FACULTY OF HEALTH AND SOCIAL SCIENCES
Biological Sciences
Title: Cardiovascular disease: reducing the risk by vascular enzyme inhibition
Cardiovascular diseases are the leading cause of death; a third of all deaths in 2005 were due to heart disease.
Annual European cardiovascular disease costs are £73 billion. Atherosclerosis is the main contributory factor in
cardiovascular disease development. Early intervention to reduce the development of atherosclerosis will
greatly alleviate the burden that cardiovascular disease poses to society. Semicarbazide-sensitive amine
oxidase (SSAO) is expressed by vascular cells and increased activity is implicated in the pathogenesis of
atherosclerosis, type I and II diabetes, smoking, inflammatory diseases, obesity and cerebrovascular disease
and strokes. The relationship between SSAO activity and disease processes is poorly understood; direct
damage to cells via oxidative stress and aldehyde toxicity has been proposed as a key mechanism. Oxidation of
low density lipoprotein (LDL, "bad cholesterol") by SSAO is an important early step in the development of
atherosclerosis that has not been studied.
Human SSAO will be expressed in eukaryotic cells. Purification of protein from these cells will allow
characterisation of SSAO at a molecular level. The student will determine enzyme kinetic parameters;
substrate inhibitors (semicarbazide, methylhydrazine) and effectors will be explored. Potential substrates
known to be associated with increased risk of cardiovascular disease, such as aminoacetone, methylamine and
allylamine, will be tested to understand their role in LDL oxidation. In vivo activity of SSAO will be assessed by
culturing cells in the presence of LDL and plasma lipids and substrates. The ability of inhibitors to prevent or
reduce oxidation of lipids will be evaluated.
The results from these studies will determine factors associated with an increased risk of cardiovascular
disease. A development pathway for a new class of drugs aimed at reducing the risk of cardiovascular disease;
together with high throughput screening of drugs will identify inhibitors of SSAO with clinical and commercial
potential. This project is a novel approach to develop cardiovascular disease treatments, providing a rationale
for prevention in known high risk groups, which will be published in Circulation Research or Biochemical
Pharmacology.
Please contact Dr John Skamarauskas for further details: J.T.Skamarauskas@leedsbeckett.ac.uk
Biological Sciences
Title: Combating inheritable heart disease: Functional and Molecular characterisation of mitochondrial
ADP/ATP carrier
Mitochondrial adenine nucleotide carriers are polytopic proteins located in the inner mitochondrial
membrane. They are essential for life as they are one of the main sources of ATP supply for the cell thanks to
their ability to export matrix ATP, synthesised by the respiratory chain, and import cytosolic ADP, thus
providing new substrate for the ATP synthase. Three tissue specific isoforms exist in humans. Point mutations
in the human cardiac isoform (hANC1) have been shown to be involved in two main pathologies: myopathies
and autosomal dominant progressive external ophtalmoplegia (adPEO). The first affect muscles and the
second is clinically characterized by exercise intolerance, ptosis and muscle weakness. The mechanisms
underlying the mitochondrial pathologies caused by hANC1 mutations remain largely poorly understood.
In the light of extensive biochemical studies, it is admitted that these transporters switch between at least four
conformations (ADP binding/release and ATP binding/release), two of which can be frozen by the use of
specific inhibitors called carboxyatractylate (CATR) and bongkrekic acid (BA). In 2003, the molecular structure
of the bovine homologue of hAnc1 has been solved in a CATR conformation, allowing to understand one of the
conformations the transporter adopt during the ADP/ATP exchange. The complete cycle, however, has not
been yet understood due in part to the inability to isolate a BA-inhibited conformation. In addition, although
more than 20 years of biochemical data tend to indicate that these carriers function as dimers, the crystal
structure only showed a monomer and opened an endless debate.
The aim of the project is to isolate and structurally characterise the adenine nucleotide translocators through
the solving of their structure in a BA conformation as well as isolating the transporter in his native lipidic
environment. Unlike conventional techniques using detergents which have failed to produce crystal of this
transporter in its 'BA conformation', this study will use a cutting edge methodology which take advantage of
the SMALP (styrene maleimic acid lipoparticles) technique.
To maximise the applicant chance of success, the human ANC1 protein will be studied together with the yeast
homologue ANC2. Both isoforms genes are readily available from Dr. Postis previous work (de Marcos Lousa et
al., 2003; Postis et al., 2004). These will need to be cloned in an adequate vector for expression in mammalian
cells such as HEK cells, which have been demonstrated to be a reliable system for membrane protein
expression. Molecular studies using SMALP technology has been pioneered by Dr Postis (Postis et al., 2015).
