oxygenase. The aim of the present study is to characterise the effects of different PGs i.e. compare the actions of PGD2 PGF2� and 15deoxy-PGJ2 to PGE2 on the proliferation and death of glioma cells derived from human tumour tissue. This will be carried out by using a variety of agonists and antagonists of the specific PG-receptor subtypes and different inhibitors of PG-synthesis will also be used. If time permits the production of PGs from the cells will be evaluated using ELISA and radiolabel tracer techniques. Techniques to be used: Cell isolation, ELISA, thin layer chromatography, flow cytometry, radiotracer labelling References: [1] Payner, T., Leaver, H. A., Knapp, B., Whittle, I. R., Trifan, O. C., Miller, S. and Rizzo, M. T.
(2006). Microsomal prostaglandin E synthase-1 regulates human glioma cell growth via prostaglandin E(2)-dependent activation of type II protein kinase A. Mol Cancer Ther 5: 1817-26.
[2] Baryawno, N., Sveinbjornsson, B., Eksborg, S., Orrego, A., Segerstrom, L., Oqvist, C. O., Holm, S., Gustavsson, B., et al. (2008). Tumor-growth-promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets. Neuro Oncol 10: 661-74.
[3] Bertagnolli, M. M., Eagle, C. J., Zauber, A. G., Redston, M., Breazna, A., Kim, K., Tang, J., Rosenstein, R. B., et al. (2009). Five-year efficacy and safety analysis of the Adenoma Prevention with Celecoxib Trial. Cancer Prev Res (Phila Pa) 2: 310-21.
BMS 12 Project Title: Investigating assays for lipid peroxidation in plasma: a European approach Primary Supervisor: Dr Corinne Spickett Email: [email protected] Secondary Supervisor: David Flint Email: [email protected] Project Area: (one or more of the following) Biochemistry/Biomedical Analysis Project Description:
Unsaturated lipids are susceptible to oxidative damage by a process called lipid peroxidation, which is recognized to be important in inflammation and other diseases where reactive oxidants are produced by immune cells or other mechanisms. In view of the importance of this process, a European collaborative action group (COST Action B35) has been set up to study the process in diseases and biological models, and to compare different methods for analysing lipid peroxidation in biological samples. Dr Spickett is responsible for overseeing and coordinating this research, and some of the analysis methods are being tested in her laboratory. There is potential for a biochemically-orientated student to participate in this project, and to extend it by investigating additional assay methods.
This MRes project builds on work over the last year in establishing protocols for the analysis of 2 lipid peroxidation products: malondialdehyde (MDA) and 4-hydroxy-trans-2-nonenal (HNE). There are many assays for MDA, but the most common involve reaction with thiobarbituric acid to form thiobarbituric acid reactive substances (TBARS), which can be assayed directly by spectrophotometer, or separated by HPLC. An ELISA for HNE has recently been developed and is being tested. These methods are being used to determine the level of lipid peroxidation in sets of human plasma samples produced and randomized in a laboratory in Germany. The project will also involve work with purified phospholipids and plasma oxidized at Strathclyde to test additional analytical methods. Depending on the ability of the student, this may include electrospray mass spectrometry interfaced with liquid chromatography to measure phospholipid oxidation products.
Please consult Dr Spickett for further information on the project, in particular regarding progress with the COST Action B35 programme, and the methodology involved.
Techniques to be used: Treatment of pure lipids and human plasma with oxidants Extraction of biological samples Spectrophotometric assays and HPLC analysis. Possibly also electrospray mass spectrometry and liquid chromatography-mass spectrometry References: 1.A novel HPLC method for the measurement of thiobarbituric acid reactive substances (TBARS). A comparison with a commercially available kit. Seljeskog E, Hervig T, Mansoor MA (2006) Clin. Biochem. 39: 947-954. 2. Enzyme-linked immunosorbent assay for 4-hydroxynonenal-histidine conjugates. Borovic S, Rabuzin F, Waeg G, Zarkovic N (2006) Free Radical Research, 40:809-820 3. Studies of phospholipid oxidation by electrospray mass spectrometry: from analysis in cells to biological effects. SPICKETT, C.M. and Dever, G. (2005) Biofactors 24: 17-31.
