Medication adherence, compliance, satisfaction and health related quality of life after myocardial infarction: EMMACE 3 and 4 trials
Supervisor(s): Chris Gale, Theresa Munyombwe, Bernadette Dondo, Teumzghi Mebrahtu
Key words: Medication adherence, Health related quality of life, Myocardial Infarction
Summary: Previous research has shown that patients with Myocardial Infarction (MI) experience decreased health related quality of life (HRQoL) compared to the general population (Lewis et al. 2014; Schweikert et al. 2008). The American Heart Association recommends the inclusion of patient’s HRQoL as an important measure of cardiovascular health among patients with cardiovascular disease (Rumsfeld et al. 2013). Medication adherence is necessary for effective therapy and there is evidence to suggest insufficient observed and self-reported adherence to medications (Krack et al. 2018). To improve adherence, its determinants need to be known. There are a few studies that have looked at the associations between medication adherence, patient satisfaction with medicines, and patient health related quality of life using longitudinal data. Therefore the aim of this project is to investigate associations of HRQoL, patient satisfaction with medicines, and medication adherence among AMI patients. We hypothesize that decreased HRQoL is associated with decreased medication adherence.
Data sources:The project will use EMMACE 3 and 4 data. EMMACE-3 is a national, multi-centre, prospective, longitudinal cohort of patients hospitalised with Acute Coronary Syndrome (ACS) in England. 5556 patients were recruited between 1st November, 2011 and 17th September, 2013. Patient reported data at admission, 1 month, 6 months and 1 year follow-up comprised: Morisky Medication Adherence, Single Question Medicine Adherence, and Beliefs about Medicine, The Adherence Estimator and Problems with Taking medications, Satisfaction with Information about Medicines Scale, and EQ-5D. All patients were consented to enter the study and for their data to be linked to electronic health record data.
EMMACE-4 is also a national, multi-centre, prospective, longitudinal cohort of patients hospitalised with ACS in England. 9343 patients were recruited between October 2013 to 24th June 2015. Patient reported outcome measures data at admission, 1 month, 6 months and 1 year follow-up comprised of Satisfaction with Information about Medicines Scale, List of Medications, Care Quality Commission Pickering Inpatient PROM, Brief Illness Perception, and EQ-5D.
KRACK, G., R. HOLLE, I. KIRCHBERGER, B. KUCH, U. AMANN and H. SEIDL. 2018. Determinants of adherence and effects on health-related quality of life after myocardial infarction: a prospective cohort study. BMC geriatrics, 18(1), p136.
LEWIS, E. F., Y. LI, M. A. PFEFFER, S. D. SOLOMON, K. P. WEINFURT, E. J. VELAZQUEZ, R. M. CALIFF, J.-L. ROULEAU, L. KOBER and H. D. WHITE. 2014. Impact of cardiovascular events on change in quality of life and utilities in patients after myocardial infarction: a VALIANT study (valsartan in acute myocardial infarction). JACC: Heart Failure, 2(2), pp.159-165.
RUMSFELD, J. S., K. P. ALEXANDER, D. C. GOFF JR, M. M. GRAHAM, P. M. HO, F. A. MASOUDI, D. K. MOSER, V. L. ROGER, M. S. SLAUGHTER and K. G. SMOLDEREN. 2013. Cardiovascular health: the importance of measuring patient-reported health status: a scientific statement from the American Heart Association. Circulation, 127(22), pp.2233-2249.
SCHWEIKERT, B., M. HUNGER, C. MEISINGER, H.-H. KÖNIG, O. GAPP and R. HOLLE. 2008. Quality of life several years after myocardial infarction: comparing the MONICA/KORA registry to the general population. European heart journal, 30(4), pp.436-443.
4-Year International PhD Academy
Cardiovascular & Metabolic Disease
The impact of dietary factors on tinnitus severity
Supervisor(s): Dr Sarah Isherwood, Dr Laura Hardie
Key words: Tinnitus, diet
Summary: Tinnitus is a common medical condition with a prevalence rate of approximately 10 – 15% of the adult population. It can be defined as the perception of sound in the absence of external acoustic stimulus. Whereas many tinnitus sufferers are able to cope well with the condition for others it can lead to significant distress and a reduced quality of life. The cause of tinnitus is often idiopathic and there is no agreed singular cure for the condition. Available treatments and management options for tinnitus commonly include hearing aids, counselling, sound enrichment, the promotion of relaxation and regular exercise, and a consideration of dietary factors. The identification of modifiable determinants of tinnitus and tinnitus severity would provide a significant advance in the treatment and management options for this condition.
