School Research Meeting 2015

Medical School

Abstract Book

CONTENTS

Page no. Content

3 Welcome

4 Invited Guest Bio-sketches

5– 9 Programme

10 – 14 List of Poster Presentations (Numerical)

15 – 20 List of Poster Presentations (Departmental)

21 – 34 Abstracts of Oral Presentations

35 – 115 Abstracts of Poster Presentations

116 Acknowledgements

117 – 118 Notes

WELCOME

Welcome to the Medical School’s 11th Annual Research Meeting.

The aim of the meeting is to bring together all members of the Medical School, to encourage our junior researchers to present their data to a wider audience, and to showcase the breadth of clinical and scientific research ongoing in the School.

This year, the theme for the first day of the meeting is ‘Achieving a successful career beyond University’. The meeting begins with the keynote speaker, Professor Allan Pacey and his talk on ‘Communicating Science’. This is then followed by poster tours and in the afternoon a series of breakout sessions organised by young researchers, to showcase the different career options available following University. Monday is also an opportunity to meet a number of other guests from the pharmaceutical industry, who are here to sample the research environment that the Medical School provides.

On Tuesday, we have a full day of research talks selected from submitted abstracts. At Midday, Sussan Nourshargh, Professor of Microvascular Pharmacology, Barts and the London Medical School will give the Research Meeting Key Note Lecture entitled “Neutrophil-vessel wall interactions in vivo: Mode Mechanisms and Pathogenesis”. Professor Nourshargh will judge the oral and poster presentations in the lunchtime poster session with the assistance of our internal judge, Sherif El-Khamisy, Professor of Molecular Medicine, Department of Molecular Biology and Biotechnology.

As a new addition for 2015, we also have a ‘New Staff Seeking Collaboration’ exhibition, please do take the time to visit the exhibition during the lunch break. We are also piloting questions by twitter, in case people are too shy to raise their hand, however priority will be given to the floor and please remember there is no such thing as a bad question. If you wish to take part, please use the Twitter hashtag #SRM2015.

On behalf of the organising committee we hope you enjoy the meeting, that it is a stimulating experience and that new collaborations develop from interactions with colleagues and guests.

Best wishes

Dr Adrian Higginbottom Chair of Research Meeting Working Group

INVITED GUEST BIO-SKETCHES

Professor Sussan Nourshargh Professor of Microvascular Pharmacology, Barts and the London Medical School Sussan Nourshargh is a pharmacologist who studied at University College London (BSc) and King’s College London (PhD) and became Professor of Immunopharmacology at Imperial College London in 2006. In 2007 she joined Barts and The London Medical School, Queen Mary, University of London, UK, to establish and head a new Centre focusing on Microvascular Research. Her research focuses on unravelling the molecular and cellular events involved in leukocyte trafficking and through the application of advanced in vivo imaging modalities she has made seminal contributions to the field of leukocyte transmigration for which her group is internationally respected. Sussan Nourshargh is a Wellcome Trust Senior Investigator and Fellow of the UK Academy of Medical Sciences, British Pharmacological Society and UK Society of Biology. She is frequently invited to present at international meetings and is a committee member for numerous funding bodies, societies and scientific advisory boards.

Professor Sherif El-Khamisy Professor of Molecular Medicine, University of Sheffield Sherif El-Khamisy is a Wellcome Trust investigator studying the mechanisms underpinning cellular dysfunction due to defective DNA repair. He identified a defect in repairing chromosomal single-strand breaks (SSBs) in two distinct human neurological diseases in 2005 and 2006. This has led to the hypothesis that SSB repair is critical for the genetic integrity in post-mitotic tissue. His lab then focused on how mammalian cells repair protein-linked DNA breaks and their impact on tissue viability. This has led to the discovery of the enzyme that repairs topoisomerase 2 - mediated DNA damage, which was subsequently named TDP2 (Nature 2009). Last year, together with several international and national collaborators, Sherif discovered the first human disease defective in TDP2 (Nature Genetics 2014) and studied DNA repair and ageing in mouse models of ALS (Hum Mol Genet 2014). His lab is exploiting knowledge gained from studies in neurological diseases to improve cancer diagnosis and treatment (Nature Rev Cancer 2015). He has recently been awarded a prestigious Welcome Trust investigator Award and a Lister Institute Research Fellowship.

11th Annual Medical School Research Meeting 15th – 16th June 2015 PROGRAMME

Monday 15th June 2015

Venue: Octagon Centre

09:00-10:00 Poster presenters to hang posters, stand holders and “New Staff” exhibition to set up.

Venue: Student Union Auditorium

10:00-11:00 Plenary Lecture - “Communicating Science” Professor Allan Pacey (Head of Unit, Reproductive and Developmental Medicine)

Venue: Octagon Centre

11:00 -12:00 Hot Research Topics - Poster Technique Tours Organised by Medicine, Dentistry and Health Research Staff Association (MDHRSA)

12:00-13:00 Lunch with the Speakers (for poster tour participants, organisers, speakers and stand hosts only)

13:00 – 15:00 Achieving a successful career beyond University - Breakout session 1

NHS – Meeting Room 1, Octagon Centre 13:00 – Dr Hazel Wilkinson – Operations Manager, UK NEQAS for Immunology 13:30 – Miss Sarah Hawtree – Trainee Healthcare Scientist 14:00 – Dr Keith Hunter – Reader in Oral & Maxillofacial Pathology, Academic Director Oral & Dental, UoS 14:30 – Mrs Samantha Walmsley – Research Support & Management, Clinical Research Network

Academia – Student Union Auditorium, Student Union Building 13:00 – Dr Neil Harris – Research Strategy Manager, Research & Innovation Services, UoS 13:30 – Professor Simon Foster – Director of Krebs Institute, UoS 14:00 – Dr Rosemary Gault – European Project Coordinator, Advanced Manufacturing Park, UoS 14:30 – Dr Isobel Gowers – Senior Lecturer in Animal Therapy, Writtle College

Industry – Council Chamber, Octagon Centre 13:00 – Dr Samantha Decombel – Chief Operating Officer & co-founder MuscleGenes.com 13:30 – Ms Mariam Zare – General Manager – Sales & Marketing, Advanced Medical Solutions 14:00 – Dr Kirstie Bennett – Senior Pharmacologist, Heptares Therapeutics Ltd 14:30 – Dr James Lynch – Clinical Pharmacology Scientist, Astra Zeneca

15:00 – 15:30 Refreshment break

15:30 – 16:30 Achieving a successful career beyond University - Breakout session 2

NHS – Meeting Room 1, Octagon Centre 15:30 – Dr Richard Battle – Senior Healthcare Scientist, National Histocompatibility and Immunogenetics Lab Manager – Scottish National Blood Transfusion Service 16:00 – Dr Cher Cartwright – Senior Information Analyst, Health and Social Care Information Centre

Academia – Student Union Auditorium, Student Union Building 15:30 – Dr Alison Graham – Teaching Fellow, School of Biology, Newcastle University 16:00 – Dr Laura Ferraiuolo – Marie Curie Research Fellow, Neuroscience, UoS

Industry – Council Chamber, Octagon Centre 15:30 – Dr Sam Whitehouse – Chief Operating Officer, QuantuMDx 16:00 – Dr Lee Allen – Purification Research and Technology Lead, Lonza Pharma / Biotech

Venue: Octagon Centre

16:30-17:30 Wine reception and Poster Bay Tours

Tuesday 16th June 2015

Venue: Student Union Auditorium

08:40 Registration for Oral and Poster Presenters

08:55 Welcome and introduction Professor Tony Weetman – Pro-Vice Chancellor, Medicine, Dentistry & Health

Session 1: Chaired by Professor Paul Ince – Head of Department, Neuroscience Co-chaired by Dr Rachel Waller, MDHRSA

09:00 Dr Iwan Evans [Infection & Immunity] Sir Henry Dale Fellow Draper/CED-1 mediates an ancient damage response to control inflammatory blood cell migration in vivo

09:15 Dr Pallai Shillo [Human Metabolism] PhD Student – 2nd Year Low Vitamin D Levels in Patients with Painful Diabetic Peripheral Neuropathy: A Potential Role in Pathogenesis?

09:30 Ms Charlotte Rowan [Oncology] PhD Student – 2nd Year A subset of peripheral blood monocytes exhibit an M2-skewed phenotype after doxorubicin treatment: are these the precursors of relapse-promoting M2 TAMs?

09:45 Mr Simeon Mihayloy [Neuroscience] PhD Student – 2nd Year Investigating the RNA-Binding Activity of PGC-1[alpha] in Neurodegeneration and Ageing

10:00 Mrs Emma MacInnes [Oncology] PhD Student – 2nd Year MRI and mammographic screening in young women at increased familial breast cancer risk

10:15 Dr Paul Morris [Cardiovascular Science] PhD Student – 3rd Year The Development and Validation of an In Silico Model of Coronary Arterial Physiology for Diagnostic Use

10:30 – 11:00 COFFEE / TEA BREAK (OCTAGON CENTRE)

Session 2: Chaired by Professor Sheila Francis – Head of Department, Cardiovascular science Co-chaired by Mr Matthew Sellwood, MPGS

11:00 Dr Esther Hobson [Neuroscience] PhD Student – 2nd Year Facilitating access to specialist care for patients and carers affected by Motor Neurone Disease using telehealth

11:15 Mr Joshua Griffiths [Infection & Immunity] BMedSci Student Role of specific pro resolving mediators in inflammation resolution and regeneration in an in vivo zebrafish model

11:30 Dr Magdalena Adamczyk [Human Metabolism] Arthritis Research UK Foundation Fellow Are P2X7R-/- mice protected from IL-1β-mediated TG2 upregulation and rapid release?

11:45 Dr Alex Rothman [Cardiovascular Science] MRC Clinical Research Training Fellow miR140-5p is a key pathological mediator of pulmonary arterial hypertension and modulates BMP signalling through SMURF1

Keynote Speaker Introduction - Professor Nicola Brown – Deputy Head of Department, Oncology

12:00 KEYNOTE LECTURE: “NEUTROPHIL-VESSEL WALL INTERACTIONS IN VIVO: MODE, MECHANISMS AND PATHOGENESIS” Professor Sussan Nourshargh, Professor of Microvascular Pharmacology, Barts and the London Medical School

13:00 – 15:00 LUNCH, POSTER VIEWING & NETWORKING – OCTAGON CENTRE  13:00-15:00 trade stands  13:30-14:30 2nd Year postgraduate poster presentation assessment  13:30-14:00 viewing of even numbered posters  14:00-14:30 viewing of odd numbered posters  14:30-15:00 New staff seeking collaboration exhibition

Session 3: Chaired by Professor Tim Skerry – Head of Department, Human Metabolism Co-chaired by Miss Claire Bradshaw, MDHRSA

15:00 Dr Clare Howarth [Psychology] Vice Chancellor’s Advanced Fellow Mechanisms of neurovascular coupling: imaging from cell level to whole brain

15:15 Mr Felix Horn [Cardiovascular Science] PhD Student – 4th Year Quantitative 3D lung function measurement -results from patient studies and predictive modelling of global multiple breath washout

15:30 Dr Victoria Stern [Human Metabolism] PhD Student – 2nd Year Assessing the Risk of Spontaneous Premature Birth by Electrical Impedance Spectroscopy of the Cervix: The ECCLIPPx Study

15:45 Dr Fiona Taylor [Oncology] PhD Student – 2nd Year Non-invasive molecular profiling of circulating cell-free DNA in lung cancer

16:00 Dr Jonathan Cooper-Knock [Neuroscience] PhD Student – 3rd Year Antisense and sense RNA foci derived from hexanucleotide repeat expansions of C9ORF72 have similar protein-interactions but distinct neuronal expression patterns

16:15 Dr Andrew Street [Infection & Immunity] Senior Research Fellow Polycystin-1 trafficking is regulated by cAMP dependent phosphorylation of the PLAT domain

16:30 Presentation of Prizes for Oral Presentations & Posters Professor Christopher Newman – Faculty Director for Research & Innovation

16:45 Closing Remarks by Professor Sussan Nourshargh & Professor Sherif El-Khamisy

List of Poster Presentations (In numerical order)

1 Dr Alenka Brookes “I can cope right now, because I know where I have come from”; a qualitative PhD Student - 2nd Year exploration of the lived experience of young people with inflammatory bowel disease 2 Ms Sofia Granados-Aparici Foxl2 and Smad3 transcription factors during early follicle development: PhD Student - 2nd Year localisation and regulation of factors involved in granulosa cell proliferation 3 Miss Rachel Bell Characterisation of the second C5a anaphylatoxin receptor C5a2. PhD Student - 2nd Year 4 Mr Atiqur Rahman Using protein kinase inhibitor compounds to reverse dysregulated neutrophil PhD Student - 2nd Year function in COPD 5 Miss Laura Ratcliffe Investigating impaired IGF1 signalling in human astrocytes and its role in PhD Student - 2nd Year Alzheimer’s Disease Progression 6 Dr Nehal Hussain Interventricular septal angle can be used to predict which patients have combined PhD Student - 2nd Year postcapillary or precapillary pulmonary hypertension in left heart disease 7 Mrs Felicity Fletcher Exploring the impact of a simulation based educational intervention (IMASS: PhD Student - 2nd Year Integrated Medical and Surgical Simulation course) on 5th year medical students confidence as a marker of readiness to engage with Foundation Year 1 (FY1) 8 Mr Jack Green Shear stress dependent regulation of P2X4 and P2X7 receptors in the PhD Student - 2nd Year endothelium 9 Dr Hadel Ghaban Development of a Long- Acting Granulocyte Colony Stimulating Factor Molecule PhD Student - 2nd Year 10 Mr Bandar Baothman Characterization of the isoform of cyclooxygenase that mediates the generation of PhD Student - 2nd Year prostaglandin D2 from human lung mast cells 11 Mr Alejandro Lorente Pons Investigating the mechanisms underlying oligodendrocyte dysfunction in C9ORF72 PhD Student - 2nd Year ALS 12 Miss Marie-Therese Haider The tumour microenvironment – still underestimated as a mediator of anti-cancer PhD Student - 2nd Year therapy? 13 Dr Peter Mooney The clinical and phenotypic assessment of Ultra-Short Coeliac Disease PhD Student - 2nd Year 14 Mr Jude Akinwale Generation of Longer-Acting Coagulation Factor VIII for the Treatment of PhD Student - 2nd Year Haemophilia A 15 Mr Ahmed Al-Obaidy The effect of fluorescence protein tagging on the function of receptors PhD Student - 2nd Year 16 Dr Rohit Bazaz Increased atherosclerotic plaque macrophage content following Streptococcus PhD Student - 2nd Year pneumoniae pneumonia. 17 Miss Irina Vazquez-Villasenor Neuronal senescence as a contributor to neurodegeneration PhD Student - 2nd Year 18 Mr Mohammed Aldughaim The use of a novel peptide to specifically target therapeutic drugs to tumors PhD Student - 2nd Year 19 Mr David Randall Refinement in image processing for detection of abdominal adhesions using cine- MD Student - 2nd Year MRI 20 Miss Hind Naffadi The role of adrenomedullin in breast cancer metastasis to bone MD Student - 2nd Year 21 Ms Furaha Florence Asani Detection of subtle immune defects in patients at risk of pneumococcal disease PhD Student - 2nd Year 22 Dr Leon Lewis Prevalence of work-related respiratory symptoms & obstructive lung function in a Phd Student - 2nd Year cohort of UK-based foundry workers 23 Mr Wen-Yo Tu Selective Vulnerability of Motor Neurones in Spinal Muscular Atrophy PhD Student - 2nd Year 24 Mr Meshal Alhajimohammed Identification of Cancer Stem Cells in Sarcoma PhD Student - 2nd Year 25 Dr Jivendra Gosai POSTER WITHDRAWN PhD Student - 2nd Year 26 Miss Lucy Evans Clinical evaluation of a salivary 25-hydroxyvitamin D method by liquid PhD Student - 2nd Year chromatography tandem mass spectrometry for the assessment of vitamin D status. 27 Dr Ruth Wiggins Respiratory ill-health in woodworkers: a systematic review PhD Student - 2nd Year 28 Miss Chloe Marshall Staphylococcus aureus interactions with human airway glygoproteins PhD Student - 2nd Year

29 Dr Sheila Ramjug Long-term intravenous iloprost in pulmonary arterial hypertension PhD Student - 2nd Year 30 Miss Karla Robles Lopez The role of TIGAR in Parkinson’s Disease PhD Student - 2nd Year 31 Mr Neil Bowden Disturbed flow induces expression of the negative NF-kappaB regulator Cezanne PhD Student - 2nd Year 32 Miss Dingyi Yang Identifying novel immune modulating factors in a genome-wide S. aureus screen in PhD Student - 2nd Year human neutrophils 33 Mr Joseph Ford The Role of Nitric Oxide in Streptococcus pneumoniae Induced Lysosomal Phd Student - 2nd Year Membrane Permeabilisation 34 Miss Jodie Stephenson Characterisation of a novel pre-clinical model of Motor Neurone Disease (MND) PhD Student - 2nd Year 35 Mr Andrew Nichols Single nucleotide polymorphisms of the IL-1RI co-receptor, TILRR, IMPACT IL-1R1 PhD Student - 2nd Year controlled responses 36 Dr Emma Walkinshaw POSTER WITHDRAWN PhD Student - 2nd Year 37 Dr Fatma Hamed Inhibitors of IFN-g signalling pathway to restore immune privilege and enhance hair PhD Student - 2nd Year re-growth in alopecia areata patients 38 Ms Natalie Rounding Zebrafish C9orf72 loss of function models of Amyotrophic Lateral Sclerosis (ALS) PhD Student - 2nd Year 39 Mr Luke McKenzie A novel transition metal complex for use as a photosensitizer in the photodynamic PhD Student - 2nd Year therapy of cancer 40 Md Martina Sciola Quantitative computational evaluation of effect of coronary lesions on PhD Student - 2nd Year cardiovascular physiology 41 Miss Andreea Ciuntu The role of tetraspanins in neutrophil apoptosis and phagocytosis PhD Student - 2nd Year 42 Mr Taiwo Oguntona Reducing Renal fibrosis using a selective carboxypeptidase U inhibitor UK-396082 PhD Student - 2nd Year 43 Ms Evangelina Karyka RNA changes in SMN-deficient experimental cell models of Spinal Muscular PhD Student - 2nd Year Atrophy 44 Mr Simon Webster Detection Of Large Exonic And Intergenic Deletions In The Vwf Locus Of Vwd PhD Student - 2nd Year Patients Using Array Comparative Genomic Hybridisation (aCGH) 45 Dr Preethi Rao Testosterone Replacement Therapy Has Beneficial Effects on Cardiovascular Risk PhD Student - 2nd Year Factors and Liver Function in Hypogonadal Men 46 Miss Laura Vergoz Identification of new candidate gene and miRNA networks in the pathogenesis of PhD Student - 2nd Year ADPKD 47 Dr Ruth Payne Safety and Immunogenicity of the Blood-stage Plasmodium vivax Vaccine ChAd63- PhD Student - 2nd Year MVA PvDBP 48 Mr Harvey Leung Exploring the anti-inflammatory mechanism of electrical vagus nerve stimulation. PhD Student - 2nd Year 49 Dr Tariq Aabed Radiation effects on the differentiation of mesenchymal stromal cells into Phd Student - 2nd Year myofibroblasts and pericytes and development of fibrosis in soft tissue sarcoma. 50 Dr Rob Morton Randomised control trial to investigate whether electronic adherence monitoring Phd Student - 2nd Year with feedback and reminder alarms can improve adherence and outcomes in childhood asthma. 51 Dr Ascanio Tridente Association between trends in clinical variables and outcome in intensive care PhD Student - 2nd Year patients with faecal peritonitis: analysis of the GenOSept cohort 52 Dr Shamsa Ihmed Identification of specific genetic changes associated with Conjunctival Melanoma PhD Student - 2nd Year 53 Dr Melissa Hale Randomised comparison of a standard protocol using metoclopramide versus a PhD Student - 2nd Year hand held magnet to enhance gastric emptying of the small bowel capsule. 54 Mrs Jehan Alrahimi The Role of Tetraspanins in Bacterial Pathogenicity PhD Student - 2nd Year 55 Miss Roxanne Lau The streptococcal nuclease: a potential drug target PhD Student - 2nd Year 56 Dr Arash Assadsangabi The Fate Of Epithelial Keratins In Active Ulcerative Colitis PhD Student - 2nd Year 57 Mrs Amira Zawia Investigating the role of macrophage subsets in MacLow mouse and in patients PhD Student - 2nd Year with pulmonary arterial hypertension. 58 Ms Sarah Almaghrabi Gene therapy for an experimental autoimmune polyglandular syndrome 1 model PhD Student - 2nd Year caused by mutations in AIRE 59 Dr Victoria Gordon Multi-Centre Audit Of Management And Outcome In Autoimmune Hepatitis (AIH) – PhD Student - 2nd Year preliminary results

60 Mrs Khlood Mehdar HDAC6 inhibition as a therapeutic approach in Hereditary Spastic Paraplegia PhD Student - 2nd Year 61 Dr Karl Pang Identification and diagnostic performance of a small RNA within the PCA3 and PhD Student - 2nd Year BMCC1 gene locus that potentially targets mRNA 62 Miss Sayali Haldipurkar Role of ECF sigma factors in stress responses of Burkholderia cenocepacia PhD Student - 2nd Year 63 Miss Jessica Johnson Assessment of plaque macrophage phenotype in situ by multicolour fluorescence PhD Student - 2nd Year microscopy. 64 Miss Lucy Morris The effect of macrophage polarisation and delayed apoptosis in the innate immune PhD Student - 2nd Year response to S. pneumoniae infection 65 Dr Hamad Alzahrani Apathy may lead to dementia in patients with Parkinson’s Disease Postdoctoral Fellow 66 Ms Lauren Edwards Using High-Resolution Peripheral Quantitative Computed Tomography (HRpQCT) BMedSci Student - 4th Year to Better Understand the Skeletal Response to Exercise 67 Mr Alfred Kamuyango How do fungal pathogens initiate and modulate immune signalling? PhD Student - 1st Year 68 Dr Alex Wheeler Exploring Prescribing Habits in the Emergency Department- a Need For a Change Foundation Year 2 in Culture 69 Dr Alisdair McNeill SOX11 deletions and mutations in human developmental disorders Senior Clinical Fellow 70 Ms Joanna Chowdry Novel Predictive Biomarkers of Energy Intake Research Technician 71 Miss Hannah Dudhill The potential for community-based alternative care pathways for patients after BMedSci Student - 4th Year suspected seizures 72 Dr Mabruka Alfaidi Dietary Docosahexaenoic Acid Reduced Experimental Atherosclerosis by Inducing PhD Student - 4th Year Protective Hemodynamic Conditions. 73 Miss Heledd Brown-Wright Metabotropic glutamate receptor 5 (mGlu5) as a novel drug target in motor PhD Student – 1st Year neuron disease (MND). 74 Mr Mahmoud Habibullah Epitopes, Specificity, Functional Effects and Immunoglobulin G Subclasses of PhD Student - 3rd Year Calcium-Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 75 Mr Joseph Abrams Analysis of fungemic and non-fungemic Candida isolates from the Sheffield Special BMedSci Student - 3rd Year Care Baby Unit 76 Dr Paul Morris Methicillin-resistant Staphylococcus aureus (MRSA) USA300 Evades Eradication Academic Clinical Fellow Following Phagocytosis by Human Macrophage Cell Line 77 Dr Julie Simpson Neuronal DNA damage response-associated dysregulation of signalling pathways Research Fellow and cholesterol metabolism at the earliest stages of Alzheimer-type pathology 78 Ms Marwa Mahmoud Disturbed flow promotes atherogenesis through the activation of endothelial- PhD Student – 3rd Year mesenchymal transition 79 Miss Sarah Rennicks Anakinra inhibits growth of breast cancer cells in mice; in vitro and in vivo effects Placement Student on breast cancer bone metastasis. 80 Mr Billy Bryan Can feedback informed by self-regulatory microanalysis improve skills PhD Student - 1st Year performance? Insights from a systematic review 81 Mr Zabran Ilyas miRNA202 Is A Novel Regulator Of Tribbles-1 Expression PhD Student - 1st Year 82 Ms Jess Warrington Using CRISPR knockout technology to explore the role of adrenomedullin in PhD Student - 1st Year tumour metastasis 83 Ms Aylin Metzner Drug discovery for ADPKD using a zebrafish pkd2 model Marie Curie Early Researcher 84 Dr Claire Garwood The insulin signalling pathway in human astrocytes in vitro and in vivo; Research Associate characterisation, subcellular localisation and modulation at the receptors. 85 Mr Steven Bradbury The role of IL-1β in breast cancer bone metastasis. Placement Student 86 Miss Katrina Williams Investigating interactions between human spermatozoa and human PhD Student – 3rd Year Cytomegalovirus (CMV). 87 Mr David Rhodes Computational modelling of NF-κB activation by IL-1RI and its Co-receptor TILRR, Research Associate predicts a role for cytoskeletal sequestration of IκBα in inflammatory signalling. 88 Mr Paris Avgoustou Targeting Adrenomedullin (AM) as a treatment of pancreatic cancer PhD Student - 1st Year 89 Dr Zainal Abedin Are Leptin, DPP-IV, CXCL-16 and IL-8 useful biomarkers of CKD outcomes? PhD Student - 4th Year 90 Dr Rachel Waller Blood-based miRNAs potential biomarkers in motor neuron disease Research Associate

91 Mr Lewis Quayle Developing a Novel In Vitro Model for Study of Molecular Mechanisms Implicated PhD Student - 1st Year in Breast Tumour Cell Dormancy 92 Dr Steven Reynolds Direct arterial injection of hyperpolarised substrate for rapid detection of Research Fellow metabolism with minimal substrate dilution 93 Dr Steven Reynolds Investigating the properties of silk formation in Bombyx mori silkworms using T1 Research Fellow and T2 image maps. 94 Miss Hana Alloub Validating Finite Element Model of Children's Bones Medical Student 95 Dr Philip Elks Hypoxia Signaling Modulates Neutrophil Nitric Oxide in a Zebrafish Tuberculosis Sir Henry Dale Fellow Model 96 Dr Matteo De Marco Apolipoprotein E ε4 Allele Influences Functional Connectivity in Mild Cognitive Postdoctoral Researcher Impairment 97 Dr Shobna Silva Cell-free DNA as a non-invasive marker of relapse/progression in melanoma Clinical Fellow 98 Dr Shuang Feng Disturbed flow induces glycolysis enzymes at atheroprone sites Research Associate 99 Dr Robin Rumney Lysyl oxidase drives osteolytic bone lesions in a breast cancer model via RANK Postdoctoral Research Associate ligand independent effects on osteoclasts: a new player in the vicious cycle? 100 Ms Nelly Wagner Possible modulation of the innate immune response by the cell wall of PhD Student - 3rd Year Staphylococcus aureus. 101 Dr Andrea Turolla Role of BDNF Val66Met polymorphism on neuroanatomy of motor recovery after PhD Student - 3rd Year stroke. 102 Dr Safar Kheder Importance of the OCT1 transporter to the anti-cancer effect of metformin in PhD Student - 3rd Year thyroid cancer 103 Dr Nikolay Ogryzko Zebrafish tissue injury causes up-regulation of interleukin-1 and caspase Post-Doctoral Research Associate dependent amplification of the inflammatory response. 104 Dr Karan Shah Mechanosensing in osteocytes following exposure to clinically relevant Post-Doctoral Research Associate concentrations of metal ions after hip replacement 105 Miss Charlotte Gee The regulation and role of Pellino-1 in human monocyte-derived macrophages in Research Technician COPD 106 Mr Luke Flannery The Effects of Anti-Cancer Therapies on the Bone Microenvironment Placement Student 107 Dr Daniel Kelly Evidence that testosterone improves glucose utilisation and a ‘buffering’ effect of Post-Doctoral Research Associate subcutaneous fat to protect against ‘overspill’ of lipid deposition into visceral fat and non-adipose tissues. 108 Dr Khondoker Akram An innate defence role for BPIFA1/SPLUNC1 against influenza-A virus infection Postdoctoral Research Associate 109 Dr Joby Cole Changes in histone post-translational modifications arising from Streptococcus Clinical Research Training Fellow pneumoniae infection in primary macrophages. 110 Miss Caroline Carta Automatic versus executive semantic memory impairment in early Alzheimer’s PhD Student - 3rd Year disease: A possible diagnostic tool? 111 Mr Tim Ingham-Dempster A Multi-Scale Agent Based Model of Colon Carcinogenesis PhD Student - 1st Year 112 Dr Sarah Smith Altered Regulation of the inhibitor IκBα and NF-κB regulated genes in the TILRR KO Research Associate mouse 113 Miss Priyanka Anujan Towards developing in vitro models for primary ciliary dyskinesia (PCD) PhD Student - 1st Year 114 Dr Adebowale Adekoya Investigating endothelial dysfunction in patients with ADPKD PhD Student - 3rd Year 115 Mr Jack Hurrell An in-vitro model for studying low dose ionising radiation-induced damage in PhD Student - 1st Year isolated cardiac endothelial cells. 116 Miss Rebecca Collins Synthesis and hyperpolarisation of [13C]-Combretastatin A1 PhD Student - 4th Year 117 Miss Rowena Speak A long-acting growth hormone antagonist for the treatment of acromegaly BMedSci Student - 4th Year 118 Miss Justyna Serba POSTER WITHDRAWN PhD Student - 4th Year 119 Mr Jonathan Atkinson A preliminary proteomic analysis of the response of human bone metastatic breast Work Placement Student cancer cells to the loss of DOCK4 120 Mr Ho-Fung Chan Lobar comparison of CT densitometry and 3He diffusion MRI models of lung PhD Student - 1st Year microstructure in asthmatics 121 Dr Miguel Debono Modelling the salivary cortisone to serum cortisol inter-relationship to predict Clinical Lecturer serum cortisol under physiological conditions and after hydrocortisone 122 Miss Renata Caikauskaite Host defence functions of BPIFA1/SPLUNC1 PhD Student - 1st Year 13

123 Dr Jules Westbrook Molecules and mechanisms central to breast cancer metastasis to bone: A Postdoctoral Researcher quantitative proteomics approach – preliminary evidence 124 Dr Marni Greig Brain perfusion in painful diabetic neuropathy PhD Student - 3rd Year 125 Miss Hannah Roberts Identifying therapeutic targets for muscular dystrophy following macrophage Masters Student depletion 126 Dr Katherine Henry Integrating human and zebrafish studies to find new drug targets in the neutrophil Post-Docorate kinome 127 Dr Peter Novodvorsky Generation and characterisation of a stable klf2a mutant zebrafish MRC Clinical Fellow 128 Miss Maria Digby High resolution peripheral quantitative computed tomography for the assessment PhD Student - 4th Year of bone microstructure in mild and severe osteogenesis imperfecta 129 Mr MD Mohasin Pneumococcal infection exacerbates chronic obstructive pulmonary disease PhD Student - 1st Year (COPD) through changing metabolic status and phenotype of macrophages 130 Mr Henry Greenslade An immunohistochemical study investigating the interaction of P2X7 and LOX in BMedSci Student - 1st Year mammary tumours and the pre-metastatic niche in bone 131 Miss Apoorva Mulay Role of BPIFA1 in Otitis media PhD Student - 3rd Year 132 Miss Emily Fisk Bone marrow-derived macrophage responses following detection of PhD Student - 1st Year Streptococcus pneumoniae infection via pattern recognition receptors 133 Dr Emma Johnson Co-operation during Staphylococcus aureus pathogenesis Clinical Research Fellow 134 Dr Martin Bewley COPD alveolar macrophages show reduced apoptosis and bacterial killing linked to PhD Student a failure to induce mitochondrial reactive oxygen species upon Spn challenge 135 Mr Thomas Kennelly Comparison of the proteoglycan and integrin binding interaction with fibronectin PhD Student using single molecule force spectroscopy 136 Mr Paolo Prosseda The role of primary cilium stability in the pathogenesis of autosomal polycystic PhD Student – 3rd Year kidney diesease

List of Poster Presentations (In Departmental order)

Department of Cardiovascular Science

6 Dr Nehal Hussain Interventricular septal angle can be used to predict which patients have combined PhD Student - 2nd Year postcapillary or precapillary pulmonary hypertension in left heart disease 8 Mr Jack Green Shear stress dependent regulation of P2X4 and P2X7 receptors in the endothelium PhD Student - 2nd Year 13 Dr Peter Mooney The clinical and phenotypic assessment of Ultra-Short Coeliac Disease PhD Student - 2nd Year 19 Mr David Randall Refinement in image processing for detection of abdominal adhesions using cine-MRI PhD Student - 2nd Year 25 Dr Jivendra Gosai POSTER WITHDRAWN PhD Student - 2nd Year 31 Mr Neil Bowden Disturbed flow induces expression of the negative NF-kappaB regulator Cezanne PhD Student - 2nd Year 35 Mr Andrew Nichols Single Nucleotide Polymorphisms Of The IL-1RI Co-Receptor, TILRR, IMPACT IL-1R1 PhD Student - 2nd Year Controlled Responses 40 Ms Martina Sciola Quantitative computational evaluation of effect of coronary lesions on cardiovascular PhD Student - 2nd Year physiology 44 Mr Simon Webster Detection Of Large Exonic And Intergenic Deletions In The Vwf Locus Of Vwd Patients PhD Student - 2nd Year Using Array Comparative Genomic Hybridisation (aCGH) 53 Dr Melissa Hale Randomised comparison of a standard protocol using metoclopramide versus a hand PhD Student - 2nd Year held magnet to enhance gastric emptying of the small bowel capsule. 57 Mrs Amira Zawia Investigating the role of macrophage subsets in MacLow mouse and in patients with PhD Student - 2nd Year pulmonary arterial hypertension. 63 Miss Jessica Johnston Assessment of plaque macrophage phenotype in situ by multicolour fluorescence PhD Student - 2nd Year microscopy. 72 Dr Mabruka Alfaidi Dietary Docosahexaenoic Acid Reduced Experimental Atherosclerosis by Inducing PhD Student - 4th Year Protective Hemodynamic Conditions. 78 Ms Marwa Mahmoud Disturbed flow promotes atherogenesis through the activation of endothelial- PhD Student – 3rd Year mesenchymal transition 81 Mr Zabran Ilyas miRNA202 Is A Novel Regulator Of Tribbles-1 Expression PhD Student - 1st Year 87 Mr David Rhodes Computational modelling of NF-κB activation by IL-1RI and its Co-receptor TILRR, Research Associate predicts a role for cytoskeletal sequestration of IκBα in inflammatory signalling. 92 Dr Steven Reynolds Direct arterial injection of hyperpolarised substrate for rapid detection of metabolism Research Fellow with minimal substrate dilution 93 Dr Steven Reynolds Investigating the properties of silk formation in Bombyx mori silkworms using T1 and Research Fellow T2 image maps. 98 Dr Shuang Feng Disturbed flow induces glycolysis enzymes at atheroprone sites Research Associate 103 Dr Nikolay Ogryzko Zebrafish tissue injury causes up-regulation of interleukin-1 and caspase dependent Post-Doctoral Research Associate amplification of the inflammatory response. 112 Dr Sarah Smith Altered Regulation of the inhibitor IκBα and NF-κB regulated genes in the TILRR KO Research Associate mouse 116 Miss Rebecca Collins Synthesis and hyperpolarisation of [13C]-Combretastatin A1 PhD Student - 4th Year 120 Mr Ho-Fung Chan Lobar comparison of CT densitometry and 3He diffusion MRI models of lung PhD Student - 1st Year microstructure in asthmatics 124 Dr Marni Greig Brain perfusion in painful diabetic neuropathy PhD Student - 3rd Year 127 Dr Peter Novodvorsky Generation and characterisation of a stable klf2a mutant zebrafish MRC Clinical Fellow 135 Mr Thomas Kennelly Comparison of the proteoglycan and integrin binding interaction with fibronectin PhD Student - 2nd Year using single molecule force spectroscopy

Department of Human Metabolism

2 Ms Sofia Granados-Aparici Foxl2 and Smad3 transcription factors during early follicle development: localisation PhD Student - 2nd Year and regulation of factors involved in granulosa cell proliferation 9 Dr Hadel Ghaban Development of a Long- Acting Granulocyte Colony Stimulating Factor Molecule PhD Student - 2nd Year 14 Mr Jude Akinwale Generation of Longer-Acting Coagulation Factor VIII for the Treatment of Haemophilia PhD Student - 2nd Year A 20 Miss Hind Naffadi The role of adrenomedullin in breast cancer metastasis to bone PhD Student - 2nd Year 26 Miss Lucy Evans Clinical evaluation of a salivary 25-hydroxyvitamin D method by liquid chromatography PhD Student - 2nd Year tandem mass spectrometry for the assessment of vitamin D status. 36 Dr Emma Walkinshaw POSTER WITHDRAWN PhD Student - 2nd Year 45 Dr Preethi Rao Testosterone Replacement Therapy Has Beneficial Effects on Cardiovascular Risk PhD Student - 2nd Year Factors and Liver Function in Hypogonadal Men 50 Dr Rob Morton Randomised control trial to investigate whether electronic adherence monitoring with PhD Student - 2nd Year feedback and reminder alarms can improve adherence and outcomes in childhood asthma. 66 Ms Lauren Edwards Using High-Resolution Peripheral Quantitative Computed Tomography (HRpQCT) to BMedSci Student - 4th Year Better Understand the Skeletal Response to Exercise 74 Mr Mahmoud Habibullah Epitopes, Specificity, Functional Effects and Immunoglobulin G Subclasses of Calcium- PhD Student - 3rd Year Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 82 Ms Jess Warrington Using CRISPR knockout technology to explore the role of adrenomedullin in tumour PhD Student - 1st Year metastasis 86 Miss Katrina Williams Investigating interactions between human spermatozoa and human Cytomegalovirus PhD Student – 3rd Year (CMV). 88 Mr Paris Avgoustou Targeting Adrenomedullin (AM) as a treatment of pancreatic cancer PhD Student - 1st Year 94 Miss Hana Alloub Validating Finite Element Model of Children's Bones Medical Student 99 Dr Robin Rumney Lysyl oxidase drives osteolytic bone lesions in a breast cancer model via RANK ligand Postdoctoral Research Associate independent effects on osteoclasts: a new player in the vicious cycle? 104 Dr Karan Shah Mechanosensing in osteocytes following exposure to clinically relevant concentrations Post-Doctoral Research Associate of metal ions after hip replacement 107 Dr Daniel Kelly Evidence that testosterone improves glucose utilisation and a ‘buffering’ effect of Post-Doctoral Research Associate subcutaneous fat to protect against ‘overspill’ of lipid deposition into visceral fat and non-adipose tissues. 117 Miss Rowena Speak A long-acting growth hormone antagonist for the treatment of acromegaly BMedSci Student - 4th Year 121 Dr Miguel Debono Modelling the salivary cortisone to serum cortisol inter-relationship to predict serum Clinical Lecturer cortisol under physiological conditions and after hydrocortisone 125 Miss Hannah Roberts Identifying therapeutic targets for muscular dystrophy following macrophage Masters Student depletion 128 Miss Maria Digby High resolution peripheral quantitative computed tomography for the assessment of PhD Student - 4th Year bone microstructure in mild and severe osteogenesis imperfecta 130 Mr Henry Greenslade An immunohistochemical study investigating the interaction of P2X7 and LOX in BMedSci Student - 1st Year mammary tumours and the pre-metastatic niche in bone