BMS 15 Project Title: Improving cancer therapy by synergising chemotherapy and radiotherapy Primary Supervisor: Dr Marie Boyd Email:[email protected] Secondary Supervisor:Dr Anthony McCluskey and Dr Annette Sorensen Email:[email protected] [email protected] Project Area: (one or more of the following) Biochemistry/Biomedical Analysis/Food Science/Immunology/Microbiology/Molecular Biology/Parasitology/Pharmacology Project Description: Currently radiotherapy and chemotherapy are used in combination for treatment of many cancers. It is likely that radiotherapy could be curative in cancer if sufficient dose could be administered to the tumour. However this is limited by normal tissue toxicity which result in unacceptable damage to normal cells and organs. Thus therapeutic schemes are sought which maximise the efficacy of radiotherapy by limitation of normal tissue exposure, in particular by combining radiotherapy with chemotherapy drugs in ways which radiosensitise the tumour cells so lower doses are required to kill the cancer cells. There have however been few comprehensive studies elucidating the molecular and cellular basis of the design of such schemes with respect to choice of radiation/ radiosensitising drug, the combination of which is likely to be dose/quality and scheduling dependant. We have previously demonstrated enhanced efficacy of targeted radionuclide therapy for neuroendocrine tumours by rational combination of radiopharmaceuticals with radiosensitisers. These laboratory investigations have now been translated into clinical practice for the treatment of the childhood cancer neuroblastoma. There are a variety of chemotherapy agents which act on various signal transduction pathways specifically or preferentially in cancer cells. Many of these pathways are also involved in a cells response to radiation exposure and indeed are activated as mechanisms for cells to survive radiation insult. This project will therefore investigate whether combination with such agents can enhance the efficacy of targeted radiotherapy and investigate the mechanisms behind synergy in a range of tumour cell lines. We will investigate rational combinations of drugs and radiation and employ isobologram and combination index analysis to identify promising schedules. The mechanisms behind these schemes will be analysed via molecular biology and cell biology techniques. It is likely that any promising schemes will be taken forward for in vivo analysis and may be proposed to our clinical colleagues in due course for clinical evaluation. There are several factors which can result in cancers displaying resistance to therapy. Radiotherapy is a more potent form of toxic insult to cancer cells than chemotherapy, however recently a sub population of cancer cells, designated cancer stem cells have been shown to display chemo- and radioresistance. The second part of this project will therefore involve isolation of cancer stem cells from tumour cell lines which have shown a high proportion of such cells such as melanoma, glioma and pancreatic cell lines. These tumours are infamously resistant to therapy so they are ideal cancers to examine the role of cancer stem cells in drug and radio resistance as the presence of
significant numbers of resistant cancer stem cells may be the key to failure of therapy. Once isolated, these cancer stem cells will be examined for their response to chemotherapy drugs and radiation alone or in combination and inhibitors of key pathways which have been reported to play a role in this resistance will be utilised in an attempt to render these cells amenable to such therapies. This project will employ a range of techniques utilised in cancer research and translational biology. It will utilise molecular biology, cell biology, biochemistry and pharmacology assays and offers the student training in a range of laboratory techniques. The project is part of a wider programme of research within the laboratory and the results will help us to formulate novel treatment schemes for cancer. Techniques to be used: Cell culture 2 and 3d cancer cell models Clonogenic assay Isobologram and combination index analysis FACS Immunohistochemistry Western Blotting General molecular biology Statistical analysis Data collection and analysis References: 1. McCluskey et al, 2005. [131I]MIBG and Topotecan Combination treatment of tumours expressing the noradrenaline transporter. Clin Canc Res 11: 7929-7937. 2. Current Medicinal Chemistry-Anti-Cancer Agents. Volume 3 issue 5; whole journal. 3. Rich JN (2007). Cancer Stem cells in radiation resistance. Cancer Research 67, 8980,