Hence this project will combine basic molecular biology and biochemistry, ideal techniques to be learned by a
PhD student for future employability.
This project will give the opportunity to the PhD student's to develop skills in a cutting edge methodology
highly looked at by the pharmaceutical industry. Finally, the structure resolution will be solved in collaboration
with prof. Goldman, which is a renowned scientist in the crystallography field
Please contact Dr Vincent Postis for further details: V.L.Postis@leedsbeckett.ac.uk
Biological Sciences
Title: Development of low cost, therapeutic antibody treatment for flaviviurs encephalitis
For the last twenty years, RNA viruses are the biggest emerging pathogen threat. It is environmental change
coupled with tropical urbanisation and globalization that makes flaviviruses a worldwide danger to public
health. Japanese encephalitis is the most important emerging encephalitis furthermore, the WHO report that
half of the world is at risk from Dengue infection. Currently no treatments exist for any flavivirus disease.
Extraction of protective antibodies from Asian blood donors makes intravenous immunoglobulin (antibody)
treatments expensive and difficult to produce.
The flavivirus envelope (E) protein is responsible for virus attachment to the cell and triggering cellular
infection. Human vaccination against flavivirus encephalitis has shown that flaviviruses share common
protective epitopes in their E proteins (Mansfield et al., 2011). The screening of artificial antibody and antibody
mimetic libraries will allow isolation of molecules that will prevent flavivirus infection. Additionally solving the
atomic structure of the E protein will help to understand virus particle assembly and therefore permit
informed drug design.
This project will use a model flavivirus that is not associated with human disease: the langat virus (LGT). The E
protein will be crystalised after generation of recombinant protein. Academic and commercial libraries will be
screening for molecules that blocks binding of the viral particles to cells. Potential antibodies will be tested for
their ability to neutralize virus using the plaque reduction neutralization test.
Please contact Dr Sereen Galbraith for further details: S.E.Galbraith@leedsbeckett.ac.uk
Biological Sciences
Title: Development of novel membrane protein purification technology for structural studies
Unlike soluble protein, membrane protein structural studies have been hindered for many years due to various
limitations in protein expression and purification. Protein expression issues have been addressed; however the
purification of membrane proteins is still problematic. The main issue is the use of detergents for protein
solubilisation and downstream processes such as reconstitution and biophysical characterisation. Not only is
the right detergent formulation required to keep the protein in a native functional state, but the detergent
must be easy to remove to limit interference with protein assays.
The use of protein scaffolds has revolutionised membrane protein purification. Two types of scaffold are
currently employed to maintain integrity of the membrane protein in lipoparticule: protein scaffold
(nanodiscs) and polymer scaffold (SMALPs). Prior to reconstitution in nanodisc, membrane proteins require to
be solubilised by detergents. SMALP are on the other hand employed as molecular cutters allowing copurification of the membrane proteins with their natural lipidic environment. Each approach has its pros and
cons, the use nanodiscs or SMALPs is restricted by the type of protein and the nature of the downstream
biophysical assays.
In order to understand the advantages and limitations of each technique, this project aims at studying two
model proteins and characterise them through a range of biophysical techniques such as Mass Spectrometry,
-helical protein,
-barrel protein, OmpX, have been chosen for these
studies. Both membrane proteins are well characterised and expression conditions are already established.
AcrB is involved in antibiotic resistance while OmpX is a bacterial porin. Both proteins will be solubilised in
classical conditions (detergent) as a control. The proteins will be reconstituted in nanodiscs and isolated in
SMALP. Subsequently the particles will be tested for structural studies. The protein expression plasmids
required for this project are ready for use and technical training will be provided by Dr. Postis who has
extensive expertise in membrane protein purification.
Please contact Dr Vincent Postis for further details: V.L.Postis@leedsbeckett.ac.uk
Biological Sciences
Title: Effects of Plant extracts on the attachment of pathogenic and commensal bacteria in an in-vitro gut
model
Gastrointestinal (GI) infections are a widespread and costly public health problem, causing significant
morbidity and mortality. Interest in probiotics, prebiotics and food-derived antimicrobial compounds as a way
to protect against the attachment and subsequent invasion of enteropathogens is a growing area of research.