Department of Infection & Immunity

3 Miss Rachel Bell Characterisation of the second C5a anaphylatoxin receptor C5a2. PhD Student - 2nd Year 4 Mr Atiqur Rahman Using protein kinase inhibitor compounds to reverse dysregulated neutrophil function PhD Student - 2nd Year in COPD 10 Mr Bandar Baothman Characterization of the isoform of cyclooxygenase that mediates the generation of PhD Student - 2nd Year prostaglandin D2 from human lung mast cells 15 Mr Ahmed Al-Obaidy The effect of fluorescence protein tagging on the function of receptors PhD Student - 2nd Year 16 Dr Rohit Bazaz Increased atherosclerotic plaque macrophage content following Streptococcus PhD Student - 2nd Year pneumoniae pneumonia. 21 Ms Furaha Florence Asani Detection of subtle immune defects in patients at risk of pneumococcal disease PhD Student - 2nd Year 22 Dr Leon Lewis Prevalence of work-related respiratory symptoms & obstructive lung function in a PhD Student - 2nd Year cohort of UK-based foundry workers 27 Dr Ruth Wiggans Respiratory ill-health in woodworkers: a systematic review PhD Student - 2nd Year 28 Miss Chloe Marshall Staphylococcus aureus interactions with human airway glygoproteins PhD Student - 2nd Year 29 Dr Sheila Ramjug Long-term intravenous iloprost in pulmonary arterial hypertension PhD Student - 2nd Year 32 Miss Dingyi Yang Identifying novel immune modulating factors in a genome-wide S. aureus screen in PhD Student - 2nd Year human neutrophils 33 Mr Joseph Ford The Role of Nitric Oxide in Streptococcus pneumoniae Induced Lysosomal Membrane PhD Student - 2nd Year Permeabilisation 37 Dr Fatma Hamed Inhibitors of IFN-g signalling pathway to restore immune privilege and enhance hair re- PhD Student - 2nd Year growth in alopecia areata patients 41 Miss Andreea Ciuntu The role of tetraspanins in neutrophil apoptosis and phagocytosis PhD Student - 2nd Year Reducing Renal fibrosis using a selective carboxypeptidase U inhibitor UK-396082 42 Mr Taiwo Oguntona Identification of new candidate gene and miRNA networks in the pathogenesis of PhD Sudent - 2nd Year ADPKD 46 Miss Laura Vergoz Identification of new candidate gene and miRNA networks in the pathogenesis of PhD Student - 2nd Year ADPKD 47 Dr Ruth Payne Safety and Immunogenicity of the Blood-stage Plasmodium vivax Vaccine ChAd63-MVA PhD Student - 2nd Year PvDBP 51 Dr Ascanio Tridente Association between trends in clinical variables and outcome in intensive care patients PhD Student - 2nd Year with faecal peritonitis: analysis of the GenOSept cohort 54 Mrs Jehan Alrahimi The Role of Tetraspanins in Bacterial Pathogenicity PhD Student - 2nd Year 55 Miss Roxanne Lau The streptococcal nuclease: a potential drug target PhD Student - 2nd Year 58 Ms Sarah Almaghrabi Gene therapy for an experimental autoimmune polyglandular syndrome 1 model PhD Student - 2nd Year caused by mutations in AIRE 59 Dr Victoria Gordon Multi-Centre Audit Of Management And Outcome In Autoimmune Hepatitis (AIH) – PhD Student - 2nd Year Preliminary Results 62 Miss Sayali Haldipurkar Role of ECF sigma factors in stress responses of Burkholderia cenocepacia PhD Student - 2nd Year 64 Miss Lucy Morris The effect of macrophage polarisation and delayed apoptosis in the innate immune PhD Student – 2nd Year response to S. pneumoniae infection 67 Mr Alfred Kamuyango How do fungal pathogens initiate and modulate immune signalling? PhD Student - 1st Year 75 Mr Joseph Abrams Analysis of fungemic and non-fungemic Candida isolates from the Sheffield Special BMedSci Student - 3rd Year Care Baby Unit 76 Dr Paul Morris Methicillin-resistant Staphylococcus aureus (MRSA) USA300 Evades Eradication Academic Clinical Fellow Following Phagocytosis by Human Macrophage Cell Line 83 Ms Aylin Metzner Drug discovery for ADPKD using a zebrafish pkd2 model Marie Curie Early Researcher 89 Dr Zainal Abedin Are Leptin, DPP-IV, CXCL-16 and IL-8 useful biomarkers of CKD outcomes? PhD Student - 4th Year 95 Dr Philip Elks Hypoxia Signaling Modulates Neutrophil Nitric Oxide in a Zebrafish Tuberculosis Model Sir Henry Dale Fellow

100 Ms Nelly Wagner Possible modulation of the innate immune response by the cell wall of Staphylococcus PhD Student - 3rd Year aureus. 105 Miss Charlotte Gee The regulation and role of Pellino-1 in human monocyte-derived macrophages in COPD Research Technician 108 Dr Khondoker Akram An innate defence role for BPIFA1/SPLUNC1 against influenza-A virus infection Postdoctoral Research Associate 109 Dr Joby Cole Changes in histone post-translational modifications arising from Streptococcus Clinical Research Training Fellow pneumoniae infection in primary macrophages. 113 Miss Priyanka Anujan Towards developing in vitro models for primary ciliary dyskinesia (PCD) PhD Student - 1st Year 114 Dr Adebowale Adekoya Investigating endothelial dysfunction in patients with ADPKD PhD Student - 3rd Year 118 Miss Justyna Serba POSTER WITHDRAWN PhD Student - 4th Year 122 Miss Renata Caikauskaite Host defence functions of BPIFA1/SPLUNC1 PhD Student - 1st Year 126 Dr Katherine Henry Integrating human and zebrafish studies to find new drug targets in the neutrophil Post-Docorate kinome 129 Mr MD Mohasin Pneumococcal infection exacerbates chronic obstructive pulmonary disease (COPD) PhD Student - 1st Year through changing metabolic status and phenotype of macrophages 131 Miss Apoorva Mulay Role of BPIFA1 in Otitis media PhD Student - 3rd Year 132 Miss Emily Fisk Bone marrow-derived macrophage responses following detection of Streptococcus PhD Student - 1st Year pneumoniae infection via pattern recognition receptors 133 Dr Emma Johnson Co-operation during Staphylococcus aureus pathogenesis Clinical Research Fellow 134 Dr Martin Bewley COPD alveolar macrophages show reduced apoptosis and bacterial killing linked to a PhD Student - 2nd Year failure to induce mitochondrial reactive oxygen species upon Spn challenge 136 Mr Paolo Prosseda The role of primary cilium stability in the pathogenesis of autosomal polycystic kidney PhD Student - 2nd Year diesease

Department of Medical Education

7 Mrs Felicity Fletcher Exploring the impact of a simulation based educational intervention (IMASS: PhD Student - 2nd Year Integrated Medical and Surgical Simulation course) on 5th year medical students confidence as a marker of readiness to engage with Foundation Year 1 (FY1) 80 Mr Billy Bryan Can feedback informed by self-regulatory microanalysis improve skills performance? PhD Student - 1st Year Insights from a systematic review

Department of Neuroscience

5 Miss Laura Ratcliffe Investigating impaired IGF1 signalling in human astrocytes and its role in Alzheimer’s PhD Student - 2nd Year Disease Progression 11 Mr Alejandro Lorente Pons Investigating the mechanisms underlying oligodendrocyte dysfunction in C9ORF72 ALS PhD Student - 2nd Year 17 Miss Irina Vazquez-Villaseñor Neuronal senescence as a contributor to neurodegeneration PhD Student - 2nd Year 23 Mr Wen-Yo Tu Selective Vulnerability of Motor Neurones in Spinal Muscular Atrophy PhD Student - 2nd Year 30 Miss Karla Robles Lopez The role of TIGAR in Parkinson’s Disease PhD Student - 2nd Year 34 Miss Jodie Stephenson Characterisation of a novel pre-clinical model of Motor Neurone Disease (MND) PhD Student - 2nd Year 38 Ms Natalie Rounding Zebrafish C9orf72 loss of function models of Amyotrophic Lateral Sclerosis (ALS) PhD Student - 2nd Year 43 Ms Evangelia Karyka RNA changes in SMN-deficient experimental cell models of Spinal Muscular Atrophy PhD Student - 2nd Year 48 Mr Harvey Leung Exploring the anti-inflammatory mechanism of electrical vagus nerve stimulation. PhD Student - 2nd Year 60 Mrs Khlood Mehdar HDAC6 inhibition as a therapeutic approach in Hereditary Spastic Paraplegia PhD Student - 2nd Year 65 Dr Hamad Alzahrani Apathy may lead to dementia in patients with Parkinson’s Disease Postdoctoral Fellow 18

69 Dr Alisdair McNeill SOX11 deletions and mutations in human developmental disorders Senior Clinical Fellow 73 Miss Heledd Brown-Wright Metabotropic glutamate receptor 5 (mGlu5) as a novel drug target in motor neuron PhD Student – 1st Year disease (MND) 77 Dr Julie Simpson Neuronal DNA damage response-associated dysregulation of signalling pathways and Research Fellow cholesterol metabolism at the earliest stages of Alzheimer-type pathology 84 Dr Claire Garwood The insulin signalling pathway in human astrocytes in vitro and in vivo; Research Associate characterisation, subcellular localisation and modulation at the receptors. 90 Dr Rachel Waller Blood-based miRNAs potential biomarkers in motor neuron disease Research Associate 96 Dr Matteo De Marco Apolipoprotein E ε4 Allele Influences Functional Connectivity in Mild Cognitive Postdoctoral Researcher Impairment 101 Dr Andrea Turolla Role of BDNF Val66Met polymorphism on neuroanatomy of motor recovery after PhD Student - 3rd Year stroke. 110 Miss Caroline Carta Automatic versus executive semantic memory impairment in early Alzheimer’s disease: PhD Student - 3rd Year A possible diagnostic tool?

Department of Oncology

1 Dr Alenka Brookes “I can cope right now, because I know where I have come from”; a qualitative PhD Student - 2nd Year exploration of the lived experience of young people with inflammatory bowel disease 12 Miss Marie-Therese Haider The tumour microenvironment – still underestimated as a mediator of anti-cancer PhD Student - 2nd Year therapy? 18 Mr Mohammed Aldughaim The use of a novel peptide to specifically target therapeutic drugs to tumors PhD Student - 2nd Year 24 Mr Meshal Alhajimohammed Identification of Cancer Stem Cells in Sarcoma PhD Student - 2nd Year 39 Mr Luke McKenzie A novel transition metal complex for use as a photosensitizer in the photodynamic PhD Student - 2nd Year therapy of cancer 49 Dr Tariq Aabed Radiation effects on the differentiation of mesenchymal stromal cells into PhD Student - 2nd Year myofibroblasts and pericytes and development of fibrosis in soft tissue sarcoma. 52 Dr Shamsa Ihmed Identification of specific genetic changes associated with Conjunctival Melanoma PhD Student - 2nd Year 56 Dr Arash Assadsangabi The Fate Of Epithelial Keratins In Active Ulcerative Colitis PhD Student - 2nd Year 61 Dr Karl Pang Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 PhD Student – 2nd Year gene locus that potentially targets mRNA 70 Ms Joanna Chowdry Novel Predictive Biomarkers of Energy Intake Research Technician 79 Miss Sarah Rennicks Anakinra inhibits growth of breast cancer cells in mice; in vitro and in vivo effects on Placement Student breast cancer bone metastasis. 85 Mr Steven Bradbury The role of IL-1β in breast cancer bone metastasis. Placement Student 91 Mr Lewis Quayle Developing a Novel In Vitro Model for Study of Molecular Mechanisms Implicated in PhD Student - 1st Year Breast Tumour Cell Dormancy 97 Dr Shobna Silva Cell-free DNA as a non-invasive marker of relapse/progression in melanoma Clinical Fellow 102 Dr Safar Kheder Importance of the OCT1 transporter to the anti-cancer effect of metformin in thyroid PhD Student - 3rd Year cancer 106 Mr Luke Flannery The Effects of Anti-Cancer Therapies on the Bone Microenvironment Placement Student 111 Mr Tim Ingham-Dempster A Multi-Scale Agent Based Model of Colon Carcinogenesis PhD Student - 1st Year 115 Mr Jack Hurrell An in-vitro model for studying low dose ionising radiation-induced damage in isolated PhD Student - 1st Year cardiac endothelial cells. 119 Mr Jonathan Atkinson A preliminary proteomic analysis of the response of human bone metastatic breast Work Placement Student cancer cells to the loss of DOCK4 123 Dr Jules Westbrook Molecules and mechanisms central to breast cancer metastasis to bone: A Postdoctoral Researcher quantitative proteomics approach – preliminary evidence

Academic Unit of Primary Medical Care

71 Miss Hannah Dudhill The potential for community-based alternative care pathways for patients after BMedSci Student - 4th Year suspected seizures

Sheffield Teaching Hosptials NHS Trust

41 Dr Alex Wheeler Exploring Prescribing Habits in the Emergency Department- a Need For a Change in Foundation Year 2 Culture

Monday 15th June 2015

Oral Presentation Abstracts (In programme order)

9:00AM Dr Iwan Evans Emma Armitage, Frederico Rodrigues, Will Wood and Iwan Evans Sir Henry Dale Fellow Infection & Immunity Contact: [email protected]

Draper/CED-1 mediates an ancient damage response to control inflammatory blood cell migration in vivo

Tissue damage leads to a robust and rapid inflammatory response whereby leukocytes are actively drawn towards the wound. Hydrogen peroxide has been shown to be an immediate damage signal essential for the recruitment of these inflammatory blood cells to wound sites in both Drosophila and vertebrates. Recent studies in zebrafish have shown that wound-induced hydrogen peroxide is detected by the redox-sensitive Src family Kinase (SFK) Lyn within the responding blood cells. Here we show the same signalling occurs in Drosophila inflammatory cells in response to wound-induced hydrogen peroxide with mutants for the Lyn homologue Src42A displaying impaired inflammatory migration to wounds. We go on to show that activation of Src42A is necessary to trigger a signalling cascade within the inflammatory cells involving the ITAM domain-containing protein Draper-I (the fly homologue of CED-1) and a downstream kinase, Shark, that is required for migration to wounds. The Src42A-Draper-Shark mediated signalling axis is homologous to the well-established SFK-ITAM-Syk signalling pathway used in vertebrate adaptive immune responses, consequently our results suggest that adaptive immunoreceptor signalling pathways important in distinguishing self from non-self appear to have evolved from a more ancient damage response and changes the role of hydrogen peroxide from an inflammatory chemoattractant to an activator signal that potentially primes immune cells to respond to damage cues via the activation of damage receptors such as Draper and other ITAM-containing protein

9:15AM Dr Pallai Rappai Shillo P.Shillo1, D. Selvarajah4 , M. Greig1, I. D. Wilkinson2, G. Rao3, S. Tesfaye1 PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Low Vitamin D Levels in Patients with Painful Diabetic Peripheral Neuropathy: A Potential Role in Pathogenesis?

Aim Recent studies have reported an association between low vitamin D levels and painless and painful diabetic peripheral neuropathy (DPN). However, these studies had not assessed major confounding factors including seasonal sunlight exposure and daily activity. There is thus a clear rationale for a study to evaluate Vitamin D levels accounting for these potential confounders in carefully phenotyped DPN subjects. Methods 45 T2DM Caucasian subjects (17 Painful-DPN, 14 Painless-DPN and 14 No-DPN) and 14 healthy volunteers (HV) underwent detailed clinical, neurophysiological and lower limb skin intra-epidermal nerve fibre density (IENFD) assessments. T2DM subjects were divided into three groups based on the neuropathy composite score [NIS(LL)+7 tests]. 25(OH)-Vitamin D was measured between May-September and all subjects had seasonal sunlight exposure and daily activity measured. Results DPN subjects had higher body mass index(BMI) and were older in age. [BMI [HV 26.1(4.6), No-DPN 30.1(6.8), Painless-DPN 31.2(5.5), Painful-DPN 32.9(5.7); ANOVA p=0.02]. After adjusting for age and BMI, vitamin D levels (nmol/l) were significantly lower in Painful-DPN subjects [35.8(17.1)] compared to the other groups [HV 60.9(28.1), No-DPN 46.8(19.2), Painless-DPN 56.1(28.8), ANCOVA p=0.02]. Pairwise comparisons revealed group differences between Painful- vs Painless-DPN (p= 0.03) and Painful-DPN vs HV (p= 0.003). Furthermore, there was a significant correlation between lower Vitamin D levels and higher painful neuropathic symptom (DN4) scores (r= 0.36, p = 0.04). However, there was no relationship between Vitamin D levels and IENFD. Conclusions We have demonstrated a significant reduction of vitamin D levels, measured under careful conditions in subjects with painful-DPN. This suggests a possible role of vitamin D in the pathogenesis of painful-DPN. Further studies are now required to evaluate the impact of vitamin D supplementation on painful neuropathic symptoms.

9:30AM Ms Charlotte Rowan Charlotte L Rowan, Russell Hughes, Carolyn A Staton, Gill M Tozer & Claire E Lewis PhD Student - 2nd Year Oncology Contact: [email protected]

A subset of peripheral blood monocytes exhibit an M2-skewed phenotype after doxorubicin treatment: are these the precursors of relapse-promoting M2 TAMs?

Rationale & Hypothesis: Blood monocytes are continuously recruited into malignant tumours, where they differentiate into tumour associated macrophages (TAMs) and promote tumour progression. TAMs have also been shown to limit the efficacy of various cytotoxic drugs in mouse tumours. Recent studies in our group have shown that a subset of alternatively (‘M2’) activated TAMs accumulate in perivascular regions of mouse tumours following chemotherapy, and then stimulate their subsequent regrowth. These cells express high levels of both the M2 marker, MRC1, and the CXCL12 receptor, CXCR4. Furthermore, treatment of tumour-bearing mice with chemotherapy and the CXCR4 inhibitor AMD3100, prevented the accumulation of these cells and markedly delayed tumour relapse. However, the origin of these relapse-promoting, perivascular TAMs remains to be determined.

Aim of Study: To investigate whether this M2-skewed, relapse-promoting TAM subset in chemotherapy-treated tumours is derived from a subset of M2-skewed monocytes in the circulation.

Methodology: A MMTV-PyMT cell line, TS1, was injected into the mammary fat pads of female FVB/N mice and allowed to develop into tumours of 500mm3. Mice were then treated with doxorubicin (DOX) or its vehicle, PBS, and peripheral blood collected for flow cytometric analysis.

Results: Within 48h of DOX administration, there was no change in the proportions of Ly6Chi (‘classical’) or Ly6Clo (‘non-classical’) monocyte subsets in the blood of tumour-bearing mice. However, DOX significantly increased the proportion of Ly6Clo monocytes expressing MRC1. In DOX treated mice, there was also a significantly higher proportion of Ly6Clo monocytes expressing CXCR4 than Ly6Chi monocytes and an increased level of CXCR4 expression on this subset.

Conclusion: These data suggest that DOX stimulates circulating Ly6Clo monocytes to express a more M2-skewed phenotype - and that these cells may be the precursors of the perivascular, MRC1+ CXCR4+ TAMs driving relapse after chemotherapy. Cell tracking studies are now underway to investigate this further.

9:45AM Mr Simeon Mihayloy Mihaylov, Oliver Bandmann, Guillaume Hautbergue PhD Student - 2nd Year Neuroscience Contact: [email protected]

Investigating the RNA-Binding Activity of PGC-1[alpha] in Parkinson’s Disease Models

Background: Increasing evidences for mitochondrial dysregulation in neurodegenerative diseases suggest a central pathophysiological role of the organelle. It is unclear whether mitochondrial abnormalities precede disease onset or are consequences of impaired upstream pathways. The transcriptional factor PGC-1[alpha] has been identified as a key regulator of mitochondrial biogenesis and maintenance. PGC-1[alpha] contains three distinct domains. The amino-terminal and middle domains are involved in tissue- specific transcriptional functions. The C-terminal domain of PGC-1[alpha] has been the least studied. It exhibits an Arginine/Serine-rich (SR-rich) region flanked by a RNA Recognition Motif (RRM) typical of splicing factors. We hypothesise that this domain directly binds RNA and may play a role in the processing of PGC-1[alpha]-activated transcripts. Aims: To functionally investigate the potential role of PGC-1[alpha] in RNA-processing and the potential alteration of this function in mitochondrial homeostasis during ageing and neurodegeneration. Methods: Biochemical approaches were used to purify recombinant PGC-1[alpha] from E. coli and investigate interactions with various RNA oligonucleotide sequences using RNA:protein UV cross-linking assays. In addition, pull down and immunopreciptation assays of PGC-1[alpha] were performed from transfected HEK cell extracts. Fluorescence microscopy was used to assess the cellular distribution of PGC-1[alpha] and potential colocalisation with transcription activators and splicing factors. Results: In this study, we show that direct binding of PGC-1[alpha] to RNA is mediated via the carboxyl-terminal domain. Furthermore, the integrity of this domain appears to be important to the cell metabolism. Conclusions: For the first time, these results suggest that the putative carboxyl-terminal domain of PGC-1[alpha] directly binds RNA. We set to next establish how important this function is in cell/animal models and in neurodegeneration using mutated sequence of PGC- 1[alpha] in complementation studies, qRT-PCR and mitochondrial assays.

10:00AM Mrs Emma MacInnes E MacInnes, J Morgan, K Collins, R Seaton, K Mehta, S Jeyakumar, C Ingram, L Wyld PhD Student - 2nd Year Oncology Contact: [email protected]

MRI and mammographic screening in young women at increased familial breast cancer risk

Rationale & Hypothesis: Familial breast cancer accounts for one quarter of all breast cancers in the UK, with some women at a substantially increased lifetime risk of developing cancer. Management options include bilateral mastectomy, chemoprophylaxis or enhanced surveillance. UK NICE guidelines recommend that such women be offered enhanced screening with mammography and/or MRI, based on individual risk.

Hypothesis: Screening women at increased familial breast cancer risk with MRI and-or mammography at a younger age than NHS breast screening does not detect early stage cancers.

Objectives: MRI and mammographic screening for women at increased familial risk were retrospectively assessed. Sensitivity and specificity for each modality and for combined screening were compared with predicted cancer incidences.

Methodology: Patients enrolled in enhanced familial risk mammographic and MRI screening in a single UK tertiary centre were included for a 5 year period. Each screening episode and all cancers diagnosed in this group were analysed.

Findings: 529 women were MRI and-or mammographically screened over 5 years. Risk levels for women MRI and-or mammographically screened differed in line with UK NICE eligibility guidelines. Malignant recall rates were 1.4% for MRI, 0.1% for mammographic and 0.5% for dual screening, in keeping with other published series. The interval cancer detection rate in mammographically screened women was high and the screen detection rate low raising concern that mammographic screening in the increased familial risk 40-50 age group, may have a low efficacy. Conversely MRI had high sensitivity detecting cancers at an early stage with no interval cancers detected in the screening period.

Conclusion: Outcomes from five years of MRI and mammographic surveillance for women at increased familial breast cancer risk in this tertiary centre demonstrate the known efficacy of MRI. It does, however, raise questions as to the benefit derived from mammography in the lower risk profile group.

10:15AM Dr Paul Morris Morris PD, Zwierzak I, Lawford PV, Hose DR, Gunn JP PhD Student - 3rd Year Cardiovascular Science Contact: [email protected]

The Development and Validation of an In Silico Model of Coronary Arterial Physiology for Diagnostic Use

Rationale & Hypothesis: Coronary revascularisation guided by physiological assessment with fractional flow reserve (FFR) improves outcomes and expenditure but, due to practical limitations, is used in <10% of percutaneous coronary interventions (PCI) and close to no diagnostic angiograms. Aims: We developed an in silico model of coronary physiology which computes FFR using computational fluid dynamics (CFD) with data from coronary angiography (CAG), which additionally computes real-time coronary flow. Methods: We studied 40 patients with coronary artery. The in silico workflow reconstructed coronary geometry from CAG images. The 3-D domain was coupled to an algebraically encoded zero-D model representing microvascular resistance. Simulated pressure and flow were validated against patient data. Workflow development enabled flow simulation from measured data which were validated in a benchtop flow circuit comprising patient-specific 3-D printed coronary arterial models, a blood analogue, and patient-specific flow patterns using a programmable pump. To reduce computation time, a novel mathematical method was developed and investigated. Results Using completely generic boundary conditions, the initial model computed ‘virtual’ FFR (vFFR) with ±0.06 error and diagnosed physiological lesion significance (FFR<0.80) with 97% accuracy. Ultra-precise, transient CFD took 24 hours to compute, whereas the novel method reduced computation to <4 mins on a standard PC. Using artery- specific, patient averaged values yielded ±0.01 error. The workflow computed flow results (5.3% error) within 3 minutes.

Fig 1. A patient case (a) with pressure and flow simulation results (b and c) across a severe right coronary stenosis.

Conclusions Our in silico model computes intra-coronary physiology (pressure and flow), in timescales practical for clinical use in the catheter laboratory. Virtual modelling of FFR and hyperaemic stenosis resistance (HSR) is now feasible. This may facilitate increased uptake and impact of physiologically-guided revascularisation planning for every patient undergoing CAG, rather than the minority undergoing selective PCI. Improved accuracy will follow from better characterisation of the individual’s microvascular resistance.

11:00AM Dr Esther Hobson Hobson E V, Walsh T, Baird W, Cooper C, Mawson S, Shaw P, McDermott C. PhD Student - 2nd Year Neuroscience Contact: [email protected]

Facilitating access to specialist care for patients and carers affected by Motor Neurone Disease using telehealth.

Background Patients with motor neurone disease require specialist care provided by multidisciplinary teams. As their disease progresses patients struggle to attend hospital to access the care they need. Telehealth may improve access to care by using technology to monitor, educate and communicate with patients, their carers, and their healthcare team. The TiM (Telehealth in Motor neurone disease) telehealth system was designed by the Sheffield Institute for Translational Neurosciences to allow patients and their primary informal carer to enter information about their condition on a weekly basis. This is relayed to the Sheffield specialist care team who are alerted to any changes.

Aim This is a pilot study of the TiM telehealth system assessing feasibility, acceptability and safety of the telehealth system in clinical practice and the feasibility of conducting a full study of the system.

Methods A randomized controlled mixed methods pilot study of the TiM telehealth system will recruit forty patients and their primary informal carer. Twenty patients and their informal carer will be assigned to use the TiM system for a minimum of six months (intervention) and twenty patients will be assigned usual care (control). Quantitative outcome data will be collected at intervals and qualitative interviews with participants and staff and analysis of the system in use will enable a process evaluation of the system and the trial methodology. It will also assess the safety of the system in a clinical setting. Results of this pilot will determine whether a large, multi-centre full trial is appropriate and enable further development of the TiM system. It is proposed that the TiM system could be adopted throughout the UK and offer improvements in quality of life, clinical outcomes and health resource use.

Results Recruitment commenced in October 2015. Early results will be presented in this meeting.

11:15AM Mr Joshua Griffiths Joshua Griffiths, Catherine Loynes, Stephen Renshaw BMedSci Student Infection & Immunity Contact: [email protected]

ROLE OF SPECIFIC PRO RESOLVING MEDIATORS IN INFLAMMATION RESOLUTION AND REGENERATION IN AN IN VIVO ZEBRAFISH MODEL

Aim Resolution of inflammation is a pre-requisite for normal wound healing and tissue regeneration and an active process in which neutrophils and macrophages play a crucial role. Specialised pro-resolving mediators (SPMs), including resolvins and maresins hold therapeutic promise in resolving inflammation and improving regeneration. Using transgenic zebrafish tail-transection as a model for inflammation, we can observe the effects of SPMs, specifically Maresin-(R)-1 and Resolvin D2, on neutrophil and macrophage behaviour and wound regeneration.

Methods Tail-transection was performed on transgenic zebrafish and SPMs introduced via Intravenous injection or exogenous addition to fish water. Neutrophil and macrophage counts were performed over 24 hours. Regeneration of the caudal tail fin was assessed 24 hours post injury (hpi).

Results Maresin-(R)-1 and resolvin D2 both reduce neutrophil numbers at the site of injury 8 hpi, promoting inflammation resolution. Control injected larvae average neutrophil counts were 25.9 +/- SEM 1.0 and 19.7 +/- SEM 0.83 in maresin-(R)-1 injected larvae (p<0.05).Resolvin D2 treated Larvae showed 25.1 +/- SEM 1.2 when vehicle control groups demonstrated 31.7 +/- SEM 1.3 (p<0.05) Neutrophil counts of Maresin-(R)-1 significantly increases macrophage numbers recruited to the site of injury, from as early as 4 hpi (14.8+/- SEM 0.7) compared to control larvae (10.8+/- SEM 0.8) (p<0.05). Resolvin D2 showed no effect on macrophage numbers at the site of injury. Maresin-(R)-1 incubated larvae also show significant fin regeneration over 24 hours following tail transection with an area increase of 56.8% +/- SEM 6.5 compared to control larvae with an area increase of 39.3% +/- SEM 4.8 (p<0.05) but resolvin D2 showed no such difference.

Conclusions These data indicate that both maresin-(R)-1 and resolvin D2 improve inflammation resolution, yet only Maresin- (R)-1 increases regeneration. Both lipid mediators may prove to have therapeutic potential.

11:30AM Dr Magdalena Adamczyk Magdalena Adamczyk 1,2, Daniel Aeschlimann1, Bronwen Evans 3 and Alison Gartland 2 Arthritis Research UK Foundation Fellow Human Metabolism Contact: [email protected]

Are P2X7R-/- mice protected from IL-1β-mediated TG2 upregulation and rapid release?

The abundant upregulation of transglutaminase 2 (TG2) in the extracellular space can promote chondrocyte hypertrophy [1] and cartilage calcification [2], which might be important aspect of osteoarthritis initiation. TG2 is a non-conventionally exported protein and the mechanism of release has been elusive [3,4]. IL-1beta is known to upregulate TG2 extracellular activity in chondrocytes [5] and thereby might have a synergistic effect on TG2 secretion. We have previously observed that P2X7 receptor regulates rapid export of TG2 in THP-1 monocytes differentiated into macrophage-like cells. We hypothesize that the same mechanism of TG2 release might occur in chondrocytes or bone marrow derived macrophages (BMDMs) but not visible in the absence of P2X7R. The objective of the study it to identify whether P2X7R-/- mice are protected from IL-1β-mediated TG2 release.

The immunohistochemistry of the whole knee joints of wildtype BalbC and P2X7R -/- BalbC mice was performed to study TG2 expression. The murine femoral head caps were used to investigate TG2 release into conditioned medium upon treatment with P2X7R agonist (BzATP) or in the presence of IL-1β. TG2 secretion was monitored by Western blotting. GAG release was quantified with DMMB assay and Safranin O staining. Additionally, BMDMs were isolated and polarized into M2 type to upregulate TG2 expression.

Immunohistochemistry staining revealed TG2 accumulation in articular and growth plate chondrocytes of wildtype mice and this was reduced in P2X7R knockouts. On the other hand, P2X7R -/- M2 macrophages displayed higher TG2 intracellular levels than wildtype cells suggesting a different regulation of TG2 expression than in chondrocytes. The BzATP stimulation of IL-1β treated femoral heads showed enhanced TG2 medium localization but this was also to some degree observed in femoral heads from P2X7R -/- mice. It remains to be seen whether another P2X receptor might be involved in TG2 release in cartilage. Further effort is needed to confirm the differences between wild-type and P2X7R knockouts.

We gratefully acknowledge the support of Arthritis Research UK in the form of a Foundation Fellowship.

[1] Huebner, J. L. et al. (2009), Osteoarthritis Cartilage 17(8), pp. 1056-1064; [2] Cecil, D. L. and Terkeltaub, R. 2008, J Immunol 180(12), pp. 8378-8385. [3] Antonyak, M. A. et al. (2011), Proc Natl Acad Sci U S A 108(12), pp. 4852-4857 [4] Zemskov, E.A et al (2011), PLoS One 6, p. e19414 [5] Johnson, K., Hashimoto, S., Lotz, M., Pritzker, K., Terkeltaub, R. 2001; Am J Pathol 159(1), pp.149-163.

11:45AM Dr Alex Rothman Rothman AMK, Arnold NA, Pickworth JA, Dawson S, Iremonger J, Casbolt H, Southwood M, Morrell N, Lawrie A MRC Clinical Research Training Fellow Cardiovascular Science Contact: [email protected]

miR140-5p is a key pathological mediator of pulmonary arterial hypertension and modulates BMP signalling through SMURF1

Rationale and hypothesis: Pulmonary arterial hypertension (PAH) is a progressive, life limiting disease. Loss of the growth suppressive effects of Bone Morphogenetic Protein (BMP) signaling impair function of pulmonary arterial endothelial and smooth muscle cells (PASMC), leading to increased pulmonary vascular resistance and right heart failure. Current treatments are limited to pharmacological vasodilatation, however proliferative remodelling continues and many patients require lung transplantation. We have previously identified the reduction of miR-140-5p in patients with PAH and hypothesize that investigation its cellular effects will identify novel new therapeutic targets.

Objectives: To investigate the biochemical and phenotypic consequences of reduced miR-140-5p in PAH for the identification of novel therapeutic targets.

Methodology: Whole blood miR-140-5p, and target mRNA, expression was determined by qPCR. PASMCs were transfected with miR-140-5p to determine effect on proliferation and migration. Target mRNA were identified by bioinformatic analysis validated by mRNA/protein level and 3’UTR luciferase assay. Effects on BMP signalling were determined by BMP response element luciferase assay. Therapeutic delivery of nebulised miR-140-5p, and the effect of genetic ablation of target genes, were investigated in experimental models of PAH.

Findings: Patient miR-140-5p levels correlated with pulmonary artery pressure, right atrial pressure and NT- proBNP and identified those with reduced survival. miR-140-5p altered proliferation and migration of hPASMC. Prophylactic and therapeutic administration of nebulised miR-140-5p successfully attenuated experimental PAH. Biochemical analysis identified, and validated, SMURF1 as a key regulator of BMP signaling and PAH pathology. Genetic ablation of SMURF1 conferred an allele dependent protection from experimental PAH. Finally, SMURF1 was increased in whole blood mRNA, and present in remodeled pulmonary arteries, of patients with PAH.

Conclusions: These studies demonstrate that miR-140-5p and SMURF1, a E3 ubiquitin ligase that negatively regulates BMP signalling, play a central role PAH pathogenesis. Both miR-140-5p and SMURF1 represent novel therapeutic targets for the treatment of PAH.

3:00PM Dr Clare Howarth C. Howarth, B.A. Sutherland, H.B. Choi, C. Martin, B.L. Lind, L. Khennouf, J.M.P. Pakan, G.C.R. Ellis-Davies, M.J. Laurtizen, N.R. Sibson, A.M, Buchan, B.A. MacVicar. Vice Chancellor’s Advanced Fellow Psychology Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

3:15PM Mr Felix Horn Felix Horn, Juan Parra-Robles, Jim Wild PhD Student - 4th Year Cardiovascular Science Contact: [email protected]

Quantitative 3D lung function measurement -results from patient studies and predictive modelling of global multiple breath washout

Rationale & Hypothesis: Quantitative measurement of regional lung function is a crucial method to measure ventilation heterogeneity and progress of disease. Quantitative metrics can also be used for predictive modelling that might help to interpret results from tests in the pulmonary function lab.

Objectives: To demonstrate advantages of quantitative lung MRI in assessing early stages of disease in patients, before global lung function measurements detect changes and the ability to predictively model outcome of multiple breath washout as measured in the pulmonary function lab.

Methodology: Multiple breath washout imaging using hyperpolarised gas MRI was performed in healthy volunteers (n=6) and in patient cohorts including patients with early stage CF (n=12) and a group of mild to severe asthma patients (n=30).

Figure 1: (A) Quantitative 3D ventilation map measured in units of fractional ventilation r (as gas turned over with each breath) as acquired for all patients. (B) Prediction of gas volumes in different parts of the lung as calculated from fractional ventilation maps.

Findings: Findings show that quantitative metrics from multiple breath wash-out imaging are sensitive to early changes in lung disease. Statistical testing shows significant difference between mild CF patients and age-matched healthy volunteers in the imaging derived metric, where pulmonary function testing metrics of forced expiratory volume in 1 second (FEV1) and the lung clearance index (LCI) do not show significant differences. Disease severity as measured with FEV1 correlates very well (r = 0.7, P<0.001) with imaging derived metrics. In this preliminary study we have also shown that quantitative ventilation images can predict the outcome of clinically used multiple breath wash-out, an important pre-requisite for the subsequent regional interpretation of ventilation heterogeneity that multiple breath washout from the pulmonary function lab is thought to be sensitive to.

3:30PM Dr Victoria Stern Victoria Stern, Jamie Healey, Georgina Jones, Simon Dixon, Stephen Walters, Brian Brown and Dilly Anumba PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

3:45PM Dr Fiona Taylor F Taylor, J Bradford, P J Woll, M D Teare, A Cox PhD Student - 2nd Year Oncology Contact: [email protected]

Non-invasive molecular profiling of circulating cell-free DNA in lung cancer

Rationale & Hypothesis: In lung cancer, tumour derived genetic changes have been detected in circulating cell-free DNA (cfDNA) in blood plasma. Molecular blood biomarkers may aid the detection of early stage cancer in asymptomatic people. Our hypothesis is that we will detect more copy number abberations (CNA) in cfDNA of lung cancer cases compared to high risk controls.

Objectives: To establish proof of principle that SCNA are identified in cfDNA of lung cancer cases by Next Generation Sequencing (NGS. To determine the lowest limit of detection of tumour derived SCNA by spiking different proportions of FFPE extracted DNA into cfDNA of healthy volunteers.

Methodology: Blood and tumour samples from 8 lung cancer cases were collected in the Sheffield Resolucent study. Standard protocols were followed to process, extract and quantify matched cfDNA, FFPE DNA and genomic lymphocyte DNA. DNA was prepared for low coverage whole genome NGS on the Illumina HiSeq platform with the NEBNext Ultra library kit. Raw reads were mapped to the human reference genome (hg19) and SCNA identified using CNAnorm, an R Bioconductor package optimised for low read depth.

Findings: The mean total number of reads was 18,867,390, of which ~88% mapped to the human genome after filtering. CNA were detected in the cfDNA of all cases. For 3 advanced stage cases, there was positive correlation between matched cfDNA and FFPE tumour DNA copy number profiles (both normalised to genomic DNA; Spearman Rank=0.71, 0.73, 0.33). The estimated tumour content identified in cfDNA varied from 2.5% to 17% (N=6). We were able to detect FFPE derived copy number aberrations with a tumour fraction as low as 10% in cfDNA. Our preliminary results demonstrate successful non-invasive detection of tumour derived CNA in advanced lung cancer cases. Further optimisation of this approach will be required to identify SCNA in cfDNA of early stage cases.

4:00PM Dr Johnathan Cooper-Knock Johnathan Cooper-Knock, Adrian Higginbottom, Matthew J Stopford, J Robin Highley, Paul G Ince, Stephen B Wharton, Stuart Pickering-Brown, Janine Kirby, Guillaume M Hautbergue and Pamela J Shaw PhD Student - 3rd Year Neuroscience Contact: [email protected]

Antisense and sense RNA foci derived from hexanucleotide repeat expansions of C9ORF72 have similar protein-interactions but distinct neuronal expression patterns

Introduction GGGGCC-repeat expansion of C9ORF72 represents the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It is suggested that toxicity may be caused directly by RNA foci transcribed from the repeat sequence or indirectly via repeat associated non-ATG translation of dipeptide repeat proteins (DPRs). RNA foci are formed by sense and antisense transcription; we aimed to determine whether the location and behaviour of these species are distinct.

Method Pathological material was obtained from the Sheffield Brain Tissue Bank. Sense and antisense RNA foci were visualized by RNA fluorescence in-situ hybridization (FISH). Interaction with proposed foci binding partners and with TDP-43 was examined by immunohistochemistry (IHC). DPRs were also examined by IHC. Direct binding to the sense and antisense repeat sequences was examined by UV-crosslinking.

Results C9ORF72-ALS is associated with pathology of motor and non-motor areas. In the cerebellum the cellular distribution of sense and antisense RNA foci are relatively distinct: sense foci are more abundant in the granule neurons (p<0.05) whereas antisense foci are more abundant in the Purkinje cells (p<0.05). In the motor neurons of the ventral horn, which are the primary target for pathology in ALS, antisense foci are present at a higher frequency (p<0.05). The presence of antisense (chi2, p<0.05) but not sense (chi2, p=0.75) RNA foci correlates with nuclear loss of TDP-43 in motor neurons. Moreover, sense-RNA derived DPRs are present at a higher frequency than antisense-RNA derived DPRs within neuronal inclusions located in cerebellar granule cells, but the opposite is true in motor neurons (p<0.01). Protein interactions were not different between sense and antisense foci.

Conclusions Our data suggests that if sequestration of protein binding partners is important to disease pathogenesis then sense and antisense RNA foci should be equally toxic. However nuclear loss of TDP-43 in motor neurons, which correlates directly with neurodegeneration, is associated with the presence of antisense but not sense RNA foci. This suggests the increased frequency of antisense foci in motor neurons may be key to pathogenesis. Factors determining antisense transcription of the repeat expansion are unknown but may represent a novel therapeutic target.

4:15PM Dr Andrew Streets Andrew J. Streets, Yaoxian Xu, Albert C. M. Ong Senior Research Fellow Infection & Immunity Contact: [email protected]

Polycystin-1 trafficking is regulated by cAMP dependent phosphorylation of the PLAT domain.