BMS 16 Project Title: Transcriptional regulation of type VI secretion in Pseudomonas aeruginosa. Primary Supervisor: Nick Tucker Email: Secondary Supervisor: Paul Hoskisson Email: [email protected] Project Area: (one or more of the following) Biochemistry/Microbiology/Molecular Biology Project Description: Pseudomonas aeruginosa is a broad host range, opportunistic pathogen of humans, insects, plants and the nematode worm Caenorhabditis elegans. In terms of human disease, P. aeruginosa is the main pathogen of the lungs of cystic fibrosis sufferers and infects the damaged skin of burns victims. Bacteria use at least six types of transport machine to export molecules such as toxins and proteins across cell membranes. Types III and IV are capable of transporting effector molecules into the cytosol of the target cell whilst other secretion systems are capable of merely exporting molecules out of the bacterial cell. Recently, a sixth secretion system (T6SS) has been identified that is thought to have clinical significance. For example, cystic fibrosis patients produce antibodies against a component of the P. aeruginosa T6SS. Since it’s initial characterisation in Vibrio cholerae, the transcriptional regulation of the T6SS has received little or no attention. Bioinformatic analysis suggests that the P. aeruginosa genome encodes three T6SS islands, although the reason for this redundancy is unknown. Two of these T6SS islands encode transcriptional activators of the enhancer binding protein family, PA1663 and PA2359. Using molecular microbiology, biochemistry and genetics you will investigate how the genes encoding the T6SS in P. aeruginosa are regulated. Techniques to be used: Molecular microbiology, protein purification, protein biochemistry, protein-DNA interaction studies. References: 1. Hsu et al. TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa. Mol Microbiol (2009) vol. 72 (5) pp. 1111-25 2. Boyer et al. Dissecting the bacterial type VI secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources? BMC Genomics (2009) vol. 10 pp. 104 3. Filloux et al. The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology (Reading, Engl) (2008) vol. 154 (Pt 6) pp. 1570-83 4. Pukatzki et al. Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci USA (2007) vol. 104 (39) pp. 15508-13
BMS 17 Project Title: Effect of AMP-activated protein kinase (AMPK) on neuromuscular transmission Primary Supervisor: Dr Edward Rowan Email: [email protected] Secondary Supervisor: Prof Alan Harvey Email:[email protected] Project Area: (one or more of the following) Molecular Biology/Pharmacology Project Description: AMP-activated protein kinase (AMPK) is a key enzyme in the regulation of energy metabolism. AMPK is robustly activated during skeletal muscle contraction and pharmacological activation of AMPK increases glucose transport and fatty acid oxidation in skeletal muscle. It is currently thought that chronic activation of AMPK may be beneficial in the treatment of obesity and type 2 diabetes. Drugs that activate AMPK (AICAR) are believed to mimic the changes in cellular metabolism induced by prolonged exercise and have been termed “exercise mimetics”. The vast majority of the data obtained in the study of AMPK has been biochemical in nature, in addition to numerous in vivo studies on metabolism. However, there is a major gap in our knowledge of the functional effects of AICAR on skeletal neuromuscular function especially in the area of neuromuscular function. Pilot experiments (University of Strathclyde) have shown that AICAR increases twitch tension on mouse diaphragm preparations bathed in low calcium solutions. The increase in neuromuscular function would appear to be presynaptic in origin and may be due to an increase in the release of acetylcholine. Acetylcholine release is controlled by a number of key ion channels, whose role is to keep a tight rein on the levels of cytoplasmic calcium. Potassium channels are one of the key players ensuring that the membrane depolarisation Hence, the aim of this project will be to clarify whether AICAR augment twitch height by increasing nerve evoked release of acetylcholine. Techniques to be used: Twitch tension, electrophysiological, calcium imaging References: 1. Prior C, Tian L, Dempster J, Marshall IG. (1995). Prejunctional actions of muscle relaxants:
synaptic Vesicles and transmitter mobilization as sites of action. General Pharmacology. 26: 659-666.
2. Towler MC, Hardie DG (2007). AMP-activated protein kinase in metabolic control and insulin signaling. Circulation Research. 100:328-341.
3. Carey AL and Kingwell BA. (2009). Novel pharmacological approaches to combat obesity and insulin resistance: targeting skeletal muscle with ‘exercise mimetics’. Diabetologia. [Epub ahead of print].
4. Hawley, JA., Holloszy, JO. (2009) Exercise: it's the real thing! Nutrition Reviews. 67:172-178. 5. ViolletB., et al., (2007).Targeting AMP-activated protein kinase as a novel therapeutic approach
for the treatment of metabolic disorders. Diabetes and Metabolism. 33:395-402.