Certain types of probiotics have shown a degree of inhibition on a range of enteropathogens in vitro (Bernet et
al., 1994; Candela et al., 2005; Stöber et al., 2010) and a varying ability to reduce the duration and severity of
gastroenteritis without adverse effects in clinical trials (Allen et al., 2010). However, the results from these
studies also show that probiotics fall far behind antibiotics and little work has been carried out on agents that
may facilitate probiotic bacteria’s action in preventing attachment. Natural food-derived antimicrobial agents
and prebiotics exist in many different spices, herbs and other foods, such as garlic, oregano, coriander and
turmeric. Garlic is of great interest as not only is it an established antibacterial agent (Harris et al., 2001); it
also possesses a prebiotic effect for commensal gut microbiota due to its high concentration of fructans
(Kyung, 2012). Crude garlic extracts have been seen to facilitate antibiotics against otherwise resistant strains
of enteropathogenic bacteria (Eja et al., 2007); however, most studies exploring garlic’s and other food’s
antibacterial properties against enteropathogens have been carried out in vitro, which cannot reflect the
intricate mechanisms by which bacteria adhere to gut epithelium in vivo. Previous studies have produced
clear evidence that both probiotics and isolated compounds from garlic, coriander, oregano and turmeric,
either alone or in conjunction with an antibiotic inhibit the growth of enteropathogens in basic in vitro studies,
however, no study has yet actively assessed the combined effect of these agents and ‘beneficial’ commensal
strains on the attachment and penetration of enteropathogenic bacteria to intestinal epithelial cells.
The study will evaluate the effects of natural food-derived antimicrobials and prebiotics on the attachment of
enteropathogenic, indigenous commensal and probiotic bacterial strains to in vitro intestinal models.
Objectives:
Optimise bacteria-cell models to accurately assess the extent of attachment and penetration of
pathogens
Investigate the antimicrobial and prebiotic potential of a range of food-derived antimicrobial agents,
including compounds derived from garlic, oregano, coriander and turmeric
Investigate the effect of isolated compounds on the attachment and penetration of a range of
enteropathogens using established intestinal cell line models
Assess the competitive attachment of probiotic Lactobacillus and Bifidobacterium strains and
enteropathogens including Listeria monocytogenes, Escherichia coli, Salmonella Sp., and
Campylobacter jejuni in the presence of isolated compounds
Evaluate the role of agents protecting or enhancing commensal gut microflora and the combined
effect this has on enteropathogen establishment and attachment to gut models
This will be the first study to investigate the facilitation of commensal and probiotic strains with specific agents
to prevent proliferation, attachment and penetration of enteropathogenic bacteria. It will direct and focus the
research evaluating the potential of non-antibiotic prophylactic alternative for bacterial gastroenteritis
potentially leading to novel therapeutic pro-biotic interventions.
The gut model proposed is based on a cell culture system of epithelia from the small and large intestine. Caco2 and HT29, cell lines from the small and large intestine respectively, have been selected as they differentiate
forming tight junctions and in the case of HT29 cells express goblet cells which produce mucin. Characteristics
of the model such as permeability of the epithelial layer will be determined. The models developed provide a
system in which attachment of specific bacterial strains to human gut epithelium can be assessed. Common
invasive enteropathogens such as Salmonella sp., Escherichia coli, Listeria monocytogenes and Campylobacter
sp. will be assessed, as this will allow the study of different mechanisms of attachment and invasion.
Quantification of cell attachment will be carried out by the counting of attached bacteria. The nature of
attachment will be determined by atomic force microscopy (AFM).
Garlic has already been shown to have an effect on the attachment of Salmonella thompson utilising a CaCo-2
gut model (Sinclair et al., in preparation). There are a number of other plant extracts that could have similar
activity, which include oregano, coriander, turmeric all of which are herbal in origin and hence food-grade
extracts can be obtained. The model will provide a system in which the effects of such phytochemicals upon
attachment of specific bacterial species can be assayed within this project.
Strains such as Lactobacillus acidophilus and Bifidobacterium breve have both shown to adhere to CaCo-2 cells
and inhibit enteropathogenic bacteria to some degree (Bernet et al., 1994; Candela et al., 2005; Stöber et al.,
2010). Further tests will be carried out to determine the plant extracts previously tested enhance their
inhibiting effect on pathogen attachment.
Please contact Dr Margarita Gomez Escalada for further details: m.gomez-escalada@leedsbeckett.ac.uk
Biological Sciences
Title: Epigenetic control and the development of antimicrobial resistance and cross-resistance to antibiotics
Triclosan is a widely used antimicrobial that has been associated with resistance development and
development of antibiotic cross-resistance, following the discovery of its competitive inhibition of enoyl
reductase, which is involved in fatty acid biosynthesis. This issue is timely as the US Food and Drug
Administration recently published an announcement that it will review Triclosan’s licence because of these
concerns which may be applicable to a range of other antimicrobial agents. A number of publications have
reported the development of antibiotic resistance (ABR), and in some cases multiple antibiotic resistances
(MARs), following exposure to sub-inhibitory concentrations of Triclosan. While it is thought that the
mechanisms of resistance may be acquired through efflux, the mechanisms of this acquired ABR/MAR have
never been fully elucidated.