INTRODUCTION AND AIMS: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease affecting the kidney (incidence 1 in 1000) and is caused by mutations in two genes, PKD1 (85- 90%) or PKD2 (10-15%). Around 10% of patients on renal replacement therapy have ADPKD and it is estimated that this treatment costs £140 million per annum in the UK alone. Polycystin-1 (PC1), the protein product of the PKD1 gene, localizes to the plasma membrane and primary cilia where it can function as a mechanosensitive or ligand-activated receptor. The PLAT (PC1, Lipoxygenase and Alpha Toxin) located in the 1st intracellular loop of polycystin-1, represents the most evolutionarily conserved domain in PC1 and is a signature domain of the PC1 superfamily. In previous work we observed that cAMP mediated phosphorylation of the isolated PLAT domain regulates its localisation to the plasma membrane and hypothesised that it may be of critical importance for the dynamic trafficking of full length PC1.

METHODS: The dynamics of PC1 membrane trafficking was visualized by live cell staining of PC1 and expression of YFP tagged PLAT fusion proteins. The interaction of PLAT with its binding partners was confirmed by immunoprecipitation and GST pull down assays. siRNA knockdown was used to determine the functional importance of PLAT binding proteins in PC1 trafficking.

RESULTS: A PLAT-YFP fusion protein as well as full length PC1 localised to the cell surface in transfected kidney epithelial cell lines. Endocytosis of PC1 is regulated by protein kinase A (PKA) mediated phosphorylation of S3164 in the PLAT domain. Interestingly, a pathogenic mutation at R3162>C disrupts dynamic trafficking of PC1 by preventing phosphorylation at S3164. We have identified a novel protein complex between the PLAT domain, β- arrestin1/2 and AP2 which we believe is responsible for mediating the dynamic trafficking of polycystin-1 in response to cAMP. siRNA knockdown of β-arrestin1 and 2 and AP2 disrupts this process.

CONCLUSIONS: We propose a model for the cAMP dependent internalisation of PC1 mediated by β-arrestin1/2 and AP2 binding to PLAT. These results reveal new roles for PC1-PLAT in renal epithelial cells and explain why excessive PKA stimulation could lead to disease through loss of PC1 surface expression.

Poster Presentation ABSTRACTS (In numerical order)

Poster: 1 Dr Alenka Brookes A. J. Brooks, G. Rowse, A.Ryder, P. Narula, B. M. Corfe, P. Norman, A. J. Lobo PhD Student - 2nd Year Oncology Contact: [email protected]

“I can cope right now, because I know where I have come from”; a qualitative exploration of the lived experience of young people with inflammatory bowel disease

Introduction

Young people (YP) living with inflammatory bowel disease (IBD) are at risk of increased psychological morbidity whilst adjusting to living with IBD as an adult. This is in the context of a globally rising incidence in paediatric Crohn’s disease (CD) with peak onset in adolescence. Despite this there remains little research exploring YPs perceptions and experiences of living with IBD. The aim of this first qualitative study was to explore the lived experiences of YP with IBD.

Methods

YP aged 19-21years with a diagnosis of ulcerative colitis (UC) or CD were recruited for a qualitative interview. Interviews were conducted following a semi-structured interview schedule regarding; receiving a diagnosis, impact on relationships, ways of coping, identity and the future. Interviews were audio-recorded, transcribed and subject to in-depth interpretative phenomenological analysis (IPA).

Results

Seven patients were interviewed (4 CD, 3 UC), median age 20years (range 19-21) with 43% (3/7) male. Through IPA the YPs accounts clustered around 4 super-ordinate themes with contradiction as an overarching theme, with several sub-themes illustrating experiences of living with IBD; 1) coping with uncertainty and unpredictability; included misdiagnosis, mislabelling, “everyone is different” and “takes years to make sense of it” 2) living with invisibility and visibility; included “losing myself”, embarrassment, disgust and use of humour. 3) Lack of compassion for self; included a contradiction of control and lack of control, being limited by yet not held back, stoic and hiding emotions. 4) Resilience; included positive impact on relationships and a positive outlook.

Conclusions

The findings of this first qualitative study to investigate the lived experience of YP with IBD highlights key areas for enabling YP to make sense of IBD. Furthermore, improving health care professionals understanding of experiences of YP may improve relationships and self-management which may inform development of high quality, YP friendly healthcare services.

Poster: 2 Ms Sofia Granados-Aparici Sofia Granados-Aparici, Isam Sharum, Kate Hardy, Stephen Franks, Sarah Waite, Neil Chapman and Mark Fenwick PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Foxl2 and Smad3 transcription factors during early follicle development: localisation and regulation of factors involved in granulosa cell proliferation

Introduction:

Primordial follicles are relatively quiescent structures that form the basis of the ovarian reserve. Maintenance of the quiescent state, and conversely, release from this state towards irreversible growth, involves mechanisms that are currently unresolved. Previous work by our group has reported granulosa cell proliferation as the earliest morphological event during primordial follicle activation. Two transcription factors have been implicated alongside this process in granulosa cells. Specifically, nuclear exclusion of Smad3 was identified in granulosa cells at the onset of follicle growth while Foxl2 is known to be a fundamental regulator of granulosa cell viability and proliferation during early follicle development. Since Smad3 is known to interact with Foxl2 in other models, we aimed to determine whether these two transcription factors co-localise in nuclei of granulosa cells, and if both can directly regulate genes involved in granulosa cell proliferation, such as the cell cycle regulator CyclinD2.

Methods:

Immature mouse ovaries at 4 and 8 days of age, containing high proportions of small (primordial and growing) follicles were used in this study. To study the protein expression pattern of Smad3, Foxl2 and CyclinD2 throughout the different follicle stages, sections from paraffin-embedded ovaries were immuno-fluorescently localised, imaged by confocal microscopy and analysed using Image J. In samples of whole ovaries, ChIP-PCR was used to determine if Foxl2 and Smad3 directly bound to the gene promoter of CyclinD2.

Results and Discussion:

Image analysis of immuno-fluorescent ovarian sections showed clear relationships between Smad3, Foxl2 and CyclinD2 during early follicle development. In addition, CyclinD2 promoter fragments were detectable in Foxl2 and Smad3-bound chromatin complexes. Current studies are now focussing on determining if a direct or indirect molecular interaction occurs between Smad3 and Foxl2. As Foxl2 and Smad3 co-operate in other models to regulate gene transcription, their relationship with CyclinD2 in the ovary suggests that a similar mechanism may trigger cell cycle regulation in granulosa cells thereby contributing to primordial follicle activation.

Poster: 3 Miss Rachel Bell R Bell, PN Monk PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Characterisation of the second C5a anaphylatoxin receptor C5a2.

Rationale & Hypothesis: C5a is a by-product of complement activation and is a potent inflammatory chemokine. Elevated levels of C5a have been found in a number of inflammatory diseases such as asthma and arthritis. Thus far two C5a receptors are known. The first is C5a1 and is a G-protein coupled receptor with the classic 7- transmembrane helical structure. The second, C5a2, shares a similar 7-transmembrane helical structure but cannot couple to G proteins. C5a is rapidly metabolised in serum to form the less potent C5a des-Arg by removal of the C-terminal Arginine residue. It has been previously shown that C5a2 has a greater affinity for C5a des-Arg than C5a1 despite the two sharing similar affinities for C5a and so the receptors appear to have different ligand binding mechanisms. The function of the C5a2 receptor controversial with reports of both anti-inflammatory and pro-inflammatory roles in vivo. The main objective of the work is to determine the binding mechanism for recombinant human receptor ligands, C5a and C5a des-Arg, to C5a2, using receptor mutations suggested by structural modelling. We hypothesise that mutations in human C5a2 in equivalent positions to those previously studied in C5a1 will have different effects on ligand binding. Binding to 11 different human C5a2 receptor mutants, is being studied, using both fluorescently labelled C5a and C5a des-Arg measured using flow cytometry. To date, results show that there are differences in effect of some mutations between C5a1 and C5a2 for binding of C5a and C5a des-Arg. This data will be used to develop selective ligands for C5a2 for use in definitive studies of the role of this receptor in the control inflammatory responses.

Poster: 4 Mr Atiqur Rahman Atiqur Rahman, David Sammut, Richard Budd, David Dockrell, Katherine Henry, Moira Whyte, Stephen Renshaw, Lynne Prince PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Using protein kinase inhibitor compounds to reverse dysregulated neutrophil function in COPD

Rationale: Persistent neutrophilic inflammation is observed in a number of inflammatory conditions, including chronic obstructive pulmonary disease (COPD), which is considered as the fourth leading cause of death worldwide and has no effective therapies. There is emerging evidence that macrophages from COPD patients are less efficient at phagocytosis than healthy macrophages. Delayed neutrophil apoptosis and defective innate immune functions including neutrophil phagocytosis may contribute to COPD pathophysiology. Uncovering the detailed mechanism of these defects may be considered as the foremost strategy to develop effective therapies.

Objectives: Here, we studied neutrophils from the disease group to identify any defect in neutrophil phenotypes and also aimed to screen a protein kinase inhibitor library to identify pathways that are targeted in inflammatory diseases.

Methodology: Neutrophils were isolated from peripheral blood of healthy donors and COPD patients by percoll gradient centrifugation. Neutrophils were cultured for 5 hours with or without GMCSF [50 U/ml] and pyocyanin [50µM] followed by an assessment of apoptosis by light microscopy. Phagocytic Index (PI) following incubation with heat-killed Staphylococcus aureus was determined. Protein kinase inhibitors (compounds) screening on neutrophil apoptosis was performed by flow cytometry.

Results: Neutrophil apoptosis with GMCSF treatment was significantly lower in COPD compared to HC neutrophils [3.16%±0.53% and 7.41%±1.86%, respectively, n=6 (COPD), 5 (HC), p=0.0173]; whereas pyocyanin treatment resulted significantly higher apoptosis in HC compared to COPD [70.18%± 5.75% and 45.82%±4.13%, respectively, n=6 (COPD), 5 (HC), p=0.0087]. Furthermore, COPD neutrophils had a defect in S. aureus phagocytosis compared to healthy neutrophils (n=5, p=0.0317). From compound screening, 60 compounds were found to accelerate neutrophil apoptosis by ≥2 fold.

Conclusion: These functional defects in COPD neutrophils may contribute to poor immune function and inflammation in the disease group. Further wok to identify regulatory mechanism of the kinase targets on neutrophil phenotypes is in progress.

Poster: 5 Miss Laura Ratcliffe L.Ratcliffe, C.J.Garwood, J.Simpson, P.G.Ince, S.B.Wharton PhD Student - 2nd Year Neuroscience Contact: [email protected]

Investigating impaired IGF1 signalling in human astrocytes and its role in Alzheimer’s Disease Progression

Introduction: Astrocytes play a key role in essential brain functions including homeostasis, metabolism and synaptic activity. The insulin/insulin like growth factor (IGF) signalling pathway is downregulated in astrocytes in the ageing Alzheimer’s brain (Simpson et al. 2011). The specific role of IGF1 signalling in these cells is unclear although signalling through this pathway is believed to exert neuroprotective effects in the brain and removal of IGF1R enhances longevity in a wide range of in vivo models. We are investigating the impact of reduced IGF1 signalling in astrocytes in vitro and in an astrocyte-neuronal co-culture model.

Methodology: Cultured human primary astrocytes were grown in media containing serum and/or 13.2nM of human recombinant IGF1 to characterize the IGF signalling pathway in these cells. To reduce signalling through the IGF pathway an IGF1 monoclonal antibody (MAB391) has been used to induce receptor degradation. Cell viability and oxidative stress assays are being used to determine how loss of IGF1 signalling affects astrocytic function. A co-culture system using human astrocytes and differentiated Lund Human mesencephalic cells (LUHMES) is also being established. This astrocyte-neuron co-culture system will be used to assess whether IGF1-impaired astrocytes are able to adequately support neurons.

Findings: We have characterized the IGF1 signalling pathway in primary human astrocytes and shown that a monoclonal antibody against IGF1R can be used to induce degradation of IGF1R and reduce downstream signalling through Akt. Astrocytes compensate for loss of IGF1R by restoring Akt signalling at later timepoints. Loss of IGF1R does not affect cell viability, astrocytic reactivity or induce a stress response but may enhance susceptibility to oxidative stress. Using our novel human astrocyte and neuronal co- culture we will assess how impaired IGF1 signalling in astrocytes may affect neuronal support and thus reveal how reductions in IGF1 affects astrocytic function.

Poster: 6 Dr Nehal Hussain Hussain N, Capener D, Elliot C, Condliffe R, Wild J, Kiely D, Swift A PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Interventricular septal angle can be used to predict which patients have combined postcapillary or precapillary pulmonary hypertension in left heart disease

Rationale & Hypothesis: A transpulmonary gradient (TPG)>12mmHg is thought to represent evidence of vascular change beyond that expected from passive pulmonary venous congestion in patients with pulmonary hypertension and left heart disease (PH-LHD). However recent studies found those with a diastolic pressure gradient (DPG) >6 to have a worse survival. This has led to a change in the recent guidelines suggesting 2 types of PH-LHD: “isolated post-capillary PH” (pulmonary arterial wedge pressure (PAWP)>15 mm Hg and DPG<7mmHg) and “combined postcapillary PH and precapillary PH” (PAWP>15 mm Hg and DPG≥7 mmHg). It would be advantageous if a non-invasive method of predicting DPG could be found for both prognostication and to identify potential patients for clinical trials of targeted therapies

Objectives: Our aim was investigate the utility of cardiac magnetic resonance (CMR) imaging for estimation of DPG in patients with PH-LHD

Methodology: Patients with suspected pulmonary hypertension underwent CMR imaging at our unit between April 2012 and April 2014. Classification followed systematic evaluation with multimodality imaging and right heart catheterization (RHC). Patients were diagnosed as PH-LHD if no other causes of PH could be identified and they fulfilled the following criteria: signs and symptoms of heart failure; mean pulmonary artery pressure (mPAP) ≥25mmHg at rest and pulmonary arterial wedge pressure >15mmHg by RHC. TPG was defined mPAP–PAWP, and DPG was defined as diastolic PAP–PAWP. A number of parameters were analysed including: right and left ventricular indexed volumes, ejection fractions and mass; aortic and pulmonary flow, pulmonary arterial area, and the inter-ventricular septal angle in systole and diastole

Findings: 89 patients where found to have a diagnosis of PH-LHD. The average age was 70yrs with 58% being female. Of these 89 patients, 67% were found to have a TPG>12mmHg, but only 26% had a DPG >6mmHg. No patients were found to have a raised TPG, but normal DPG. The average mPAP=44mmHg (SD±11), TPG=23mmHg (SD±11), and DPG=5 (SD±9). Systolic septal angle was the only CMR marker with a significant association with DPG, (r=0.69, p<0.0001). Receiver operating characteristics (ROC) curve of systolic septal angle against DPG>6mmHg yielded an area under the curve (AUC) of 0.87. ROC curve analysis established a systolic septal angle of >135o to be the optimal threshold for distinguishing a normal DPG from those with a DPG>6mmHg (sensitivity 100%, specificity 79%). In addition linear regression was used to derive coefficients to allow formulation of an equation estimating DPG from systolic septal angle: DPG=0.359xsystolic septal angle–51

Conclusion: Systolic septal angle can be used to estimate whether a patient has “isolated post-capillary” or “combined postcapillary and pre-capillary” PH

Poster: 7 Mrs Felicity Fletcher Fletcher, F.A, Marshall, M., Vivekananda-Schmidt, P., Rosario, D.J. PhD Student - 2nd Year Medical Education Contact: [email protected]

Exploring the impact of a simulation based educational intervention (IMASS: Integrated Medical and Surgical Simulation course) on 5th year medical students confidence as a marker of readiness to engage with Foundation Year 1 (FY1)

Rationale: Medical undergraduates express low confidence about their preparedness for FP. Null hypothesis: Simulation-based interventions have no effect on confidence.

Objective: 1. Track impact of simulation based experience (IMASS) on confidence. 2. Explore qualitatively, perceived value of simulation for undergraduates.

Methods: A mixed methods study: evaluation of repeated measures over time with exploration of emergent themes. Pilot study informed a power calculation for quantitative data. Final year students (n=94) from two Universities underwent a simulation course designed in a mock clinical environment with structured debriefing, a Sim Man 3G®, part task trainers with 7 faculty members. Learning outcomes mapped to Tomorrows’ doctors and FP curriculum. Self-expressed confidence scores in 19 domains set out by the FP curriculum were collected at pre-determined intervals as surrogate markers of preparedness for FP. Baseline scores were collected immediately prior to half-day course (1 = None, 10 = very), repeated immediately after (n = 94), one-week post course (n = 79) and one month into FP (n = 58). A global rating scale allowed individual formative assessment. Thematic analysis was performed on facilitated focus group data. Data was analysed on SPSS v21 with ANOVA and Bonferroni correction for repeated testing.

Results/ Findings: Confidence scores increased significantly, post course (F(2.48,148) = 49.98, p = <0.001). Mean baseline confidence score was 5.88 (SD 1.2) (range 3 - 8), increased to 7.22 (SD 1) immediately post course (p<0.001), 7.4 (SD 0.8) at one week (p = 0.655) and 7.8 (SD 1) one month in to FP (p = 0.15).

The main emergent themes from focus groups were the importance of ‘practise under pressure’, ‘responsibility’, inter-professional learning, specific personalised debriefing and realism.

Conclusions: A simulation based intervention had a positive impact on confidence with effect maintained into FP. Students value small group, planned high fidelity simulation with personalised structured debriefing.

Poster: 8 Mr Jack Green Jack P. Green, Paul C. Evans and Heather L. Wilson PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Shear stress dependent regulation of P2X4 and P2X7 receptors in the endothelium

Introduction Regions of the arterial tree exposed to disturbed blood flow with low wall shear stress (WSS) are prone to atherosclerotic plaque development, whereas areas under undisturbed flow with high WSS are considered protected. The endothelium expresses the ATP-gated cation channels P2X4 and P2X7, which can be activated by shear stress- induced ATP release. As shear stress influences atherosclerotic plaque development and ATP release, we hypothesised that P2X4 and P2X7 receptors are differentially regulated by shear stress and thereby contribute to the focal nature of atherosclerosis.

Methods and Results Human umbilical vein endothelial cells (HUVECs) were exposed to different levels of shear stress for 72 hours. P2X4 and P2X7 splice variants were identified in the endothelium using PCR or western blotting. Expression of P2X7 splice variants was significantly increased under high shear undisturbed flow relative to low shear disturbed flow. mRNA expression of P2X4 splice variants significantly increased under disturbed flow, but protein levels remained unchanged. Immunocytochemistry confirmed that P2X7 was expressed on the cell surface and in the ER/golgi of HUVECs. Calcium imaging of HUVEC showed the presence of functional P2X4 and P2X7 channels, which could be modified pharmacologically. The P2X7 agonist BzATP induced calcium responses in endothelial cells that were modulated according to pre-conditioning with either high WSS, low WSS or oscillating low WSS, indicating that P2X7 responses may be altered after exposure to different WSS.

Conclusions These studies show that P2X7 expression is augmented under high shear undisturbed flow. P2X7 activity may also be differentially regulated in endothelial cells conditioned to different WSS conditions. Thus P2X7 may contribute to atheroprotection at sites of high shear by controlling downstream calcium signalling pathways. Our future studies will test this concept and determine how shear stress conditions alter P2X receptor trafficking and regulatory interacting proteins.

Poster: 9 Dr Hadel Ghaban Hadel Ghaban, Ian Wilkinson, & Richard Ross PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Development of a Long- Acting Granulocyte Colony Stimulating Factor Molecule

Background: Granulocyte Colony Stimulating Factor (G-CSF) is a glycoprotein which has the primary function of regulating neutrophil production. G-CSF has proven successful in the treatment of chemotherapy induced neutropenia, and stem cell mobilisation. The key problems with the first generation G-CSF therapy are short half-lives, and the necessity for daily injections. A number of technologies are in development to generate a long acting G-CSF. Pegylated G-CSF (pegfilgrastim) is the only second generation G-CSF available on the market. The long term side effects of using PEG products remain unknown, therefore, there is a need for new delayed clearance G-CSF products.

Aim: The aim of this project is to use a protein fusion technology (ProFuseTM) to generate long-acting G-CSF. A G-CSF molecule will be linked to an inactive growth hormone binding protein (GHBP) via a flexible Glycine-serine linker. To prevent GH binding, a mutation in the GHBP moiety at tryptophan-104 to alanine (W104A) will be introduced.

Hypothesis: The G-CSF fusion protein is predicted to have a prolonged circulating half-life through increased protein size, whilst retaining biological activity.

Methods: Two fusion proteins were constructed using recombinant DNA techniques. GCSF fusions with (GCSF_A1) and without (GCSF_A2) the W104A mutation, were expressed in a Chinese hamster ovary (CHO) cell line. Protein expression was determined by ELISA and western blotting. GCSF_A1 was purified using Immobilised Metal Affinity Chromatography (IMAC), and in vitro bioactivity tested using an in house AML-193 proliferation assay.

Results: Construction of the fusion proteins was confirmed by sequencing. Both constructs were positive for G-CSF using ELISA. The inclusion of the GHBP increased the molecular weight from 18.8 kDa to ~ 50-70 kDa as judged by western blot. Purified GCSF_A1 showed increased bioactivity (EC50=0.039) compared to native G-CSF (EC50= 0.046 nM).

Conclusions: It is possible to clone & express G-CSF fusion proteins in a CHO cell line, & these molecules show an increase in molecular weight whilst retaining biological activity.

Poster: 10 Mr Bandar Baothman Bandar K Baothman, Jennifer Smith and Peter Peachell PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Characterization of the isoform of cyclooxygenase that mediates the generation of prostaglandin D2 from human lung mast cells

Background: The mast cell is strongly linked to allergic diseases. Mast cells are mainly activated through IgE- dependent mechanisms but more recently stem cell factor (SCF) has been shown to be an effective activator of human lung mast cells by interacting with the c-kit receptor. Mast cells are known to be the principal source of histamine in the body but they are also a very rich source of prostaglandin D2 (PGD2). PGD2 is generated by the action of the enzyme cyclooxygenase (COX) on arachidonic acid, generating intermediates that are then acted on by PGD2 synthase. COX exists as two isoforms, COX-1 and COX-2, and both have the ability to generate intermediates leading to the generation of prostanoids. Aim: To identify which COX isoform is responsible for PGD2 generation in lung mast cells. Methods: Mast cells were isolated from human lung tissue and purified by flotation over Percoll and immunomagnetic bead separation. The cells were activated with SCF or anti-IgE and the release of histamine, PGD2 and cysteinyl-leukotrienes was assessed by spectrofluorometry and ELISA. The effects of COX inhibitors, Indomethacin (non-selective), FR122047 (COX-1 selective) and Celecoxib (COX-2 selective) on mediator release were determined. Purified mast cells were solubilised and used to determine the expression of COX-1 and COX-2 by Western blotting. Results: Indomethacin and FR122047 completely abrogated (P<0.05, n=4) the production of PGD2 from lung mast cells activated by SCF or anti-IgE while Celecoxib was ineffective (P>0.05, n=4). These drugs did not inhibit histamine or leukotriene generation. Western blotting studies showed that COX-1 was strongly expressed in all mast cells preparations while the expression of COX-2 was more variable (n=4). Conclusions: These findings indicate that COX-1 is the principal isoform involved in PGD2 generation from human lung mast cells

Poster: 11 Mr Alejandro Lorente Pons A. Lorente-Pons, J.D. Wood, P.J. Shaw, P.G. Ince, J. Cooper-Knock, A. Ramachandran, J. Kirby, J.R. Highley PhD Student - 2nd Year Neuroscience Contact: [email protected]

Investigating the mechanisms underlying oligodendrocyte dysfunction in C9ORF72 ALS

Rationale & Hypothesis: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by motor neuron degeneration. Oligodendrocyte dysfunction has also been shown to be a feature of ALS. Moreover, pathological ubiquitylated cytoplasmic inclusions (UCIs) are well documented in oligodendroglia in addition to neurones. Myelin basic protein (MBP) is translated in oligodendrocyte processes as it is toxic when present in cell somata. MBP messenger RNA (mRNA) must therefore be transported to processes while inhibiting its translation. The C9ORF72 gene has been found to harbour the expansion of an intronic GGGGCC repeat in many ALS cases. The GGGGCC motif binds a number of proteins including heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), which is essential for the assembly of MBP mRNA transport granules. This may lead to sequestration of hnRNPA2, reducing its availability to bind to MBP mRNA, thereby causing myelin dysfunction in C9ORF72 cases (C9ALS).

Patients and methods: We used immunohistochemistry (IHC) for p62 to quantify oligodendroglial UCIs in motor and frontal cortices and spinal cord (SC) from 62 sporadic cases (sALS), 17 C9ALS cases and 6 healthy controls (Ct). IHC for hnRNPA2 was used to quantify hnRNPA2-positive nuclei in the white matter of the motor cortex (MCx WM) and in the pyramidal tracts of the spinal cord in 15 sALS, 11 C9ALS and 7 Ct cases.

Findings: C9ALS cases showed increased numbers of glial UCIs in the cortex compared to sALS and Ct cases. Both C9ALS and sALS cases show increased UCIs in the SC in comparison with Ct cases, suggesting that glial pathology in the spinal cord is a feature of both sALS and C9ALS. IHC for hnRNPA2 revealed that C9ALS cases with higher number of UCI have a lower proportion of hnRNPA2-labelled glial nuclei, suggesting that hnRNPA2 sequestration is related to UCIs in C9ALS.

Poster: 12 Miss Marie-Therese Haider MT. Haider, HK. Brown, K. Hunter, NJ. Brown, I. Holen PhD Student - 2nd Year Oncology Contact: [email protected]

The tumour microenvironment – still underestimated as a mediator of anti-cancer therapy?

Rationale & Hypothesis: The tumour microenvironment is increasingly recognized as a key component of tumourigenesis and response to therapy. Breast cancer preferentially metastasizes to bone, hence calling for therapeutic agents that target not only the tumour but also the bone microenvironment (BME). We hypothesize that increased knowledge about how the BME responds to anti-cancer therapy is detrimental to treatment success or failure.

Objectives: Our experiments aimed to establish a better understanding of how current and newly developed anti-cancer therapies modulate their treatment response and to evaluate new therapeutic opportunities for patients diagnosed with cancer-induced bone disease, which still remains incurable.

Methodology: Experiments were performed in a variety of animal models including male and female BALB/c nude and mice expressing GFP positive osteoblastic cells. Effects of both Zoledronic acid (100µg/kg, i.p.,single dose) and CBZ (30mg/kg, o.g., 5 and 8 administrations) on the BME were determined using histomorphometry, µCT-analysis, ELISA, RT- PCR and confocal microscopy. Effects of ZOL on tumour cell homing to bone was analysed using multiphoton microscopy.

Results: Both anti-cancer therapies caused significant and rapid modification to key cells of the BME including but not limited to alterations in osteoblast and osteoclast number and activity as well as epiphyseal growth plate composition. ZOL-induced alterations to the BME appeared to change the preferential of breast cancer cells to home to specific areas of trabecular bone. CBZ administration in addition altered the bone marrow composition by increasing the number of megakaryocytes and disrupting vascular integrity and structure as well as altering the expression of genes associated to bone remodelling.

Conclusion: The in vivo BME is rapidly modified by anti-cancer therapy highlighting its potential as key mediators of treatment response and the need to establish treatment modalities that target pathways involved in tumour and bone biology.

Poster: 13 Dr Peter Mooney Peter Mooney, Matthew Kurien, Simon Wong, Alex Johnston, Eleanor Rosario, Andrew Hopper, David Sanders PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

The clinical and phenotypic assessment of Ultra-Short Coeliac Disease

Rationale & Hypothesis: An additional duodenal bulb (D1) biopsy may increase the diagnostic yield for coeliac disease (CD) by up to 10%. However it is not clear if these patients with Ultra-Short Coeliac Disease (USCD) have the same phenotype or are at risk of the same consequences as conventional CD.

Objectives: We aimed to assess the clinical phenotypes of patients with USCD compared to those with conventional disease.

Methodology: Patients attending a specialist CD endoscopy list had duodenal biopsy taken as routine. Standard quadrantic biopsies were taken from the second part of the duodenum (D2) and at least one biopsy taken from D1. Biopsies were analysed separately according to the Marsh classification system. Marsh 3 disease was required to diagnose CD. Patients had concurrent tissue transglutaminase (tTG) and endomysial antibodies (EMA). Patients were followed up in the CD specialist clinic where routine bloods and DXA scans were requested. Haematology and biochemistry results were compared to a control group of patients that had CD excluded with normal serology and histology.

Findings: 1378 new presentations (62% female; mean age 50.3) underwent duodenal biopsy. 268 (19.4%) new diagnoses of CD were made (66% female; mean age 41.6). 25/268 (9.3%) of new CD patients had USCD. Univariate analysis showed fewer USCD patients had diarrhoea than conventional CD (3.8 vs 24.0%, P<0.0001). Decision tree analysis to identify USCD showed the absence of diarrhoea was the single discriminating factor (Adj P=0.018). Ferritin deficiency (P=0.007) and folate deficiency (P=0.003) rates were higher in conventional CD than USCD and controls. On multivariate analysis, patients with USCD were younger than those with conventional CD 36.6 vs. 42.1 (AOR 0.97 (0.94 – 0.998) P=0.03), had lower tTG titres (AOR 0.89 (0.81 - 0.98) P=0.02) and had higher folate levels (AOR 1.17 (1.01 - 1.36) P=0.03) compared to conventional disease.

Poster: 14 Mr Jude Akinwale Jude Akinwale, Ian Wilkinson and Richard Ross PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Generation of Longer-Acting Coagulation Factor VIII for the Treatment of Haemophilia A

Rationale & Hypothesis: Haemophilia A is a sex-linked clotting disorder predominantly affecting males. The clinical manifestations of this disease include spontaneous bleeding and excessive bleeding following a trauma/surgery. Current treatment of haemophilia A is by replacement therapy with recombinant factor VIII (FVIII) on demand or as a prophylaxis. However, due to the relatively short circulating half-life of current therapeutic products, multiple weekly injections are required to manage the haemophiliacs. This is an expensive treatment, costing about £250,000/patient/year, and also hampers the quality of life of the afflicted. This research study aims to improve the treatment of haemophilia A by generating novel recombinant FVIII molecules (nrFVIII) with prolonged half-life. Details of nrFVIII are confidential as subject for patent filing.

Objectives: (i) Design and express novel recombinant FVIII (nrFVIII) constructs in mammalian cell lines. (ii) Test nrFVIII proteins for biological activity. (iii) Purify nrFVIII for in vivo studies. (iv) Provide proof of concept that nrFVIII has delayed plasma clearance in animal models.

Methods: nrFVIII molecules were cloned and, stable and transient expression constructs generated. The expressed proteins were analysed by western blotting, tested for biological activities in a Coamatic chromogenic bioassay and purified by ion exchange and immobilized-metal affinity chromatography.

Results: Two nrFVIII proteins could be expressed under serum free conditions in suspension and adherent cells. Expression was confirmed by western blot using an anti-FVIII antibody that confirmed expression of the heavy chains. Both proteins were biologically active when tested in the Coamatic chromogenic assay demonstrating approximately 60% and 25% activities compared to FVIII control. Purification by affinity chromatography was achieved however it requires optimization to minimize loss of proteins.

Conclusions: It was possible to express nrFVIII in CHO cells and retain biological activity. Current objective is to optimise purification and determine the plasma half-life of the nrFVIII in animal model.

Poster: 15 Mr Ahmed Al-Obaidy Ahmed Al-obaidy, Peter Monk PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 16 Dr Rohit Bazaz R.Bazaz, L.Williams, H.Marriott, S.Francis, D.Dockrell PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Increased atherosclerotic plaque macrophage content following Streptococcus pneumoniae pneumonia.

Rationale & Hypothesis: Clinical studies consistently find an increase in the risk of acute coronary syndrome (ACS) within 1-2 weeks after a respiratory infection. It has yet to be established whether a causal relationship exists. We hypothesise that the systemic inflammatory response to pneumonia leads to acute localised inflammatory changes within established atherosclerotic plaques, favouring plaque instability, thereby resulting in ACS.

Objectives: Our objective was to investigate the effect of resolving pneumonia on atherosclerotic plaque composition.

Methodology: 11-13 week old male ApoE-/- mice, a well-established model of atherosclerosis, were fed an atherogenic diet for 7- 8 weeks before intranasal infection with 5x105cfu type 4 Streptococcus pneumoniae or mock infection. All mice received 3 doses of 100 mg/kg subcutaneous amoxicillin, 12 hourly, the first dose given at 24 hours post infection. Mice were sacrificed 2 weeks post infection. Formalin fixed, paraffin embedded aortic sinus and brachiocephalic artery atherosclerotic plaque sections were analysed by histological and immunohistochemical staining.

Results: This model of atherosclerotic plaque development following pneumonia resulted in high rates of bacteraemia at 24 hours post infection and high survival rates at 5 days post infection (92% and 83% respectively). Streptococcus pneumoniae infection was associated with significantly increased aortic sinus atherosclerotic plaque macrophage content (18.1 vs. 8.0%; p<0.05). No significant differences in aortic sinus plaque burden, smooth muscle or collagen content were found following infection. Relatively few mice developed significant atherosclerosis at the level of the brachiocephalic artery and no significant differences were found between the two groups in plaques at this site.

Conclusion: In this murine model, pneumococcal pneumonia resulted in increased atherosclerotic plaque macrophage content, a marker of plaque instability. Pneumonia may therefore lead to an increased propensity for atherosclerotic plaques to rupture, although further work is required to assess the polarisation of macrophages in atherosclerotic plaques post pneumonia.

Poster: 17 Miss Irina Vazquez-Villaseñor I Vazquez-Villasenor, JE Simpson, C Garwood, PG Ince, P Heath, SB Wharton PhD Student - 2nd Year Neuroscience Contact: [email protected]

Neuronal senescence as a contributor to neurodegeneration

Rationale & Hypothesis: Cellular senescence and the senescence associated secretory phenotype (SASP) could contribute to neurodegeneration. Recent findings in vivo suggest that neurones can undergo senescence in association with persistent DNA damage (Jurk et al., 2012; Simpson et al., 2014). If senescent neurones express a SASP in response to persistent DNA damage, this could affect surrounding cells and contribute to neurodegeneration.

Objectives: To establish a model of senescence in a post-mitotic neuronal cell culture model and to determine whether senescence may play a role in MND.

Methodology: LUHMES cells were differentiated into post-mitotic neurones and stressed with increasing concentrations of

H2O2. Cell viability was assessed at different time points to identify a sub-lethal concentration of H2O2 that promotes a persistent DNA damage response and senescence. Expression of senescence-associated β-galactosidase (SA- β-gal) and γH2AX was also evaluated. Markers of senescence p16, p21 and SA-β-gal, were investigated in the frontal and motor cortex of human autopsy MND and control cases from the Sheffield Brain Tissue Bank.

Results: Post-mitotic LUHMES showed a significant decrease in cell viability after 4 hours of exposure to low H2O2 concentrations; 75 and 100 μM H2O2 were lethal. Activity of SA-β-gal was found in untreated post-mitotic LUHMES, possibly related to stress in culture. In vivo, p16 predominantly associated with glial cells, whereas p21 and SA-β-gal also associated with neurones. Higher glial p16 expression (p=0.017) and higher neuronal p21 expression (p=0.029) were detected in MND versus control cases; no significant differences were found in the number of SA-β-gal+ neurones.

Conclusions: The response to oxidative stress of a post-mitotic neuronal culture model has been characterised and senescence markers have been detected. Preliminary results in vivo indicate that senescent neurones and glia are present in MND, whilst differential expression of senescence markers suggests that pathways may differ between neurons and glia.

Poster: 18 Mr Mohammed Aldughaim Mike Barker, Mohammed Aldughaim PhD Student - 2nd Year Oncology Contact: [email protected]

The use of a novel peptide to specifically target therapeutic drugs to tumors

Tissue inhibitor of metalloproteinases 3 (TIMP3) is an extracellular matrix (ECM) protein with a number of novel properties, including the ability to inhibit vascular endothelial growth factor receptor 2 (VEGFR2), a key mediator of angiogenesis. We have identified several potential VEGFR2 binding sequences within the C-terminal domain of TIMP3 including a short 16 amino acid peptide sequence (p700). We aim to more precisely localize the VEGFR2 binding site and utilize P700 as a novel delivery vehicle for cytotoxic drugs to treat cancer.

Methods: Specific residues in TIMP3, identified as potential VEGFR2 binding sites, have been introduced into the C-terminus of a TIMP3-TIMP4 chimeric cDNA sequence with the aim of restoring its ability to inhibit VEGFR2. The sequence was cloned into pcDNA3 and will be screened by transfection of HUVEC cells to determine its effect on VEGF-induced VEGFR2 phosphorylation by western blotting. Additionally stable ARPE19 cell lines have been produced and ECM from these cells will be tested for its ability to inhibit VEGF-induced invasion of HUVECs. Codon optimised cDNA corresponding to carboxypeptidase 2 (CPG2 - an enzyme that converts non-toxic drugs into cytotoxic nitrogen mustards) has been synthesised and subcloned into pET28a bacterial expression vector ± the p700 sequence.

Result / Future Work: The modified TIMP3-TIMP4 chimera has been successfully cloned into the pcDNA3 mammalian expression vector and transfected into ARPE19 cells, however expression of the recombinant protein was inconclusive due to endogenous TIMP3 expression. To address this a V5-[His]6 tag has been added to the C- terminus of the constructs by PCR. The CPG2-p700 fusion protein is currently being expressed and purified and will screened for its ability to bind to HUVECs and tumour cells and to activate pro-drug.

Poster: 19 Mr David Randall D. Randall, K. D. Bardhan, R. Gillott, P. A. Spencer, J. W. Fenner PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Refinement in image processing for detection of abdominal adhesions using cine-MRI

Introduction: Smooth visceral sliding of the abdominal contents against the abdominal wall is required for healthy intra- abdominal movement. Peritoneal adhesion formation is expected to disrupt visceral sliding motion. Adhesions are a common complication of abdominal surgery and are associated with chronic pain, bowel obstruction, reoperation complications and female infertility. Non-invasive diagnosis of adhesions has the potential to improve patient management by determining the cause of symptoms. Cine-MR (cMR) has been used for this purpose [1] but has proven labour intensive and highly operator dependent. We previously presented a mathematical interpretation of abdominal movement in cMR to infer gross abnormalities [2]. The work presented here discusses a refinement of our approach for detection of subtle abnormalities.

Objective: To develop a refined technique for improved detection of abdominal wall adhesions by analysis of visceral sliding.

Methodology: The method operates on sagittal cMR images by calculating shear as an analogue for visceral slide. The principle driving the methodology is the expectation that the resistance of an adhesion to visceral slide produces a quantifiable reduction in shear at that location. Specifically the process involves: 1. Defining a line of demarcation between the sliding regions, typically the abdominal contents and the abdominal wall. 2. Independent interrogation of the respective modes of motion in these separate regions using image registration software. 3. Deduction of the relative motion at the boundary between the two regions. 4. Extraction of the magnitude of the shear and its presentation as a ‘sheargram’.

Findings: Testing of the technique on semi-idealised test-objects has demonstrated accurate calculation of shear. Application to clinical images has revealed localisations of reduced shear at areas corresponding to surgically confirmed adhesions. Supporting interpretation also comes from the absence of pathological shear in patients without anterior abdominal wall adhesions. Application to a larger patient cohort is underway to strengthen these results.

References: [1] Lienemann et al., Radiology, vol. 217, no. 2, pp. 421-5, 2000 [2] Fenner et al., Phys Medica, vol. 30, no. 4, pp. 437-447, 2014

Poster: 20 Miss Hind Naffadi Hind Naffadi, Dr. Gareth Richards, , Dr. Jules Westbrook , Prof Janet Brown, Prof Tim Skerry PhD Student - 2nd Year Human Metabolism Contact: [email protected]

The role of adrenomedullin in breast cancer metastasis to bone

Rationale & Hypothesis: 80% of advanced breast cancer patients develop osteolytic bone metastases. Patients suffer from extreme skeletal complication which will increase the morbidity. Bone metastasis is currently incurable. Cross talk between the tumour cell and bone microenvironment stimulate a vicious cycle, in which secreted several factors including tumour secreted factors. Inhibiting the activity of signalling pathways of tumour secreted factors promise a novel therapeutic target for bone metastasis. One hormone has been found involved in carcinogenesis and tumour progression is Adrenomedullin (AM) a 52 a.a peptide hormone. AM is involved in tumour initiation and progression by promoting tumour cells proliferation, angiogenesis, and inhibition of apoptosis. AM binds to two specific receptors composed of calcitonin receptor-like receptor (CLR) and accessory proteins known as receptor activity modifying proteins 2 and 3 forming AM1(CLR/RAMP2) and AM2 (CLR/RAMP3). We hypothesized that AM and its receptors are involved in metastasis of breast cancer to bone.