BMS 18 Project Title: PURIFICATION AND IDENTIFICATION OF ANTIMICROBIAL NATURAL PRODUCTS FROM TRADITIONAL HERBAL REMEDIES Primary Supervisor: Dr Veronique Seidel, [email protected] Secondary Supervisor: Dr Geoff Coxon, [email protected] Project Area: Biomedical Analysis /Microbiology Project Description: There is now an urgent need to discover and develop some novel antimicrobial agents, particularly to combat “neglected diseases” (e.g. tuberculosis) and to tackle infections caused by drug-resistant bacteria (e.g. meticillin-resistant Staphylococcus aureus or MRSA).1,2 Investigating natural sources is an approach which offers a unique source of chemically-diverse molecules that could become new drug leads. Only a small proportion of these sources have been investigated thoroughly for their chemical constituents and/or biological activity. Therefore, many natural products with potentially useful applications in therapeutics remain to be discovered.3 As part of a research programme investigating the antimicrobial activity of natural remedies,4,5 we have identified several medicinal plants traditionally used in the treatment of tuberculosis and other infectious diseases. The exact nature of the active chemical constituent(s), however, remains to be identified. The purpose of this project is to;
• Purify a number of natural products (i.e phytochemicals) from selected crude extracts using a range of chromatographic procedures
• Identify the chemical structures of the natural products isolated using a combination of spectroscopic techniques
• Screen all isolated compounds for in vitro activity against a panel of mycobacteria, bacteria, and yeasts
Techniques to be used: Analytical techniques; High-pressure liquid chromatography (HPLC), liquid-liquid partition, vacuum liquid chromatography (VLC), gel filtration, open column chromatography (CC), ion-exchange chromatography, thin-layer chromatography (TLC), Infra-red and ultra-violet spectroscopy (IR, UV), mass spectrometry (MS) and high-field nuclear magnetic resonance (NMR) spectroscopy. Microbiological techniques; In vitro antimicrobial screening assays using broth and agar dilution methods. References: 1. Casenghi M, Cole ST, Nathan CF. (2007). New Approaches to Filling the Gap in Tuberculosis Drug Discovery, PLoS Medicine 4: 1722-5. 2. Talbot GH, Bradley J, Edwards JE, Gilbert Jr.D, Scheld M, Bartlett JG. (2006) Bad Bugs Need Drugs: An Update on the Development Pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America, Clinical Infectious Diseases 42: 657–68. 3. Clardy J, Walsh C. (2004) Lessons from natural molecules. Nature 432: 829-37. 4. Liu M, Katerere DR, Gray AI, Seidel V. (2009). Phytochemical and antifungal studies on Terminalia mollis and Terminalia brachystemma Fitoterapia 80: 369-73. 5. Gordien AY, Gray AI, Ingleby K, Franzblau SG, Seidel V. (2009) Activity of Scottish plant, lichen and fungal endophyte extracts against Mycobacterium aurum and Mycobacterium tuberculosis.” Phytother Res. (in press)
BMS 20 Project Title: Phenotypic differences between Varicella Zoster Virus isolates obtained from herpes zoster patients with and without Post-Herpetic Neuralgia Primary Supervisor: Dr Edward Rowan Email: [email protected] Secondary Supervisor: Prof Alan Harvey Email:[email protected] Project Area: (one or more of the following) Molecular Biology/Pharmacology Project Description: Varicella-Zoster virus (VZV) causes chickenpox (varicella) as a primary infection following which the virus becomes latent in sensory ganglia and reactivates causing herpes zoster (shingles).The most important complication of zoster is post-herpetic neuralgia (PHN) which causes severe dermatomal pain which is very difficult to treat. Our core hypothesis is that PHN but not non-PHN-associated VZV induce alterations in neuronal sodium channel activity, and that this is highly relevant to PHN causation and treatment. We will answer the following questions:
1) Do PHN and non-PHN VZV isolates, as well as vaccine strains, show different phenotypes in terms of altering the expression of neuronal voltage-gated sodium channels in neuronal cell lines?
Does the neurophysiological phenotype of the different VZV strains depend on the particular neuronal sodium channel? This will be determined by transfecting different types of sodium channels to determine whether this alters the channel-modulating phenotype of the different VZV isolates. Techniques to be used: Tissue culture, molecular biology, electrophysiological, calcium imaging References: 1. Kennedy PGE, Grinfeld E, Gow JW. Latent varicella-zoster virus is located predominantly in neurons in human trigeminal ganglia. Proc Natl Acad Sci, USA. 1998;95:4658-4662. 2. Vafai A, Wellish M, Gilden DH. Expression of varicella-zoster virus in blood mononuclear cells of patients with postherpetic neuralgia. Proc Natl Acad Sci U S A. 1988;85:2767-2770 3. Lai, J, Porreca F, Hunter, J C, Gold, M,S. Voltage-gated sodium channels and hyperalgesia. Annu.Rev.Pharmacol.Toxicol. 2004;44:371-97 4. Cummins TR, Dib-Hajj SD, Waksman SG. Electrophysiological properties of mutant Na v1.7 sodium channels in a painful peripheral neuropathy. J.Neurosci. 2004; 24:8232-8236