This PhD will explore the association between dietary factors and tinnitus. In particular whether certain dietary factors are able to exacerbate or alleviate the experience of tinnitus. Evidence is beginning to show a possible link between diet and hearing health however fewer studies have specifically explored the link between diet and tinnitus. The few studies exploring this area have revealed that there may be a small but reliable benefit to modifying diet to circumvent persistent tinnitus symptoms. However whether a specialised type of diet will produce the most significant decrease in tinnitus symptoms, such as the Mediterranean diet or the dietary approach to stop hypertension (DASH), is still unknown. Research has found that following a diabetic diet results in decreased tinnitus severity in patients with hyperinsulinemia. This PhD will explore potentially modifiable dietary factors for tinnitus via systematic review of the literature and empirical research.
Basut, O., Ozdilek, T., Coskun, H., Erisen, L., Tezel, I., Onart, S. & Hizalan, I. (2003). The incidence of hyperinsulinemia in patients with tinnitus and the effect of a diabetic diet on tinnitus. Kulak burun bogaz ihtisas dergisi: KBB= Journal of ear, nose, and throat, 10, 183-187.
Baguley, D., McFerran, D. & Hall, D. 2013. Tinnitus. The Lancet, 382, 1600-1607
Heeren, A., Maurage, P., Perrot, H., De Volder, A., Renier, L., Araneda, R., Lacroix, E., Decat, M., Deggouj, N. & Philippot, P. 2014. Tinnitus specifically alters the top-down executive control sub-component of attention: evidence from the Attention Network Task. Behav Brain Res, 269, 147-54.
Henry, J. A., Dennis, K. C. & Schechter, M. A. 2005. General review of tinnitus: prevalence, mechanisms, effects, and management. Journal of speech, language, and hearing research, 48, 1204-1235.
McCormack, A., Edmondson-Jones, M., Mellor, D., Dawes, P., Munro, K. J., Moore, D. R. & Fortnum, H. 2014. Association of dietary factors with presence and severity of tinnitus in a middle-aged UK population. PLoS ONE, 9 (12).
Spanovich, C., Bishop, C., Johnson, M. F., Elkins, A., Su, D., Lobarinas, E. & Le Prell, C. G. 2017. Relationship between dietary quality, tinnitus and hearing level: data from the national health and nutrition examination survey, 1999–2002. International journal of audiology, 56, 716-722.
Gender-imbalance and the future audiology workforce
Supervisor(s): Dr Ruth Brooke and Dr Ted Killan
Key words: Audiology; gender; workforce; education; health behaviour
Summary:Audiology is the clinical discipline concerned with the diagnosis, treatment and management of individuals with hearing and balance problems. Data drawn from several years’ admissions cycles show a ratio of approximately 7:1 female to male applicants study audiology at undergraduate level. As well as having workforce planning consequences, evidence from other healthcare disciplines suggests that patients’ health behaviours can be influenced by clinician gender, with positive outcomes more likely when the clinician and patient are the same sex. This is especially the case in long-term conditions, such as hearing loss, where patients are seen repeatedly by clinicians.
The aim of this PhD is to explore questions related to the issues described above. These could include reasons for the low number of males studying audiology in the UK, whether this gender imbalance is having a negative effect on hearing loss patient behaviour, and/or what can be done to raise the attractiveness of a career in audiology to young males. A range of methodologies could be employed including systematic literature review and qualitative approaches.
Whittock, M and Leonard, L (2003). Stepping outside the stereotype. A pilot study of the motivations and experiences of males in the nursing profession. Journal of Nursing Management, 11(4), 242-249.
Nemes, JL (2005). As the gender gap widens, does audiology need a few good men? Hearing Journal, 58(3), 19-24.
4-Year International PhD Academy
Cardiovascular & Metabolic Disease
Severe to profound hearing loss and sleep quality.