Objectives:

 To determine the gene and protein expression of AM and AM receptors in human metastatic breast cancer cell lines MDA-MB-231.  To understand the mechanisms by which AM is acting through its two receptors CLR/RAMP2 and CLR/RAMP3.

Methodology: In this study, triple negative human metastatic breast cancer cell line MDA-MB-231 was used. We examined the mRNA expression of AM, Prepro-AM, Pro-AM and Proadrenomedullin N-terminal 20 peptide (PAMP), CLR and RAMPs 1, 2, 3 by reverse transcription PCR and protein identification using western blotting.

Findings: Our data showed that AM and its receptor are expressed in MDA-MB-231 cell line at mRNA levels. All receptor components proteins CLR and RAMPs 1, 2 and 3 were also detected using western blotting.

Conclusion and Future work: AM and its receptors may contribute to mediate the function of AM in tumour metastasis. For future works generating knock-out cells using CRISPR technique. Then, protein fractionation using spike in SILAC followed by mass spectrometry (MS) analysis.

Poster: 21 Ms Furaha Florence Asani Furaha Florence Asani, Thushan de Silva, Andy Heath PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Detection of subtle immune defects in patients at risk of pneumococcal disease

Rationale & Hypothesis: Non-invasive and invasive pneumococcal disease (IPD) are caused by Streptococcus pneumoniae. Spn inhabits the human nasopharynx, and whilst cumulative immunity to it is acquired over time, some individuals still succumb to disease. Young children, the elderly, and individuals who are immuno-compromised have been identified as most at risk to disease. There are currently two vaccines in routine use for disease prevention namely: pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). Vaccine failures have been reported for PCV in some children in the UK. Our lab. previously found that unvaccinated sufferers of IPD and meningococcal disease had a defect in their B cell immune response to a T-independent antigen mimic, relative to healthy controls. Further, that vaccinated individuals who had succumbed to meningococcal disease (vaccine failures) had a defect in their T cell responses to a T-dependent antigen mimic. The present study hypothesizes that this same B and/or T cell defect will be found in certain groups most at risk to disease.

Objectives: This study aims to investigate the molecular mechanisms behind the poor response registered in unvaccinated previous sufferers of IPD using polyclonal stimuli. Further, to assess responses in other risk groups (HIV patients, haematological malignancy patients) and individuals identified as vaccine failures.

Methodology: Blood samples were obtained from consenting adults and lymphocytes separated out on a ficoll gradient. Lymphocytes were cultured with a T-independent mimic (addex) and T-dependent mimic (antiCD3), alone and in combination. B and T cell activation and proliferation were assessed using standardised flow cytometry.

Findings: Preliminary results from healthy donors show a suppression of CD4+ T cell activation by IgD-stimulated B cells. Experimentation on HIV patients is currently ongoing.

Poster: 22 Dr Leon Lewis Leon Lewis PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Prevalence of work-related respiratory symptoms & obstructive lung function in a cohort of UK-based foundry workers

Introduction: It is recognised that certain lung diseases are attributable to exposures in the workplace. Chronic obstructive pulmonary disease is increasingly recognised as being such a condition. Despite this little is known about the prevalence of work-related respiratory symptoms and abnormal lung function in foundry workers. Health surveillance, including basic measures of lung function, is one method by which health care workers may be able to identify this and other conditions.

Methodology: Workers from foundries across the UK were approached to participate in a cross-sectional study. Data encompassing basic demographics, known physician diagnosed respiratory disease, current respiratory symptoms, smoking history and occupational exposures were collected. Participants were also asked whether they considered themselves to be “healthy” or “unhealthy”. Participants performed spirometry to assess the prevalence of obstructive lung disease, defined as an FEV1/FVC ratio of <0.7.

Findings: Data was collected from 350 foundry workers: 349 were male, with a mean age of 42 years. Mean duration of employment within foundries was 15 years. 190 were “ever smokers” and 92 current smokers. 45 had a prior diagnosis of asthma and six were known to suffer from COPD. The prevalence of work-related respiratory symptoms was as follows: 44 participants reported work-related cough, 26 chest tightness, 23 wheeze, 1 breathlessness. 65 participants reported ≥ 1 work- related respiratory symptom. 35 participants had obstructive lung function. Increasing age (p<0.01), a history of tobacco smoking (p=0.028), answering “unhealthy” (p=0.01) and having ≥ respiratory symptom were all significantly associated with the presence of obstructive lung function (as was, by definition, a prior diagnosis of COPD).

Conclusion: In this cohort, 19% of participants reported suffering from at least one work-related respiratory symptom, the commonest being cough, reported by 12%. A number of factors were associated with the presence of obstructive lung function, but with only a negative perception of health and increasing age retaining an independent association.

Poster: 23 Mr Wen-Yo Tu Wen-Yo Tu, Sophie Thomson, J. Robin Highley, Thomas H. Gillingwater, Paul R. Heath PhD Student - 2nd Year Neuroscience Contact: [email protected]

Selective Vulnerability of Motor Neurones in Spinal Muscular Atrophy

Rationale & Hypothesis: Spinal muscular atrophy is a neurodegenerative disease, characterised by lower motor neurone degeneration. The loss of motor neurone ultimately causes patient’s paralysis. However, many studies have revealed that such motor neurone loss is not evenly distributed in the spinal cord, raising the possibility of whether there is intrinsic factor that can modulate motor neurone pathology. A previous study showed that, in a SMA mouse model, the motor neurone innervating extensor digitorum longus (EDL) has greater resistance to the disease than those innervating anterior tibialis (TA) regarding their ability of maintaining neuromuscular junction (NMJ) during the disease progress. Thus, we put forward the existence of intrinsic disease modifying factors that account for the distinct vulnerability seen in motor neurones innervating EDL and TA.

Objectives: Using microarray analysis to identify the intrinsic factors that differentiate motor neurone vulnerability and microcapillary based cell culture to investigate these differences

Methodology: Retrograde motor neurone labelling, Laser capture microdissection (LCM), Microarray analysis, Cell culture

Findings: Previous attempt identified cholesterol synthesis is up-regulated in vulnerable motor neurones (TA group). Meanwhile, in vitro neuromuscular junction (NMJ) system has been established for further functional validation of identified disease modifying gene.

Poster: 24 Mr Meshal Alhajimohammed Meshal Alhajimohammed, Karen Sisley PhD Student - 2nd Year Oncology Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 25 Dr Jivendra Gosai Gosai J, Gunn J, Purva M PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

A survey of medical trainees’ experience and attitudes to simulation training in the United Kingdom.

Rationale & Hypothesis: There has been considerable investment and development in simulation training (SBT). There has to date been no large scale study exploring the experience of postgraduate medical trainees.

Objectives: To survey a wide and representative sample of medical trainees on SBT they have been had, and explore how the experience was perceived, what barriers existed and how they saw the role of SBT within their training and ongoing career.

Methodology: Ethical approval to conduct this research was obtained. Survey questions were designed in consultation with experts in the subject, and medical trainees. A mixture of demographic questions, Likert scales and free text questions was employed. A pilot was conducted to ensure that completion time remained under 10 minutes.

Following this, the questions were transferred to a web based hosting service. Postgraduate deans were contacted for permission to distribute the questions to trainees. Data analysis was conducted using a mixed-methods approach. Descriptive and quantitative analysis was employed to assess demographics and Likert type answers. Qualitative analysis using a thematic and broad brush approach was used

Findings: A total of 655 valid responses were received, primarily from 4 major regions within the UK. All medical specialties and grades were represented. 540 had experienced at least one type of simulation training, and 123 had participated as teachers. There was a weakly positive attitude to considering this form of learning in the future (mean 5.23, SD 1.51 procedures, mean 5.83m SD 1.44 scenarios), and a perception that simulation has the potential to enhance healthcare quality and safety (mean 5.85 quality, 5.92 safety). Key themes within the data were curriculum integration, the limitations of current approaches and technology, positive and negative aspects of its use and the potential for future development.

Poster: 26 Miss Lucy Evans Evans L1, Naylor K1, Keevil B2, Eastell R1 PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Clinical evaluation of a salivary 25-hydroxyvitamin D method by liquid chromatography tandem mass spectrometry for the assessment of vitamin D status.

Vitamin D is essential for musculoskeletal health. Current assays measure total 25-hydroxyvitamin D (25OHD) in order to assess vitamin D status. Most 25OHD is bound to vitamin D binding protein (85-90%) or albumin (10-15%) while only a small proportion (>1%) remains unbound. Recent studies have found that free 25OHD levels are better associated than total levels with markers of bone health. Total 25OHD has also been shown to decrease during an acute inflammatory response and is therefore an unreliable biomarker in patients with inflammatory illnesses. Free 25OHD may be a better biomarker in these patients, however this is analytically challenging. Liquid chromatography tandem mass spectrometry (LCTMS) is the gold standard method of measuring total 25OHD, however only total 25OHD can be measured in this way. Bikle et al. (1986) separated free 25OHD prior to analysis by using centrifugal ultrafiltration, we will use this as our reference method but its complexity makes it unsuitable for routine clinical use. Saliva is a source of free 25OHD, as it does not contain binding proteins. We have preliminary data where we were able to quantitate human salivary 25OHD at concentrations around 10 pmol/L. We will validate this assay in two clinical groups and compare the method with centrifugal ultrafiltration. We will measure salivary 25OHD, free serum 25OHD, total 25OHD, levels of the binding proteins, calcium, parathyroid hormone and CRP before, 2 and 5 days after lower limb arthroplasty in 25 patients to assess whether free 25OHD is affected by the inflammatory response. We will also measure the effect of high dose (300,000IU) vitamin D supplementation on 25 patients before and after 3, 7, 10 and 14 days as we predict that the binding proteins will become saturated so that salivary and serum free 25OHD concentrations increase disproportionately to total 25OHD during supplementation.

Poster: 27 Dr Ruth Wiggans Wiggans RE, Evans G, Fishwick D and Barber CM. PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 28 Miss Chloe Marshall Chloe Marshall, Lynne Bingle, Colin Bingle PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Staphylococcus aureus interactions with human airway glygoproteins

S. aureus is found as a commensal bacterium in the upper respiratory tract in around 20% of the population however airway infections are rare in healthy individuals. The upper airways employ a robust innate immune response to limit the establishment of bacterial infections. A major aspect of this innate immunity is the secretion of fluid and proteins from epithelial cells which form the airway surface liquid (ASL). The ASL acts as a physical protective barrier sitting directly above the epithelial cells with many of the proteins present in the ASL having proposed anti-bacterial functions. We are interested in the role that protein components of the ASL play in the initial interactions with S. aureus.

Two of the most abundant secretory airway proteins, BPIFA1 and BPIFB1, were used in bacterial-protein pull-down assays to detect interactions with S. aureus wild-type and a series of adhesion mutant strains from the Nebraska transposon mutant library. Interactions between S. aureus and these airway proteins were visualised using western blotting techniques with specific polyclonal antibodies. Each of the S. aureus strains examined showed strong interactions with BPIFA1 or BPIFB1 following the 1 hour incubation period.

To overcome issues relating to the complex protein mixture found in ASL we will study individual recombinant proteins. We have shown the BPIFA1 and BPIFB1 expressed as V5-His epitope tagged constructs in Chinese hamster ovary (CHO) cells also allows for studies of S. aureus interactions via pull-downs. We are currently establishing lines expressing a range of additional proteins including, secretoglobins (SCGB1A1, SCGB3A2), mucins (MUC7) and peptidoglycan recognition protein. We will use these constructs in S. aureus pull-down assays to investigate the specific interaction of these airway proteins with the bacteria.

Poster: 29 Dr Sheila Ramjug S Ramjug, J Hurdman, N Hamilton, C Elliot, D Kiely, R Condliffe PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Long-term intravenous iloprost in pulmonary arterial hypertension

Introduction Although intravenous (iv) iloprost is used in several large pulmonary hypertension (PH) centres there are relatively few data describing long-term outcome in patients with pulmonary arterial hypertension (PAH). We have therefore reviewed dosage, response to therapy and survival in PAH patients treated at a large UK PH centre.

Methodology Hospital databases, including the ASPIRE registry, were interrogated to identify all PAH patients treated as an out-patient with intravenous iloprost between 1999-2013.

Results 61 patients were treated: 33 (54%) idiopathic PAH (IPAH), 22, (36%) connective tissue disease-associated PAH (CTD- PAH), 2 portopulmonary hypertension and 4 PAH-CHD. Within the IPAH and CTD-PAH groups, 25 (45%) died and 4 (7%) underwent lung transplantation. Baseline characteristics and PAH therapy at commencement of iv iloprost for IPAH and CTD-PAH are described in table 1. Median dose of iv iloprost at initial discharge and maximum dose achieved in IPAH was 2.8ng/kg/min and 4.7ng/kg/min, and in CTD-PAH was 2.5ng/kg/min and 5.2ng/kg/min respectively. At one month following commencement of therapy 67% of IPAH and 45% of CTD-PAH patients had improved by ≥1 functional class. Median incremental shuttle walking distance pre iv iloprost therapy was 140m in IPAH and 110m in CTD-PAH. Median shuttle walking distance within six months of commencing therapy had increased by 40m in both IPAH patients (p=0.001) and CTD-PAH patients (p=0.054). 1, 3 and 5 year survival to census date or transplantation from commencement of iv iloprost was 81%, 72% and 64% in IPAH and 64%, 23% and 17% in CTD-PAH (log rank p=0.002).

Conclusion Intravenous iloprost is associated with functional improvement in both IPAH and CTD-PAH. Survival in patients with IPAH treated with iv iloprost is superior to that previously described. This may be, in part, related to higher doses used in the present study. Patients with CTD-PAH had markedly poorer survival than IPAH, in keeping with the previously observed more aggressive clinical course in CTD-PAH.

Poster: 30 Miss Karla Robles Lopez Karla L Robles Lopez, Guillaume Hautberge, Heather Mortiboys, Oliver Bandmann PhD Student - 2nd Year Neuroscience Contact: [email protected]

The role of TIGAR in Parkinson’s Disease

Rationale & Hypothesis: Parkinson disease (PD) is the second most common neurodegenerative disease amongst the elderly. The pathological hallmark of disease is the loss of dopaminergic neurons. Common causes of early onset PD are mutations in parkin and PINK1 genes. Upregulation of tigarb (human orthologue of TIGAR (TP53 –Induced Glycolysis and Apoptosis Regulator) was demonstrated in a zebrafish pink-/- model with dopaminergic (DA) cell loss and mitochondrial dysfunction. Knockdown of tigarb resulted in rescue of dopaminergic neurons and recovery of mitochondrial function. Parkin and PINK1 function in the same pathogenic pathway. Better understanding of TIGAR-related mechanisms in early onset PD due to either parkin or PINK1 mutations will help us understand whether TIGAR may be a promising target for disease-modifying therapy.

Objectives: To determine the role of TIGAR in PD mutant patient tissue, namely in parkin-mutant fibroblasts.

Methodology: First, TIGAR mRNA and protein levels were assessed in fibroblasts of PD patients with homozygous or compound heterozygous parkin mutations and matched controls, using q-PCR and Western blots. Next, we optimised the transfection conditions and assessed the efficiency of RNAi mediated knockdown of TIGAR and parkin in control fibroblasts by either siRNA or miRNA. Mitochondrial function and morphology were assessed using ATP assays and live cell imaging in one control and one parkin-mutant fibroblasts.

Findings: TIGAR is expressed and translated in fibroblasts with a wide range in protein levels in parkin-mutant fibroblasts. Efficient siRNA mediated knockdown was achieved in fibroblasts. In addition, miRNA-mediated gene knockdown was achieved for both parkin and TIGAR in HEK 293 cells. Outlook: Having validated all relevant tools, we will now further determine the role of TIGAR in PD patient tissue and additional suitable model systems.

Poster: 31 Mr Neil Bowden N Bowden, H Duckles, M Mahmoud, S Hsiao, S E Francis & P C Evans PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Disturbed flow induces expression of the negative NF-kappaB regulator Cezanne

Introduction Atherosclerosis is a chronic inflammatory disease occurring at arterial sites exposed to disturbed flow (DF). Endothelial cells (ECs) at these sites transduce haemodynamic forces into inflammatory signals through the NF- kappaB pathway, leading to lesion development. The deubiquitinase Cezanne negatively regulates NF-kappaB activity. In order to understand the potential role of Cezanne in regulation of focal vascular inflammation, we characterised the expression of Cezanne under different flow patterns.

Methods and results Human umbilical vein ECs (HUVECs) were cultured under DF (+/-5 dyn/cm squared) and non-DF (13 dyn/cm squared) in vitro. Quantitative real-time PCR revealed that Cezanne mRNA was expressed at significantly higher levels in ECs exposed to DF compared to non-DF (p<0.01). Western blotting confirmed an increase in Cezanne protein expression in response to DF, and revealed that Cezanne was expressed as multiple isoforms. Additionally, murine aortae were stained en face for Cezanne and analysed by laser scanning confocal microscopy. This demonstrated an increase in Cezanne expression at a DF region of the inner aortic arch in comparison to non-DF regions in the outer arch and descending aorta.

Conclusions We conclude that Cezanne expression in EC was enriched at an atheroprone site in the murine aorta. The mechanism is related to local haemodynamics since DF induced Cezanne in cultured ECs. It is plausible that Cezanne functions to limit inflammation at disease-prone sites by inhibiting NF-kappaB.

Poster: 32 Miss Dingyi Yang Dingyi Yang, John Connolly, Simon Foster, Lynne Prince PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Identifying novel immune modulating factors in a genome-wide S. aureus screen in human neutrophils

Rationale & Hypothesis: Staphylococcus aureus is a highly adaptive and widespread bacterial pathogen causing numerous clinical problems due to widespread antibiotic resistance. Recent human-pathogen interaction studies reveal multiple evasion mechanisms of S. aureus which they use to survive or even replicate within neutrophils which are thought to be the primary cellular defence against S. aureus. In addition, S. aureus induces rapid and profound neutrophil necrosis, which further disables the immune response.

Objectives: To identify novel immune modulators regulating neutrophil function through a genome-wide screen S. aureus mutants.

Methodology: Primary human neutrophils were incubated with individual S. aureus mutants at an MOI of 10 for 3 hours before ToPro-3 staining and assessment of cell loss via flow cytometry.

Findings: Assessment of ToPro-3 negativity and viable cell counts identified 118 S. aureus strains that showed attenuated neutrophil cell death. This was reduced to 34 after further validation by focused secondary screens (n=3). A number of internal controls with known pro-death functions were among the identified attenuated strains including lukAB, lukGH, agrA and saeS. Current studies to validate the mutations include transduction of gene of interest followed by gene complementation. Phenotypes of these strains in functional neutrophil assays will confirm their role in neutrophil cell death.

Poster: 33 Mr Joseph Ford Joseph F. Ford, Martin A. Bewley, Simon A. Johnston, David H. Dockrell PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The Role of Nitric Oxide in Streptococcus pneumoniae Induced Lysosomal Membrane Permeabilisation

Rationale & Hypothesis: A key phase in the macrophage response to S. pneumoniae is a program of host-mediated apoptosis. This occurs after the macrophage has engulfed bacteria, but becomes overwhelmed. The cell then follows an apoptotic death pathway in order to contain the infection and increase late stage killing of the bacteria. The apical event in this pathway is lysosomal membrane permeabilisation (LMP). This is caused by a currently unknown mechanism that involves the bacterial toxin pneumolysin (PLY). PLY is known to contribute to nitric oxide (NO) production. We hypothesised that PLY-induced NO production by the macrophage contributes to LMP.

Objectives: To deduce the role of nitric oxide in LMP in the context of Spn infected macrophages

Methodology: Primary human monocyte derived macrophages (MDMs) were isolated from healthy blood samples and rested in RPMI 1640 for 14 days before infection. MDMs were infected at an MOI of 10 using opsonised D39 S. pneumoniae, a PLY negative mutant (Stop), or latex beads. At 16hpi MDMs were stained using acridine orange (AO) and analysed by FACS flow cytometry. Cell fractionation and western blots were performed to confirm LMP. In some experiments MDMs were treated with inhibitors of ROS (Trolox, DPI) and NO (1400W), NO donors SNAP or NOC-13 and/or TLR agonists lipoteichoic acid (LTA) and lipopolysaccharide (LPS).

Findings:. D39 infected MDM displayed significantly higher levels of LMP than Stop infected cells. Incubation with 1400W and Trolox significantly reduced LMP in D39 infected cells, but DPI had no effect. Conversely, incubation with SNAP and NOC-13 was able to restore LMP in Stop infected cells. Further dissection of the pathways leading to LMP revealed that NO was necessary but not sufficient for LMP, with toll-like receptor (TLR) 2 and 4 activation, and phagosomes formation also required. These data identify a novel role for NO in LMP and host-pathogen interactions. 55

Poster: 34 Miss Jodie Stephenson Jodie Stephenson, Dr Richard Mead, Dame Professor Pamela Shaw PhD Student - 2nd Year Neuroscience Contact: [email protected]

Characterisation of a novel pre-clinical model of Motor Neurone Disease (MND)

Rationale & Hypothesis: Motor Neurone Disease (MND) is a progressive degenerative disease of motor neurones usually leading to death within 2-3 years. Approximately 10% of MND is familial and several causal mutations have been identified in genes such as SOD1, TDP43, FUS and C9ORF72. Current mouse models are based on SOD1 mutations. There is a need for more models to improve translation to the human disease. We are currently investigating a new mouse model which is transgenic for human TDP43 with a Q331K mutation and hypothesise that this model may be more representative since TDP43 protein is heavily implicated in the pathogenesis of sporadic MND which accounts for 90% of cases.

Objectives: To fully characterise the TDP43-Q331K mouse model of MND using a variety of quantitative measures of disease progression.

Methodology: Two mouse colonies were established, TDP43WT transgenic, and TDP43Q331K transgenic. Both genotypes were investigated for phenotypic characteristics of MND using rotarod, automated gait analysis, weight and neurological scoring.

Findings: The TDP43-Q331K mice showed signs of spasticity, tremor, muscle loss and weight gain. Male mice showed significantly worsening rotarod performance from 9 weeks of age (39.4%-61.5% reduction in TDP-43Q331K mice, between weeks 9 to 27, P<0.05, two way ANOVA) and female mice from 12 weeks (30.5%-51.5% reduction in TDP-43Q331K mice, between weeks 12 to 24, P<0.05, two way ANOVA). Preliminary analysis of mice at 6 months of age indicated that they have altered gait parameters. In addition male and female TDP43Q331K transgenic mice showed significantly increased weight despite a reduction in muscle mass. Spasticity, tremor and muscle loss support the validity of this model of MND, whereas the weight gain is unexpected.

Conclusion: TDP43-Q331K mice show promise as a model of MND as they demonstrate a progressive motor degenerative phenotype. Further investigation is required to establish whether the mice show pathological signs of MND.

Poster: 35 Mr Andrew Nichols Andrew Nichols, Sarah Smith, Caroline Gray, Nikolay Ogryzko, Timothy Chico,Heather Wilson & Eva Qwarnstrom PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 36 Dr Emma Walkinshaw E Walkinshaw, SA Little, A Bernjak, A Lubina Solomon, E Chow, JAM Shaw, SR Heller PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Cardiac autonomic regulation during experimental hypoglycaemia in type one diabetes with impaired awareness of hypoglycaemia.

Background: Impaired awareness of hypoglycaemia (IAH) affects 25% of patients with Type 1 diabetes (T1DM) and increases the risk of severe hypoglycaemia (SH). Previous studies report decreased vagal outflow during hypoglycaemia in T1DM, potentially predisposing increasing vulnerability to arrhythmias. Our aim was to investigate the effect of experimental hypoglycaemia on cardiac autonomic regulation in patients with IAH.

Methodology: Ten participants with T1DM and intact hypoglycaemia awareness, eight with IAH (Gold score≥4), six with restored awareness (Gold score<4) and ten without diabetes underwent a stepped hyperinsulinaemic hypoglycaemic clamp study. Glucose was held at 5mmol/l at time 20 minutes (T20) and reduced step-wise to a nadir of 2.4mmol/l at time 180 minutes (T180). Heart rate variability was measured during each step.

Results: Heart rate (HR) increased (p=0.008 T20 vs T180) with decreased high frequency (HF) power, suggesting vagal withdrawal (p=0.038 T20 vs T180) in participants with intact hypoglycaemia awareness. Normalised low frequency (LFnorm) slightly increased, indicating elevated sympathetic contribution. Subjects without diabetes showed smaller increases in HR and LFnorm and a reduction in HF power which recovered to baseline by T180. Participants with IAH showed a maximum increase in HR at 100 minutes (p=0.041) with a reduction towards baseline at T180. HF power reduced until 140 minutes and then showed signs of recovery. There was a reduction in sympathetic outflow and which corresponded to reduced metanephrine levels in comparison to the other participant groups seen in this study. Participants with recovered awareness showed an increase in HR, reduction in HF power and increase in LF norm which peaked at 60 minutes with recovery by T180.

Conclusions: Cardiac autonomic regulation during hypoglycaemia is time dependent and differs according to self-reported awareness of hypoglycaemia. In participants with intact awareness, reduced vagal outflow during hypoglycaemia could predispose to cardiac arrhythmia. In subjects with IAH, hypoglycaemia has a diminished effect, potentially providing cardiovascular protection.

Poster: 37 Dr Fatma Hamed Fatma Hamed, Andrew J McDonagh, Thomas Lovewell, Andrew G Messenger, Rachid Tazi-Ahnini PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Inhibitors of IFN-g signalling pathway to restore immune privilege and enhance hair re-growth in alopecia areata patients

Alopecia areata (AA) is an autoimmune disease of hair follicles (HF). The exact pathogenesis is unclear, but it is believed to result from immune privilege (IP) collapse in the HF. IP collapse is induced by IFN resulting in the upregulation of MHC expression in HF keratinocytes leading to lymphocytic attack towards HFs. Ruxolitinib has restored hair growth in AA patients and supressed the IFN signalling pathway by inhibiting phosphorylation of STAT proteins. However, Ruxolitinib is expensive, potentially toxic with a broad target spectrum. Therefore our aim is to select more specific molecules with reduced toxicity; specifically molecules capable of inhibiting the IFN signalling pathway leading to the restoration of IP in affected HFs. We have tested a selection of natural products and small molecules which inhibit STAT-1 phosphorylation. EGCG, a green tea extract, was found to have an inhibitory effect on IFN signalling pathway in HaCat and JURKAT cell lines, affecting the expression of STAT-1 and downstream genes. Our next objectives are to test other inhibitors and measure the effect of EGCG on T-cells isolated from blood and skin of healthy and AA candidates. Our results can eventually lead to a clinical pilot study to measure the efficacy of the EGCG on restoring hair growth.

Poster: 38 Ms Natalie Rounding Natalie Rounding, Kurt De Vos, Andrew Grierson PhD Student - 2nd Year Neuroscience Contact: [email protected]

Zebrafish C9orf72 loss of function models of Amyotrophic Lateral Sclerosis (ALS)

Background: Expansions of a noncoding G4C2 hexanucleotide repeat in C9orf72 are the most common genetic defect found to date in both Amyotrophic Lateral Scleorsis (ALS) and Frontotemporal Lobar Dementia (FTLD), providing the most direct molecular link between these two disorders (DeJesus-Hernandez et al., 2011; Renton et al., 2011). It is not known how the repeat expansion causes disease, however three principal mechanisms have been suggested: haploinsufficiency, RNA toxicity and repeat-associated, non-ATG (RAN) translation.

Hypothesis: This project will test the hypothesis that haploinsufficiency of C9orf72 causes ALS in zebrafish, by a mechanism involving defective autophagy and protein homeostasis.

Methods: Transcription activator-like effector nucleases (TALENs) were designed against a target sequence within exon 7 of zebrafish C9orf72 (zC9orf72) to generate zC9orf72 loss of function alleles. Single cell stage wild type zebrafish embryos were injected with the designed TALEN and mutations were confirmed in one day old embryos via PCR and restriction enzyme digest. This was the method used for screening throughout the genotyping process. Survival characterisation was performed during larval stages. Tanks were inspected twice per day and dead larvae removed to permit genotyping.

Results: Four F2 lines have been raised carrying a range of insertion and/or deletion (INDEL) mutations within exon 7 of zC9orf72. All of these mutations are predicted to result in a truncated form of zC9orf72. Identified heterozygous carriers from each line were incrossed and survival of the resulting offspring monitored up to 21 days. Survival characterisation revealed that these mutations in zC9orf72 do not lead to early loss in viability.

Conclusion: Ultimately, we aim to generate a stable C9orf72 knockout zebrafish. If future characterisation reveals that these mutants develop ALS phenotypes, it would allow for the investigation into C9orf72-linked disease mechanisms and suggested function(s) of the uncharacterised protein.

Poster: 39 Mr Luke McKenzie Luke McKenzie, Gareth Williams, Julia Weinstein, Helen Bryant PhD Student - 2nd Year Oncology Contact: [email protected]

A novel transition metal complex for use as a photosensitizer in the photodynamic therapy of cancer

Abstract: A novel transition metal compound was investigated for use as a drug in photodynamic therapy, PDT. The chemical, photochemical and biological action of the compound was assessed.

Rationale & Hypothesis: Photodynamic Therapy, PDT, is a disease treatment whereby a drug is administered in a non-active form, only to be activated when irradiated by a specific wavelength of light. A novel class of metal- based compounds had been shown to have photodynamic properties in solution and to be cell permeable. We hypothesised that these compounds could therefore be used as light-activated chemotherapeutic agents.

Objectives: To assess the toxicity and mechanism of action of a novel transition metal compound as a chemotherapeutic.

Methodology: The photochemical properties of the compound were assessed, its singlet oxygen yield and lifetimes as well as cellular localisation. An assessment of its dark vs light toxicity in cells, cellular localisation and incubation times was conducted. The mode of cell death was assessed by flow cytometry.

Findings: The compound was found to have excellent photodynamic therapeutic effect at the wavelength of excitation of 410nm. It was found that the treatment with 1 μM of compound and 3 minutes of 20 mW light led to near complete killing of a number of cell lines whilst the toxicity in the dark was low across all cell lines.

Conclusion: The compound showed high light induced toxicity in a range of cell lines whilst showing very low dark toxicity. This suggests that the compound may be viable as a drug in PDT. 58

Poster: 40 Ms Martina Sciola M. Sciola, P. Lawford, D.R. Hose, J.Gunn PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Quantitative computational evaluation of effect of coronary lesions on cardiovascular physiology

Rationale & Hypothesis: Coronary artery disease is diagnosed with the aid of imaging techniques such as angiography. To determine the physiological significance of a lesion, coronary flow at rest is compared with flow under an extreme condition of pharmacologically-induced hyperaemia (administration of adenosine). This enables the calculation of an index, the Fractional Flow Reserve (FFR), used by clinicians to decide if the Percutaneous Coronary Intervention (PCI) is necessary. Whilst FFR is a valuable indicator of stenosis severity, depending on specific patient characteristics, the decision to treat can still be subjective, especially where FFR values are close to the treatment threshold.

Objectives: This project aims to obtain a more meaningful measure of lesions severity for the individual patient using a lumped- parameter model of cardiovascular system incorporating the relevant regulatory mechanisms. The model can be personalised based on physiological data collected during the patient’s daily activities. It is anticipated that this will provide a more objective measure to aid decision making by predicting the impact of the coronary lesion for the specific patient.

Methodology: A 0-D model developed by Korakianitis1 has been extended (Open Cor software) to include the coronary circulation, effects of ventricular contraction and the blood and oxygen distribution. Patient-specific total blood volume is calculated from the Nadler formula. As a first step, the model is being used to simulate adenosine-induced hyperaemia and thus IV adenosine pharmacokinetics have been incorporated into the model. Myocardial resistance is a function of adenosine concentration. The model will be validated against pressure data obtained during angiography, using optimisation processes, to give a patient-specific model.

Findings: Preliminary outputs from the model configured for a generic individual gave physiologically representative pressures, flows and oxygen saturation. The model is able to predict the distribution of adenosine and the myocardial response to hyperaemia. Different lesions can be represented by adjusting coronary parameters. Future work will include the introduction of regulation pathways, and validation against clinical data. Most importantly the model will be personalised further using data acquired during the patient’s daily activities, and will be used to predict the effect of a specific coronary lesion and the effect of the eventual PCI.

Poster: 41 Miss Andreea Ciuntu Andreea Ciuntu, Dr. Lynda J. Partridge, Dr. Lynne Prince PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The role of tetraspanins in neutrophil apoptosis and phagocytosis

Rationale & Hypothesis: Inflammatory lung diseases are characterized by chronic inflammation, and represent a major economic burden worldwide. Neutrophils are innate immune cells that play a major role in the pathogenesis of inflammatory lung disease. Their prolonged survival in the tissue results in tissue damage and prolonged tissue inflammation. Tetraspanins are a family of four membrane spanning proteins that have key roles in immune functions including cell adhesion, motility and proliferation as well as bacterial adhesion, although little is known in the neutrophil. We hypothesize that tetraspanins play a role in neutrophil function.

Objectives: Determine the roles of the tetraspanins CD63 and CD151 in neutrophil apoptosis and phagocytosis in in vitro studies.

Methodology: Primary human neutrophils were isolated from peripheral blood of healthy volunteers via Percoll gradient centrifugation. Anti-CD63 and anti-CD151 antibodies and/or blocking peptides were used to probe tetraspanin functions. Neutrophil apoptosis was assessed by bright field microscopy and flow cytometry while phagocytosis of heat killed Staphylococcus aureus and Streptococcus pneumonia was assessed by bright field and fluorescent microscopy.

Findings: Anti-CD63 antibodies and fusion proteins from the CD63 extracellular domain significantly delayed neutrophil apoptosis. Anti-CD151 antibodies inhibit the phagocytosis of heat killed Staphylococcus aureus, but not Streptococcus pneumoniae. Additional studies to understand the mechanisms underpinning the roles of tetraspanins in neutrophil function are in progress

Poster: 42 Mr Taiwo Oguntona Taiwo S Oguntona, Peter W Gurney, Phil Watson, David J Buttle PhD Sudent - 2nd Year Infection & Immunity Contact: [email protected]

Reducing Renal fibrosis using a selective carboxypeptidase U inhibitor UK-396082

Rationale & Hypothesis: In in vitro and in vivo rat models renal fibrosis can be reduced by increasing plasmin activity through inhibition of carboxypeptidase U activity using a selective carboxypeptidase U inhibitor-UK396082

Objectives: To assay uPA, tPA, plasmin, carboxypeptidase U, matrix metalloproteinases, and ECM in rat kidney epithelial, fibroblast and mesangial cells in the presence and absence of fibrin. Determine if the treatment of in vitro and in vivo models of diabetes with UK-396082 will result in reduction of ECM accumulation in kidney fibrosis.

Methodology: Carboxypeptidase U, plasmin, urokinase-type and tissue-type plasminogen activators were assayed in the presence and absence of UK-396082 in rat kidney epithelial, fibroblast and mesangial cells cultures, with and without fibrin gel.

Findings: Results obtained have confirmed carboxypeptidase U, tissue–type and urokinase-type plasminogen activators and plasmin activities in rat kidney epithelial, fibroblast and mesangial cells. Higher carboxypeptidase U, tissue–type and urokinase-type plasminogen activators and plasmin activities were found in the presence of fibrin.

Conclusion: This study will help to determine whether CPU is a potential therapeutic target for treating fibrosis.

Poster: 43 Ms Evangelia Karyka Evangelia Karyka, Kurt De Vos, Guillaume Hautbergue, Mimoun Azzouz PhD Student - 2nd Year Neuroscience Contact: [email protected]

RNA changes in SMN-deficient experimental cell models of Spinal Muscular Atrophy

Background: Spinal Muscular Atrophy (SMA) is a fatal neurodegenerative disease affecting primarily lower motor neurons. It is the major cause of death during infancy with no effective treatment to date. SMA is associated with homozygous deletion or mutation of the Survival Motor Neuron 1 gene (SMN1), which encodes the ubiquitously expressed protein SMN. SMN plays a crucial role in pre-mRNA splicing and it is also involved in axonal trafficking of mRNA-binding proteins and their target mRNAs. How SMN deficiency leads to selective loss of motor neurons is still unclear.

Objectives: In this study we aim firstly to identify transcriptome perturbations in SMN-deficient motor neurons using next generation RNA-sequencing (RNA-seq). Secondly, we plan to investigate any potential axonal transport defects of candidate mRNAs using live cell imaging.

Methods and Results: To analyse splicing defects we have developed methods to extract RNA suitable for RNA- seq from whole cell extracts and cytoplasmic fractions of SMA type I human fibroblasts and healthy controls as well as primary cortical and motor neurons in which SMN expression is ablated by adeno-associated serotype 9 (AAV9) mediated RNAi. To analyse the effect of SMN deficiency on axonal transport of mRNAs identified through RNA-seq we have established an mRNA visualization system suitable for live imaging in primary neurons based on a bidirectional lentiviral vector.

Poster: 44 Mr Simon Webster Simon J. Webster1, Daniel J. Hampshire1, Bimal Theophilus2, Reinhard Schneppenheim3,Daniel B. Bellissimo4, Paula D. James5, Ian Peake1, Anne C. Goodeve1 and on behalf of the EU-VWD and ZPMCB-VWD study groups PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

DETECTION OF LARGE EXONIC AND INTERGENIC DELETIONS IN THE VWF LOCUS OF VWD PATIENTS USING ARRAY COMPARATIVE GENOMIC HYBRIDISATION (aCGH)

Rationale & Hypothesis: von Willebrand disease (VWD) is caused by mutations in von Willebrand factor (VWF), a large multimeric glycoprotein, essential for platelet dependent primary haemostasis and binding and transporting factor VIII. Mutations in the VWF gene (VWF) result in qualitatively defective (type 2), or quantitatively deficient (types 1 and 3) VWD. In type 1 VWD, ~35% of patients have no causative VWF mutation following exon and intron-exon boundary sequencing. Copy number variation (CNV) consisting of large exonic deletions/duplications within VWF has been reported, but little is known about the pathogenicity of CNV in VWF non-coding regions. We hypothesise that deep intronic and flanking untranslated region (UTR) CNV contribute to VWD. Objectives: To use aCGH to identify novel CNV across the VWF locus, including introns, 5’ and 3’ UTR that may contribute to VWD pathogenesis. Methodology: Custom 8x15K and 8x60K microarrays were designed (Agilent Technologies) containing ~1800 probes spanning the VWF locus. 18 patients with known CNV were selected for validation including whole-gene deletions, 6 pseudogene region deletions (exons 28, 32-34, 33-34), deletions of exons 3, 4-5, 17-18 and a duplication of exons 9-10. 20 test patients (no identified CNV) were selected to check for novel CNV. Data were analysed using Agilent CytoGenomics software and CNV frequencies checked against the database of genomic variants (DGV). Findings: Known CNV were detected in 16/18 patients, where array breakpoints mapped to within a minimum of ~26bp and a maximum of ~1.3kb of known breakpoints. In 2 patients, a novel ~4kb 5’UTR deletion was also detected. In 2/20 test patients, a further novel 5.1kb 5’UTR deletion was detected. Custom aCGH is an effective strategy for CNV screening in VWF. In addition to known CNV, two novel 5’UTR deletions of ~4kb and ~5kb, approximately 11kb and 23kb upstream of VWF were detected. These findings could have important implications for our understanding of VWF levels in VWD and in the normal population.

Poster: 45 Dr Preethi Rao Preethi Rao*1, Jonathan C Brooke2, Debbie Walter2, Amrita Dhutia2, David McLaren2, Muraleedharan Vakkat2 and Thomas Hugh Jones1 PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Testosterone Replacement Therapy Has Beneficial Effects on Cardiovascular Risk Factors and Liver Function in Hypogonadal Men

Rationale & Hypothesis: Low testosterone levels are associated with cardiovascular morbidity and an increased risk of non-alcoholic steatohepatitis (NASH). NASH is closely associated with insulin resistance and atherosclerosis. We studied the effect of long-term testosterone replacement therapy (TRT) on liver function and cardiovascular risk factors in hypogonadal men.