BMS 21 Project Title: Mast cells in gastrointestinal helminth infection Primary Supervisor: Dr Catherine Lawrence Email: [email protected] Secondary Supervisor: Email: Project Area: (one or more of the following) Immunology/Microbiology/Parasitology/ Project Description: Potent Th2 responses concomitant with profound mastocytosis are characteristics of infection with gastrointestinal nematodes. Mast cells have long been recognised as effector cells playing key roles in the pathology associated with inflammatory diseases ranging from allergy to GI helminth infection. Following activation, MC contribute to inflammation by excessive and/or inappropriate release of pre-formed and de novo-synthesized mediators, including cytokines, which may also amplify and perpetuate responses via recruitment of inflammatory cells. Infection with gastrointestinal (GI) nematode parasites, most notably T. spiralis, is associated with mastocytosis accompanied by the systemic release of the �-chymase, mouse mast cell protease-1 (mMCP-1). Depletion of MC or infection of MC deficient W/Wv mice has demonstrated a role for MC in the expulsion of some GI nematode infections with expulsion being accompanied by intestinal inflammation mediated by MC and their products. We have previously demonstrated amelioration of the intestinal pathology accompanying the loss of T. spiralis following infection of W/Wv or mMCP-1 deficient mice. The aim of this project is to analyse the effects of infecting a newly described mast cell deficient mouse W-sh with the helminth Trichinella spiralis Techniques to be used: ELISA, histology, parasite infection, in vivo model, FACS, tissue culture References: Ierna, M. X., Scales, H. E., K L Saunders, K. L. and C E Lawrence. (2008) Mast cell production of IL-4 and TNF may be required for protective and pathological responses in gastrointestinal helminth infection. Mucosal Immunol. 1:147-155 Lawrence, C.E., Paterson, Y.Y.W, Wright, S.H., Knight, P.A. & Miller H.R.P. (2004) Mouse mast cell protease-1 is required for the intestinal inflammatory response associated with infection with Trichinella spiralis. Gastroenterology. 127:155-65 Knight, P. A., Wright, S. H., Lawrence, C, E, Paterson, Y, Y, Miller, H. R. (2000) Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1. J Exp Med. 192,1849-56.
BMS 22 Project Title: Identifying possible mechanism of fistulae failure in renal dialysis patients: Effect of needle injury on vascular neointimal proliferation Primary Supervisor: Dr. Paul Coats Email: [email protected] Secondary Supervisor:Prof Roger. M. Wadsworth Email: [email protected] Project Area: (one or more of the following) Biochemistry/Biomedical Analysis/Food Science/Immunology/Microbiology/Molecular Biology/Parasitology/Pharmacology Project Description: Surgical creation of a vascular access point in the forearm is called an arteriovenous fistula. This is the favoured method used in renal patients to permit blood dialysis. Good blood flow function of this vascular access point is critical for dialysis and adds greatly to the patient’s quality of life. Unfortunately a significant number of these access points fail due to accelerated growth within the fistula lumen. This is a significant problem and presently little is known about the cellular growth that accumulates leading to blockage and the proliferative mechanisms involved. Based on preliminary data generated in the cardiovascular lab, we propose that the cellular hyperplasia observed in arteriovenous fistulae (AVF) is driven by the MAP kinase growth pathways and these mechanisms are promoted by the local release of pro-inflammatory cytokines. The objective of this project is to help validate this hypothesis by helping to clarify the mechanisms of hyperplasia and identify the possible interaction of pro-inflammatory-mediators on cell/ tissue hyperplasia (proliferation) in AVFs compared with saphenous vein control. You will study the effect of cannulation injury of the vein wall (analogous to needle cannulation used in haemodialysis) in our established blood vessel culture model1. Lengths (2-3cm) of human saphenous vein will be cannulated, pressurised (90mmHg; nominal flow rate 15-20mls hour-1) and kept in sterile culture conditions for 7 days. During this period the vein will be cannulated with a 17 gauge sterile needle on days 3 and 5. The cannula will be left in situ for 4 hours. We anticipate that the needle cannulation will evoke an inflammatory response favouring pro-neointimal growth and pro-inflammation. In the pressure cultured vein the effect of repeated 17 gauge needle cannulation alone, cannulation in the presence of pro-inflammatory mediators LPS (1-ng/ml), AS 101 (10nM) and IL-1B (10iu/ml) and cannulation with inhibitors of TNF-� (Pentoxifylline, 10nM) and NFkB (Bay 11-7085, 10nM & SC514, 10uM) will be studied2. The effect of these agents on vascular structure and cell proliferation will be studied. Likewise the effect of these agents of MAP kinase p44/42, p38, JNK and MK2 activity will be assessed by western blotting3. By pre-incubating the cultured tissues with LPS and IL-1B we would anticipate measuring exaggerated responses to needle injury Techniques to be used:
1. Tissue culture of blood vessels; cell culture of isolated cells from intact blood vessels 2. Histology of blood vessels; H & E, Alcian blue 3. Immunohistochemistry of section blood vessels (MAP Kinases; ICAM, VCAM, MCP-1) 4. Biochemistry; 3HThymidine incorporation to measure cell proliferation 5. Western blotting for MAP Kinases
References: 1. Coats P. et al (2008) Atherosclerosis; 197: 515-23 2. MacKenzie CJ. et al (2007). Cell Signal; 19: 75-80 3. Landry DB. et al (1997). Am J Pathol; 151: 1085-95