Supervisor(s): Dr Ted Killan, Dr Paul Baxter, Dr Jane Blackwell and Mr Simon Carr
Key words: Hearing loss; sleep quality; cochlear implants
Summary:Hearing loss affects approximately 1 in 6 individuals in the UK. There is growing evidence suggesting that individuals with hearing loss experience are more likely to experience reduced sleep quality to normally hearing peers. A potential cause for this association is that living with a hearing loss causes high levels of anxiety or stress, which in turn can affect sleep quality. As sleep plays an important role in physical and mental health, and given the high prevalence of hearing loss, further investigations are warranted in order to better understand this association. Further, it is possible that an as yet undescribed benefit of interventions for hearing loss, such as hearing aids or cochlear implants (CIs), is improvement in sleep quality (and subsequently improved mental and physical health outcomes). Any positive effects will be useful in informing ongoing debates on CI candidacy.
This PhD project aims to describe sleep behaviours and experiences in patients with severe to profound hearing loss referred for cochlear implants. It will also investigate whether successful use of CIs lead to improvements in sleep quality. A range of methodologies could be employed including self-report and objective measures of sleep quality. You will be expected to design and implement primary data collection (following obtaining appropriate ethical approval) and analyse and interpret a range of different data types.
Kim, S.Y., Kim, H.-J., Kim, M.-S., Park, B., Kim, J.-H., Choi, H.G., 2017. Discrepancy between self-assessed hearing status and measured audiometric evaluation. PLoS One. https://doi.org/10.1371/journal.pone.0182718
Jansson-Frojmark, M., Linton, S.J., Flink, I.K., Granberg, S., Danermark, B., Norell-Clarke, A., 2012. Cognitive-behavioral therapy for insomnia co-morbid with hearing impairment: a randomized controlled trial. Journal of Clinical Psychology in Medical Settings 19, 224–234.
A machine learning approach to understand the disease trajectories of atrial fibrillation
Supervisor(s): Jianhua Wu and Chris Gale
Key words: Machine learning, atrial fibrillation, big data, health informatics, disease trajectory
Summary: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, with a lifetime risk of 1 in 4 in the general population and an increasing prevalence as the population ages. Effective treatment of patients with AF includes not only rate control and prevention of stroke, but also management of cardiovascular risk factors and comorbid diseases. Although AF is associated with increased risk of major cardiovascular events such as stroke and heart failure, the absolute and relevant event rates of these competing outcomes are not well described. The disease trajectories and the transitions among disease states after AF are lesser known given the uprising use of direct oral anticoagulants (DOACs) in recent years comparing with traditional anticoagulants, such as warfarin.
The aim of the project is to study, using big health data and health informatics techniques, the disease trajectories of patients with atrial fibrillation. Patients with atrial fibrillation will be identified from Hospital Episode Statistics (estimated analytical cohort n ~ 1.2 million) and linked to primary care for comorbidities and prescription data. A machine learning approach through hierarchical clustering or neural networks will be used to explore the disease trajectory of atrial fibrillation through this big electronic health database. This project will provide a national overview of the profile of patients with atrial fibrillation and quantify the subsequent major cardiovascular and non-cardiovascular events and disease trajectories. Accessing national individual patient data will provide a high resolution and granularity to this AF population.
John Camm et al. Guidelines for the management of atrial fibrillation: The Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC), European Heart Journal, 2010; 31 (19): 2369–429, https://doi.org/10.1093/eurheartj/ehq278
David Conen. Epidemiology of atrial fibrillation, European Heart Journal, 2018; 39(16): 1323-4, https://doi.org/10.1093/eurheartj/ehy171
Paulus Kirchhof. The future of atrial fibrillation management: integrated care and stratified therapy. Lancet, 2017; 290: 1873-87
Intercellular signalling in the heart: paracrine signalling between cardiac fibroblasts and cardiomyocytes.
Supervisor(s): Dr Neil Turner & Dr Azhar Maqbool
Key words: cardiac fibroblasts - cardiomyocytes - signalling - cardiac remodelling
Summary:Cardiac fibroblasts are one of the most prevalent non-myocyte cell populations in the heart. Although traditionally viewed solely in relation to extracellular matrix remodelling, fibroblasts are now acknowledged as being important nodal regulators of multiple aspects of cardiac function under both physiological and pathophysiological conditions [1-3]. An important role for cardiac fibroblasts in stimulating cardiomyocyte hypertrophy has emerged in recent years whereby fibroblasts secrete paracrine growth factors and other signalling molecules that drive cardiomyocyte hypertrophy [4-5]; and recent work in our laboratory has implicated interleukin-6 in this process . This laboratory-based project will utilise a variety of cellular, molecular and pharmacological approaches to explore the signalling mechanisms regulating fibroblast-myocyte communication and seek to identify novel targets for therapeutic intervention for reducing adverse cardiac remodelling.