Objectives: To assess the effects of testosterone therapy on liver function and cardiovascular risk factors in hypogonadal men

Methodology: A long-term retrospective analysis was carried out of 505 hypogonadal men receiving TRT as part of normal clinical practice with a mean follow-up of 4.94 years. 82.8% of the patients were on testosterone gels and the rest were on testosterone injection. Levels of testosterone, estradiol and PSA were monitored at 3, 6 and 12 months and yearly thereafter. BMI, waist circumference, blood pressure (BP), hemoglobin (Hb), hematocrit (HCT), lipid profile and liver function tests were also recorded. Changes in medications for diabetes and cholesterol were documented. Data from the most recent appointments represented the primary endpoint.

Findings: Mean age was 59.9+14.4 years and mean baseline testosterone was 7.09+2.53nmol/l. 46.1% of the cohort had type 2 diabetes mellitus; 33.9% were on metformin, 20.0% on insulin and 56.0% were on statins. TRT was associated with a significant decrease in AST between baseline and the primary endpoint (28.5 vs. 25.8u/l; -2.68u/l, p=0.014). ALT was also significantly lowered (35.2 vs. 30.4u/l; -4.77u/l, p=0.004). Patients with raised liver transaminases at baseline (>40u/L) had the greatest response to TRT. Hemoglobin levels were significantly higher at the primary endpoint (14.30 vs. 15.06g/dl; +0.76g/dl, p<0.001) but HbA1C levels were significantly reduced (7.24% vs. 6.90%; -0.34%, p=0.009). Diabetes medication was increased in 52 patients and decreased in 28. TRT reduced total cholesterol levels (over and above the effect of statins in 56.0% of the cohort) at the primary endpoint (4.48 vs. 4.18mmol/l; -0.30mmol/l, p<0.001) and non-fasting triglycerides were also significantly lowered (2.39 vs. 2.10mmol/l; -0.29mmol/l, p=0.006). LDL levels significantly fell at the primary endpoint (2.36mmol/l vs. 2.16mmol/l; -0.20mmol/l, p=0.014) and a modest fall in HDL levels was observed (1.10 vs. 1.05; -0.049mmol/l, p=0.035). No significant changes in BMI, waist circumference or BP were identified.

This is the first study to demonstrate the long-term beneficial effects of TRT on cardiovascular risk factors and liver function tests after 5 years. This is the largest and most comprehensive study of TRT to date, representing over 2467 patient years of TRT.

Poster: 46 Miss Laura Vergoz Laura Vergoz, Andrew J Streets and Albert CM Ong PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Identification of new candidate gene and miRNA networks in the pathogenesis of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is the most commonly inherited renal disorder with a prevalence of 1/1000. ADPKD pathology has been linked to mutations in two genes: PKD1 and PKD2, encoding Polycystin-1 and Polycystin-2 respectively. However its pathogenesis is variable, even between patients carrying the same PKD1 or PKD2 mutation, suggesting that epigenetic factors such as microRNAs play a role in the phenotype of the disease. MiRNAs are very short non-coding nucleic acids which role is to regulate gene expression. Some have already been linked to ADPKD, but the large amount of data generated by network analyses suggests that many have not yet been identified.

This project is part of the TranCYST network, a Marie Curie Initial Training Network (ITN) focused on ADPKD which goals are to broaden our understanding of the disease pathogenesis, improve its diagnosis and monitoring and eventually find new treatments.

We carried out parallel miRNA and mRNA microarrays on 4 cystic and 2 normal cell lines. 11 miRNAs were significantly dysregulated in the cystic lines, and 2 were then confirmed by qPCR (mir-193b and mir-582). By correlating alterations in miRNAs and their potential target genes, we were able to identify a number of significantly altered target genes. Luciferase reporter assays and other functional assays are being performed to study the interactions between the selected miRNAs and their target genes, and their functional effects in vitro. These results will help us to better comprehend the mechanisms that could explain the often marked phenotypic variability underlying ADPKD pathogenesis.

Poster: 47 Dr Ruth Payne Ruth Payne, Kathryn Milne, Sean Elias, David Llewellyn, Thomas Rawlinson, Nick Edwards, Simone de Cassan, Anna Goodman, Chetan Chitnis, Alfredo Nicosia, Sarah Moyle, Eleanor Berrie, Alison Lawrie, Adrian Hill and Simon Draper PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Safety and Immunogenicity of the Blood-stage Plasmodium vivax Vaccine ChAd63-MVA PvDBP

There has been comparatively little research into vaccines for Plasmodium vivax in the past, with only two antigens previously reaching Phase Ia clinical trials. More recently, the importance of a vaccine for P. vivax has been recognised and included in the 2013 update to the Malaria Vaccine Technology Roadmap. Here we report on the first blood-stage P. vivax vaccine Phase Ia clinical trial, carried out in Oxford in 2013 - 2014. This used recombinant simian adenovirus ChAd63 and poxvirus MVA encoding the P. vivax antigen PvDBP (Duffy-binding protein region II) in a heterologous prime-boost regimen. The P. vivax parasite requires an interaction between the Duffy-binding protein ligand and its host receptor, the Duffy antigen receptor for chemokines (DARC), in order to invade reticulocytes, making PvDBP a promising antigen for vaccine development.

Twenty-four healthy volunteers were enrolled in this Phase Ia dose escalation study. The ChAd63 PvDBP vaccine was given at doses of 5 x 109 - 5 x 1010 vp and MVA PvDBP was given to fifteen volunteers 8 weeks after ChAd63 PvDBP prime at doses of 1 x 108 – 2 x 108 pfu. The primary objective of the study was safety, and there were no safety concerns following vaccination although the higher dose of MVA PvDBP was more reactogenic than the lower dose. The secondary objective was humoral and cellular immunogenicity, assessed using assays including PvDBP IFN-γ T cell ELISPOT, B cell ELISPOT, PvDBP IgG antibody ELISA and functional antibody analysis. ChAd63- MVA PvDBP is immunogenic, with increases in the cellular and humoral responses seen particularly after MVA PvDBP boost. Functional antibody analysis of sera from volunteers vaccinated with ChAD63-MVA PvDBP demonstrated inhibition of binding of recombinant PvDBP_RII to DARC in vitro. Following these promising results, a Phase IIa efficacy study is planned in the near future.

Poster: 48 Mr Harvey Leung Mr Harvey Leung, Dr Lorena Preciado-Llanes, Dr Saurabh Jain, Prof Marcus Reuber, Prof Andrew Heath, Prof Arshad Majid PhD Student - 2nd Year Neuroscience Contact: [email protected]

Exploring the anti-inflammatory mechanism of electrical vagus nerve stimulation.

Rationale & Hypothesis: The vagus nerve is the major nerve of the parasympathetic nervous system and innervates many organs in the body. Electrical vagal nerve stimulation (VNS) has also been shown to have a beneficial anti-inflammatory effect in animal models of inflammation (Levine Y et al 2014, Yamakawa et al., 2013, and Ay et al., 2009, 2011); and is currently being trialled to treat patients suffering from rheumatoid arthritis. Since VNS, in the form of an implantable device, has been clinically used to treat refractory epilepsy for over 20 years; we have a unique opportunity to observe the effects of VNS on the immune system in humans. We hypothesise that VNS produces an observable anti-inflammatory effect in blood, and will correlate with the therapeutic outcome of VNS in epilepsy, thus providing further insight into their mechanisms.

Objectives: This pilot study will observe the frequency and function of peripheral blood mononuclear cell (PBMC) populations and cytokine levels from blood before and after VNS in epilepsy patients. These markers will be compared with seizure control.

Methodology: Blood samples will be collected 3-weeks before, and up to 6 months after VNS insertion. Cell population frequency and activity will be analysed via flow cytometry, and key cytokines identified from cytokine arrays will be further quantified by enzyme-linked immunosorbent assay (ELISA). The Liverpool seizure severity scale questionnaire will be used to monitor seizure control.

Findings: Recruitment is ongoing for the current study. Although there is insufficient data to suggest the influence of VNS on the inflammatory profile; we expect to identify upregulated markers of anti-inflammatory responses. This study investigates the potential use of VNS to treat inflammatory diseases.

Poster: 49 Dr Tariq Aabed Tariq Aabed, Debayan Mukherjee, Gill Tozer and Chryso Kanthou PhD Student - 2nd Year Oncology Contact: [email protected]

Radiation effects on the differentiation of mesenchymal stromal cells into myofibroblasts and pericytes and development of fibrosis in soft tissue sarcoma.

Rationale & Hypothesis: Sarcomas are locally aggressive tumours that metastasise. Treatment is with radiotherapy (pre or post-operative) and chemotherapy. Metastatic sarcoma is difficult to treat, so there is a need to improve current treatments by using a combination of targeted agents like vascular targeting agents (VTAs) with radiation. Some studies showed that irradiation induced recruitment of mesenchymal stromal cells (MSCs) to tumours. MSCs can differentiate into pericytes and myofibroblasts (MFs). The exact origin of MFs and pericytes in tumours has not been established. Presence of activated MFs in tumours is associated with poor prognosis and an aggressive phenotype. MFs contribute to fibrosis that makes vessels impermeable to macromolecules and resistant to further treatment with VTAs or chemotherapy. It is not clear how radiation recruits MSCs and/or drives their differentiation into MFs and pericytes.

Objectives: To determine whether radiation influences recruitment of MSCs to tumours and their differentiation into MFs and pericytes.

Methodology: Cultured C3H/10T1/2 mouse MSCs were used to study differentiation into MFs/pericytes. The cells were irradiated with different doses (0, 2, 4 and 8 Gy); As a control cells were treated with TGF-β, a known inducer of MF differentiation. C3H/10T1/2 differentiation was assessed after direct irradiation or after incubation with media collected from irradiated fibrosarcoma cells. Detection of differentiation markers expressed by pericytes and/or MF such as α-SMA, desmin, NG-2, RGS5 and PDGFR-β was performed by western blotting. Immunohistochemical analysis of irradiated and control mouse fibrosarcoma solid tumours was performed for α-SMA.

Findings: Preliminary results showed that C3H/10T1/2 cell express α-SMA, desmin and PDGFR-β, but were negative for NG2 and RGS5. Ongoing experiments are establishing whether radiation alters the levels of expression of these markers. Immunohistochemistry showed presence of α-SMA positive cells in both control and irradiated tumours. Quantification of staining will be performed using Aperio image analysis.

Poster: 50 Dr Rob Morton Morton RW, Everard ML, Elphick HE PhD Student - 2nd Year Human Metabolism Contact: [email protected]

Randomised control trial to investigate whether electronic adherence monitoring with feedback and reminder alarms can improve adherence and outcomes in childhood asthma.

Rationale & Hypothesis: Inhaled corticosteroids are the principal treatment for asthma, but adherence levels have been shown to be sub- optimal in the paediatric population. Sub-optimal adherence causes multiple consequences including increased morbidity, unnecessary escalations of treatment, increased health care utilisation and associated cost.

Electronic adherence monitoring enables accurate data to be openly discussed between the patient, family and medical team. Studies have shown that this monitoring and discussion can improve adherence, although they have been under-powered to show an increase in clinical outcomes. Reminder alarms provide a direct prompt to take medication and have been shown to also improve adherence levels, but not clinical outcomes.

We hypothesize that a combination of these two interventions will improve adherence and outcomes in childhood asthma.

Objectives: To determine whether a complex intervention comprising electronic adherence monitoring with feedback and reminder alarms can improve adherence and clinical outcomes in childhood asthma.

Methodology: 90 children aged 6-16 with asthma were randomised to either intervention or control groups, and followed up for 12 months. Intervention participants had their adherence to inhaled steroids electronically monitored, and fed- back every 3 months. Their devices also played daily reminder alarms. Control participants had their adherence monitored, but received no feedback or alarms. The primary outcome was asthma control, as measured by the Asthma Control Questionnaire (ACQ). Secondary outcomes were number of rescue doses of oral steroids, number of unscheduled medical attendances for asthma, quality of life, school days missed, and lung function.

Findings: Interim analysis shows that at 12 months there is no difference in the ACQ between the two groups. Participants in the intervention arm of the study have higher mean rates of adherence (83% vs 34%), higher lung function (100% vs 87%), and require fewer rescue courses of oral steroids (1.7 vs 2.7) than those in the control arm.

Poster: 51 Dr Ascanio Tridente Ascanio Tridente, Geraldine M Clarke, Andrew Walden, Anthony C Gordon, Paula Hutton, Jean- Daniel Chiche, Paul A H Holloway, Gary H Mills, Julian Bion, Frank Stuber, Christopher Garrard and Charles Hinds PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 52 Dr Shamsa Ihmed Shamsa Ihmed, Ian G Rennie, David W Hammond, Karen Sisley PhD Student - 2nd Year Oncology Contact: [email protected]

Identification of specific genetic changes associated with Conjunctival Melanoma

Rationale & Hypothesis: Conjunctival melanoma (ConM) is a relatively rare ocular malignancy that is likely to recur and carries an overall mortality rate of approximately 30%. The incidence, like that of skin melanoma, is however increasing and sunlight exposure is considered to be a factor in its development. Most ConMs arise from atypical neavi, but infrequently malignant transformation may take place in a conjunctival naevus and in some cases arise de novo. Poor prognosis has been associated with increased tumour thickness, high mitotic count, cell morphology and lymphatic invasion. Little is known about the genetic changes that are associated with this malignancy, but mutations of the BRAF gene have been found. There have been few other genetic studies of ConM, although it seems they are more like cutaneous melanomas rather than other ocular melanomas.

Objectives: To improve understanding of the associated genetic alterations, and relate if possible to prognosis.

Methodology: Fresh samples of ConM have been established as short term cultures including Mel 621, Mel 635 and Mel 610. Cytogenetics, array comparative genomic hybridization (aCGH) and Fluorescence In Situ Hybridization (FISH) has been performed on both uncultured and cultured specimens.

Findings: Multiple abnormalities affecting chromosomes 1, 4, 6, 8, 9, 10, 11, 13, 15, 16, 19, 20, 21 and 22 were identified. We are working now to confirm findings with FISH and cytogenetics where feasible, and determine BRAF status in all cases.

Poster: 53 Dr Melissa Hale MELISSA F HALE, KAYE DREW, REENA SIDHU, MARK E MCALINDON PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Randomised comparison of a standard protocol using metoclopramide versus a hand held magnet to enhance gastric emptying of the small bowel capsule.

INTRODUCTION: Delayed gastric emptying can be a significant factor in incomplete small bowel capsule endoscopy (SBCE) examinations. This has been addressed with the use of prokinetic agents, albeit with the need for cannulation or intramuscular (IM) injections and the small risk of adverse drug reactions. Recently a manoeuvrable small bowel (SB) capsule has become available using a handheld magnet to control movement. We compared this magnetically steerable method to a standard protocol using metoclopramide for enhancing gastric transit of the SB capsule.

METHODS: Single centre, randomised controlled trial involving 121 patients attending for SBCE using MiroCam Navi (Intromedic, Seoul, Korea). Patients were randomised to either the standard (control) group or the steerable capsule (intervention) group. The control group were mobilised for 30 minutes after ingestion of the capsule, followed by IM metoclopramide 10mg if the SBCE had failed to enter the SB. The intervention group ingested a gastric distention volume of 1000mls of water with 5 drops of simethicone prior to SBCE ingestion. Positional change and magnetic steering was used to manipulate the SBCE into the duodenum. If unsuccessful after 30 minutes, the patient was transferred to the standard protocol. Patient demographics, clinical and medication history was also collected. 60 patients per group were required to detect an improvement in capsule endoscopy completion rate (CECR) of 20%, with 80% power. Ethics approval (13/YH/0358).

RESULTS: 121 patients were recruited (60 to the control group, 61 to the intervention group: mean age 49 years (range 21- 85), 61 females). Two patients were excluded due to gastric retention of the capsule endoscope. Gastric transit time (GTT) was longer in the intervention group but this did not reach statistical significance (median 23 vs 51 minutes Mann-Whitney U=1487, p=0.116). There was no significant difference in CECR between the two groups (χ² p=0.395). Examining the intervention sub-group: no correlation between body mass index (BMI) and GTT (r=0.002) or waist-hip ratio (WHR) and GTT (r=5.98) was demonstrated. Similarly no significant difference between WHR (p=0.938) or BMI (p=0.507) and procedure completion rate was noted.

CONCLUSION: Magnetic steering of a small bowel capsule is unable to overcome pyloric contractions to enhance gastric emptying. However, mobility and image clarity within the gastric cavity was excellent and therefore with further improvements this technique could potentially be harnessed to provide targeted movements within the gastric cavity to enable capsular examination of the stomach.

Poster: 54 Mrs Jehan Alrahimi Jehan Alrahimi, Mark Thomas and Peter Monk PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The Role of Tetraspanins in Bacterial Pathogenicity

It has been observed that common pathogens are increasingly multi drug resistant and this poses a problem in disease treatment and eradication. Bacterial internalization and colonisation of the host cells and tissues involves the adherence to membrane proteins on the host cell surface. Prevention of this process would play a major role in developing new therapeutic strategies for reducing infectious diseases. Tetraspanins are highly conserved proteins, which interact with other proteins resulting in tetraspanin-enriched micro domains (TEM). These domains play a major role in the fusion, adhesion and trafficking of cells. Studies carried out on Neisseria meningitidis have shown the role of tetraspanins in the inhibition of pathogen attachment to the host cell. Pseudomonas aeruginosa is a Gram-negative bacterium and the causative agent of serious infection in immunocompromised individuals such as cystic fibrosis patients, burns units patients, and cancer patients.

The aim of this study is to investigate whether tetraspanins play a role in adhesion of P. aeruginosa in human macrophages and epithelial cells and subsequently, to investigate the potential of using recombinant tetraspanin extracellular domains (EC2s) as anti-adhesives in treating P. aeruginosa infections by inhibit adherence to human epithelial cells, if tetraspanins are involved in adhesion.

Conclusion: we have made fluorescent bacteria and we have developed a flow cytometry assay to measure bacterial adherence. Synthetic peptides have also been used to try to inhibit adherence. The project will aid the identification of which tetraspanin, if any, is involved in bacteria-host cell adhesion and the possible use of recombinant tetraspanin as therapeutics for treating P. aeruginosa infection.

Poster: 55 Miss Roxanne Lau Roxanne Lau PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The streptococcal nuclease: a potential drug target

Drug-resistant and multidrug-resistant strains of Streptococcus pneumoniae are among some of the most concerning pathogens to confront modern medicine. Today, the pressing need for new drugs demands the innovation of new strategies. One such strategy is the identification of novel drug targets in highly conserved components of bacterial machinery, in the hope that drugs developed against these will be more resilient to antibiotic resistance. The streptococcal 5’ nuclease represents a potential target since it plays a crucial role in DNA replication. Preliminary data reveal the potent exonuclease and endonuclease activities of the purified recombinant streptococcal 5’ nuclease, indicating that highly active enzyme preparations can be produced. Further biochemical work will compare the activities of the wildtype with active site mutants and possible inhibition with small molecules, while the structure will be resolved by x-ray crystallography. Not only will this add to the growing number of nuclease structures, but it will also supply some of the first structural data for this protein from pathogenic organisms, thereby facilitating long-term possibilities for future drug design.

Poster: 56 Dr Arash Assadsangabi A. Assadsangabi, C. Evans, D. Majumdar, S. S. Cross, B. M. Corfe, A. J. Lobo PhD Student - 2nd Year Oncology Contact: [email protected]

THE FATE OF EPITHELIAL KERATINS IN ACTIVE ULCERATIVE COLITIS

Introduction: Intermediate filaments (IF), which mainly consist of keratins (K), are one of the main components of the human cell cytoskeleton. K8, K18 and K19 constitute the main keratins in the intestinal epithelial cells. Keratin alterations may play a role in the pathophysiology of ulcerative colitis (UC). We have previously shown reduced expression of Keratins in IF fraction from distal active UC relative to proximal inactive mucosa as well as controls using proteomic iTRAQ-based analysis (1). Whether this reduced expression is due to change in solubilisation, reduced total expression and/or degradation, is unknown. We aimed to clarify the fate of epithelial keratins during UC disease activity.

Method: Soluble proteome fractions were extracted from rectal biopsies in patients (n=10) with active distal colitis (ACT) as well as endoscopically and histologically uninflamed proximal colonic mucosa (INACT) in the same patients. Median histological activity index in ACT and INACT were 2 (range 1-3) and 0 (range 0). Control colonic biopsies (n=7) from normal individuals were fractionated similarly. An iTRAQ-compatible extraction protocol for soluble proteins was developed and applied. Labelled peptides from pooled patients in each group were analysed by SCX-LC-MS/MS and data reconstituted in GeneBio Phenyx. Inter-group comparisons were made using in-house algorithms based on t-testing with multiple test correction. Validation of iTRAQ results was carried out with immunoblotting on pooled and individual samples using monoclonal antibody against K8. These outcomes were then compared with our previous insoluble proteome analyses (1).

Results: Comparative iTRAQ analysis of the soluble fraction showed significantly lower log fold changes in K8, K18 & K19 levels in ACT relative to INACT (0.42, 0.53, 0.42) and normal controls (0.61, 0.76, 0.41), respectively. The results were similar to our recent iTRAQ analysis of the IF fraction (1). Immunoblotting further confirmed these findings: median relative K8 concentration in IF/soluble proteome fractions in individual ACT and INACT samples was 0.18/1 (p=0.02) and 1.21/1.02 (p=0.02), respectively.

Conclusion: This study suggests relative total reduction in expression of epithelial keratins in actively inflamed colonic mucosa of UC patients compared to un-inflamed proximal mucosa and normal controls. Keratins may have utility as a tissue biomarker of UC disease activity.

References: 1. Corfe BM, Majumdar D, Assadsangabi A, Marsh AMR, Cross SS, Connolly JB, Evans CA, Lobo AJ. Inflammation decreases keratin level in ulcerative colitis; inadequate restoration associates with increased risk of colitis associated-cancer. BMJ Open Gastroenterology. In press.

Poster: 57 Mrs Amira Zawia A Zawia, N Arnold, J Pickworth, K Hopkinson, G Miller, A Lawrie PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Investigating the role of macrophage subsets in MacLow mouse and in patients with pulmonary arterial hypertension.

Rationale & Hypothesis: Pulmonary arterial hypertension (PAH) is a devastating condition with high morbidity and poor life expectancy. Pathologically PAH is characterised by the medial thickening of the small distal pulmonary arteries. Early endothelial cell (EC) dysfunction and apoptosis, and the subsequent abnormal proliferation and migration of pulmonary artery smooth muscle cells are thought to be a major contributing factor. Macrophages are proposed to play an important role in regulating these processes and are recruited to remodelled pulmonary arteries but the exact role of macrophages, and whether they are required for this remodelling remains unclear. We have recently developed a mouse model (MacLow) where approximately 50% of macrophages are depleted and now aim to investigate whether MacLow mice would demonstrate a different pulmonary hypertension phenotype in presence and absence of hypoxia, when compared to non-macrophage ablated littermates.

Objectives: understanding the role of macrophages/ macrophage subsets in the pulmonary arterial hypertension and whether they required for the pulmonary vascular remodelling (and/ or reverse remodelling).

Methodology: Macrophage ablation was induced in CD68-rtTA-eGFP/tetDTA double transgenic mice (MacLow) where macrophage-specific (CD68) induction of the cytotoxic diphtheria toxin A chain (DTA) is achieved by administration of doxycycline. All mice were phenotyped for PH by echocardiography followed by closed chest cardiac catheterisation. Heart and lung tissue were harvested for morphological, immunohistochemical and biochemical analyses. Findings: male MacLow mice with induced macrophage ablation displayed a spontaneous PAH phenotype even before exposing to hypoxia. The changes in RVSP were accompanied by appropriate changes in RVH. Macrophage count showed no significant changes in macrophage population especially within the lung tissue. These data suggest that macrophages play a modulating role in pulmonary vascular remodelling but further work is required to explore the mechanisms involved in this phenotype.

Poster: 58 Ms Sarah Almaghrabi Sarah Almaghrabi, Thomas Lovewell, Mimoun Azzouz and Rachid Tazi-Ahnini PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Gene therapy for an experimental autoimmune polyglandular syndrome 1 model caused by mutations in AIRE

Autoimmune Polyglandular Syndrome 1 (APS-1) is an autosomal disorder characterized by systemic multiorgan manifestations, caused by loss-of-function mutations in the Autoimmune Regulator gene (AIRE). AIRE is a transcription factor that directs the promiscuous gene expression of tissue-specific antigens in the thymus, allowing their presentation to developing T-cells during the process of central tolerance.

Our aim is to develop an efficient gene therapy approach to restore Aire expression in an Aire-/- (Aire knockout) mouse model of APS-1. Our first objective is to make and evaluate expression cassettes under control of various promoters. This would allow selection of a promoter that would give an optimal level of AIRE expression in the thymus. Constructs with either Aire or eGFP under the control of murine tissue-specific promoters Aire, Srgn and Csn2, as well as constitutive promoters CMV, PGK and CAG were made and assessed using in-vitro gene expression assays in HEK-293 and thymus cell line TEC-1A3. AIRE under either a moderate promoter or a strong promoter will be used to generate gene therapy viral vectors. Viral particles will be then directly injected into the thymus of Aire-/- mice using an ultrasound probe for image guidance. This study will form the basis for a gene therapy approach to treat APS-1 patients.

Poster: 59 Dr Victoria Gordon V.Gordon and the Autoimmune Hepatitis Group (UK) PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

MULTI-CENTRE AUDIT OF MANAGEMENT AND OUTCOME IN AUTOIMMUNE HEPATITIS (AIH) – PRELIMINARY RESULTS

Rationale: There are few data on presenting features of AIH outside large centres.

Objectives: To report data from an outcome audit of AIH in 28 UK centres of varying size.

Methods: We collected all prevalent cases since 2000 and incident cases since 2007 by searching electronic patient letters, histology databases and hospital coding. Case validation was by 1999 International AIH Group diagnostic criteria. We used a web-based data collection system.

Results: Of 1109 patients (227 prevalent, 882 incident) with/treated as AIH, 80% were women. Median age at diagnosis was 55(8-86)years. There were 91% Caucasian, 7% Asian, 1% Afro-Caribbean and 0.1% Chinese patients. 39% had a personal/family history of autoimmune disease: 14% thyroiditis/hypothyroidism, 10% PBC, 4% IBD and 3% coeliac disease. HBV/HCV serology was negative in 1061(96%) patients and undocumented in 42(4%). Six patients (0.5%) had acute hepatitis IgM antibodies (4 CMV, 1 EBV and CMV, 1 HEV). ANA was present in 57%, ASMA in 47% and Anti-LKM1 in 2% and AMA in 9%. Serum IgG/globulin was raised in 78%. Seventy four patients (7%) did not meet IAIHG diagnostic criteria.

Presenting symptoms: none 22%, jaundice/itching 42%, fatigue 35%, nausea14%, weight loss 13%, abdominal pain 16%, joint aches 12%, flu-symptoms 6%, rash 3%,amenorrhoea 0.4%, others 8%. Time from first abnormal liver tests to diagnosis was 3(0-167)months and shorter in patients presenting with jaundice/pruritus: 1 vs 5months; (p=<0.0001). At presentation, 4.4% had ascites, 5.5% oedema, 1.9% encephalopathy, 0.7% variceal bleeding and 8.4% had clinical decompensation. In another 13%, MELD score was >15. 96% of patients had a diagnostic liver biopsy; 23% had cirrhosis.

Conclusions: In this large multicentre cohort of UK AIH patients, 22% had liver decompensation at presentation. Delay in diagnosis was often many months, especially in those without jaundice. 4% had undocumented HBV/HCV serology and 7% did not meet IAIHG criteria.

Poster: 60 Mrs Khlood Mehdar Khlood Mehdar, Gary Shaw , Kurt J De Vos , Andrew J Grierson PhD Student - 2nd Year Neuroscience Contact: [email protected]

HDAC6 inhibition as a therapeutic approach in Hereditary Spastic Paraplegia

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of inherited neurological disorders that are characterised by lower limb spasticity and weakness. The gene, which is responsible for 40% of HSP, is spastin (Spast). Spast encodes a microtubule-binding protein that has several important roles in the cell including microtubule severing. Mutations in Spast cause reductions in axonal transport that lead to mitochondrial accumulation in axonal swellings in the corticospinal tract. Mice carrying a human pathogenic mutation develop a progressive gait defect. Microtubule acetylation has been shown to regulate axonal transport; increased acetylation correlates with upregulation of axonal transport. One way to increase microtubule acetylation is to inhibit the key enzyme responsible for reducing microtubule acetylation, histone deacetylase6 (HDAC6). Inhibition of HDAC6 to restore axonal transport has been shown to be neuroprotective in several neurodegenerative disease models.

The aim of this project is to test the hypothesis that increasing microtubule acetylation via chemical or genetic inhibition of HDAC6 will restore axonal transport and correct the gait defect in the mouse model of HSP. (i) To generate cohorts of Spast+/+ and SpastΔE7/ΔE7 (null mutant) mice for in vivo studies with pharmacological HDAC6 inhibition, (ii) To breed a cohort of double mutant mice that are HDAC6 null and SpastΔE7/ΔE7 to test the effect of genetic silencing of HDAC6, and (iii) To test the effects of HDAC6 inhibition on axonal transport in primary neurons. Crosses have been set up to generate double mutant mice that are HDAC6 null and SpastΔE7/ΔE7. HDAC6 inhibitor compounds have been used for a pilot dosing experiment and showed increased tubulin acetylation in CNS and peripheral tissues. Cohorts of Spast+/+ and SpastΔE7/ΔE7 mice have been generated and gait is being studied every month using a Catwalk apparatus. The mice were randomly assigned to treatment and control groups, to be treated with HDAC6 inhibitor. We will determine the effect of treatment on the HSP-related gait defect.

Poster: 61 Dr Karl Pang Karl Pang, Ross M Drayton, Ishtiaq Rehman, Raymond Clarke, Saiful Miah, Robert Stoehr, Arndt Hartmann, Sheila Blizard, Martin Lavin, Helen E. Bryant, Elena S. Martens-Uzunova, Guido Jenster, Freddie C. Hamdy, Robert A. Gardiner and James W.F. Catto PhD Student - 2nd Year Oncology Contact: [email protected]

Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 gene locus that potentially targets mRNA

Rationale & Hypothesis: Prostate Cancer Antigen-3 (PCA3) is a non-coding RNA (ncRNA) upregulated in prostate cancer (PCa) used to stratify biopsy. Physician acceptance of PCA3 has been limited through vulnerability of the mRNA during transportation to the laboratory and the fact that the function of PCA3 is unclear. Long ncRNAs may be processed into smaller active species. We hypothesized this role for PCA3 and searched for short-RNAs (sh-RNA) within this gene and evaluated their translational role.

Objectives: 1) To identify a short-RNA within PCA3, 2) Measure its expression in cell lines and patient urinary samples, 3) Investigate potential roles within PCa oncogenesis.

Methodology: We computed RNA hairpins within the BMCC1 gene (encompasses PCA3) and searched a prostate transcriptome, generated through deep sequencing, for RNAs derived from these hairpins. We measure their expression using qPCR in three cohorts: PCa tissues (n=60), exfoliated urinary cells (cancer, n=355; control, n=166) and cell lines (n=22). We used in silico predictions and RNA knock-up to identify potential mRNA targets of the sh-RNAs.

Findings: We predicted 13 hairpins, including PCA3-shRNA2 that was the majority species expressed in a prostate transcriptome. PCA3- shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues (T-test p<0.01), exfoliated urinary cells from men with PCa (13-273 fold change, T-Test p<0.003) and closely correlated to PCA3 expression (r=0.84 to 0.93, p<0.001). PCA3-shRNA2 (C-index 0.75-0.81) and PCA3 (C-index 0.78) could identify the presence of cancer from urinary samples of most men. Following PCA3-shRNA2 knock-down, altered expression of several mRNAs in PCa cell line DU145, including COPS2, SOX11, WDR48, TEAD1, Noggin was identified. When validating this, SOX11 expression was found to be reduced in urine samples obtained from men with PCa (p=0.03).

Poster: 62 Miss Sayali Haldipurkar Sayali Haldipurkar & Mark S. Thomas PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

Role of ECF sigma factors in stress responses of Burkholderia cenocepacia

Rationale & Hypothesis: Burkholderia cenocepacia is an opportunistic pathogen that causes infections in CF and CGD patients. Pathogens encounter ‘stressful’ conditions inside the host such as low iron environments and oxidative stress. One strategy used by bacteria to respond to such stresses is through the use of extracytoplasmic function (ECF) sigma factors. We are interested in identifying roles of each of the 13 B.cenocepacia ECF sigma factors in stress resistance. Here we elucidate the DNA sequence requirements for promoter utilisation by one ECF sigma factor, OrbS, that responds to iron starvation. Moreover, unlike other ECF sigma factors, OrbS is not regulated by an anti-sigma factor that senses the stress. Therefore we also investigate whether OrbS responds to iron availability by another mechanism.

Objectives: Establishment of the DNA recognition sequence of OrbS will facilitate the identification of the OrbS regulon. Transcription of orbS is iron-regulated by the Fur repressor. E.coli and B. cenocepacia fur mutants will be used to investigate the presence of an alternative mechanism for iron sensing by OrbS. To identify roles of the remaining ECF sigma factors, the putative promoters will be identified using bioinformatic analysis and ChIP-seq.

Methodology: The effect of site-directed mutations on the activity of an OrbS-dependent promoter were investigated using reporter fusions. Phenotypic characterisation of a B. cenocepacia fur mutant was carried out. Putative ECF sigma- dependent promoters identified by bioinformatic analysis were validated by measuring promoter activity.

Findings & conclusions: We demonstrate that OrbS recognises two tetranucleotide motifs (-35 and -10 regions) separated by a GC rich spacer whose length and conformation appear to be important for promoter recognition. Results obtained with the E. coli fur mutant suggest the presence of an alternative iron-sensing mechanism for OrbS which need to be confirmed in a B. cenocepacia fur mutant. Putative promoters have been identified for two novel ECF sigma factors.

Poster: 63 Miss Jessica Johnston Jessica Johnston, Adrienn Angyal, Mabruka Alfaidi, Sheila Francis & Endre Kiss-Toth PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Assessment of plaque macrophage phenotype in situ by multicolour fluorescence microscopy.

Rationale & Hypothesis: Macrophages are a heterogeneous and diverse population of cells with an important role in immunity and also in the development of diseases including atherosclerosis. Macrophages exhibit a spectrum of phenotypes, from pro- inflammatory (M1) to anti-inflammatory (M2) cells. These cells are highly plastic and can switch from one phenotype to another depending on environmental cues. Despite considerable work aiming to characterise the polarisation of macrophages in disease, there have been few studies that have elucidated macrophage phenotype in situ and our knowledge of their complexity in vivo is only partial. Here we wished to establish a robust analysis pipeline to assess macrophage phenotype in plaques of high-fat diet fed mice. This approach will provide a unique insight into macrophage phenotype and will advance our understanding of phenotypic switching of macrophages in vivo.

Methodology: Using multicolour immunohistochemistry, plaques from high-fat diet fed (ApoE-/- and LDLR-/-) mice were simultaneously stained for Mac-3 (CD107b), iNOS (M1 marker) and Arginase I (M2 marker) in situ, and imaged using fluorescence microscopy. The resulting images were analysed by Image J. Briefly, individual Mac-3 positive cells were selected as the region of interest (ROI) and corresponding iNOS and ArgI positive staining was evaluated. The analysis allowed consideration for the spectrum of M1/M2 associated marker expression and enabled characterisation of individual cells based on staining intensity. Using this approach it is possible to reveal and quantify complex populations of plaque macrophages in situ.

Findings: We have successfully optimised a simultaneous staining protocol for macrophage phenotype and have created a robust, semi-automated pipeline to analyse macrophage phenotype and validated this approach by comparing murine models of experimental atherosclerosis. We envisage that our platform provides a novel tool to gain an in- depth understanding of macrophage phenotype in atherosclerosis and we will use this to elucidate the action of modulators of macrophage polarisation in vivo.

Poster: 64 Miss Lucy Morris Morris, L., Marriott, H.M., Dockrell, D.H. PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The effect of macrophage polarisation and delayed apoptosis in the innate immune response to S. pneumoniae infection

Rationale & Hypothesis: Shifts in macrophage polarisation are associated with COPD pathogenesis. Macrophages undergo apoptosis when infected with Streptococcus pneumoniae, a common cause of COPD exacerbations, as part of the innate immune response. This apoptosis is reduced in alveolar macrophages from COPD patients associated with increased expression of the anti-apoptotic protein Mcl-1. We hypothesise that macrophages from transgenic mice which over express Mcl-1 (CD-68 Mcl-1), will demonstrate altered polarisation resulting in reduced host defence to pneumococcal infection.

Objectives: Determine any differences in basal polarisation of wild-type and CD68 Mcl-1 BMDMs. Explore phagocytosis, bacterial killing and apoptosis in polarised wild type and CD68 Mcl-1 BMDMs and MDMs after S. pneumoniae infection. Understand how cytokine and chemokine repertoire changes in response to S. pneumoniae infection in polarised wild-type and CD68 Mcl-1 BMDMs and MDMs.

Methodology: Bone marrow derived macrophages (BMDM) from Mcl-1-transgenic and wild-type littermates were stimulated for 24 hours with cytokines; IL-4 (M2a), IL-10 (M2c) or IFNγ+LPS (M1) and polarisation determined by western blotting, ELISA and RT-PCR. Polarised macrophages were infected with S. pneumoniae for defined times and bacterial internalisation and survival assessed. Nuclear fragmentation of mock infected versus infected macrophages was quantified by staining with DAPI. Supernatants were collected and assessed for various cytokines and chemokines by MSD analysis. Cell viability and hyplodiploid DNA were assessed by MTT assays and PI staining respectively.

Findings: M1 polarisation caused increased levels of hyplodiploid DNA and decreased cell viability compared to other polarisation states. Assessment of S. pneumoniae internalisation and intracellular survival over a 2 – 4 hour time course showed increased bacterial clearance by M1 wild type and transgenic macrophages. There was also increased apoptosis of M1 macrophages after S. pneumoniae infection 16 hours onwards. M1 polarisation prior to infection increased proinflammatory cytokine and chemokine repertoire in BMDMs, however this effect was not replicated in MDMs.

Poster: 65 Dr Hamad Alzahrani Hamad Alzahrani, Annalena Venneri Postdoctoral Fellow Neuroscience Contact: [email protected]

Apathy may lead to dementia in patients with Parkinson’s Disease

Background Apathy refers to a combination of behavioural, emotional and cognitive features that lead to reduced interest and participation in daily life activities. The prevalence of apathy in Parkinson’s Disease (PD) is approximately 40%. Few studies have investigated the cognitive and neuroanatomical correlates of apathy in PD. From apathy studies in Alzheimer’s disease and other neurological disorders we expected to find deficits in frontal regions and in cognitive functions associated with frontal and temporal structures.

Materials and Methods Forty PD patients in the early stages of the disease (18 with apathy and 22 without apathy) underwent extensive neuropsychological screening, neuropsychiatric assessment using the Neuropsychiatric Inventory, structural MRI scanning and neurological examination. A voxel-based multiple regression analysis was used to measure negative correlation between grey matter volumes and apathy scores.

Results Higher apathy scores correlated with lower grey matter volume in several brain areas including the left insula, left inferior/middle/medial frontal gyrus, right anterior cingulate and the left superior temporal gyrus. Patients with apathy had lower scores in all cognitive tests. However, significant impairments were found in tests assessing executive functions and a trend-level significant difference was observed in memory tests in patients with apathy when compared with patients without apathy.

Discussion Apathy was associated with greater levels of atrophy in the frontal cortex, temporal cortex and anterior cingulate as well as overall lower level of cognitive performance, particularly in executive function and memory skills. These findings are in line with results from studies of other neurological conditions. For instance, a similar pattern of executive dysfunction has been reported in apathetic patients with PD with dementia and apathetic patients with Alzheimer disease. Apathy appears to be a risk factor for cognitive impairments in PD and might be a useful clinical indicator of dementia risk in PD.