BMS 23 Project Title: Examination of the anti-inflammatory properties of the herb lovage Primary Supervisor: Dr Val Ferro Email: [email protected] Secondary Supervisor: Dr Sandy Gray Email: [email protected] Project Area: (one or more of the following) Biochemistry/Biomedical Analysis/Food Science/Immunology/Microbiology/Pharmacology Project Description: Lovage (Levisticum Officinale), is a fragrant perennial and a close relative of celery. The medicinal properties of this plant have been acknowledged for centuries, though never scientifically recognised, with the roots and fruits of the plant being commonly used to treat digestive problems. It has also been used as a diuretic and in treating bladder infections. Previous work has shown that the plant leaves have anti-inflammatory and anti-microbial properties. The aim of this project is to ascertain scientifically whether the roots have similar properties. Techniques to be used: In this project, solvent extracts will be made from the roots of the plant. Analytical tests such as TLC, flash chromatography, gel filtration, NMR and spectroscopy will be carried out on the extracts and fractions. Active fractions will be further purified. Anti-inflammatory effects of the purified compounds will be assessed using bacterial stimulation assays of human immune cells and immunoassays to determine cytokine production. Anti-microbial properties will be examined on a range of gram positive and gram negative bacteria. References: 1. Composition of flavonoids in fresh herbs and calculation of flavonoid intake by use of herbs in traditional Danish dishes. Justesen U.1; Knuthsen P. Food Chemistry, Volume 73, Number 2, May 2001, pp. 245-250(6) 2. Determination of quantitative composition of poliphenolic compounds occur in anatomically different parts of Levisticum officinale Koch. Agnieszka Najda, Tadeusz Wolski, Jan Dyduch, Tomasz Baj. Electronic Journal of Polish Agricultural Universities, Horticulture, Vol 6 (1), http://www.ejpau.media.pl 3. Botanical medicines for the urinary tract. Yarnell E. World J Urol. 2002 Nov;20(5):285-93. Epub 2002 Oct 17.
BMS 24 Project Title: Use of glycogen synthase kinase 3 beta from Leishmania mexicana as a drug target Primary Supervisor: Martin Wiese Email: [email protected] Secondary Supervisor:Alan Harvey Email: [email protected] Project Area: (one or more of the following) Pharmacology/Biochemistry/Parasitology Project Description: Protein kinases are important regulators of all cells and play a role in vital processes like differentiation, proliferation, adaptation and motility. The human parasite Leishmania mexicana like other eukaryotes relies on signal transduction via reversible phosphorylation. As such parasite protein kinases are potential drug targets to be used to treat leishmaniasis which dependent on the parasite species and the immunological background of the host can be a fatal disease. We have shown that glycogen synthase kinase 3beta is essential for the parasite in both life stages, the promastigotes living in phlebotomine sand fly and the amastigotes residing in the parasitophorous vacuole of macrophages of the infected host. In this project, we want to use the available bacterial expression system to produce recombinant GSK3beta in sufficient amounts and purity to use it in screening assays employing the plant extract library of SIDR (Strathclyde Innovations in Drug Research). Hits will be analysed and validated in Leishmania cell culture. Moreover, recombinant protein will be produced for crystallisation in collaboration with Prof. Malcolm Walkinshaw from Edinburgh University. Techniques to be used: protein purification, enzymatic assays, cell culture, inhibitor studies References:
1. Eldar-Finkelman H. Glycogen synthase kinase 3: an emerging therapeutic target. Trends Mol Med. 2002 Mar;8(3):126-32. 2. Wiese, M.. A mitogen-activated (MAP) protein kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO J., 17, 2619-2628 (1998)
BMS 26 Project Title: Use of the green fluorescent protein (GFP) to tag Leishmania protein kinases and their substrates for in vivo and in vitro analyses Primary Supervisor: Dr Martin Wiese Email: [email protected] Secondary Supervisor: Dr Craig Roberts Email: [email protected] Project Area: (one or more of the following) Biochemistry/Microbiology/Molecular Biology/Parasitology Project Description: Protein kinases are important regulators of all cells and play a role in vital processes like differentiation, proliferation, adaptation and motility. The human parasite Leishmania mexicana like other eukaryotes relies on signal transduction via reversible phosphorylation. As such parasite protein kinases are potential drug targets to be used to treat leishmaniasis which dependent on the parasite species and the immunological background of the host can be a fatal disease. In this project protein kinases and/or their substrates fused to the green fluorescent protein will be expressed in Leishmania parasites and visualised by fluorescence microscopy. This will provide us with valuable information about their cellular localisation. Moreover, the tagged proteins will be enriched from cell lysates and their activity and phosphorylation state assessed. The information gained will help to understand the role of the kinase and its suitability as a drug target. Techniques to be used: Cell culture, electroporation, protein purification, gene cloning, microscopy, kinase assay, immunoblot analysis. References: 1. Wiese, M. Leishmania MAP kinases – familiar proteins in an unusual context. Internat. J. Parasitol. 37(10), 1053-62 (2007) 2. Wiese, M. A mitogen-activated (MAP) protein kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO J., 17, 2619-2628 (1998)