Porter KE, Turner NA. Cardiac fibroblasts: at the heart of myocardial remodeling. Pharmacol. Ther. 2009;123:255-78.
van Nieuwenhoven FA, Turner NA. The role of cardiac fibroblasts in the transition from inflammation to fibrosis following myocardial infarction. Vascul. Pharmacol. 2013;58:182-8.
Turner NA. Inflammatory and fibrotic responses of cardiac fibroblasts to myocardial damage associated molecular patterns (DAMPs). J. Mol. Cell. Cardiol. 2016;94:189-200.
Kamo T, Akazawa H, Komuro I. Cardiac nonmyocytes in the hub of cardiac hypertrophy. Circ. Res. 2015;117:89-98.
Fujiu K, Nagai R. Fibroblast-mediated pathways in cardiac hypertrophy. J. Mol. Cell. Cardiol. 2014;70:64-73.
Bageghni SA, Hemmings KE, Porter KE, Denton C, Ainscough JFX, Drinkhill MJ, Turner NA. Cardiac fibroblast-specific p38 MAPK promotes cardiac hypertrophy via a putative paracrine IL-6 signalling mechanism. FASEB J. 2018 (in press).
Development of novel non-invasive techniques for the measurement of the input function for dynamic positron emission tomography
Supervisors: Dr Charalampos Tsoumpas, Professor David L. Buckley, Dr David Tuch
Key words: Positron Emission Tomography, Dynamic Imaging, Input Function, Kinetic Modelling
Summary:Positron emission tomography (PET) is a quantitative imaging modality that can probe a number of functional and biological processes, depending on the radiolabelled tracer used . Standard clinical PET imaging is followed by analysis using semi-quantitative indices, such as the standardised uptake value, which is used in the majority of clinical assessments in which PET has a role . However, considerably more information can be extracted from dynamic image acquisition protocols, followed by application of appropriate radiotracer kinetic modelling techniques, but the latter approaches have mainly been restricted to drug development and clinical research applications due to their complexity in terms of both protocol design and parameter estimation methodology . One of the key limitations to translate dynamic PET imaging in clinical practice is the need of obtaining arterial blood samples which, in most cases, are essential for kinetic modelling.
This project proposes a novel approach by measuring non-invasively the radioactivity in the blood stream using a recently developed imaging system. In particular, LIGHTpoint Medical has developed a Hand-Held RadioLuminescence Imaging (HARLI) device which is the world’s first system for hand-held imaging of PET tracers . The technology employs direct conversion of beta particles to an optical image. This system could be potentially utilised to provide dynamic quantitative measures of the radioactivity in blood eliminating the need of an arterial catheterisation.
The candidate will perform dynamic experiments on 3D printed phantoms  and in vivo preclinical experiments using the HARLI system. A variety of radiotracers will be used to demonstrate feasibility of the concept and different preclinical models. Data analysis and software development will be required to ensure a precise and accurate measurement of the input function extraction is achieved from the raw data. The candidate will need to undertake extensive training in preclinical imaging techniques. In addition, s/he will benefit from prior knowledge of the basic principles of radiation and particle physics as well as experimental and software development skills for data manipulation and modelling.
Phelps MA (2000) Positron emission tomography provides molecular imaging of biological processes PNAS 97(16): 9226-9233; doi:10.1073/pnas.97.16.9226
Schmidt KC, Turkheimer FE (2002) Kinetic modeling in positron emission tomography,” Q J Nucl Med 46:70–8.