Poster: 66 Ms Lauren Edwards Edwards LS, Paggiosi M, Skerry T, Offiah A BMedSci Student - 4th Year Human Metabolism Contact: [email protected]

Using High-Resolution Peripheral Quantitative Computed Tomography (HRpQCT) to Better Understand the Skeletal Response to Exercise

Objectives To use HRpQCT to investigate the effects of short term but intense exercise on the bone architecture of the distal radius in exercise-naïve women, with the ultimate aim of developing exercise regimes for children that will maximise their peak bone mass. Methods We have recruited 17 exercise-naïve women, aged 18-25, for a 12-week exercise study. The exercise consists of supervised hammering of a metal plate, using their dominant arm, 3 days a week for 12 weeks. Each session consists of 40 hammer blows separated by a 10 second rest between each blow. Baseline HRpQCT of both wrists will be compared to scans obtained after the last exercise session (April 2015). Results The mean baseline age of participants was 21.4 (±1.165 SD) years. The mean baseline BMI was 22.1 (±2.584 SD) kg/m2. A paired t-test of baseline average bone density, trabecular bone density, compact bone density, trabeculae number, trabecular thickness and cortical thickness showed no significant difference between the right and left radii at baseline (P>0.05). Differences between baseline and post-exercise HRpQCT values will be tested for significance using a dependent t-test. Assessment of least significant change will indicate whether the changes are real or due to inherent variability. Conclusion The study will show what effect a relatively short but intense exercise regimen has on bone microarchitecture in exercise-naïve women and will indicate the feasibility of such a study in children and young people.

Poster: 67 Mr Alfred Kamuyango Alfred A Kamuyango, Robin May & Simon Johnston PhD Student - 1st Year Infection & Immunity Contact: [email protected]

How do fungal pathogens initiate and modulate immune signalling?

C. neoformans causes life threatening meningoencephalitis in immunocompromised individuals particularly those with AIDs. Macrophages are known to have a significant role in host defences against C. neoformans. The balance between classically and alternatively activated macrophages has the potential in determining C. neoformans disease progression or resolution. Experiments in vitro have shown that outcomes of intracellular cycle of C. neoformans include fungal replication, killing, latency, nonlytic exocytosis and lateral transfer of the fungus between macrophages. We aim to addressing the following questions: What are the outcomes of C. neoformans following an encounter with macrophage in vivo? What is the polarisation state of macrophages before and during encounter with cryptococci? How long does the macrophage polarisation state last? And lastly, does macrophage polarisation determine the outcome of the macrophage interaction?

Zebrafish have become a useful model to study pathogen-phagocyte interaction because they are almost transparent enabling the capturing of images in real time. Previous experiments in our lab to study C. neoformans-macrophage interactions have been done by injecting C. neoformans into the zebrafish blood stream. However, with this technique it is difficult to continually track individual macrophages as they are highly motile and efficiently move between tissues. To solve this problem, we recently established intramuscular injections in larval zebrafish that allows continuous imaging.

Using live imaging we can characterise the intracellular interactions of cryptococci with macrophages over three days of infection. As a first step to characterizing the immune polarisation we have examined the activation of the pro-inflammatory inducible nitroreductase (iNos) pathway in macrophages. Furthermore, we have identified iNos (NOS2A), mannose receptor 1 (mrc1) and arginase 1 (arg1) as potential macrophage polarisation markers in zebrafish suitable to generate imaging reporters. Both mrc1 and arg1 are duplicated genes in zebrafish and we have undertaken sequence analysis to confirm their relationship in zebrafish and in comparison to mammalian immune genes.

In conclusion, our intra-muscular injection model is suitable for long term analysis of cryptococcal interactions with macrophages and we have identified zebrafish genes to construct fluorescent immune reporters.

Poster: 68 Dr Alex Wheeler Dr Alex Wheeler, Dr Lauren Pout Foundation year 2 Sheffield Teaching Hospitals NHS Trust Contact: [email protected]

Exploring Prescribing Habits in the Emergency Department- a Need For a Change in Culture

Rationale & Hypothesis: Drug prescribing errors are recognised as being widely prevalent in the Emergency Department. An updated emergency department case notes record was introduced in 2014. Many of the updates to it had been centred on the prescription chart. We wanted to audit current prescribing practice in the Emergency Department against current Sheffield Teaching Hospitals prescription writing standards, whilst comparing both old and new case notes.

Objectives: To determine if the introduction of the new Northern General Hospital Emergency case notes record has improved prescribing practice and whether this meets the standards set by Sheffield Teaching Hospitals. To identify areas of substandard drug prescribing so that these can be highlighted and addressed.

Methodology: 100 randomly selected case notes were reviewed in total; 50 from prior to the introduction of the new case notes, 50 from the new case notes. Each set was reviewed against standards set by the Sheffield Teaching Hospitals NHS Foundation Trust prescription writing standard 2013.

Findings: Overall, the new case record was a significant improvement on previous versions, and reduced prescribing errors and incorrect dispensing by improving correct documentation of patient demographics from around 25% to 100%. However, results showed that medical staff were still generally poor at documenting allergy status, meaning over 50% of dispensed medication in the Emergency Department had officially been incorrectly prescribed. There were also instances where medications for which the prescriptions had been unsigned had been dispensed, and issues with prescriptions not being timed. Overall, a need for a culture shift was identified - if the prescription is not correct, the drug should not be dispensed - being in a busy Emergency Department is no excuse for this not to happen. We identified changes need to be made by not only clinicians prescribing but also by nurses that are dispensing.

Poster: 69 Dr Alisdair McNeill Alisdair McNeill PhD MRCP (UK), Susanne Kuhl PhD, Annmarie Hempel PhD. Senior Clinical Fellow Neuroscience Contact: [email protected]

SOX11 deletions and mutations in human developmental disorders

Rationale & Hypothesis: A chromosome deletion which removed SOX11 was identified in a clinical referral. The pathogenic significance and clinical relevance of this was unclear. We hypothesised that SOX11 deletion/mutation may be associated with human developmental disorders and that further deletions/mutations could be identified in affected children.

Objectives: 1. to identify cases of SOX11 deletion and define the phenotype in children carrying the deletion 2. to identify cases of SOX11 mutation and define the phenotype in children carrying a mutation 3. to examine brain development in a xenopus system with SOX11 knockdown

Methodology: Clinical case reports of patients with SOX11 deletions (found on clinical array comparative genomic hybridisation) ascertained via the DECIPHER collaboration and clinical referrals, and SOX11 mutations ascertained via the Deciphering Developmental Disorders (DDD) exome study. Generation of a xenopus model of SOX11 deficiency with morpholino knockdown.

Findings: We report 5 children with chromosome deletions which include SOX11, they had a common phenotype of microcephaly and developmental delay. Two children with de novo SOX11 mutations were identified from the first 1000 parent-child trios sequenced in DDD. They had microcephaly and developmental delay. SOX11 knockdown in xenopus induced microcephaly in the morphants. Taken together these findings suggest that SOX11 plays an important role in human brain development and that loss of function (through mutation/deletion) can be associated with a developmental disorder.

Collaborations sought: I would like to establish collaboration with research groups using animal models to investigate developmental phenotypes. These collaborations would investigate the developmental role of genes identified as being deleted/mutated in clinical referral series and large scale genomics projects.

Poster: 70 Ms Joanna Chowdry Joanna Chowdry1, Caroline Evans1, Carmen Diaz Toledo2, Roberta Re2, Martin Wickham2, Martin Yeomans3, Bernard Corfe2 Research Technician Oncology Contact: [email protected]

Novel Predictive Biomarkers of Energy Intake

Rationale & Hypothesis: To address the growing obesity epidemic, health claims made by the food industry should be objective and substantiated. Subjective appetite ratings (AP) are widely used as predictors of energy intake (EI). However, we have previously shown that AP and EI do not correlate. Other candidate biomarkers include circulating levels of gut hormones, however their assay often requires invasive blood sampling, itself influencing appetite making measurements difficult to quantify.

Objectives: Saliva is a potential source of biomarkers and collection is simple and non-invasive. Proteomics workflows can objectively profile the protein content and complexity of samples using iTRAQ (isobaric tags for absolute and relative quantification). We previously demonstrated an increase in thioredoxin following a pre-load containing the omega 3 fatty acid DHA which corresponded to reduced appetite measurements in saliva samples. The current is aim to identify novel biomarkers of EI in saliva samples, in response to a pre-load challenge.

Methodology: 34 healthy volunteers participated in a 3-way randomised crossover study to assess the effect on EI of three different pre-loads varying in calorific content and sensory properties. Volunteers provided subjective appetite ratings, saliva samples and energy intake data (Kcals) pre- and post-exposure to each challenge. Samples were pooled for iTRAQ analysis according to ranked EI data, treatment and time-point, allowing for potential discovery of fundamental physiological biomarkers, as well as labile or stable markers in response to the challenge.

Findings: Targets for cross-validation of all un-pooled samples will be undertaken by ELISA and western blot based on maximal differences in iTRAQ and availability of antibodies. Proof of concept for novel EI biomarkers has the potential to impact significantly on the food industry, and speed up the EFSA regulatory approval process. Candidate biomarkers will represent significant IP jointly held between UoS and LFR. 77

Poster: 71 Miss Hannah Dudhill Hannah Dudhill* Louise Taylor* Markus Reuber# Richard A Grunewald# Jane Shewan$ Peter Mortimer$ Jon M Dickson* BMedSci Student - 4th Year Academic Unit of Primary Medical Care Contact: [email protected]

The potential for community-based alternative care pathways for patients after suspected seizures

Rationale & Hypothesis: The estimated prevalence of active epilepsy in the UK is 1%. 13-18% of these patients will attend an emergency department (ED) each year, resulting in a significant number of hospital admissions and creating large costs to the NHS. It has been found that patients with epilepsy who repeatedly attend emergency departments report poorer quality of life, greater psychological distress, and greater perceived stigmatisation. A large proportion of these attendances may be preventable through alternative care pathways, which could result in significant cost savings and improvement in patient care.

Objectives/Methodology: We conducted a literature review looking at the emergency hospital management of seizures to find out current knowledge and to help to inform the cohort study. A previous study (EPIC1) by the current authors retrospectively reviewed the ambulance notes of 214 incidents where an emergency 999 call was made to Yorkshire Ambulance Service for a suspected seizure. In this study (EPIC2), we are reviewing the clinical notes (emergency department, in-patient and epilepsy clinic) of the 98 incidents where patients were taken to hospital. We will describe the characteristics of the patients, their clinical management, and their outcomes.

Results: The literature review emphasised the magnitude of the costs associated with emergency hospitalisation in epilepsy and the negative effects on patients. It also highlighted potentially preventable factors associated with ED use. Data collection and analysis for the cohort study has not yet been completed. The results will be available to present at the School Research Meeting in June.

Conclusion: There is uncertainty about how to optimise care, and further research is needed to assess whether these attendances can be avoided and to inform potential alternative care pathways. The EPIC2 cohort study will provide the first data of this sort that we are aware of in the medical literature.

Poster: 72 Dr Mabruka Alfaidi Mabruka A. Alfaidi1, Torsten Schenkel 2, Paul C. Evans1, Janet Chamberlain1, Sheila E. Francis1. PhD Student - 4th Year Cardiovascular Science Contact: [email protected]

Dietary Docosahexaenoic Acid Reduced Experimental Atherosclerosis by Inducing Protective Hemodynamic Conditions.

Rationale & Hypothesis: Dietary omega-3 fatty acids have been associated with protection from atherosclerosis. However, the underlying mechanisms are incompletely understood. Blood flow generates a frictional force on endothelial cells called wall shear stress (WSS) that alters vascular function. We hypothesized that docosahexaenoic acid (DHA) modulates vascular wall inflammation by WSS mechanism.

Objectives: The aim of this study was to determine whether DHA modulates vascular wall inflammation, blood flow velocity and WSS in experimental atherosclerosis.

Methodology: ApoE-/- mice were fed either high fat diet (control) or high fat diet plus DHA (300mg/kg/day) for 12 weeks (n=12/group). Blood pressure was recorded using a Visitech tail-cuff system. Atherosclerosis was measured in whole aortae, aortic roots and brachiocephalic arteries by en face staining using oil red O. Computational fluid dynamics (CFD) was used to map wall shear stress (WSS) magnitude and oscillations in the aorta. Plasma cholesterol levels were quantified by gas chromatography.

Findings: Plasma high density lipoprotein (HDL)/total cholesterol ratio was increased in DHA-treated mice compared to controls (10.77±1.86 vs. 6.63±1.04, p<0.05). DHA fed mice exhibited a 4-5 fold reduction in distal aortic and brachiocephalic atherosclerosis (p<0.001) whereas lesion burden in the aortic arch was similar between groups. Dietary supplementation using DHA led to a reduction in blood pressure (119.5±7.33mmHg (+DHA) vs 159.7±2.482mmHg (controls), p<0.001 and a 12% decrease in aortic blood flow. CFD revealed that oscillatory shear in the descending aorta was reduced in DHA-fed mice compared to controls. Our study suggests that dietary DHA can act systemically by enhancing levels of HDL. It can also act locally by reducing oscillatory shear stress in the descending aorta. Dietary DHA reduced lesion formation specifically in the descending aorta, an effect that can be explained by its dual effects on oscillatory shear and HDL.

Poster: 73 Miss Heledd Brown-Wright Heledd Brown-Wright, Kirstie Bennett, Prof Pamela Shaw, Richard Mead. PhD Student - 1st Year Neuroscience Contact: [email protected]

Metabotropic glutamate receptor 5 (mGlu5) as a novel drug target in motor neuron disease (MND).

MND is a fatal disease and at present there are no effective disease modifying therapies available. Glutamate-mediated excitotoxicity is a recognised mechanism of neuronal injury. Riluzole, the only drug that has been shown to modestly prolong survival in MND, exerts its therapeutic effect through the indirect antagonism of glutamate. There is accumulating evidence to support a role of the metabotropic glutamate receptor type 5 (mGlu5) in MND. Astrocytes expressing mutant SOD1 [a common mutation causing MND] are more vulnerable to increased glutamate levels and undergo cell death mediated by mGlu5. Further, the mGlu5 antagonist, MPEP, has been shown to delay onset of disease and extend survival in the SOD1G93A mouse model. It is conceivable that modulating glutamate neurotransmission through targeting mGlu5 could provide a more effective therapeutic target for the treatment of MND.

Objectives: Defining the expression profile of mGlu5 in in vitro and in vivo models of mutant SOD1 related motor neuron disease with a view to validating mGlu5 as a therapeutic target.

Methodology: An immunofluorescent staining method was established for mGlu5 and specificity of staining confirmed in peptide antigen competition experiments. Tissue was then collected from SOD1G93A transgenic mice (a model of MND) and mGlu5 expression characterised with dual staining for astrocytes. The expression of mGlu5 was also characterised in human iAstrocytes derived from MND patients with SOD1 mutations.

Findings: mGlu5 expression was predominant in substantia gelatinosa (Rexed’s lamina 2 ) of lumbar spinal cord in both wild-type and SOD1G93A transgenic mouse spinal cord although weaker staining in grey matter parenchyma and in dorsal motor neurons was also apparent. There was no obvious co-localisation between astrocytes and mGlu5 positive processes. Co-staining with motor neuronal markers is underway. The data generated thus far contradicts literature reports of increased expression of mGlu5 in astrocytes in the SOD1G93A transgenic mouse model of MND.

Poster: 74 Mr Mahmoud Habibullah Mahmoud Habibullah, Nicolas Kluger, Annamari Ranki, Kai J.E. Krohn, Anthony P. Weetman, E. Helen Kemp PhD Student - 3rd Year Human Metabolism Contact: [email protected]

Epitopes, Specificity, Functional Effects and Immunoglobulin G Subclasses of Calcium-Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1

Rationale & Hypothesis: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disease characterised by multiple autoimmune endocrinopathies and results from mutations in the autoimmune regulator (AIRE) gene. Approximately 80% of patients present with hypoparathyroidism which is suggested to result from autoimmune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of PTH, has been identified as a parathyroid autoantibody target in APS1.

Objectives: The aim of the study was to characterise patient CaSR autoantibodies in relation to their epitopes, specificity, IgG subclass and effects upon CaSR function.

Methodology: Phage-display; ELISA; bioassays.

Findings: CaSR autoantibody binding sites (epitopes) were identified between amino acids 41–69, 114–126, 171–195 and 260- 340 in the extracellular domain of the receptor. Absorption experiments confirmed the specificity of CaSR autoantibodies in recognising their respective epitope. CaSR autoantibodies were analysed for their ability to increase both Ca2+-dependent extracellular signal-regulated kinase phosphorylation and inositol phosphate accumulation in HEK293 cells expressing the CaSR. The results indicated that two APS1 patients had CaSR-activating autoantibodies, suggesting that although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, there may be a small minority of patients in whom the hypoparathyroid state is the result of functional suppression of the parathyroid glands. Autoantibodies against CaSR epitopes 41–69, 171-195 and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. The study provides a detailed analysis of the characteristics of CaSR autoantibodies in APS1 patients, although further investigations are required to determine the exact role played by the autoimmune response against the CaSR in the pathogenesis of APS1-associated hypoparathyroidism.

Poster: 75 Mr Joseph Abrams Joseph JB Abrams, David Partridge & Simon Johnston BMedSci Student - 3rd Year Infection & Immunity Contact: [email protected]

Analysis of fungemic and non-fungemic Candida isolates from the Sheffield Special Care Baby Unit

Candidiasis is a fungal infection that occurs due to yeasts which belong to the genus Candida, there are over 20 species of Candida which are known to cause infection in humans the most common being Candida albicans. Invasive candidiasis occurs when the fungus enters previously sterile sites such as the bloodstream. This is usually not a problem in immunocompetent hosts, Candida is a commensal in healthy humans particularly on the skin, gastrointestinal, and gastrourinary tracts, but in those with risk factors such as hospitalized patients, low birth weight infants and people with compromised immune systems it can pose a high mortality rate.

We aim to identify any difference between strains of Candida that have caused invasive candidiasis in newborns and those that have not. Both invasive and non-invasive samples have been collected from the Sheffield Children’s Hospital, the majority of which are Candida albicans with some C. glabrata samples.

To identify any differences in the infection phenotypes of strains ex vivo we are using a zebrafish model. Zebrafish have a similar immune system to mammals and have been previously characterised as a Candida infection model. To measure invasiveness we have injected Candida into the posterior of the swimbladder, an enclosed epithelial structure, and assessed survival and dissemination.

To measure differences in immune response we have used intramuscular injection and assessed neutrophil and macrophage recruitment and phagocytic responses. Thus, our approach means we can rapidly assess if our strain collection contains pathogens determined invasive phenotypes distinct from host variation.

Further investigations will be performed based on the preliminary data produced which indicates that there is no differences between collected strains in terms of invasiveness.

Poster: 76 Dr Paul Morris Morris P, Dockrell D, Peden A Academic Clinical Fellow Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 77 Dr Julie Simpson Julie E. Simpson, Paul G. Ince, Paul R. Heath, Fiona Matthews, Carol Brayne, Pamela J. Shaw, Magnus Rattray, Claire Garwood, Emily Goodall, Stephen B. Wharton Research Fellow Neuroscience Contact: [email protected]

Neuronal DNA damage response-associated dysregulation of signalling pathways and cholesterol metabolism at the earliest stages of Alzheimer-type pathology

Rationale & Hypothesis: Oxidative damage and an associated DNA damage response (DDR) are evident in mild cognitive impairment and early Alzheimer’s disease, therefore we hypothesised that neuronal dysfunction resulting from oxidative DNA damage accounts for the cognitive impairment not fully explained by classical Alzheimer-type pathology in the ageing brain.

Methodology: Frontal cortex (Braak stage 0-II) was obtained from the Medical Research Council’s Cognitive Function and Ageing Study cohort. Neurones were isolated from 10 cases (5 high and 5 low DDR) by laser capture microdissection and changes in the transcriptome identified by microarray analysis. Candidate genes were validated by qPCR, and the protein they encode by immunohistochemistry and western blotting.

Results: 2378 genes were significantly differentially expressed (1690 up-regulated, 688 down-regulated, p<0.001) in cases with a high neuronal DDR. Functional grouping identified dysregulation of cholesterol biosynthesis, insulin and Wnt signalling, and up-regulation of GSK3B. Cerebrospinal fluid levels of 24(S)-hydroxycholesterol associated with neuronal DDR across all Braak stages (rs=0.30, p=0.03).

Conclusions: A persistent neuronal DDR may result in increased cholesterol biosynthesis, impaired insulin and Wnt signalling, and increased GSK3β, thereby contributing to neuronal dysfunction independent of Alzheimer-type pathology in the ageing brain.

Poster: 78 Ms Marwa Mahmoud Marwa Mahmoud, Rosemary Kim, Sarah Hsiao, Ruoyu Xing, Kim Van der Heiden, Akiko Mammoto, Jing Chen, Ismael Gauci, Shuang Feng, Paul C.Evans PhD Student - 3rd Year Cardiovascular Science Contact: [email protected]

Disturbed flow promotes atherogenesis through the activation of endothelial-mesenchymal transition

Rationale & Hypothesis: Atherosclerosis is influenced by local blood flow patterns, which exert wall shear stress (WSS) on endothelial cells (EC). Low, oscillatory WSS promotes atherosclerosis by influencing EC permeability and proliferation, while high WSS is athero-protective. Our recent microarray study of EC at athero-prone or athero-protective WSS regions of the porcine aorta revealed differential expression of GATA4 and Twist1. These transcriptional-activators can promote endothelial-mesenchymal transition (EndMT). We hypothesise that GATA4 and Twist1 may promote atherogenesis at sites of disturbed flow by inducing EndMT.

Methods & Results: Quantitative RT-PCR and en face staining confirmed elevated GATA4, Twist1 and EndMT effector gene (Snail, Slug and N cadherin) expression at the inner curvature (lower-WSS) compared to the outer curvature (higher-WSS) of porcine and murine aortae (p<0.05). Snail expression at the inner curvature of the murine aorta was reduced by EC deletion of Twist1 (Tie-2 Twist1 KO) compared to controls (Twist1 fl/fl; p<0.05). WSS was modified in murine carotid arteries using a constrictive cuff, this elevated Twist1 expression at the low WSS site and enhanced GATA4 and Snail expression at the low, oscillatory WSS site. Similarly, GATA4, Twist1 and EndMT effector genes were induced in PAEC or HUVEC exposed to low, oscillatory WSS using an orbital plate system (p<0.05). Gene silencing demonstrated that GATA4 and Twist1 are required for Snail induction in EC exposed to low, oscillatory WSS, and chromatin immunoprecipitation revealed GATA4 interaction with Twist1 and Snail. Low, oscillatory WSS promoted EndMT-characteristic changes including N-cadherin induction, VE-cadherin disorganization and enhanced proliferation (p<0.05). Silencing of GATA4, Twist1 and Snail significantly reduced these processes alongside EC permeability (p<0.05).

Conclusion: Low WSS induces EndMT and subsequent EC proliferation and permeability through GATA4 and Twist1 induction. Our observations illuminate for the first time, the role of EndMT in arterial biomechanics and injury. Future studies should define the role of EndMT in focal atherosclerosis.

Poster: 79 Miss Sarah Rennicks Sarah Rennicks, Steven Bradbury, Mary Eraso-Bastidas, Alyson Evans, Ingunn Holen and Penelope Ottewell Placement Student Oncology Contact: [email protected]

Anakinra inhibits growth of breast cancer cells in mice; in vitro and in vivo effects on breast cancer bone metastasis.

Rationale & Hypothesis: We have recently identified IL-1β as a potential biomarker for identifying breast cancer patients who are likely to develop metastatic bone disease. Furthermore, in mouse models, we have found upregulation of IL-1β and its receptor (IL-1R1) in breast cancer cells that metastasise to bone compared with cells that do not metastasise. This project aims to establish the effects of blocking activity of IL-1β with the IL1-R1 antagonist Anakinra on MDA-MB-231 breast cancer cell growth and metastasis. We hypothesise that inhibiting IL-1β activity will reduce breast cancer bone metastases.

Objectives: Establish the effects of Anakinra on MDA-MB-231 growth and bone metastasis in vitro and in vivo.

Methodology: Nude mice received subcutaneous or intra-cardiac injection of MDA-MB-231-IV cells and 1mg/kg/day Anakinra/placebo was administered 3-days before (preventative) or 7-days after (treatment). Tumour volume was measured using calipers, apoptosis, proliferation and angiogenesis by TUNEL, Ki67 and CD34 immunohistochemistry. Effects of Anakinra/placebo on proliferation, migration and invasion were monitored in vitro by cell counting, scratch assays and transwell assays.

Findings: Anakinra significantly reduced growth of subcutaneous tumours from 656.68mm3 (placebo) to 160.47mm3 (treatment; P < 0.005) and 31.08mm3 (preventative P < 0.005). Anakinra also reduced the number of mice that developed bone metastases from 90% (placebo) to 40% (treatment) and 10% (preventative). Interestingly, inhibition of tumour growth was not a result of induced apoptosis. Instead, Anakinra significantly reduced proliferation in subcutaneous tumours from 247.8 cells/mm2 (placebo) to 205.2 cells/mm2 (treatment; P < 0.05) and 134.6 cells/mm2 (preventative; P < 0.001). In vitro analysis revealed that Anakinra did not exert direct effects on MDA-MB-231 cell growth or migration.

Conclusions: Anakinra inhibits MDA-MB-231 breast cancer growth and bone metastasis in vivo via indirect effects on the tumour microenvironment.

Poster: 80 Mr Billy Bryan Billy Bryan PhD Student - 1st Year Medical Education Contact: [email protected]

Can feedback informed by self-regulatory microanalysis improve skills performance? Insights from a systematic review

Rationale & Hypothesis: Self-regulated students are able to exert control over their own behaviours and performance in the environment in which they pursue their goals; these individuals are significantly more successful than those who do not exhibit self-regulatory characteristics. Feedback relating to self-regulatory processes is rarely given or recognised as feedback in medical education which is reflected in clinical skills training. There has been no review in this area and little research investigating microanalysis (targeted questioning) and it’s applications for skills performance feedback in any context. The hypothesis is that the intervention will yield significantly higher performance outputs than other interventions in the literature including ‘usual best practice’ feedback (e.g. Pendleton’s rules or feedback sandwich).

Objectives: The overarching objective is to find out whether this style of feedback helps to improve performance and future learning. This review aims to investigate whether SRL microanalysis informed feedback can be delineated to identify ‘what works and why’ to improve skills performance. Additionally, this review looks to explore contexts where SRL microanalysis is being utilised and its perceived usefulness.

Methodology: A Systematic review using pre-specified search terms, specific inclusion/exclusion criteria and eligibility screening. More specifically, a two-stage qualitative review including descriptive mapping of the research leading to a narrative empirical synthesis. Research areas explored included: education, sport and healthcare in order to provide a rich, contextual element.

Findings: Preliminary findings suggest that feedback informed by self-regulatory microanalysis improves performance in skills based and reasoning activities compared to other feedback practices. Microanalysis in sporting contexts is typically used to improve discrete skills e.g. dart throwing. In other contexts, the intervention has been used in diagnostic reasoning and decision making tasks. Three areas of self-regulation typically utilised to elicit improvement are goal setting, self-efficacy and self- appraisal. However, there is little data showing perceived usefulness and impact on future learning at this stage.

Poster: 81 Mr Zabran Ilyas Zabran Ilyas, Adrienn Angyal, Iquo E Offiong, Endre Kiss-Toth PhD Student - 1st Year Cardiovascular Science Contact: [email protected]

miRNA202 IS A NOVEL REGULATOR OF TRIBBLES-1 EXPRESSION

Introduction: Tribbles-1 (trib-1) pseudokinase is a regulatory protein that has been shown to be a protective gene in a number of cell types and processes, relevant to the development of atherosclerosis. These include inhibition of vascular smooth muscle cell proliferation, polarisation of macrophages towards an alternatively activated phenotype and lowering the production and release of LDL in hepatic tissues. Therefore, understanding the molecular mechanisms, which regulate trib-1 expression are of significant interest. Our group have identified miRNA202 as a controller of trib-1 levels.

Rationale: Trib-1 mRNA is highly unstable with a half-life of less than 1 hour; the 3’ UTR encodes for a number of putative binding sites for miRNAs, including miRNA202. This study aimed to experimentally validate the importance of miRNA202 in the control of trib-1 expression.

Methods: 1. We have used a luciferase reporter to demonstrate the role of trib-1 3’UTR in mRNA stability and to characterise the impact of miR202 on trib-1 3’UTR in hepatic cells (HepG2). 2. qRT-PCR was used to quantify endogenous trib-1 and miRNA202 levels upon stimulation of IL-1 and in high glucose media. 3. Western blotting was used to elucidate the effect of miRNA202 on trib-1 protein levels.

Results: 1. Overexpression of miR202 reduced luciferase – trib1-3’UTR reporter expression. 2. The endogenous trb-1 mRNA was also modulated by miR202. Furthermore, stimulation of IL-1 in HepG2 cells has shown trb-1 levels to reduce by >70% and miR202 levels to increase by 2 fold. 3. Trib-1 protein levels have shown a reduction upon overexpression of miRNA202. Conversely, miRNA202 inhibitor increased trib-1 protein levels.

Discussion: miR202 is a novel regulator of trib-1 expression and may represent a target by which trib-1 levels could be raised in vivo, thereby providing a mechanism to augment the anti-atherosclerotic effects of this protein.

Poster: 82 Ms Jess Warrington JI Warrington, GO Richards, TM Skerry PhD Student - 1st Year Human Metabolism Contact: [email protected]

Using CRISPR knockout technology to explore the role of adrenomedullin in tumour metastasis

Background CRISPR is a new genetic engineering technique that excises short DNA sequences using a guide RNA and a Cas9 endonuclease. This system allows gene knockout by introducing a double-stranded cut at the DNA target site. I have used CRISPR to knockout expression of adrenomedullin (AM) signaling components in cells so that we can determine the role of AM in tumour metastasis. AM is a peptide known to promote different aspects of tumorigenesis and metastasis but the mechanisms involved are not fully understood. The alteration of AM functions will provide insight into its associated signaling pathways and the potential effects of AM inhibition both in-vivo and in-vitro.

Aim To generate CRISPR knock-out cell lines lacking components of the two heteromeric AM receptors to determine the effect of AM receptor knockdown on tumour progression and associated pathways.

Methods CRISPR constructs were transfected by lipofectamine into SAOS-2 cells at a dose found to produce the highest level of GFP expression, which is linked with expression of the Cas9 enzyme. Cells were then sorted using flow cytometry into single cell clones which were grown in conditioned media. Primers were designed for each CRISPR site, including a restriction site to aid validation. DNA was extracted from all clones on 96-well plates using Phire Tissue® Direct PCR Mastermix. This allowed for a simplified DNA extraction direct from the plate and high speed PCR reaction. Clones were then ready for knockout validation with restriction enzymes and Sanger sequencing.

Conclusions I have developed a CRISPR protocol and generated several cell lines for the study of AM’s role in tumour growth and development. These cells will be used throughout the rest of my PhD investigating the effects of AM inhibition on tumour progression.

Poster: 83 Ms Aylin Metzner Aylin Metzner, Freek van Eeden, Albert CM Ong Marie Curie Early Researcher Infection & Immunity Contact: [email protected]

Drug discovery for ADPKD using a zebrafish pkd2 model

Background: Autosomal dominant polycystic kidney disease (ADPKD), is the most common inherited kidney disease with a reported incidence of 1/1000, thus potentially affecting 12 million people worldwide. 10% of all end-stage renal disease is attributed to ADPKD, which translates to ~50,000 patients on renal replacement across Europe costing 1.5 billion Euros p.a. Currently there are no approved treatments available for this devastating disease. As part of the EU FP7-funded initiative, TranCYST, we have conducted a high-throughput screen utilising the zebrafish pkd2hu2173 mutant as the first step towards developing new treatments for ADPKD patients.

Materials and Methods: pkd2hu2173 mutants with a truncated PC2 protein develop a range of ADPKD-related features including abnormal heart looping, cardiac oedema and a curly tail phenotype. The latter is thought to be caused by deregulated collagen deposition, also a hallmark of human ADPKD, and is the most accessible and penetrant phenotype for high-throughput screening. We have now completed a screen with the Spectrum library (2000 FDA-approved and natural compounds). For each of the compounds, initially three pkd2hu2173 embryos were exposed to 10 µM compound for 24 h starting at 28 hpf. Following the first-pass screen, all hits were validated in confirmatory screens.

Results and Discussion: To date 14 compounds of interest have been identified in the Spectrum library which either aggravate or alleviate the phenotype. Interestingly, most of the compounds cluster into three distinct chemical classes. Future work will seek to select the most promising of these hits by in silico analysis (bioinformatics and literature searching), in vitro assays (cyst cultures) prior to testing in rodent models.

Poster: 84 Dr Claire Garwood Claire J. Garwood, Laura E. Ratcliffe, Julie E. Simpson, Paul R. Heath, Paul G. Ince, and Stephen B. Wharton Research Associate Neuroscience Contact: [email protected]

The insulin signalling pathway in human astrocytes in vitro and in vivo; characterisation, subcellular localisation and modulation at the receptors.

Background: Our group has previously shown that astrocyte hypertrophy and injury occur early in the progression of AD neuropathology and using microarray analysis, that the insulin/IGF-1 signalling (IIS) pathway is downregulated as Alzheimer-type pathology develops. We therefore examined these pathways in human astrocytes and developed methods to reduce IIS at the levels of the insulin receptor.

Methods: The IIS pathway, including insulin receptor (IR) isoform expression, were characterised in human primary astrocytes cultured with/without serum and/or insulin. To validate expression patterns observed in culture, expression of pathway components was assessed on post-mortem AD and control tissue using immunohistochemistry. Impaired signalling was induced in astrocytes using a combined insulin-fructose treatment and confirmed by immunoblotting for IR as well as the downstream targets Akt and ERK1/2. Astrocyte growth/viability was determined using Cyquant® and MTT assays and changes in astrocyte reactivity by immunoblotting for GFAP.

Results: IIS is present and functional in human astrocytes, which express the IR-B receptor subtype. The phosphorylation status of IRS1 affects its subcellular localisation. Insulin+fructose treatment resulted in decreased expression of IR, and decreased phosphorylation of the downstream target Akt. There was no effect of insulin+fructose on cell growth/viability or GFAP immunoreactivity.

Conclusions: IIS is functional in astrocytes. Expression of the IR-B receptor subtype may be important in differential susceptibility of astrocytes and neurons to insulin resistance. The variable presence of IRS-1 in the nucleus, dependent on phosphorylation pattern, suggests the function of signalling molecules is not confined to cytoplasmic cascades. Down-regulation of IR signalling results in decreased downstream signalling, though the lack of effect on viability suggests that astrocytes can compensate for changes in single pathways. Understanding the impact of changes in astrocytes signalling is important as these cells play a key role in neuronal homeostasis and astrocyte dysfunction is implicated in a number of neurodegenerative diseases.

Poster: 85 Mr Steven Bradbury Steven Bradbury, Sarah Rennicks, Mary Eraso-Bastidas, Diane Lefley, Alyson Evans, Ingunn Holen and Penelope Ottewell Placement Student Oncology Contact: [email protected]

The role of IL-1β in breast cancer bone metastasis.

Rationale & Hypothesis: Elevated IL-1β has been observed in a number of cancers including breast and prostate. Increased IL-1β is associated with a more aggressive phenotype, higher rates of bone metastases and an increased risk of death. In the current study we have investigated the effects of blocking IL-1β activity with the IL-1 receptor antagonist Anakinra on breast cancer bone metastases in mouse models. We have also investigated the direct effects of IL-1β on growth, migration and invasion of MDA-MB-231 breast cancer cells in vitro. We hypothesise that high concentrations of IL-1B promote a metastatic phenotype in breast cancer cells.

Objectives: Establish the effects of IL-1β on MDA-MB-231 growth and bone metastasis in vitro and in vivo.

Methodology: Nude mice received intra-cardiac injection of MDA-MB-231-IV cells 3 days before (preventive protocol) or 7-days after (treatment protocol) administration of 1mg/kg/day Anakinra/placebo for 4 weeks. Tumour take was monitored by GFP imaging and tumour volume measured on H&E stained histological sections. Effects on bone were measured by μCT and bone histomorphometry following TRAP staining. Direct effects of IL-1β on proliferation, migration and invasion were monitored in vitro by cell counting, scratch assays and transwell assays.

Findings: Anakinra significantly reduced the number of mice that developed bone metastasis from 90% (placebo) to 40% (treatment) and 10% (preventative). In mice that developed bone metastases, Anakinra reduced tumour volume from 1.61 ± 0.5mm2 2 2 (placebo) to 0.74 ± 0.49mm (treatment) and 0.06 ± 0.06mm (preventive), accompanied by increased bone volume. Administration of Anakinra to mice without tumours caused a small increase in bone volume (12.17% in control v.s.14.42% preventative). Interestingly, direct addition of IL-1β to MDA-MB-231 cells had no effect on migration but reduced proliferation compared to control.

Conclusions: IL-1B stimulates breast cancer growth and bone metastasis in vivo via indirect effects on bone. 86

Poster: 86 Miss Katrina Williams Williams, K; Chapman, N.R; Griffiths, P; Milne, R; Pacey, A.A PhD Student - 3rd Year Human Metabolism Contact: [email protected]

Investigating interactions between human spermatozoa and human Cytomegalovirus (CMV).

Rationale: Human Cytomegalovirus (CMV) is a common herpesvirus found in 60% of the population. Normally, it poses no risk, however it can have consequences for unborn babies. This is of concern when donor sperm is used in assisted conception, as CMV is present in semen. The risk of transmission from a positive donor is unknown; hence why professional guidelines recommend that it is preferable to recruit CMV negative donors. Whether or not sperm can act as a vector for transmission, by interacting directly with the virus, is unknown.

Hypothesis: CMV will interact with human sperm and sperm will be a vector for viral transmission. Objectives: To determine if CMV is directly interacting with human sperm by investigating if co-incubation with the virus has an effect on sperm function parameters, such as motility, viability, DNA damage and acrosome status.

Methodology: CMV (AD169) was grown in culture by infecting MRC-5 cells and harvesting supernatant every 2-3 days once infection was established. Viral load was quantified by qPCR and infectious dose assessed using a plaque assay. Motile sperm were obtained by density gradient centrifugation, using an 80:40% (v/v) PureSperm gradient, to separate sperm from seminal fluid. The concentration of sperm was adjusted to ~20x106/ml and infected with either virus- infected supernatant, or mock-infected supernatant, at a ratio of 2 virus particles:1 spermatozoon down to 0.25:1. A media control was also included. Samples were incubated at 37°C for 6 hours before assessing sperm motility and viability, acrosome status and DNA damage levels.

Findings: No significant effect on sperm motility or viability was observed at these viral concentrations in comparison to a mock-infected supernatant control and media control. Further investigation will determine any effects on the acrosome status of the sperm and the levels of DNA damage in response to viral exposure.

Poster: 87 Mr David Rhodes Rhodes D, Smith SA, Holcombe M, Qwarnstrom EE Research Associate Cardiovascular Science Contact: [email protected]

Computational modelling of NF-κB activation by IL-1RI and its Co-receptor TILRR, predicts a role for cytoskeletal sequestration of IκBα in inflammatory signalling.

Background The transcription factor NF- B is central to control of inflammatory responses and anti-apoptotic signals. Dys-regulation of the system underlies chronic inflammatory diseases and tumour development. Activation of NF- B involves rapid phosphorylation, and degradation of the inhibitor, I B which allows NF- B to enter the nucleus to trigger gene transcription. NF- B induction of I B provides a negative feedback, crucial to pathway control. We have demonstrated that about 2/3 of the NF- B inhibitor, I B , are sequestered by the cytoskeleton in the resting cell. Here we use an interdisciplinary approach to establish the role of the cytoskeletal bound I B in regulation of NF- B.

Methods Mechanisms controlling binding and release of the cytoskeletal bound inhibitor were determined by 3-D predictive protein modelling software and in vitro experiments. In silico simulations used a comprehensive agent-based model, which we show faithfully reproduces the multiple steps comprising the NF- B pathway, to predict the role of the cytoskeletal pool of the inhibitor in control of signal amplification and NF- B activity.

Results The 3-D predictive protein modelling and in vitro assays, demonstrated that I B is sequestered to the actin/spectrin complex within the cytoskeleton of the resting cell, and released during IL-1 stimulation, through a process controlled by the IL-1RI co-receptor TILRR. Further, in the case of IL-1 stimulation, the cytoskeletal pool of I B is released in a process controlled by the co-receptor TILRR, and coinciding with Ras- induced cytoskeletal changes during activation. In silico simulations demonstrate the requirement of this process in pathway control and predict the cytoskeletal pool of I B to control signal amplification of inflammatory responses in relation to input levels.

Conclusion The results suggest that cytoskeletal sequestration of I B provides a mechanism for signal calibration, which enables efficient, activation-sensitive regulation of NF-κB and of inflammatory responses.