BMS 27 Project Title: Isolation of bacteriophage from river water as an indicator of disease outbreaks Primary Supervisor: Dr Paul Hoskisson Email: [email protected] Secondary Supervisor: Niamh Murphy (Health Protection Agency) Email: [email protected] Project Area: (one or more of the following) Biochemistry/Microbiology/Molecular Biology Project Description: Bacteriophage are amongst the most abundant biological agents on earth, and are very specific for their host bacteria. The aquatic environment appears to harbor an immensely diverse population of bacteriophage that maybe representative of the pathogenic organisms currently circulating in the population. Isolation of bacteriophage against pathogens from river water, are able to infect pathogens maybe indicative of potential outbreaks of particular diseases. The river Thames in London is sampled regularly by the Health Protection Agency for the presence of pathogens released through Sewage outlets, yet the isolation of these organisms is generally poor and doesn’t appear to correlate with recent outbreak strains. This project, in collaboration with the Health Protection Agency aims to identify bacteriophage for specific foodbourne pathogens to indicate if this is a more appropriate method for pre-empting outbreaks. The isolation of bacteriophage using this method may also isolate novel phage which can be incorporated in to phage-typing screens. Techniques to be used: microbiology, bacteriophage isolation, genetic characterisation of bacteriophage, southern blotting References:
1. Faruque et al., (2004). Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area. PNAS, 101, 2123-2128.
2. Hoskisson, P. A. & Smith M. C. M (2007). Hypervariation and phase variation in the bacteriophage
'resistome'. Curr. Op. Microbiol. 10, 396-400.
3.
BMS 28 Neuroprotection against oxidative and carbonyl stress Primary Supervisor:Elizabeth Ellis Email:[email protected] Secondary Supervisor: Eve Lutz Email:[email protected] Project Area: Biochemistry Molecular Biology Pharmacology Project Description: Oxidative stress has been implicated in a range of neurodegenerative diseases, as well as ischaemia-reperfusion type injury such as stroke. Neuronal cells have limited intrinsic ability to protect themselves against oxidants, and this may lead to cellular damage to lipids, proteins and DNA. Oxidation of lipids leads to the production of lipid peroxidation products, many of which are highly toxic aldehydes (1). Previously we have shown that the human neuroblastoma cell line SH-SY5Y cell line is extremely sensitive to carbonyl stress (2). We have also shown that treatment of cells with certain phytochemicals can lead to the induction of protective enzymes and that this is sufficient to cause increased protection against reactive aldehydes and oxidants (2). The SH-SY5Y cell line is neuronal-like and can be induced to differentiate (3,4). What is not known is to what extent differentiated cells have increased levels of protective enzymes and whether or not this can protect them against reactive carbonyls. In this study, SH-SY5Y cells will be differentiated, and the effect on protective enzyme expression measured using Western blots and RT-PCR. The effect on cell survival will be monitored using MTT assays. Techniques to be used: Mammalian cell culture Cytotoxicity assays Gene Expression studies References: 1. Ellis, E.M. 2007 Pharmacol Ther. 115:13-24. 2. Biedler et al., 1978. Cancer Res. 38:3751-7. 3. Monaghan T et al (2008) J. Neurochem. 104, 74-88
BMS 29 Molecular basis of selective toxicity of antimicrobial minor groove binders Primary Supervisor:Elizabeth Ellis Email:[email protected] Secondary Supervisor:Colin Suckling Email:[email protected] Project Area: Biochemistry Microbiology Molecular Biology Drug Delivery Project Description: Minor Groove Binders (MGB) are a class of compounds that bind to the minor groove of DNA. By doing so, they can disrupt normal cell processes. Although some MGBs are toxic to mammalian cells and can be used as anticancer drugs, we have identified a class of MGBs that can effectively kill microbial cells, yet which do not display significant toxicity to mammalian cells (1). This makes them ideal for use as antimicrobial agents. However the mechanism of action and the basis for the observed selective toxicity is not known. The aim of this project is to find out why these compounds can kill bacterial or fungal cells, but cause little damage to mammalian cells. Lines of investigation will include measuring the ability of the compounds to enter cells; to prevent key DNA-dependent processes such as DNA replication and transcription in bacterial and mammalian cells (2); to inhibit topoisomerase activity and supercoiling (3), and perturbation of membrane transport systems. Techniques to be used: Fluorescent microscopy Mammalian and microbial cell culture Cytotoxicity assays Toposiomerase, DNA cleavage and supercoiling assays References: 1. Anthony NG, Breen D, Clarke J, Donoghue G, Drummond AJ, Ellis EM, Gemmell CG, Helesbeux JJ, Hunter IS, Khalaf AI, Mackay SP, Parkinson JA, Suckling CJ, Waigh RD. 2007. Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups. J Med Chem.50:6116-25. 2. Simon H, Kittler L, Baird E, Dervan P, Zimmer C. (2000) Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration. FEBS Lett. 2000 Apr 14;471(2-3):173-6. 3. McHugh MM, Woynarowski JM, Sigmund RD, Beerman TA. (1989). Effect of minor groove binding drugs on mammalian topoisomerase I activity. Biochem Pharmacol. 38:2323-8.