Kotasidis FA, Tsoumpas C, Rahmim A (2014) Advanced kinetic modelling strategies: towards adoption in clinical PET imaging. Clin Trnsl Imaging: 2(3): 219-237, doi:10.1007/s40336-014-0069-8
Grootendorst MR, Cariati M, Pinder SE, Kothari A, Douek M, Kovacs T, Hamed H, Pawa A, Nimmo F, Owen J, Ramalingam V, Sethi S, Mistry S, Vyas K, Tuch DS, Britten A, Van Hemelrijck M, Cook GJ, Sibley-Allen C, Allen S, Purushotham A (2017) Intraoperative Assessment of Tumor Resection Margins in Breast-Conserving Surgery Using 18F-FDG Cerenkov Luminescence Imaging: A First-in-Human Feasibility Study. J Nucl Med 58(6):891-898
Filippou V, Tsoumpas C (2018) Recent advances on the development of phantoms using 3D printing for imaging with CT, MRI, PET, SPECT and Ultrasound. Med Phys: At press
The impact of obesity and metabolic disturbance on oocyte health and fertility
Supervisor(s): Prof Helen Picton and Dr John Huntriss
Key words: oocyte, PCOS, fertility, obesity, metabolism
Summary:More than six million IVF babies have been born worldwide, however, infertility treatments remain costly and inefficient as we know little about the mechanisms that confer developmental competence (quality) on human oocytes or indeed how oocyte quality is affected by advancing age and obesity. The latter is relevant as a major cause of female subfertility - Polycystic Ovarian Syndrome (PCOS) affects millions of women in the UK and worldwide. PCOS is associated with obesity, endocrine disturbance, insulin resistance, acne, hirsuitism, type 2 diabetes, high cholesterol levels and coronary heart disease. In infertile women with PCOS the pathology is characterised by the presence of ovarian cysts on ultrasound scan and may or may not be combined with obesity and altered glucose metabolism, as well as ovulation failure, altered pituitary hormone levels and abnormal androgen metabolism. These parameters may all impact on oocyte fertility and subsequent embryo quality. A detailed understanding of human oocyte biology in health, age and obesity is therefore essential if we are to quantify the impact of metabolic disturbances such as obesity and PCOS on women’s health and reproductive potential and on the success of infertility treatments in these patient groups.
This Ph.D. will integrate studies of the metabolome and transcriptome of human oocytes from women with and without PCOS with mechanistic, in vitro animal models of oocyte and embryo development. The experimental plan will address four broad objectives. (i) Quantification of the metabolic, genetic and epigenetic signatures of oocytes from fertile and infertile women of different weights, with and without PCOS. (ii) Comparison of the metabolic and molecular signatures of follicles and oocytes across al stages of oogenesis in women with PCOS relative to normal control tissues. (iii) Evaluation of how obesity alters the mechanisms conferring oocyte fertility and developmental competence. (iii) Investigation of how the manipulation of energy substrate and antioxidant levels in the in vitro environment will impact on oocyte in vitro maturation and fertilisation and subsequent embryo development in vitro.
The student will join a team of world class scientists working on human oocyte biology. He/she will be trained in a range of specialist laboratory methods including: follicle and oocyte harvest, in vitro maturation, in vitro fertilisation and embryo culture; confocal and fluorescent microscopy; analysis of the oocytes’ metabolome (mitochondrial activity measurement, quantification of energy and protein turnover), transcriptome (real time PCR and RNA Sequencing), and epigenome (pyrosequencing methylation analysis); and bioinformatics. The project will combine research on abattoir-derived ruminant oocytes and embryos with analyses of human cells donated for research under ethically approved protocols. The programme of work will combine novel, highly sensitive assays of protein and energy turnover and mitochondrial activity in individual oocytes with transcriptome and methylome analysis and quantitative PCR on the same cell to catalogue exactly how the metabolic, genetic and epigenetic signatures of oocytes change with obesity, metabolic disturbance and/or the PCOS phenotype, as appropriate. The data generated will significantly advance our understanding of human oocyte biology and it will help improve the treatment of PCOS and the management of women’s reproductive health.
Palep-Singh M, Picton HM, Yates ZR & Balen AH (2007). Polycystic ovary syndrome and the C677T single nuceotidepolymorphism of methylenetetrahydrofolate reductase: a pilot observational study. Human Fertility 10(1): 33-41.
Palep-Sing M, Picton HM, Barth JH, & Balen AH (2007). Ethnic variations in the distribution of obesity and biochemical metabolic abnormalities in fertility clinic attenders. Journal Reproductive Medicine (USA) 53(2):117-23
Harris SE, Maruthini D, Tang T, Balen AH, Picton HM (2010). Metabolism and karyotype analysis of oocytes from patients with PCOS. Human Reproduction, 25(9):2305-15
Hemmings KE, Maruthini D, Vyjayanthi S, Hogg JE, Balen AH, Campbell BK, Leese HJ, Picton HM (2013). Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment. Human Reproduction. 28(4):1031-44
Cotterill M, Harris SE, Collado Fernandez E, Lu J, Huntriss JD, Campbell BK, Picton HM (2013). The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro. Molecular Human Reproduction. 19(7): 444-50.