In order to maximise survival, clonal bacterial populations exhibit cell-cell variation. While it has been generally
assumed that mutation is the cause of such variation, it is becoming increasingly apparent that mechanisms
such as DNA methylation can also result in such. DNA methylation is the most common epigenetic change in
prokaryotes and in Gram negative bacteria, it is primarily mediated by Dam and Dcm (adenine and cytosine
methyltransferase enzymes respectively). DNA methylation has been shown to affect a range of processes
including: regulation of secretion systems in Ps. aeruginosa and enteroaggressive E. coli, and phase regulation
in uropathogenic E. coli. In addition, the methyltransferases have been linked to virulence and pathogenesis in
a range of bacteria including Y. pestis and P. multocida[9]. It has been suggested that DNA methylation may
also impact resistance development. Militelo et al. found that lack of methylation was linked to ethidium
bromide resistance through relieved repression of the small multidrug resistance protein SugE. Adam et al.
also saw increased antibiotic resistance in E. coli as a result of epigenetically-induced changes in the expression
of resistance-associated genes.
Currently, robust and user-friendly assays are available for the assessment of cytosine methylation, largely due
to the widespread study of such in humans. Although not specifically designed for bacteria, these are easily
transferable for the study of bacterial cytosine methylation. There are, however, no equivalent methods
available for the study of adenine methylation. In bacteria, the ability to assess adenine methylation is vitally
important given the range of roles it is implicated in and thus its analysis represents an area of unmet need.
In the short term we would make available a new tool for the study of epigenetic control in bacteria which
would be of benefit in a range of areas outside of AMR. In the medium term the application of the techniques
piloted here could be applied to a more inclusive range of important, clinically relevant Gram negative
pathogens such as Pseudomonas, Klebsiella and Acinetobacter. The ultimate long term goal of this work is the
identification of novel pathways for the development of antimicrobial sensitising agents or circumventing
strategies and policies to the epigenetic adaptive mechanisms. Overall this project adds knowledge to the
pathways involved in AMR, with epigenetic control being of particular importance due to its potential to
produce short term resistance that could lead to stable resistance mutations. The PhD student will work
alongside to develop and validate assays for non-E. coli species and take advantage of the sequencing data to
more fully investigate the mechanisms and key pathways involved in the development of AMR in E. coli using
gene expression analysis.
Please contact Dr Donna Johnson for further details: Donna.Johnson@leedsbeckett.ac.uk
Biological Sciences
Title: Flavivirus Replication: Drug development using high throughput enzymatic analysis
There has been unprecedented change in the geographic distribution of mosquito borne flaviviruses due to
spread of invasive mosquito species, with half the world's population at risk of infection with Dengue (DEN).
Transmission of DEN was reported in France and Croatia in 2010 and in Portugal in 2012. DEN infection causes
flu-like illness, but can develop into severe DEN, which can be lethal. There is a complete lack of effective drug
treatments. Replication enzymes (RNA polymerase) are highly conserved among flaviviruses so this proposal
will use the non-pathogenic Langat virus as a model for enzymatic analysis of replication. New drug treatments
will be identified as well as carrying out informed drug development for virus replication inhibitors.
The virus genome contains short 5' and 3’ untranslated regions (UTR) required for replication. Conserved RNA
stem loops in this UTR region are essential for binding of the RNA polymerase. Examination of the interaction
of the RNA polymerase with these stem loops using single molecule total internal reflection fluorescence
spectroscopy (smTIRF) and fluorescence resonance energy transfer (FRET) will allow investigation of the
behaviour of these interactions at a new level; revealing previously unknown information and opening new
avenues for drug design.
RNA stem loops (15-30 nucleotides long) will be synthesised with fluorophore dyes attached, kinetic and
enzymatic studies using smTIRF and FRET will be carried out to identify the important nucleotide residues for
replication. Hybridization and thermal characterization of RNA stem loops will be carried out. Ensemble
determination of global structure of the RNA stems loops and changes with and without the presence of
replication enzyme will determined. The PhD student will learn about new instrumentation and key techniques
in biochemistry and molecular biology, including data analysis, allowing employment in industry and or
academia post qualification. This proposal will generate novel data using smTIRF and FRET to examine virus
replication for publication in JACS (impact factor 11.44)and/or Journal of Virology (impact factor of 4.6)
submission to the MRC for program grant funding.