BMS 30 Project Title: Biological production of hydrogen by cyanobacteria Primary Supervisor:Dr Elizabeth Ellis Email:[email protected] Secondary Supervisor:Dr Xiongwei Ni Email: [email protected] Project Area: (one or more of the following) Biochemistry/Microbiology/Molecular Biology Project Description: Hydrogen is considered a clean fuel in terms of its use, and there is considerable interest in using it to power vehicles. However, the production of hydrogen by electrochemical or thermo-chemical processes requires energy, and is not very efficient. An alternative source is to use cyanabacteria that can naturally produce hydrogen. An advantage of this approach is that cyanobacteria can use sunlight to fix CO2 and this therefore represents a means of harnessing solar energy. In collaboration with Heriot-Watt University, we have optimized growth of cyanabacteria in batch culture and are currently establishing small scale bioreactors for hydrogen production. We would like to use systems biology approaches to optimize hydrogen production. The aim of this production is to measure some of the key parameters within the cyanboacterial cell and relate them to the production of hydrogen. Parameters include metabolites such as glycogen, as well as the expression of hydrogenase and nitrogenase components. The work will involve some travel to Heriot-Watt University to collect samples. Techniques to be used: Metabolite analysis, Quantitative RT-PCR, glycogen assays, growth curves References: 1. Angermayr SA, Hellingwerf KJ, Lindblad P, de Mattos MJ. (2009) Energy biotechnology with cyanobacteria. Curr Opin Biotechnol. 20:257-63. 2. Tamagnini P, Leitão E, Oliveira P, Ferreira D, Pinto F, Harris DJ, Heidorn T, Lindblad P. (2007) Cyanobacterial hydrogenases: diversity, regulation and applications. FEMS Microbiol Rev. 31:692-720.
BMS 31 Project Title: Cancer Chemoprevention: Transcriptional Regulation of AKR7A1 by dietary chemoprotectors Primary Supervisor:Dr Elizabeth Ellis Email:[email protected] Secondary Supervisor:Prof John Hayes, University of Dundee Email: [email protected] Project Area: (one or more of the following) Biochemistry/Molecular Biology Project Description: Chemoprevention is the process by which compounds naturally present in the diet can prevent or reduce the development of cancer. One mechanism of chemoprevention is through the induction of protective enzymes, mediated by the transcription factor Nrf2 binding to the Antioxidant Response Element or ARE that is in the promoter of induced genes. Our previous work has investigated the up-regulation of an aldo-keto reductase AKR7A1 that can protect liver cells against aflatoxin B1-induced cancer. We have cloned and analysed a 720 bp promoter region and have identified putative regulatory sequences in this promoter. We have also shown that this promoter can drive expression of a reporter construct in cell lines. More recently we have obtained BAC clones that contain a further 4kb of upstream sequence. We have subcloned regions of this promoter into reporter vectors and have shown that it is functional in cell lines. The aim of this project is to: 1. Confirm the sequence of this promoter region 2. Create defined mutations in putative ARE sequences in the promoter in order to demonstrate the role of Nrf2/ARE in regulation of AKR7A1 This work will increase our knowledge of chemoprevention and its potential as an anticancer strategy. Techniques to be used: Bioinformatics, DNA sequencing, PCR, reporter genes, cell culture References: 1. Ellis EM, Judah DJ, Neal GE, Hayes JD. (1993) An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases. Proc Natl Acad Sci U S A. 90:10350-4 2. Hayes JD, Ellis EM, Neal GE, Harrison DJ, Manson MM (1999). Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress Biochem Soc Symp. 64:141-68. 3. Ellis EM, Slattery CM, Hayes JD. (2003) Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1. Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter. Carcinogenesis. 24(4):727-37