Maruthini D, Harris SH, Balen AH, Campbell BK, Picton HM (2014) The effect of metformin treatment in vivo on acute and long-term energy metabolism and progesterone production in vitro by granulosa cells from women with PCOS. Human Reproduction 29(10): 2302–2316.
Huntriss J, Woodfine K, Huddleston JE, Murrell A, Picton HM (2015). Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®. Methods In Molecular Biology. 1315:259-70.
Balen AH, Morley LC, Misso M, Franks S, Legro RS, Wijeyaratne CN, Stener-Victorin E, Fauser BC, Norman RJ, Teede H (2016). The management of anovulatory infertility in women with polycystic ovary syndrome: an analysis of the evidence to support the development of global WHO guidance. Human Reproduction Update. 22(6):687-708.
Nicholas SL, Field HP, Calder N, Picton HM, Balen AH, Barth JH (2017). Anti-Müllerian Hormone reflects the severity of Polycystic Ovary Syndrome. Clinical Endocrinology 86(3):395-400.
Evaluation Of The Therapeutic Potential Of In Vitro-Derived Oocytes For Fertility Preservation
Supervisor(s): Prof Helen Picton and Dr John Huntriss
Key words: oocyte, in vitro growth, IVF, cryopreservation, fertility preservation
Summary:In recent years improvements in the diagnosis, management and treatment of a range of solid and haematological malignancies has lead to a marked increase in the chances of long-term survival for a significant number of children and adolescents. Unfortunately germ cells, like the cancer cells that are their intended targets, are highly susceptible to damage by radiation and by the alkylating agents and platinum compounds that are commonly used in chemotherapy formulations. Thus the radiation and chemotherapy used to cure many cancers can render patients of either sex or any age temporarily or even permanently infertile. The premature gonadal failure or total sterility that can occur as a consequence of cancer treatment is particularly relevant to young girls who, in the event of ovarian failure, are faced with the prospects of life long hormone replacement therapy. Healthcare professionals are therefore faced with the challenge of not only developing and improving the technologies that will treat the cancer and protect the individual’s wellbeing, but they must also implement strategies that will conserve the fertility of young patients.
The current options available to preserve the fertility of young female cancer patients range from no medical intervention at all to the use of invasive procedures to harvest and freeze-store tissues or isolated cells. Assisted reproductive techniques can be used for the collection and storage of mature oocytes (eggs) and embryos. However these approaches are only suitable for patients of reproductive age and they are clearly not an option for most adolescents or for prepubertal girls. An alternative approach that is suitable for both children and adults is the cryopreservation of ovarian tissue.
Ovarian tissue cryopreservation involves freezing the earliest staged primordial oocytes in situ either through freezing small pieces of the surface of the ovary or by freezing the whole ovary. The tissue fragments or ovary can then be transplanted back to the patient when she is in remission and wishes to start her family. Unfortunately, ovarian autografting is not suitable for patients with blood bourn diseases such as Leukaemia or steroid related cancers as these diseases carry a high risk of reintroducing the cancer to the body through the transplanted tissue. Where any such risk exists a safer alternative for the restoration of fertility is to grow the immature oocytes within the ovarian tissue to maturity in the laboratory through a process known as the in vitro growth (IVG) of eggs contained within ovarian follicles. Once the eggs are fully grown they can then be matured by in vitro maturation (IVM) and fertilised using in vitro fertilisation (IVF) technologies so that only embryos that are free from cancer cells are transferred back to the patient to establish a pregnancy.
The development of technologies for the complete IVG of fertile oocytes from the most abundant primordial follicles holds many attractions for assisted conception, in humans and for animal production and animal conservation. Any culture strategy designed to support the complete IVG of oocytes must mimic the sequence of events seen by these cells in vivo in terms of growth rates, gene expression patterns, protein synthesis and metabolism. This legacy of development is required to support the production of a fertile oocyte and early post-fertilisation development of the zygote. The team at Leeds has recently developed a 3D culture strategy that supports the complete in vitro development of oocytes over extended periods of up to 3-4 months.