Poster: 88 Mr Paris Avgoustou P.Avgoustou, J.O.Zirimwabagabo, M.J.Tozer, K.R.Gibson, J.P.A.Harrity, G.O.Richard, T.M.Skerry PhD Student - 1st Year Human Metabolism Contact: [email protected]

Targeting Adrenomedullin (AM) as a treatment of pancreatic cancer

Background: Pancreatic cancer accounts for 5% of cancer-related deaths in the UK. Only 22% of patients survive for 1 year, at 5 years survival is 4%. There is a profound unmet need for more effective drugs that will provide life extension and improve patients’ quality of life. Signalling between cancer cells/tumour and the host underlies the process of metastatic tumour development. One molecule known to have an important role in these communications is adrenomedullin (AM), a hormone that exerts its actions through a combination of the calcitonin-like receptor (CLR) and one of two accessory proteins, RAMP2 or RAMP3 forming AM1r and AM2r respectively. AM receptors are involved in a variety of physiological and pathological processes including vasodilation, increasing cancer cell proliferation, angiogenesis and inhibition of apoptosis. Our data have shown that specific inhibition of the AM2r with a monoclonal antibody reduced pancreatic tumour growth and development in-vivo.

Aims and objectives: This project aims to develop selective small molecule AM2r antagonists, using rational drug design based on a homology model and crystal structures of the receptors. The starting point of this work is MK-3207, an antagonist of the closely related Calcitonin gene-related peptide (CGRP) receptor. MK-3207 has a higher receptor affinity for CGRP (Ki=0.024nM) than AM2r (Ki=165nM).

Methods: We have designed and tested compounds using a cell-based competition assay (Lance TR-FRET cAMP assay) against AM and CGRP agonists.

Results: We have screened ~ 40 compounds and found the first AM2r selective antagonist with a Ki of 3.8μM compared to 46μM on the CGRPr. Subsequent iterative design and production has led to compounds with different efficacy and selectivity providing useful Structure–activity information.

Conclusion: These early leads suggest to us that we will be able to develop a series of selective efficacious AM2r antagonists for assessment in functional assays and further development.

Poster: 89 Dr Zainal Abedin Zainal Abedin, Jean Russell, François Guesdon and Meguid EL Nahas PhD Student - 4th Year Infection & Immunity Contact: [email protected]

Are Leptin, DPP-IV, CXCL-16 and IL-8 useful biomarkers of CKD outcomes?

Rationale & Hypothesis: Chronic Kidney Disease (CKD) is a major public health problem and a significant limitation of therapeutic interventions is failure of early identification. Most patients present with late stage disease (CKD3 onward) with little scope for interventions. Therefore, there is a need to search for more sensitive biomarkers for the early identification of CKD prognosis.

Objectives: We tested the hypothesis that the urine levels of Leptin, CXCL-16, Dipeptidyl-peptidase IV (DPP-IV) and IL- 8 may correlate with causes of CKD or predict outcomes or progression of the disease.

Methodology: Urine samples from 269 patients and 49 healthy individuals were assayed for leptin, CXCL-16, DPP-IV and IL-8 using ELISA (Duoset kits, R & D systems). For statistical analyses, patients were categorised by cause of disease, progression, stage of disease or outcome. Outcome categories were: CKD(S) (discharged from follow-up as CKD was stable, n=87), CKD(P) (under follow-up as CKD was progressing, n=123), ESRD/Dialysis (n=38) or death (n=21) during a 3 year follow-up period. Values were expressed per unit of urine creatinine (Cr). ANOVA and Receiver Operator Characteristic (ROC) curve analyses were used to determine the discriminatory and predictive values of each biomarker. Because the data was not normally distributed, ANOVA analyses were carried out on log-transform data.

Findings: CXCL-16 and DPP-IV were elevated at stage 5 and in patients undergoing dialysis. Given that most samples used in this study had been collected whilst patients were at stage 3a or 3b, this suggest that CXCL-16 and DDP-IV could be used at this stage to predict the outcome of the disease. However, ROC curve analyses show that none of the candidate biomarkers tested predict the progression of CKD better than the currently clinically use ACR.

Poster: 90 Dr Rachel Waller Rachel Waller, Emily Goodall, Mbombe Kazoka, Helen Wollff, Janine Kirby, Pam Shaw Research Associate Neuroscience Contact: [email protected]

Blood-based miRNAs potential biomarkers in motor neuron disease

Rationale & Hypothesis: Motor neurone disease (MND) is a fatal neurodegenerative disease leading to paralysis and death. Effective biomarkers would help to diagnose MND, aid in drug development and track disease progression and possible prognosis. microRNAs (miRNAs) are small non-coding RNAs that direct post transcriptional gene regulation. They have been shown to be dysregulated in neurodegenerative diseases including MND and are found at detectable levels in the blood, therefore having the potential to be used as biomarkers of disease. The hypothesis of this study is that candidate circulating miRNAs identified that distinguish MND patients from control cases will change over time with disease progression.

Objectives: The objective of this study was to investigate miRNA expression in MND patient serum samples to assess whether; 1. miRNA expression alters in patients taking riluozle or riluzole naïve at time of diagnosis. 2. miRNA expression alters over time with disease progression.

Methodology: miRNAs were extracted from patient serum samples at time of diagnosis (or within three months) and a subsequent serum sample was taken at least three months post diagnosis. Qiagen miScript reverse transcription, pre- amplification and qPCR was carried out to investigate the expression of miR-206, miR-143-3p and miR-374b-5p between patients taking riluzole and riluzole naïve and subsequently to establish the miRNA expression over time with disease duration.

Findings: There was no significant difference in the expression of miR-206, miR-143-3p and miR-374b-5p between patients taking riluzole or riluzole naïve patients at time of diagnosis. However, as the disease progressed a significant increase in miR- 143-3p was identified in MND patients, while a significant decrease was found in miR-374b-5p.

Poster: 91 Mr Lewis Quayle Quayle, L., Ottewell, P.D., Holen, I. PhD Student - 1st Year Oncology Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 92 Dr Steven Reynolds Steven Reynolds, Stephen Metcalf, Martyn Paley, Gillian Tozer Research Fellow Cardiovascular Science Contact: [email protected]

Direct arterial injection of hyperpolarised substrate for rapid detection of metabolism with minimal substrate dilution

Rationale: Hyperpolarised dissolution dynamic nuclear polarisation is an established pre-clinical/clinical technology for monitoring 13 in vivo metabolism kinetics by magnetic resonance spectroscopy and imaging. Hyperpolarised C1-pyruvate, has provided important insights into how in vivo tumour cell metabolism is influenced by various conventional and novel cancer treatments. Hyperpolarised substrates are rapidly administered to the animal intravenously (i.v.) and metabolite signals are acquired within a few multiples of the T1 relaxation time, before the signal decay becomes too significant. To image short T1 substrates, such as drugs, the hyperpolarised signal must be quickly acquired after dissolution. Objectives: Develop a method for rapid signal acquisition by directly injecting 13C-labelled compounds into a single tumour supplying artery. This will minimise the delivery time and maximise substrate delivery to the tumour. Methodology: BDIX rats (~300g), were implanted with 106 P22 fibrosarcoma cells into the right inguinal fat pad. The tumour received its principle blood supply directly from the superior epigastric artery. The saphenous artery up to the superior epigastric 13 branch supplying the tumour was cannulated for hyperpolarised substrate delivery. Either hyperpolarised C1-pyruvate, 13 13 Cu-glucose-d7 or C-CA1P was administered to the rats, which were positioned in a 7T MRI scanner, via a superior epigastric artery cannulation or femoral vein cannula. 13C MR spectra or images were collected every 1s and processed using Matlab. Findings: The pyruvate signal was 7.5 larger for arterial injected pyruvate compare to a venously administered route in spite of a lower arterial dose. Administering Gadolinium contrast agent confirmed that the tumour was the principal perfusion site. Glucose, which has a short T1 (~10s) was observed and a potential metabolite, alanine, was tentatively assigned. Hyperpolarised custom 13C labelled (University of Sheffield, Chemistry dept.) vascular disrupting agent CA1P signal was observed in vivo.

Poster: 93 Dr Steven Reynolds Steven Reynolds, Peter Laity, Ben Curie, Chris Holland and Martyn Paley Research Fellow Cardiovascular Science Contact: [email protected]

Investigating the properties of silk formation in Bombyx mori silkworms using T1 and T2 image maps.

Rationale: Bombyx mori form silk fibres from a proteinaceous mixture, silk dope, in two large glands. The posterior section of the gland secrete the dope as protein components (Fibroins), which are stored in an enlarged midsection before being spun through a tapered duct. Flow induced protein denaturation initiates fibre formation before exiting through the animal’s spinneret. Rheological studies on excised silk dope show that silk is stored as a viscous gel before undergoing chemical and mechanical changes during spinning. Better knowledge of this process would help develop novel fibre spinning techniques. Silk generated scaffolds are of major interest for human stem cells tissue engineering, e.g. cardiac tissue repairs. The study of fibroin aggregation is a potential model for amyloidogenesis.

Objectives: Use MRI to determine T1 and T2 maps in live silkworms as an indicator of the rheological properties of the silk dope throughout the gland and duct

Methodology: Bombyx mori were wrapped in Clingfilm and cooled to induce a state of dormancy. MRI images were obtained using a 9.4T MRI and a 10mm 1H volume coil. T1 and T2 values were measured using a RAREVTR sequence (TR=1397.83,1703.35,2094.91,2640.75,3548.71,6989.13ms, TE=12.56,25.11,37.67,50.23ms, NEX=4, FOV=15x15mm, Matrix=64x64, 25x1mm slices). Using custom MatLab software T1 and T2 relaxation time maps were estimated from reconstructed images for each slice. Mean±S.D. T1/T2 values were determined for regions of interest around the major silk ducts.

Findings: The T1 and T2 values varied smoothly along the gland. The posterior gland showed a significant difference in mean T1 compared to the midsection (ANOVA). No significant differences in T2 values were observed between the regions. The results show that changes in the T1 and T2 properties of the silk dope could be used to track changes in the rheology. Further work is ongoing to reconstruct the images as a single continuous 3D gland.

Poster: 94 Miss Hana Alloub Hana Alloub, Derek Burke, Amaka Offiah Medical Student Human Metabolism Contact: [email protected]

Validating Finite Element Model of Children's Bones

Poster: 95 Dr Philip Elks Philip M Elks1, 2, Michiel van der Vaart3, Fredericus J van Eeden2, Herman P Spaink3, Sarah R Walmsley4, Annemarie H Meijer3 and Stephen A Renshaw1,2 Sir Henry Dale Fellow Infection & Immunity Contact: [email protected]

Hypoxia Signaling Modulates Neutrophil Nitric Oxide in a Zebrafish Tuberculosis Model

Rationale & Hypothesis: Tuberculosis is on the rise due to the increasing prevalence of multi-drug resistant strains. Understanding the host response to the causative pathogen, Mycobacterium tuberculosis, is critical to identify host-derived factors as potential therapeutic targets, circumventing bacterial resistance. However, there is a lack of whole-organism, in vivo, models in which to study host- mycobacterial interactions.

Objectives: In a zebrafish mycobacterial model, I aimed to determine whether hypoxic signalling, via hypoxia inducible factor 1 alpha (Hif-1α), is a host-derived signalling pathway that modulates the host response to infection.

Methodology: Hif-1α was modulated in zebrafish embryos by expression of dominant active and negative variants. Mycobacterium marinum (Mm), a close relative of Mycobacterium tuberculosis and well-established TB model, was microinjected into Hif-1α modulated embryos and the host response to infection was investigated.

Findings: Hif-α signalling was observed in infected macrophages early in pathogenesis but was not present at later stages. Stabilisation of Hif-1α led to decreased bacterial burden in an inducible nitric oxide synthase (iNOS) dependent manner. iNOS is a leukocyte specific enzyme that produces nitric oxide (NO) as a bacterial killing mechanism. An anti-nitrotyrosine antibody assay was developed to investigate NO levels during Mm pathogenesis. Infection with Mm, live or heat-killed, increased NO production by neutrophils. Hif-1α stabilisation primed neutrophils with NO, allowing them to better deal with Mm upon infection. This was found to be neutrophil cell-autonomous when Hif-1α was specifically overexpressed in neutrophils. Live (but not heat-killed) Mm were able to decrease neutrophil NO levels during pathogenesis, indicative of a reprogramming of leukocytes by Mm to allow for permissive conditions for the bacteria.

Our data show that Hif-1α and iNOS are important host-derived immune modulators that are initially activated after Mm infection only to be downregulated later in pathogenesis. Maintaining Hif-1α/NO levels may be a novel therapeutic strategy that circumvents the problem of multi-drug resistance. 91

Poster: 96 Dr Matteo De Marco Matteo De Marco, Annamaria Vallelunga, Annalena Venneri Postdoctoral Researcher Neuroscience Contact: [email protected]

Apolipoprotein E ε4 Allele Influences Functional Connectivity in Mild Cognitive Impairment

Background: The brain circuitry of healthy elderly adults is down-regulated by the presence of the Apolipoprotein E (ApoE) ε4 allele. It has not been determined, however, whether such effect persists or mutates after the onset of the earliest symptoms of neurodegeneration.

Objectives: In this study allele-dependent differences in functional connectivity were investigated in adults diagnosed with Mild Cognitive Impairment (MCI), and specific emphasis was placed on those patterns of connectivity which are altered by Alzheimer’s disease.

Methodology: Thirteen adults diagnosed with MCI with an ApoE ε4ε3 genotype and thirteen MCI adults homozygous for the ε3 allele were included in this study. Both groups were characterised as of the amnestic MCI subtype. All participants completed a MRI protocol including resting-state fMRI. The two groups did not differ neither in their demographic status nor in their levels of cognitive functioning. Moreover, no between-group difference was found in global/regional indices of grey-matter and white-matter maps. Using a seed-based approach, the Default-Mode Network (DMN), the Salience Network (SN) and hippocampal connectivity were computed, and two-sample t tests served to compare the two groups.

Findings: ε4 carriers showed increased connectivity within the SN and between the left hippocampal seed and a fronto- insular cluster.

Discussion: The down-regulation of the DMN caused by the ε4 allele as seen in healthy senescence was no longer directly visible in early-stage neurodegeneration. However, an ε4-dependent up-regulation of its anticorrelated network (SN) was reported, indicating that the presence of the ε4 allele influences the overall balance between the two networks in MCI adults. This is also supported by the evidence of increased connectivity seen in ε4 carriers between the hippocampus and the insula, whose timecourse should normally be negatively correlated. These findings indicate that the presence of the ε4 allele in MCI is associated with a more profound functional disconnection.

Poster: 97 Dr Shobna Silva S.Silva, S. Danson, M.D. Teare, A. Cox Clinical Fellow Oncology Contact: [email protected]

Cell-free DNA as a non-invasive marker of relapse/progression in melanoma

Rationale: Melanoma is the most aggressive form of skin cancer. Once melanoma has relapsed, it is often incurable. The median survival for metastatic disease is 6-9 months. Melanoma relapse is currently detected by clinical examination and radiological imaging. If we can detect relapses early, we could start treatments earlier, and potentially improve outcomes for such patients. Fragments of tumour DNA (circulating cell-free DNA, ccfDNA) can be detected in plasma. Levels can be quantified, and genetic profiles could potentially be used to provide a non-invasive marker of relapse.

Hypothesis: We hypothesize that changes in the genetic profiles, as well as circulating levels, of ccfDNA can be utilised as biomarkers of relapse and progression in melanoma.

Methodology: We are carrying out a longitudinal study to recruit 120 melanoma patients and 120 healthy controls. Blood samples will be obtained at 3-monthly intervals to enable assessment of the relationship between changes in ccfDNA and the clinical course of the disease. A protocol for efficient extraction of ccfDNA from plasma & quantification using SYBR-green real-time PCR has already been established in our laboratory. An example of ccfDNA quantification from plasma samples of 2 melanoma patients is shown below:

Two samples analysed: ccfDNA levels 4.10ng/mL and 3.58 ng/mL respectively

Using plasma samples from melanoma patients from a previous epidemiological study (some of whom had active disease and others with recently excised primary disease), I will carry out low-coverage next-generation sequencing to map out copy number profiles and develop a genome instability score (using a data analysis pipeline which has already been established), and determine the ability of this score to accurately identify active disease.

This score will be optimised and then tested in the longitudinal study to validate its ability to reliably predict relapse/progression.

Poster: 98 Dr Shuang Feng Shuang Feng, Paul C Evans Research Associate Cardiovascular Science Contact: [email protected]

Disturbed flow induces glycolysis enzymes at atheroprone sites

Hypothesis &Objectives: Atherosclerosis is a chronic disease of arteries which is characterized by the accumulation of lipids and inflammatory cells at the inner wall of arteries. Atherosclerotic plaques develop predominantly at regions of branches and bends that are exposed to low or disturbed blood flow, which generates low wall shear stress (WSS) and also influences the transport of biomolecules and dissolved gases from blood to the vessel wall. Using microarray technology we demonstrated that hypoxia inducible factor 1(HIF1α) and several of its downstream targets (including glycolysis enzymes HK2 and ENO2) were enriched in endothelial cells (ECs) at an atherosusceptible region of the porcine aorta exposed to disturbed flow (unpublished). Thus we hypothesized that hypoxia and/or low WSS may influence EC dysfunction by inducing HIF1α-dependent altered glucose metabolism in EC.

Methodology & Findings: Endothelial collected from inner (disturbed flow) and outer (unidirectional flow) curvatures of the porcine aortic arch. qPCR demonstrated that the expression of HIF1α and downstream glycolysis enzymes HK2 and ENO2 was enhanced at the inner curvature compared to the outer curvature. Similarly, en face staining of the mouse aortic arch demonstrated that expression of HK2 and ENO2 was higher at inner curvature. Staining with pimonidazole revealed that EC at the inner curvature of the murine aortic arch were hypoxic, whereas cells at the outer curvature were not. Thus EC at the inner curvature were exposed to hypoxia and low WSS and they expressed elevated levels of glycolysis enzymes. In the HUVEC exposed to WSS for 72h in the presence or absence of DMOG western blotting revealed that the expression of ENO2 and HK2 was higher in cells exposed to low WSS compared to high WSS. Both enzymes were induced by 0.5mM DMOG (2-3 fold change), and the combination of low WSS and DMOG had a synergistic effect on ENO2 and HK2 expression (4-6 fold change). We conclude that EC at atheroprone sites of disturbed flow express HIF1a which in turn increased downstream glycolysis enzymes HK2 and ENO2 and altered glucose metabolism. This could be a fundamental reason of atherosclerosis initiation.

Poster: 99 Dr Robin Rumney R.M.H. Rumney, A. Agrawal, T. Cox, J. Erler, A. Gartland. Postdoctoral Research Associate Human Metabolism Contact: [email protected]

Lysyl oxidase drives osteolytic bone lesions in a breast cancer model via RANK ligand independent effects on osteoclasts: a new player in the vicious cycle?

Background and Rationale: As part of the ‘vicious cycle’ of bone metastasis, invading cancer cells uncouple bone remodelling to form osteolytic lesions. We have previously demonstrated that lysyl oxidase (LOX) plays a critical role in the formation of lytic lesions in bone, prior to cancer cell colonisation. In this study we provide new evidence that LOX elicits these effects by driving osteoclastogenesis independently of RANKL

Objectives: Elucidate the mechanisms by which LOX drives bone lesion formation.

Methodology: 4T1 breast cancer cells were injected into the fat pads of syngeneic BALB/c mice Sections of tibiae were stained for TRAP. The LOX effect was studied using human osteoclasts in vitro. Cultures were treated with recombinant LOX (rLOX) to quantify the effects on osteoclast number and resorption. Changes in the master regulator of osteoclasts, nuclear factor of activated T cells (NFAT)c1 were quantified by immunofluorescent labelling.

Findings: TRAP stained tibia from 4T1 injected animals demonstrated a two-fold increase in osteoclast number compared to controls (P=0.001); genetic knockdown of LOX in the primary tumour abrogated these effects (P=0.011). Human osteoclast cultures treated with rLOX in the absence of RANKL had a two-fold increase in cell number (P<0.001), a twelve-fold increase in total resorption (P<0.001) and five-fold increase in resorption per osteoclast (P<0.001) compared to RANKL treated controls. rLOX significantly increased NFATc1 translocation to the nucleus by double compared to RANKL (P<0.01). The addition of catalase to remove the by-product of LOX activity hydrogen peroxide (a potent reactive oxygen species) abrogated NFATc1 nuclear translocation (P<0.0001).

Summary: The discovery that LOX drives de novo osteoclastogenesis via a ROS mediated effect independently of RANKL increases our knowledge of disease mediated uncoupling of bone remodelling and inaugurates LOX as a novel player in the ‘vicious cycle’.

Poster: 100 Ms Nelly Wagner Nelly L Wagner, Simon J Foster, Stephen A Renshaw PhD Student - 3rd Year Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 101 Dr Andrea Turolla Andrea Turolla, Raffaella Flaibani, Michela Agostini, Paolo Tonin, Annalena Venneri PhD Student - 3rd Year Neuroscience Contact: [email protected]

Role of BDNF Val66Met polymorphism on neuroanatomy of motor recovery after stroke.

Rationale & Hypothesis: Recent evidence suggests that polymorphism (Val66Met) in brain derived neurotrophic factor (BDNF) affect negatively motor learning after stroke. Recent findings (Qin et al. 2014) with mice models showed a larger striatal volume in the contralesional hemisphere in BDNF Val66Met carriers, after stroke. We hypothesised that a similar pattern may happen in humans, after stroke.

Objectives: Investigating whether structural difference exists in the white matter between carrier and non-carrier of the BDNF Val66Met polymorphism, after stroke.

Methodology: A sample of 19 consecutive stroke survivors inpatients were recruited at San Camillo Hospital Foundation (Italy). All the patients received equal amount of rehabilitation care within group and motor function was assessed by Fugl-Meyer Upper Extremity (F-M UE) scale. Moreover 3DT1W images were acquired and polymorphism was characterised from blood samples. A VBM analysis was performed comparing (t-test) BDNF Val66Met carriers with non-carriers, with age, sex and F-M UE as covariates. The statistical threshold was set at p<0.05 and an extent threshold of 5 voxels was considered.

Findings: 10 BDNF Val66Met carriers (7 males, 3 females; 6 right hemisphere, 4 left hemisphere) and 9 non-carriers (8 males, 1 female; 4 right hemisphere, 5 left hemisphere), were enrolled and the motor function level was comparable between groups (Mann-Whitney U test, p = 0.942). The VBM analysis showed that BDNF Val66Met carriers had larger correlations in the white matter structures than non-carriers (Figure 1), with bigger clusters in the supramarginal, middle frontal and superior gyri. These preliminary results seem to confirm that BDNF Val66Met carriers may need a larger volume of fibres in the recovery process after stroke, maybe aimed to compensate the lower availability of free BDNF facilitating neural plasticity. Future studies with larger samples are needed for confirmation.

Figure 1. Structures of the white matter in BDNF Val66Met carriers compared with non-carriers. Chromatic significance of clusters are displayed in coronal (a), axial (b) and sagittal (c) sections.

Poster: 102 Dr Safar Kheder Safar Kheder, Sirwan Hadad, Karen Sisley, Saba Balasubramanian PhD Student - 3rd Year Oncology Contact: [email protected]

Importance of the OCT1 transporter to the anti-cancer effect of metformin in thyroid cancer

Rationale & Hypothesis: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment. Some sub-types however have a poor prognosis. In a previous study, we showed that metformin (an oral antidiabetic drug of the biguanide class) inhibited thyroid cancer cell proliferation in a time and concentration dependent manner. Here we investigated the expression of organic cation transporter 1 (OCT1) in thyroid cancer cells. OCT1 plays role in cellular uptake of metformin in liver cells and is highly expressed in the kidney and intestine. Hypothesis: OCT1 is expressed in thyroid cancer cells and plays an important role in facilitating the anti-cancer effect of metformin.

Objectives: Study the expression of OCT1 in thyroid cancer cell lines with and without metformin.

Methodology: Thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1 and 8305C), normal thyroid follicular epithelium cells (Nthy-ori 3-1), human hepatic carcinoma cell line (HepG2) and breast cancer cell line (MDA-231) were treated with various concentration of metformin (0, 0.3, 1 and 10mM) for two different time points (24 and 48hours). Immunocytochemistry was used to investigate OCT1 expression. The proportion and intensity of OCT1 expression was estimated using the Allred Score (range 0-8).

Results: OCT1 expression was observed in all thyroid cancer cell lines and HePG2 cells, but not in MDA-231 cells. No significant difference was observed in the proportion and intensity of OCT1 expression in metformin treated and non-treated cells.

Conclusions: The data has demonstrated the presence of OCT1 in thyroid cancer cells. This is likely to facilitate the effect of metformin as an anti-cancer effect.

Poster: 103 Dr Nikolay Ogryzko Nikolay Ogryzko, Anne Robertson, Stephen Renshaw, Heather Wilson Post-Doctoral Research Associate Cardiovascular Science Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 104 Dr Karan Shah Orton PN, Shah KM, Wilkinson JM and Gartland A Post-Doctoral Research Associate Human Metabolism Contact: [email protected]

Mechanosensing in osteocytes following exposure to clinically relevant concentrations of metal ions after hip replacement

We have previously shown that cobalt (Co) and chromium (Cr) ions at clinically relevant concentrations after hip arthroplasty affect osteoclast and osteoblast cell viability and function in-vitro1, with implications for bone health. More recently, we have observed altered dendricity and reduced viability for osteocyte-like cells when exposed to similar concentrations and combinations of Co and Cr. Here we aim to study their cellular response to mechanical stimuli following metal ion exposure.

Murine osteocyte cell-line (MLO-Y4) cultured on collagen-coated slides were treated with 50µg/L or 500µg/L of Co2+ and Cr3+ (1:1) for 30 minutes or 24 hours, with untreated cells serving as control. Cells were then loaded with a fluorescent Ca2+ indicator (Fluo-4AM) and subjected to fluid shear-stress (16 dynes/cm2) using a laminar fluid-flow chamber. Changes in cellular fluorescence was measured in real-time using a time-lapse microscope and images analysed using ImageJ. The area under the curve (AUC), a measure of cellular response, and peak intensity was calculated using GraphPad Prism6®, and expressed relative to control.

Following 30 minute and 24 hour exposure to Co and Cr, a reduction in cellular response (AUC) to mechanical stimuli was observed for 50µg/L (p<0.0001) and 500µg/L (p<0.0001) compared to untreated controls. A reduction in peak response was also observed for both 50µg/L (p<0.0001) and 500µg/L (p<0.0001) compared to controls, for both time-points. The effects were dose-dependent with 500µg/L CoCr showing reduced cellular and peak responses compared to 50µg/L following 30 minutes and 24 hour exposures (p<0.0001).

The data suggests that Co and Cr at concentrations observed in patient serum and hip aspirate following hip replacement may impair osteocyte response to mechanical stimuli and reduce the intensity of their response. This may compound the direct effects of metal ions on bone health by alteration in osteocyte-mediated regulation of bone remodelling in response to mechanical loading.

1. Andrews, RL et al. Bone (2011), 49; 717-723

Poster: 105 Miss Charlotte Gee Gee CS, Budd R, Sammut D, Dockrell DH, Whyte MKB, Sabroe I, Prince LR Research Technician Infection & Immunity Contact: [email protected]

The regulation and role of Pellino-1 in human monocyte-derived macrophages in COPD

Rationale & Hypothesis: Pellino-1 is an E3 ubiquitin ligase that has known roles in inflammatory signalling. Murine macrophage models suggest that Pellino-1 is involved in Toll-Like Receptor (TLR) signalling. However, little is known about the regulation and role of Pellino-1 in human macrophages, particularly in the context of inflammatory disease such as COPD (Chronic Obstructive Pulmonary Disease). We hypothesise that Pellino-1 participates in controlling macrophage driven inflammation in the airway, through the regulation of macrophage activation in response to TLR signalling.

Objectives: To determine if Pellino-1 expression is regulated by TLR agonists in healthy and COPD human monocyte-derived macrophages (MDMs). To consistently knock down Pellino-1 in healthy and COPD human MDMs and assess any regulation of inflammatory outputs.

Methodology: Primary human peripheral blood mononuclear cells were isolated from peripheral blood of healthy human volunteers or patients with COPD via percoll gradient centrifugation and cultured for 7 days to generate MDMs. Gene and protein expression were determined by quantitative Polymerase Chain Reaction and western blotting respectively. siRNA transfection was performed using Dharmafect4 transfection reagent. Cytokine generation was assessed by ELISA.

Results: Pellino-1 mRNA is expressed in healthy MDMs but is not regulated at 24 hours by any of the tested Toll-like/Interleukin Receptor agonists. COPD MDMs express similar levels of Pellino-1 protein at baseline, but LPS does not regulate Pellino-1 as profoundly as in healthy MDMs. In healthy and COPD MDMs LPS up-regulates Pellino-1 protein at early time-points (3-6h). Using siRNA transfection techniques we have demonstrated Pellino-1 knockdown in both healthy and COPD MDMs with concomitant reduction in IL-8 generation.

Conclusions: Our data link Pellino-1 regulation with TLR4 signalling in human macrophages. Functional studies in Pellino-1 knockdown MDMs may highlight roles for this protein in human macrophages. 98

Poster: 106 Mr Luke Flannery Flannery L, Brown HK, Evans CA, Holen I, Haider MT Placement Student Oncology Contact: [email protected]

The Effects of Anti-Cancer Therapies on the Bone Microenvironment

Rationale & Hypothesis: The majority of studies in cancer-induced bone disease are focusing on establishing effects of the therapeutics on the cancer cells. However, it is increasingly recognized that the tumour microenvironment is a potential treatment mediator, by inducing changes in bone resorption and the cellular composition of bone marrow. We hypothesize that response to anti-cancer treatments are partly mediated by modification of the bone microenvironment. Objectives: To establish the effects of Parathyroid Hormone (PTH, stimulates bone formation), Cabozantinib (CBZ, Tyrosine Kinase inhibitor), Doxorubicin (chemotherapy) and Zoledronic Acid (anti-resorptive agent) combination therapy on the bone microenvironment in vivo. Methodology: Nude mice were treated with PTH (80µg/kg daily), CBZ (30mg/kg 5x weekly), combination therapy (2mg/kg i.v. Dox, 100µg/kg i.p. Zol weekly) or control. Bone marrow and organ samples were collected. Treatment effects were quantified for variances in bone cell populations including the number of osteoblasts and osteoclasts using histological and assay-based techniques including immunohistochemistry, qtPCR and western blot analysis to detect differing protein levels. Bone samples taken for histology were stained for proliferation, apoptosis and H&E for scoring. Results were quantified and comparisons made to control groups. Findings: CBZ treatment increased the number of osteoblasts and megakaryocyte number in the bone marrow scored on H&E sections. Vasculature was also increased in bone marrow when visualised with endomucin staining. PTH treatment resulted in increased osteoblast numbers between day 5-10 and returned to control levels by day 15. Combination therapy (Dox+Zol) caused differential effects on bone and bone marrow when compared to control groups. Conclusion: The bone microenvironment is a potential mediator of anti-cancer treatments by altering its cell composition and cytokine release to create a less favourable environment for tumour cells.

Poster: 107 Dr Daniel Kelly Daniel Kelly, Samia Akhtar, Donna Sellers, Vakkat Muraleedharan, Kevin Channer, Hugh Jones Post-Doctoral Research Associate Human Metabolism Contact: [email protected]

Evidence that testosterone improves glucose utilisation and a ‘buffering’ effect of subcutaneous fat to protect against ‘overspill’ of lipid deposition into visceral fat and non- adipose tissues.

Rationale and hypothesis. Testosterone deficiency is associated with adverse effects on cardio-metabolic function promoting insulin resistance, dyslipidaemia and fat accumulation. Excess deposition of fat resulting from poor lipid and glucose control and inadequate storage capacity in subcutaneous adipose depots can lead to ‘overspill’ into visceral fat, and non-adipose tissue. Objectives. To examine the effects of testosterone status on the expression of key targets involved in glucose utilisation and lipid metabolism in muscle, liver, subcutaneous (abdominal) and visceral fat depots. Methodology. Expression of metabolic targets were assessed via qPCR in all tissues and western blotting in liver and muscle of testicular feminised (Tfm) mice, which exhibit non-functional androgen receptors (AR) and low circulating testosterone levels and compared against wild-type and testosterone treated mice. Aortic and hepatic lipid deposition was assessed histologically. Results. Testosterone deficiency was associated with aortic fatty-streak formation and hepatic adiposity in Tfm mice. Targets of glucose utilisation were negatively altered in liver, muscle and subcutaneous adipose at the gene and protein level of Tfm mice. Targets of lipid metabolism were negatively altered in liver, and gene targets in subcutaneous adipose of Tfm mice. Testosterone treatment of Tfm mice reduced lipid accumulation in the aorta and liver, and beneficially altered the expression of hepatic targets of glucose and lipid regulation; muscle targets of glucose and subcutaneous targets of lipid regulation. Conclusion. Testosterone is important in tissue-specific control of metabolism and may differentially promote metabolic function. Improving glucose utilisation in liver and muscle tissue testosterone may prevent the conversion of excess glucose to fat. Additionally, by beneficially modulating subcutaneous adipose metabolism testosterone may increase its buffering capacity to protect against energy imbalance and fat overspill into liver and the arterial vascular tree.

Poster: 108 Dr Khondoker Akram Khondoker Akram, Nathifa Moyo, Mark Tompkins, Ralph Tripp, Lynne Bingle, James P Stewart, Colin D Bingle. Postdoctoral Research Associate Infection & Immunity Contact: [email protected]

An innate defence role for BPIFA1/SPLUNC1 against influenza-A virus infection

BPIFA1 is one of the most abundant secretory proteins in the upper airways. Recent studies suggest a host defence role of this protein against bacterial infection. However, the anti-viral role of the protein remains largely unexplored although we have shown alterations in the protein in the lung of mice infected with influenza-A (Inf- A).

To further evaluate the host defence role of BPIFA1 in viral respiratory infection, we developed an in vitro tracheal model. Tracheal epithelial cells isolated from wild-type and BPIFA1-/- mice, were seeded onto transwells and differentiated in ALI culture. Day-14 differentiated epithelium was infected with Inf-A virus. Samples were collected at 2, 24, 48 and 72 hpi (hour post infection) to evaluate through RT-PCR, western blot and confocal microscopy techniques.

Our data shows that the Day 14 differentiated epithelial layer mimics the original tracheal epithelium. Abundant BPIFA1 protein was detected in the apical secretions of differentiated epithelium. Inf-A infection initiated inflammatory gene expression, where, TNF-α, IFN-β and IFN-λ2 were highly upregulated at 24 hpi. Influenza-A primarily infected ciliated cells, these then lost physical cilia and down-regulated cilia associate gene TEKT1 expression. At lowest viral dose (0.1 MOI), BPIFA1 and MUC5B expressing cells were largely uninfected. Low dose of Inf-A infection also significantly increased (p < 0.05) apical secretion of BPIF1 protein. At 24 and 48 hpi, viral load was higher in BPIFA1-/- cells with increased cell death compared to wild-type cultures. Our data suggest that BPIFA1 abrogates Inf-A viral infection; and actively plays crucial host defence role. Therefore, BPIFA1 protein could be a novel target for antiviral treatment for respiratory tract infections.

Poster: 109 Dr Joby Cole J Cole, M. Dickman and D. Dockrell. Clinical Research Training Fellow Infection & Immunity Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

100

Poster: 110 Miss Caroline Carta Caroline Carta & Annalena Venneri PhD Student - 3rd Year Neuroscience Contact: [email protected]

Automatic versus executive semantic memory impairment in early Alzheimer’s disease: A possible diagnostic tool?

Rationale & Hypothesis: An early symptom of Alzheimer’s disease (AD) is a decline in semantic memory (SM). This impairment can be tracked through linguistic tasks that require semantic processing, such as the picture naming test which is widely used in the assessment of dementia. In a healthy brain, semantic cognition involves two interacting but separable components referred to as semantic control and semantic representation. The underlying principles of SM deficit in AD are still not well understood.

Objectives: Novel instruments were devised to establish the nature of SM impairment and improve the identification of people at risk of developing AD.

Methodology: The test included 40 novel photographed pictures (20 living and 20 non-living) which allowed for the assessment of the different aspects of SM. Half of the pictures consisted of whole objects (semantic representation) while the other half contained part of an object (semantic control). A group of cognitively healthy participants and a group of patients with mild cognitive impairment (MCI) (amnestic and non-amnestic) were tested with this newly devised confrontation naming test. In both cases participants were required to recognise and name the presented pictures. The group performance on both sets of pictures was analysed.

Findings: There was a significant difference in performance between the healthy and the MCI groups. Patients were able to name fewer items compared to the healthy group and the difference was more prominent on the task that required semantic control. A difference in performance was also reported between the amnestic and non-amnestic MCI subgroups. This data contributes to the understanding of the memory impairment present in early AD. The novel picture naming test may be a useful tool to detect abnormalities earlier than the current tests do. Earlier detection of abnormal cognitive decline will have implications for the management and care of the disease as well as the testing of possible disease modifying drugs.

Poster: 111 Mr Tim Ingham-Dempster Tim Ingham-Dempster, Dr Dawn Walker, Dr Bernard Corfe PhD Student - 1st Year Oncology Contact: [email protected]

A Multi-Scale Agent Based Model of Colon Carcinogenesis

Rationale and Hypothesis: Colorectal cancer (CRC) is a major cause of cancer mortality. Crypts of Lieberkuhn are multi-cellular flask-shaped invaginations of the colonic epithelium, with stem cells at their base which differentiate into the secretory and absorptive cells conferring functionality on the epithelium. Mutations in stem cells become embedded in crypts, which themselves undergo a growth and division cycle. These processes are strongly implicated in CRC formation. Computer simulation has previously helped to elucidate processes including niche succession and passive migration. Key areas for investigation now are the mechanistic basis of crypt expansion (bottom-up vs top-down hypotheses), adenoma polyclonality, and the resolution of competing hypotheses of the niche and pedigree models of cell fate determination.

Aim & Objectives: Our aim is to develop a multi-scale model of colorectal carcinogenesis, linking the cell division and differentiation cycle on one scale to the crypt division and expansion on the upper scale. The first objective is testing a mechanical model of the cell for the lower scale component. The stretch and compression forces applied to the cell and their heterogeneity across the cell are modelled using an agent-based architecture.

Methodology: An agent-based model has been built, modelling the colonocyte as a deformable object with subcellular elements (SCEs) interacting through a range of mechanical forces. Rules simulating proliferation have been implemented. The model exists in alternative forms with pan-cellular SCEs, peripheral SCEs only and with nuclear and cytosolic SCEs distinguished by differential compressibility. The model has finite space but unbounded replicative potential.

Findings: Early results support the hypothesis that both inter-cell adhesion, and differential nuclear-cytoplasmic compressibility are required for crypt formation to occur. Future developments of the model will include the incorporation of apoptosis, and a deformable surface beneath cells to allow crypt-like structures observed in initial simulations to mature.

101

Poster: 112 Dr Sarah Smith Smith SA, Samokhin A, Murphy EC, Francis SE, Qwarnstrom EE Research Associate Cardiovascular Science Contact: [email protected]

Altered Regulation of the inhibitor IκBα and NF-κB regulated genes in the TILRR KO mouse

Rationale & Hypothesis: Inflammatory responses are key drivers in the pathogenesis of atherosclerosis. TILRR, a prominent amplifier of NF-κB controlled responses, is highly expressed in atherosclerotic lesions. We have demonstrated that administration of a blocking peptide antibody, which selectively targets TILRR amplified inflammatory responses, significantly reduces atherosclerosis and promotes plaque stability in ApoE-/- mice. Here we assess the role of specific regions of the NF-κB inhibitor, IκBα, in controlling enhanced signal amplification. Methodology: Microarray analysis of monocytes from TILRR-/- mice were carried out to determine effects on NF-κB regulated genes. IκBα mutants were selected by alanine-scanning or created by regional deletions. Protein/protein interactions were analysed by predictive 3-D modelling and co-immunoprecipitation and gene activity by IL-8 reporter assays. Results: Microarray analysis of monocytes from the TILRR-/- mice, demonstrated pronounced reductions in NF-κB controlled inflammatory genes, many known to be relevant to development of cardiovascular disease, including IL-6, IL-10 and CXCL11. TILRR knockout caused a 60 % decrease in expression of IκBα and in inhibitor degradation during IL-1 stimulation. In-vitro analysis demonstrated that deletion of ANK 2 rendered the inhibitor unable to control amplified activation of the pathway, while deletions of ANK regions 3 - 5 had no significantly impact. 3-D modelling demonstrated that the ANK 2 deletion had no effect on the overall confirmation of the protein. Alanine scanning mutagenesis of conserved residues within the ANK 2 region, identified enhancers and blockers of signal amplification, which are being assessed for effects on regulation of the IκBα/NF-κB complex and control of amplified inflammatory responses. Conclusion: This study, identifies the ANK 2 region of IκBα as a key regulatory part of the protein, and suggests that it may be specifically relevant in controlling amplified responses of NF-κB.