Please contact Dr Tara Sabir for further details: t.sabir@leedsbeckett.ac.uk
Biological Sciences
Title: Investigations into the effect of kinesiology taping on pan and muscle function
Kinesiology taping is a technique used to manage musculoskeletal pain and to improve sports performance
and involves applying an elastic cotton-based self-adhesive tape to the skin. It is claimed that kinesiology
taping causes microscopic lifting of subcutaneous tissues and ‘decompression’ of blood vessels and free nerve
endings (nociceptors) removing inflammatory exudates and reducing pain. It is also claimed that kinesiology
taping stimulates cutaneous nerves improving proprioception and kinaesthetic awareness leading to an
improvement in muscle function. Research into the effects of kinesiology taping on pain and muscle function is
in its infancy and reviews of research literature have failed to find sufficient high quality studies. Thus, there is
a need for a programme of research to investigate, in-depth, the physiological effects of kinesiology taping.
The aim of this PhD is to investigate the effects of kinesiology taping on pain and muscle function. The project
will include a scoping review to contextualise the field of study and to identify a specific gap in existing
knowledge worthy of further study. The results of this scoping review will inform the design of a series of
experimental studies on healthy volunteers using state-of-the-art equipment in our newly refurbished pain
and rehabilitation laboratory. These experimental studies are likely to investigate the effect of kinesiology
taping on pain sensitivity responses, skin microperfusion and the contractile properties of superficial skeletal
muscles.
The successful candidate will be expected to conceptualise, design and implement a project for the generation
of new knowledge through original research and the systematic acquisition of data. The project will enable the
candidate to develop a detailed understanding of psychophysiological techniques for research and advanced
academic enquiry through the use of multi-purpose data physiological acquisition systems, quantitative
sensory testing, laser doppler imaging, ultrasound imaging technology and tensiomyography.
Please contact Professor Mark Johnson for further details: m.johnson@leedsbeckett.ac.uk
Biological Sciences
Title: Novel peptide based cannabinoids for Alzheimer's
The original discovery that endogenous cannabinoids (endocannabinoids) were neuroprotective against
amyloid-beta (Aβ) toxicity (Milton 2002) suggested that activation of the endocannabinoid system has the
potential for development as a potential therapeutic route for Alzheimer’s disease (AD). Subsequent studies
have shown that cannabinoid based compounds are effective in both in vitro and in vivo models of AD (Aso &
Ferrer 2014). The pepcans are a recently discovered group of cannabinoid receptor-1 (CB1) binding peptides
derived from α- and β- chains of haemoglobin (Bauer et al 2012). The pepcans are found throughout the CNS,
and the N-terminally extended forms are specific CB1 agonists (Gomes et al 2009) that may have potential as
neuroprotective agents against Aβ as a result (Milton 2002; Milton 2005). Studies have suggested that the
levels of α- and β- haemoglobin expression in the CNS of AD patients are reduced (Ferrer et al 2011). There is
also evidence that administration of erythropoietin has the potential to increase the expression of alpha and βhaemoglobin expression in the CNS (Schelshorn et al 2009).
AD currently has no effective therapeutic and cannabinoids have been suggested as possible candidates to
take to clinic (Aso & Ferrer 2014). The pepcans have the advantages of specificity for the CB1 receptors, ability
to cross the blood brain barrier (Heimann et al 2007) and have also been found to be secreted from neuronal
cells (Hofer et al 2015); as such they represent novel endogenous cannabinoids that could be used as potential
therapeutics for AD.
The project will specifically evaluate the effect of pepcans in a neuronal cell line model of AD (Milton 2002).
Using neuronal overexpression of pepcan, haemoglobin and erythropoietin genes and/or modified sequences
the role of these compounds in protection against Aβ toxicity will be examined (Milton et al 2012; Chilumuri et
al 2013). All of the techniques to be used have previously been used in research projects by the supervisory
team and the Centre for Biomedical Research at Leeds Beckett University has all the necessary facilities to
allow the student to carry out this research.
Please contact Dr Nathaniel G N Milton for further details: n.g.milton@leedsbeckett.ac.uk
Closing date is midnight on 30th June 2015.
Details of how to apply can be found at: http://www.leedsbeckett.ac.uk/research/researchdegrees/research-studentships-and-fees-only-bursaries/