Poster: 113 Miss Priyanka Anujan Priyanka Anujan, Dr Khondoker Akram, Dr Lynne Bingle, Dr Colin D Bingle PhD Student - 1st Year Infection & Immunity Contact: [email protected]

Towards developing in vitro models for primary ciliary dyskinesia (PCD)

Background: Multiciliated airway epithelial cells play a major role in clearing the lung of inhaled pollutants and pathogens. Abnormalities in ciliogenesis leads to primary ciliary dyskinesia (PCD) and underpin multiple respiratory disorders including, COPD, asthma and CF. The transcription cascades underlying these processes are not fully characterised, however several systematic approaches have identified numerous novel markers of this process.

Objectives: To characterise the functional role of these cilia markers that would aid us to generate primary ciliary dyskinesia phenotype in vitro. In addition, through the help of in vitro models, we aim to study the interaction of cilia with pathogens that will help us to gain better understanding of pathophysiology of respiratory infections.

Methodology: Mouse tracheal epithelial cells (mTEC) and nasal epithelial cells (mNEC) were isolated from C57BL/6 mice cultured at an air liquid interface (ALI) to induce mucociliary differentiation. Differentiation was assessed by mRNA analysis of markers of airway epithelial basal, ciliated and secretory cells as well as by immunocytochemistry and western blot. In addition, mTEC and mNEC cells extracted from Dnah11iv/H (iv) mice with immotile cilia due to a missense mutation on dynein heavy chain gene DNAH11.

Results: mNEC and mTEC cells grown on ALI were successfully shown to have differentiated in to epithelial cells that mimic original nasal and tracheal epithelium respectively. Cultures were also established from iv mice. Transcriptional analysis of several novel cilia markers revealed an expression pattern that corresponds to distinct stages of ciliogenesis. We will use this model to characterise the functional role of novel cilia markers through which we will seek to understand the pathology of PCD.

102

Poster: 114 Dr Adebowale Adekoya Adekoya AO1,2, Streets A3 , Chico T2 ,Ong ACM1,3 PhD Student - 3rd Year Infection & Immunity Contact: [email protected]

Investigating endothelial dysfunction in patients with ADPKD

Rational: Autosomal dominant polycystic kidney disease (ADPKD) is associated with cardiovascular complications including early onset high blood pressure. Endothelial dysfunction (ED) is known to occur before onset of cardiovascular events but there are limited data on its pathogenesis and relationships in ADPKD.

Hypothesis: Endothelial dysfunction exists in patients with early (Chronic Kidney Disease stage 1 and 2) ADPKD

Objectives: Investigating endothelial dysfunction in patients with ADPKD

Methodology: We studied 60 ADPKD patients, 40 patients with non ADPKD CKD (other CKD) and 34 healthy volunteers (HV). Plasma levels of 8-isoprostane and Asymmetric Dimethyl Arginine (ADMA) were assayed using ELISA method. Statistical analysis was performed using Statistical Package for Social Science (SPSS) version 21

Findings: Plasma level of 8 isoprostane and ADMA were significantly higher in ADPKD patients than in HV (22.4±20.5 vs 13.23±11.2pg/ml, p<0.05 and 0.43±0.37 vs 0.27±0.15 µmol/L, p<0.05) suggesting oxidative stress and endothelial dysfunction respectively. No significant difference in levels between ADPKD and other CKD group. There was an inverse association between ADMA and kidney function (B=-22.27, t=-2.708, p<0.05)).No association was found between 8 isoprostane and kidney function. Serum urate was significantly higher in ADPKD patients (565 ± 108 vs 309.07± 85.51 µmol/L) and it correlated with kidney function (B=-0.133, t=-0.027, p<0.001)

Poster: 115 Mr Jack Hurrell Jack Hurrell, Gill Tozer, Chryso Kanthou PhD Student - 1st Year Oncology Contact: [email protected]

An in-vitro model for studying low dose ionising radiation-induced damage in isolated cardiac endothelial cells.

People are constantly being exposed to ionizing radiation from numerous natural and man-made sources. Epidemiological studies of Japanese atomic bomb survivors and workers occupationally exposed to ionising radiation as well as clinical studies of patients treated with radiotherapy have raised concerns over the cardiovascular risks of radiation. While the risks associated with exposures to moderate (0.5 -5 Gy) or high (>5 Gy) radiation doses are clearly established, the degree of risk below 0.5 Gy is more difficult to assess. Microvascular damage within the myocardium is thought to play a major role in the pathogenesis of radiation- induced heart disease. However, the molecular mechanisms or triggers of radiation-induced damage to the heart microvasculature have not been defined.

The small GTPase Rho and Rho-kinase (ROCK) signalling cascade is emerging as a central mechanism of normal tissue damage by radiation. The aim of this study is to establish whether Rho activation is a molecular trigger of low dose radiation-induced cardiac microvascular endothelial damage/dysfunction.

An in vitro cell model for studying the effects of low dose radiation on cardiac endothelial function was established. Endothelial cells were isolated from enzymatically dissociated hearts of adult mice, using magnetic bead-bound antibodies raised against specific endothelial markers (CD31 and CD105/endoglin) and then cultured in endothelial-specific media. Isolated cells were characterised by Flow Cytometry and western blotting using antibodies to CD31, CD105 and CD144.

Cultured cells displayed an endothelial morphology and grew in characteristic whirls. Westerns blotting showed that antibody-selected cells were positive for CD31, CD105 and CD144, while unselected cells were negative for these markers. Flow cytometry confirmed that culture purity at passage 2 was high: 79% of cells expressed CD31, 90% expressed CD144 and 98% expressed CD105. Ongoing experiments are testing the effects of radiation (0.01- 2 Gy) on endothelial function in in vitro migration assays ±Rho/ROCK inhibitors.

103

Poster: 116 Miss Rebecca Collins Rebecca C. Collins, Steven Reynolds, Gillian M. Tozer, Martyn Paley & Simon Jones PhD Student - 4th Year Cardiovascular Science Contact: [email protected]

Synthesis and hyperpolarisation of [13C]-Combretastatin A1

Rationale: Combretastatin-A1 (CA1) is a tumour vascular disrupting agent, currently in Phase-I clinical trials as its phosphate prodrug CA1P. A technique to investigate the rapid metabolism of CA1P in vivo would help elucidate its precise mechanism of action. MRS is an intrinsically low sensitivity technique due to the small difference in the population of the two energy spin states. Dynamic nuclear polarisation (DNP) is a hyperpolarisation technique that transfers the high polarisation of an electron to a nucleus, typically 13C, by microwave radiation. Signal enhancement of up to 105 has been observed.

Objective: To synthesise a 13C-labelled analogue of CA1P ([13C]-CA1P) for monitoring its metabolism by magnetic resonance spectroscopy (MRS).

Methodology & Results: The hyperpolarisation of CA1P first involves the synthesis of a 13C-labelled analogue. The location of the 13C-label was determined by identifying the carbon atom with the longest T1 relaxation time, as this dictates the length of hyperpolarisation and hence the imaging window. The most suitable location was a carbon atom embedded in the aromatic ring of ring B.

A suitable methodology was developed on unlabelled 12C material. The first step of the synthesis involved the insertion of iodomethane into a linear species. The iodomethane will be subsequently replaced by commercially available [13C]- iodomethane. The linear species was then cyclised and aromatised to form the core aromatic species, which will eventually bear the [13C]-label. This was converted to a benzaldehyde, which was coupled with a phosphonium ylide in a Wittig reaction to form CA1, which was then converted to the prodrug CA1P.

Conclusions: A suitable 13-step method for the synthesis of [13C]-CA1P has been established on 12C material that can now be applied to 13C-material. This methodology will enable monitoring of the rapid metabolism of CA1P in tumour tissue using hyperpolarisation and MRS.

Poster: 117 Miss Rowena Speak R. Speak, I. R. Wilkinson, S. L. Pradhananga, J. Sayers, P. Artymiuk, R. J. Ross BMedSci Student - 4th Year Human Metabolism Contact: [email protected]

A long-acting growth hormone antagonist for the treatment of acromegaly

Rationale & Hypothesis: There is need for a new medical treatment for Acromegaly. Somatostatin analogues control disease in only 55% patients and Pegvisomant controls disease in >95% cases, but is not cost effective and requires high dose daily injections. We have developed fusion technology for making a cost-effective long-acting GH. Using this technology we designed potent antagonists by fusing mutated GH to its binding protein. GH contains 2 binding sites; a G120R mutation at site 2 produces a receptor antagonist with mutations in site 1 enhancing receptor binding to generate a more powerful antagonist. We hypothesised that an additional W104A mutation in the GHBP component of the fusion protein reduces intra and intermolecular associations making the GH moiety freely available, generating a more potent antagonist.

Objectives: To produce a potent long-acting GH antagonist therapy that is able to minimise the cost of manufacturing, has fewer side effects, is easy to administer, and is acceptable to patients.

Methodology: Four antagonists were made: GHA1 with the site 2 mutation; GHA2 with site 2 and site 1 mutations; GHA3 with site 2 and W104A mutations; GHA4 with site 2, site 1 and W104A mutations.

Findings: Proteins were expressed from a CHO cell line and purified using antibody affinity chromatography. Coomassie stained SDS PAGE confirmed purity. In vitro bioactivity was tested by GH-specific luciferase dual reporter assay. Preliminary results revealed IC50s for the constructs of 59.33nM (GHA1); 201.1nM (GHA2); 96.70nM (GHA3); 13.74nM (GHA4). Introducing site 1 mutations to enhance binding (GHA2) decreases bioactivity but the additional W104A mutation in GHA4 increases bioactivity, likely by reducing intra and intermolecular binding, allowing the powerful antagonist with site 1 mutations to be more available and therefore more active.

104

Poster: 118 Miss Justyna Serba J. J. Serba, T. K. Prajsnar, S. J. Foster, S. A. Renshaw PhD Student - 4th Year Infection & Immunity Contact: [email protected]

In vivo imaging of host – pathogen interactions in Staphylococcus aureus infection

Abstract Rationale & Hypothesis: S. aureus has evolved strategies to manipulate host-pathogen interactions to its own ends, and avoid killing by professional phagocytes. It has been suggested that S. aureus can reside and survive inside professional phagocytes – mainly neutrophils and macrophages. Moreover, intracellular environment instead of being disruptive could constitute a beneficial “niche”. Macropinocytosis and autophagy are cellular processes already reported to favour pathogenesis of other bacteria and viruses. Our hypothesis is that S. aureus evade elimination by subverting one or more of internalisation and intracellular processes to escape killing by innate immune system cells.

Methodology: I am using light fluorescence and confocal microscopy to track pathogens inside of the transparent zebrafish embryo. S. aureus strains are stained before injection with pH-sensitive and insensitive fluorescent dyes to assess the stage of endosome maturation by pH decrease. We have developed transgenic zebrafish lines to specifically mark autophagosomes (LC3 fused with GFP and RFP) and macropinosomes (sorting nexin 1 fused with mTurquoise). Image acquisition is performed in vivo at various time points of infection. Imaging data is also processed in quantitative analysis.

Findings: Nomarski DIC combined with high power fluorescence microscopy enabled us to capture pathogen uptake, phagocyte-to-phagocyte interactions and bacterial acidification in vivo. Bacterial cells co-localise with LC3 protein in neutrophils. Results of whole body counts of bacteria internalised by phagocytes showed more intake into macrophages than into neutrophils. Quantification of bacteria labelled with pH sensitive bacterial cell wall dyes indicated different acidic conditions in bacteria- containing endosomes and lower acidification rates in neutrophils.

Conclusions: Bacterial presence in LC3 tagged vesicles suggest that autophagy is involved in S. aureus intracellular existence. Further analysis will be performed to reveal the numbers of bacteria colocalised with intracellular pathway markers in phagocytes. Complex quantification experiments combined with real time in vivo imaging will potentially increase our understanding of host – pathogen interactions at the different stages of infection.

Poster: 119 Mr Jonathan Atkinson Jonathan Atkinson, Jules Westbrook, Richard Beniston, Carl Smyth, Robert E Coleman, Janet E Brown Work Placement Student Oncology Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

105

Poster: 120 Mr Ho-Fung Chan Ho-Fung Chan, Juan Parra-Robles, Chris Brightling, Wim Vos, Jim Wild PhD Student - 1st Year Cardiovascular Science Contact: [email protected]

Lobar comparison of CT densitometry and 3He diffusion MRI models of lung microstructure in asthmatics

Introduction: CT can provide tissue density information from the lungs, whilst hyperpolarised 3He diffusion MRI (dMRI) enables estimation of mean alveolar dimensions. In order to reconcile the micro-structural information available from these two imaging modalities, a quantitative regional comparison is needed.

Objective: To develop a framework to compare measures of lung microstructure in lobar regions using CT densitometry and 3He dMRI in a cohort of asthma patients.

Methods: Inspiratory and expiratory CT and 3He dMRI images were obtained in a study of 23 asthmatics. Lung lobar regions were automatically segmented from CT images and manually segmented from 3He dMRI. Coronal CT slices were reconstructed and matched to the same slices as the MRI, and mean tissue density (Figure 1a) was calculated for each lobe. Estimates of mean alveolar dimension (Figure 1b) were derived in the same lobar regions using quantitative models from 3He dMRI [Parra-Robles et al., Proc. ISMRM 2014, 3529].

Results: For both inspiratory and expiratory (see Figure 2) CT lung volumes, statistically significant linear correlations (p- values <0.05) between mean CT density (HU) and measures of mean alveolar dimension (μm) from 3He dMRI were observed for each of the four lobar regions. The linear correlations for expiratory CT lobar regions were all statistically equal or stronger than those observed in inspiratory CT.

Conclusion: CT and 3He dMRI can be analysed at a lobar level to compare regional measures of tissue density and acinar microstructure, respectively. The correlations suggest that in asthmatic lungs alveolar size and mean tissue density are related. Differences in statistical correlation strength between CT lung volumes suggest that a smaller (expiratory) lung volume provides stronger correlations between lung alveolar size and mean tissue density.

Poster: 121 Dr Miguel Debono Miguel Debono, Robert F Harrison, Brian G Keevil, Martin Whitaker, Dena Digweed, Wiebke Arlt, Richard J Ross Clinical Lecturer Human Metabolism Contact: [email protected]

Modelling the salivary cortisone to serum cortisol inter-relationship to predict serum cortisol under physiological conditions and after hydrocortisone

Rationale & Hypothesis: The evaluation of both the normal circadian rhythm and adequacy of hydrocortisone replacement using multiple serum cortisol readings is an expensive, laborious process. The use of salivary tests is non-invasive and cheaper. We hypothesised that salivary cortisone predicts circulating cortisol levels and can be used as an alternative marker of serum cortisol in a physiological setting or in patients on hydrocortisone replacement.

Objectives: To demonstrate that salivary cortisone is superior to salivary cortisol as a surrogate marker of serum total cortisol levels and to develop a model to study the inter-relationship between serum cortisol and salivary cortisone.

Methodology: This was an observational study in 14 healthy male volunteers who had hourly serum cortisol measurements over 24 hours in addition to salivary cortisol/cortisone measurements from 15:00h-22:00h and from 07:00h-15:00h measured by liquid chromatography/tandem mass spectrometry. Relationships between serum and salivary cortisol/cortisone were assessed by individual correlation analysis and linear mixed effects modelling. A similar analysis was carried out after 20mg of oral and intravenous hydrocortisone.

Findings: Correlation analysis (ρ), for the relationship between endogenous log-transformed salivary cortisone or salivary cortisol with serum cortisol rhythm, ranged from 0.96-0.99;p<0.001 and from 0.84-0.99;p<0.001, respectively. Salivary cortisone was superior to salivary cortisol (range R2:0.93-0.99 vs 0.71-0.97) in predicting circulating, serum cortisol. By fitting a mixed effects model to predict serum cortisol levels from salivary cortisone [predicted serum cortisol {nmol/l}=(13.57 + individual- specific random effects) x salivary cortisone {nmol/l}] correlation ρ and R2 between measured and predicted serum cortisol were 0.91 and 0.82 under physiological conditions, 0.89 and 0.66 for 20mg oral hydrocortisone and 0.93 and 0.72 for 20mg intravenous hydrocortisone, respectively.

Conclusion: Salivary cortisone levels are strongly related to serum cortisol and repetitive measurements of salivary cortisone may be used to predict the serum cortisol rhythm and cortisol levels after hydrocortisone.

106

Poster: 122 Miss Renata Caikauskaite Renata Caikauskaite, Dr Lynne Bingle, Dr Colin Bingle PhD Student - 1st Year Infection & Immunity Contact: [email protected]

Host defence functions of BPIFA1/SPLUNC1

Rationale: BPIFA1/ SPLUNC1 is an abundantly secreted protein released by airway epithelial cells in the upper and lower respiratory tract. SPLUNC1 functions in airway defence as an antimicrobial (or bacteriostatic) agent against multiple respiratory pathogens including Pseudomonas aeruginosa and Mycoplasma pneumonia. SPLUNC1 also plays a role in the mucociliary clearance and exhibits immunomodulatory and chemotactic functions. Levels of SPLUNC1 expression fluctuates in the airways of COPD, CF and IPF patients. However, the actual role of SPLUNC1 in these respiratory diseases remains unresolved. Although existing data supports the role of SPLUNC1 in the airway defence the exact mechanisms remain undetermined.

Objectives: In this study we aim to identify functional domains by using GFP tagged proteins to visualise interactions with respiratory pathogens.

Methodology: NCI-H292 cells were transfected with human full length SPLUNC1 construct cloned in-frame into the pEGFPN1 vector and G418 was used to generate a stable cell lines. PCR was used to detect the expression of the gene and Western Blotting and IF microscopy was used to identify the protein.

Findings: We are currently generating a series of different sizes of SPLUNC1 constructs in pEGFPN1 and will establish stable cell lines with these constructs. After stable cell lines are generated, IF microscopy will be used to identify the localisation of SPLUNC1 constructs within the cells. Transformed cells will be stimulated to release SPLUNC1 protein and medium will be collected for analysis of SPLUNC1 function using a series of bacterial pull down assays. This will enable us to identify regions of the protein responsible for binding to respiratory pathogens.

Poster: 123 Dr Jules Westbrook Jules A. Westbrook1, David A. Cairns2, Caroline A. Evans3, Penny Ottewell1, David N. Perkins4, Carl Smythe5, Phillip C. Wright3, Valerie Speirs6, Andrew M. Hanby6, Ingunn Holen1, Robert E. Coleman1, Janet E. Brown1 Postdoctoral Researcher Oncology Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

107

Poster: 124 Dr Marni Greig M. Greig, I.D. Wilkinson, P. Shillo, D. Selvarajah, R. Gandhi, S. Tesfaye; PhD Student - 3rd Year Cardiovascular Science Contact: [email protected]

Brain perfusion in painful diabetic neuropathy

Background and aims: Diabetic neuropathy (DN) has been linked to abnormalities in structure and function of the brain on Magnetic Resonance (MR). This study sought to explore intracranial vascular perfusion in patients with type-1 diabetes, with and without painful DN (PDN) and those without diabetes (HV).

Materials and methods: Groups: PDN=7; T1DM without neuropathy (D-NN)=8 & HV=7. MR images were obtained at 3T (Ingenia, Philips Healthcare) using a Dynamic Susceptibility Contrast, T2*-weighted technique (TR/TE=1250/35; 72 dynamics) to assess the passage of a bolus of intravenous gadolinium-chelate passing through the cerebral vascular bed. Contrast perfusion was determined (Nordic Neurolab ICE, Bergen, Norway) to yield the rBF (cerebral blood flow), rBV (volume) and mTT (mean transit time) in the SC (sensory cortex), Thalami (Thal) and parieto-occipital white matter (POWM).

Results: Statistically significant differences were observed (ANOVA) between PDN and D-NN group mean mTT as well as PDN and HV group mean mTT [ eg, SC-mTT: PDN mean=2.87,SD=0.35; D-NN mean=3.46,SD=0.51; HV mean=3.72,SD 0.73,p=0.004; Thal-mTT:PDPN mean=2.89, SD=0.48; D-NN mean=3.41,SD=0.55; HV mean=3.82, SD=0.63, p=0.004]. BV and BF did not show significant mean differences between the 3 groups.

Conclusion: These early results (from a large on-going study) suggest cerebrovascular perfusion is altered within the brain parenchyma of subjects with PDN when compared to both diabetic and non-diabetic control groups. Microvascular dysfunction may have important implications in our understanding of the brain’s involvement in DN, the search for functional neuropathic markers and indicators of future therapeutic intervention. Further work is warranted to further characterise this cerebrovascular involvement.

Poster: 125 Miss Hannah Roberts Hannah Roberts, Rachel Doherty, Louise Nugent, Danyang Li and Gaynor Miller Masters Student Human Metabolism Contact: [email protected]

Identifying therapeutic targets for muscular dystrophy following macrophage depletion

Rationale & Hypothesis: Macrophages are versatile cells of the myeloid lineage which play an essential role in the immune response. Proinflammatory macrophages are also prevalent in diseases such as Duchenne muscular dystrophy (DMD) where they are thought to exacerbate muscle damage. We hypothesise that targeting specific macrophages (i.e. CD68+) in DMD patients may reduce muscle damage whilst avoiding some of the negative side effects observed with the use of corticosteroids.

Objectives: Previously, the MacLow mouse model was generated which allows the inducible depletion of CD68 positive (CD68+) macrophages. In the present study, we crossed the mdx model of muscular dystrophy (MD) with the MacLow model enabling induction of macrophage depletion. The aims of this study were to determine the effects of macrophage depletion on muscle pathology and gene expression in the MacLow-MD model.

Methodology: In MacLow mice, the human CD68 promoter drives doxycline-controlled transcriptional activation of the diphtheria toxin A chain, resulting in selective and inducible CD68 macrophage depletion. MacLow mice were crossed with the mdx mouse model to generate the mdx-MacLow model. 4 week old animals were fed on either doxycycline containing chow or control feed for two weeks. Sections of quadriceps muscle were stained to determine the effect on macrophage infiltration, central nucleation and necrosis. Muscle RNA was also isolated to interrogate the expression of various immune and muscle cell specific genes using RT PCR.

Findings: To date, two weeks of macrophage depletion resulted in a 50% reduction in the number of CD68+ cells in the tibialis anterior of six week old MacLow-MD mice (P<0.05). Furthermore, the number of regenerating i.e. centrally nucleated muscle fibres, in macrophage deficient MacLow-MD animals was significantly reduced by ~33% (P value <0.05). The effects of macrophage depletion on muscle necrosis and gene expression will also be described.

108

Poster: 126 Dr Katherine Henry Katherine M Henry, Anne L Robertson, Joseph Burgon, Caroline Tabor, Stuart Farrow, Bill Zuercher and Stephen A Renshaw Post-Docorate Infection & Immunity Contact: [email protected]

Integrating human and zebrafish studies to find new drug targets in the neutrophil kinome

Neutrophils are the first cells recruited to sites of inflammation where they defend against invading pathogens. However, in some cases of chronic inflammation, neutrophils contribute to tissue damage. Down-regulating neutrophil pro-inflammatory functions and enhancing neutrophil clearance may be beneficial in these circumstances. One potential source of drug target to exploit in this way is kinases. There are 518 protein kinases in humans, responsible for the phosphorylation of cellular proteins. They are involved in many cellular processes and as such they are promising drug targets. It became apparent that to identify kinase targets for potential pro-resolution therapies, an understanding of the human neutrophil kinome was required. Probing of a publically-available RNAseq database identified 284 (of 518) annotated protein kinases in human neutrophils. A subset was regulated by GM-CSF. Serum and glucocorticoid regulated-kinase 1 (SGK1) is one such kinase. Inhibition of this kinase resulted in increased neutrophil apoptosis in primary human neutrophils that was able to override the survival signal, GM- CSF. These observations were confirmed in an in vivo setting using a zebrafish model of inflammation. To assess the functional role of other kinases in neutrophilic inflammation, the Publically-available Kinase Inhibitor Set 1 (PKIS1) was screened using our zebrafish model of inflammation. Kinase profiling of hit compounds revealed potential new drug targets in the neutrophil kinome.

Poster: 127 Dr Peter Novodvorsky Peter Novodvorsky, Freek van Eeden, Tim Chico MRC Clinical Fellow Cardiovascular Science Contact: [email protected]

Generation and characterisation of a stable klf2a mutant zebrafish

Introduction & Objectives: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces the physical force of blood flow into molecular signals responsible for a wide range of biological responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies have reported a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. We therefore generated a stable klf2a mutant zebrafish line and characterised the effect on cardiovascular development.

Methods & Results: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a stable klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants demonstrated no discernible vascular patterning defects, survive to adulthood and are fertile. klf2ash317 mutants do not reproduce previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Furthermore, the klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that we have shown to be dependent on blood flow. To attempt to explain the lack of phenotype in the klf2ash317 mutants we examined expression of other klf family members in both wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but no differences in expression of any gene between wildtypes and klf2ash317 mutants.

Conclusions: The klf2ash317 mutation produces a truncated Klf2a protein but this is not associated with a detectable cardiovascular phenotype. The klf2ash317 mutant therefore fails to reproduce previously described morphant phenotypes. The reasons for this discrepancy remain unclear but our findings suggest klf2a may be dispensable for zebrafish cardiovascular and haematopoietic development.

109

Poster: 128 Miss Maria Digby Miss MG Digby1, Prof P Dimitri1,2, Dr P Arundel2, Prof N Bishop1,2, Dr MA Paggiosi1, Dr AC Offiah1,2. PhD Student - 4th Year Human Metabolism Contact: [email protected]

High resolution peripheral quantitative computed tomography for the assessment of bone microstructure in mild and severe osteogenesis imperfecta

Abstract Children with osteogenesis imperfecta (OI) currently endure invasive biopsies to assess bone integrity. High- resolution peripheral quantitative computed tomography (HRpQCT) with a resolution of 82μm offers an in-vivo alternative and a means to monitor changes in bone microarchitecture due to the disease and its treatment.

Objectives: To compare the skeletal microarchitecture of the non-dominant ultradistal radius of OI patients with age and gender matched controls. Presently, no HRpQCT studies include children with severe OI.

Methodology: We have scanned six patients with a variety of bisphosphonate treatment durations and OI types (type I, III and V).

Findings:

Type of Sex Duration of Age OI vs Control Inhomogeneity of OI Bisphosphonate (years) Trabecular Bone Treatment Trabecular Cortical Compared to Control BMD BMD (%) (mg/cm3) (mg/cm3)

Mild Group I Male 0 12 153.1 vs 186.0 514.2 vs +10.45 748.7 I Male 1 11 110.5 vs 155.4 688.3 vs -8.1 703.4 I Male 8 15 121.3 vs 143.5 750.0 vs +59.5 719.6 I Female 6 9 227.6 vs 111.0 742.6 vs -4.5 557.5 Severe Group III Female 10 10 252.4 vs 170.3 730.2 vs +170.8 610.9 V Female 10 10 170.4 vs 167.5 587.6 vs +491.7 625.4

Table 1. Showing BMD results for cortical and trabecular bone using HRpQCT in children with OI compared to age and sex-matched controls

Discussion: Trabecular inhomogeneity is linked to increased fracture risk. The increased inhomogeneity of trabecular bone in some OI patients compared to controls suggests significant disruption in normal trabecular organisation. The variation in the cortical and trabecular parameters may be as a response to the bisphosphonate therapy.

This initial HRpQCT analysis demonstrates clear variability in skeletal microstructural organisation in different OI types along with bisphosphonate therapy effects. Further work including microfinite element analysis is merited to assess the effects of disease and treatment on bone strength.

110

Poster: 129 Mr MD Mohasin Mohasin M, Fisk E, Marriott HM, Dockrell DH PhD Student - 1st Year Infection & Immunity Contact: [email protected]

Pneumococcal infection exacerbates chronic obstructive pulmonary disease (COPD) through changing metabolic status and phenotype of macrophages

Rationale & Hypothesis: Infectious stimuli have been shown to effect the metabolic signature of macrophages. Lipopolysaccharide (LPS) stimulates macrophages metabolic reprogramming from oxidative phosphorylation to glycolysis. Moreover, IFNγ/LPS polarises macrophages into pro-inflammatory macrophages and induces glutamine uptake which converted into succinate, which upregulates hypoxia inducible factor-1α, which induces glycolytic flux. This increased glycolytic rate with enhanced cellular glucose uptake and conversion of pyruvate to lactate is associated with down-regulation of the rate of oxidative phosphorylation, resulting in up-regulation of ROS. I hypothesize that these mechanisms are impaired in macrophages from patients with COPD which contributes to impaired macrophage host defence function. Objectives: My initial aims are to optimise metabolic assay conditions for use in adherent tissue macrophages and measure the metabolic reprogramming of macrophages after pneumococcal infection using extracellular flux analysis in cells with a molecular signature that phenocopies the defect in COPD macrophages. Methodology: Bone marrow from C57Bl/6 mice were cultured in DMEM medium with 10% HIFCS and 10% L929 conditional media for 14 days for differentiation into bone marrow derived macrophages (BMDM). Cells were reseeded into 24 well XF cell culture microplates at different densities for measurement of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a XP Extracellular Flux analyser. Cell number was normalised with CyQuant quantification assay. Cells were exposed to Streptococcus pneumoniae, heat inactivated at 700C for 40 minutes, MOI=10 and changes in ECAR and OCR measured. Findings: We found 2x105-3x105 BMDM/well was the optimal seeding density for metabolic measurements. S. pneumoniae challenge resulted in elevation of ECAR (6.5 mPH/min) compared to uninfected (4.0 mPH/min) with no effect on OCR, however optimisation of this is ongoing. Once conditions have been optimised we will study changes in ECAR and OCR with S. pneumoniae challenge in a murine models of COPD using macrophage specific transgenic cells.

Poster: 130 Mr Henry Greenslade Henry Greenslade, Robin Rumney, Iain Huggins, Penny Ottewell, Alison Gartland BMedSci Student - 1st Year Human Metabolism Contact: [email protected]

An immunohistochemical study investigating the interaction of P2X7 and LOX in mammary tumours and the pre-metastatic niche in bone

Rationale/Hypothesis Lysyl oxidase (LOX) has been shown to be over-expressed in various human cancers. A previous study by our collaborators has shown that LOX co-localises in mouse lung with fibronectin and VEGFR1+ cells prior to the arrival of metastatic cells, creating a “pre-metastatic niche” which promotes tumour cell recruitment. Recent work by our lab has shown that LOX increases osteolytic lesion formation in the bone prior to the arrival of metastatic cancer cells. We hypothesise that LOX effects on bone are mediated through the P2X7 receptor (P2X7R), a purinergic receptor expressed on both osteoblasts and osteoclasts. Objective The specific objective of this study was to analyse potential histological differences in primary tumours and bone between P2X7R KO and WT balb/c mice, and assess whether this is LOX dependent. Method Nine week old BALB/c and P2X7RKO mice received injections of either 4T1 or 4T1shLOX tumour cells into the mammary fat pad. After 3 weeks the mice were euthanised and primary tumours were dissected, weighed, and embedded in paraffin, with tibiae decalcified in EDTA and sent for sectioning. Tumour angiogenesis and proliferation were assessed via immunohistochemistry, with bone histomorphometry assessed by TRAP staining. LOX expression was quantified in tumour and bones utilising immunohistochemistry and the Tissue FAXS system. Results Tumour weight was significantly lower in P2X7RKO 4T1 mice compared to WT Balb/c 4T1 mice (P = 0.013). Bone histomorphometry revealed higher levels of osteoclasts in tumour bearing mice compared to age matched controls (P = 0.03). In bone, intensity of LOX staining in marrow cells was markedly reduced in the P2X7RKO mice compared to WT (P = <0.0001), indicating that a P2X7R dependent mechanism may be responsible for the effects of LOX in bone.

111

Poster: 131 Miss Apoorva Mulay Apoorva Mulay1, Michael Cheeseman4, Steve Brown 2, Derek Hood 2, Khondoker Akram1 Lynne Bingle3, Colin Bingle1 PhD Student - 3rd Year Infection & Immunity Contact: [email protected]

Role of BPIFA1 in Otitis media

SNPs in BPIFA1, the predominant member of BPI fold containing family of putative innate defence proteins, have been associated with Otitis media (OM). We have previously shown that BPIFA1 is expressed in the middle ear (ME) and nasal passages (NP) of Wt mice and its expression decreases with OM development in the Junbo (Jbo+/-) model of chronic OM. We aim to characterize a role for BPIFA1 in the ME and NP using in vivo and in vitro approaches. Auditory brainstem response and histological screening revealed that BPIFA1-/- mice are phenotypically similar to Wt mice and do not develop OM spontaneously. Intranasal challenge of BPIFA1-/- mice with the human otopathogen, NTHi does not show increased ascending bacterial infection compared to Wt mice. However, in the BPIFA1-/-Jbo+/- compound mutants, OM severity and ME mucosal thickness is significantly higher than Jbo+/- mice. Our novel in vitro otopathogenic infection model demonstrates that nasal and ME cells differentiate within 14 days at Air Liquid Interface. Transcriptional and proteomic analysis suggests that they express epithelial markers and abundant levels of BPIFA1. NTHi readily infects the system, in a dose and time dependent manner. Data from the current experiments suggests that BPIFA1 loss increases susceptibility of the cells to NTHi infection. There is also a decrease in BPIFA1 expression in Wt cells with progression of infection. We are currently studying the differences in cytokine responses in Wt and BPIFA1-/- cells post infection. Thus, we conclude that BPIFA1 plays an immunomodulatory role in the ME and NP and alteration in its levels is associated with OM development and an exaggerated phenotype in existing models of OM.

Poster: 132 Miss Emily Fisk Emily Fisk, MD Mohasin, Dr. Helen Marriott, Prof. David Dockrell PhD Student - 1st Year Infection & Immunity Contact: [email protected]

Bone marrow-derived macrophage responses following detection of Streptococcus pneumoniae infection via pattern recognition receptors

112

Poster: 133 Dr Emma Johnson Emma Johnson, Nelly Wagner, Alexander Williams, Tomasz Prajsnar, Stephen Renshaw, Simon Foster Clinical Research Fellow Infection & Immunity Contact: [email protected]

Co-operation during Staphylococcus aureus pathogenesis

Rationale Staphylococcus aureus is an invasive human pathogen associated with significant mortality. Host-pathogen dynamics during infection are poorly understood but recent work demonstrates that with high dose inocula, an immunological bottleneck allows clonal expansion of only a few bacteria, which go on to cause host damage.

Objectives We aimed to further assess such clonal events by using mixed inocula in vivo.

Methodology We tested mixed strain inocula including varying ratios of virulent and either avirulent bacteria or bacterial cell components, in the zebrafish embryo model of systemic infection.

Results The virulent strain S. aureus, SH1000 at high dose (1500 CFU) causes 50% embryo mortality. At low infective dose (150 CFU), S. aureus SH1000 is unable to cause significant mortality. Similarly, a high dose of either nonpathogenic bacterium such as Micrococcus luteus, attenuated S. aureus mutants or separated cell wall component, peptidoglycan, was unable to kill. However, when each of these was co-injected with the low infective dose of virulent S. aureus SH1000, significant mortality was observed.

ConclusionAvirulent bacteria do not proliferate within the host, yet these so-called ‘bystanders’ permit low- dose S. aureus SH1000 to undergo clonal expansion and kill the host, far in excess of that seen when low dose is injected alone. Moreover, the essential cell wall component, peptidoglycan, was also able to augment killing of zebrafish embryos. Further analysis of this synergistic interaction may help us unravel mechanisms by which co- operation between bacteria affects pathogenesis. This has important clinical significance as a polymicrobial presence is likely to occur during human infection.

113

Poster: 134 Dr Martin Bewley Dr. Martin Bewley1, Dr. Richard Budd1, Prof. Dave Singh3, Prof. Peter Barnes2 , Prof. Louise Donnelly2, Prof. David Dockrell1, and Prof. Moira Whyte1 PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

COPD alveolar macrophages show reduced apoptosis and bacterial killing linked to a failure to induce mitochondrial reactive oxygen species upon Spn challenge

Rationale & Hypothesis: Chronic obstructive pulmonary disease (COPD) patients are at increased risk from bacterial infections, which cause acute exacerbations and pneumonia. Apoptosis-associated killing (AAK) aids bacterial killing once conventional microbicidal killing is exhausted. We hypothesized that defects in macrophage- apoptosis-associated killing (AAK) result in a failure to clear bacteria in COPD.

Objectives: To assess AAK in COPD AM and examine potential mechanisms.

Methodology: Alveolar macrophages (AMs) were obtained from broncho-alveolar lavage (BAL) from non- frequent or frequent exacerbating (>/=2 episodes/yr) COPD patients or healthy donors and were challenged with opsonized serotype 14 Streptococcus pneumoniae (Spn) at an MOI of 10. Intracellular bacterial colony counts were measured 16h after challenge by gentamicin protection assay. Apoptosis was measured by examining nuclear fragmentation by fluorescence microscopy. Mitochondrial reactive oxygen species (mROS) were measured with mitoSOX and inhibited using the SOD2 mimetic MitoTEMPO. Western blotting was performed to analyze expression of the anti-apoptotic Bcl-2 family member Mcl-1 and superoxide dismutase 2 (SOD2).

Findings: COPD AM displayed elevated levels of Mcl-1 compared to healthy AM both before and after challenge with Spn. COPD AM also displayed reduced levels of apoptosis in response to Spn challenge when compared to healthy AM even after normalization of bacterial internalization. Reduction in apoptosis resulted in increased bacterial persistence in COPD AM. In addition, healthy AM demonstrated a significant induction of mROS in response to Spn challenge, and MitoTEMPO reduced AAK. SOD2 expression was elevated in resting COPD AM compared to healthy AM and the induction in SOD2 exceeded the induction in mROS for COPD AM but not healthy AM after challenge with Spn. These data suggest that, in COPD, defects in AM apoptosis and failure to induce mROS relative to SOD2 in these cells cause defective intracellular bacterial killing and contribute to bacterial persistence.

114

Poster: 135 Mr Thomas Kennelly Thomas Kennelly, Yi Cao, Mark Geoghegan & Eva Qwarnstrom PhD Student - 2nd Year Cardiovascular Science Contact: [email protected]

Abstract unavailable – presenter has not consented for the abstract to be shared

Poster: 136 Mr Paolo Prosseda Philipp Paolo Prosseda, Prof Albert Ong, Dr Andrew Streets PhD Student - 2nd Year Infection & Immunity Contact: [email protected]

The role of primary cilium stability in the pathogenesis of autosomal polycystic kidney diesease

Introduction: Autosomal Dominant Polycystic Kidney Disease belongs to a class of inherited systemic disorders which primary affects the normal function of the kidneys. Mutations PKD1 and PKD2, have been identified and held responsible in disease onset and progression. Most of the key genes are found to encode proteins localized in the primary cilium, an antenna like organelle found on most mammalian cells, thus suggesting a critical role in the development and maintenance of the kidney cells. Research into the various mechanisms governing cyst formation, in correlation to recent accepted critical involvement of the primary cilium in this context is urgently needed for the understanding and possible treatment of this disease.

Rationale & Hypothesis: Primary cilium dynamics (assembly and disassembly) may be impaired in PKD1/PKD2 deficient cell lines resulting in a disturbed primary cilium function in kidney epithelial cells, and this is what we primary are investigating in our study. This may result in aberrant signalling pathways from the cilium which lead to excessive proliferation and ultimately the formation of Cysts. Different proteins possibly linked to cilium stability are tested for their ability to interact with PC1/PC2.

Results: We showed that primary cilia assembly and disassembly is tightly coupled with the cell cycle in healthy cell models and we show this association also in different PKD models. We show that length regulation of the cilium is regulated by intracellular modulation of cAMP and Ca2+ levels independently of serum stimulation. We found that the PC1/PC2 complex act as a scaffold protein involved in binding to different proteins which might play an important role in primary cilium regulation.

Conclusion: Identification of the mechanism and signalling pathways which might impair the normal regulation of the primary cilium in PKD cells might lead in the future may lead to treatment aimed at rescuing ciliary function in ADPKD, thus formation of cysts.

115

Acknowledgments

The Medical School would like to thank the following companies for their generous support of the Research Meeting

116

NOTES

117

NOTES

118