The student will join a team of world class scientists working on human oocyte biology. He/she will be trained in a range of specialist laboratory methods including: ovarian tissue cryopreservation, follicle and oocyte harvest and culture, in vitro fertilisation and embryo culture; confocal and fluorescent microscopy; analysis of the oocytes’ metabolome (mitochondrial activity measurement, quantification of energy and protein turnover), transcriptome (real time PCR and RNA Sequencing), and epigenome (pyrosequencing methylation analysis); and bioinformatics. Due to the limited availability of human ovarian tissue for research, abattoir-derived sheep ovaries and oocytes will be used as a model for human ovary/oocytes throughout the course of this Ph.D project. The proposed experiments will grow follicles from the earliest primordial stages to maturity in the laboratory using our in-house, follicle culture system. The developmental capacity of in vitro grown oocytes from fresh and cryopreserved ovarian tissue will be tested. A series of cellular, metabolic and molecular assays will be used to evaluate the health and developmental capacity of oocytes derived using culture methods. Follicle and oocyte size will be measured and growth rates recorded. Follicle development and culture stress will be assessed by measuring follicle and oocyte metabolism and waste product production in samples of spent culture media. Molecular genetic and epigenetic indices of oocyte growth and health will be compared to in vivo derived cells. The capacity of in vitro-derived oocytes to complete meiotic maturation and produce a fertile gamete will ultimately be assessed using methods for IVM and IVF. Embryo developmental potential will be quantified by culturing embryos for up to 7 days to the blastocyst stage and recording embryo cleavage rates, blastocyst production, cell number, embryo metabolism and gene expression profiles. All of the technologies for IVG, IVM and IVF of sheep oocytes, together with analytical techniques used to assess growth and fertility are in routine use in the Leeds laboratory.
Picton HM, Kim S, & Gosden RG (2000). Cryopreservation of gonadal tissue and cells. British Medical Bulletin 56(3), 603-615
Picton HM, Harris SE, Muruvi W and Chambers EL (2008) Advances In In Vitro Follicle Development And Oocyte Maturation. Reproduction 136: 703-715.
Huntriss J & Picton HM (2008). Epigenetic consequences of assisted reproduction and infertility on human preimplantation embryos. Human Fertility (Camb).11(2):85-94.
Chambers EL, Gosden RG, Yap C, Picton HM (2010) In situ identification of follicles in ovarian cortex as a tool to quantify follicle density, viability and developmental potential in strategies to preserve female fertility. Hum Reprod 25(10):2559-68
Cotterill M, Catt SL, Picton HM (2012) Characterisation of the cellular and molecular responses of ovine oocytes to preovulatory levels of FSH and LH during in vitro maturation. Reproduction, 44:195-207.
Hemmings KE, Maruthini D, Vyjayanthi S, Hogg JE, Balen AH, Campbell BK, Leese HJ, Picton HM (2013). Amino acid turnover by human oocytes is influenced by gamete developmental competence, patient characteristics and gonadotrophin treatment. Hum Reprod 28(4):1031-44
Campbell BK*, Hernandez-Medrano J, Onions V, Pincott-Allen C, Aljaser F, Fisher J, McNeilly AS, Webb B, Picton HM (2014). Restoration of ovarian function and natural fertility following the cryopreservation and transplantation of whole adult ovaries. Human Reproduction August; 29(8): 1749–1763
Newton HL, Glaser A, Picton HM (2017) The feasibility, safety and outcome of fertility preservation treatment options in pre-pubertal girls. Minerva Ginecol. 69(6):568-586. doi: 10.23736/S0026-4784
Martinez F (2017) Update on fertility preservation from the Barcelona International Society for Fertility Preservation-ESHRE-ASRM 2015 expert meeting: Indications, results and future perspectives. Hum Reprod, 32(9): 1802-1811.
Matthews SJ, Picton H, Ernst E, Andersen CY. (2018) Successful pregnancy in a woman previously suffering from β-thalassaemia following transplantation of ovarian tissue cryopreserved before puberty. Minerva Ginecol 70(4):432-435.
The diagnostic and prognostic biomarkers of stroke
Supervisor(s): Dr Kirti Kain, Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine, University of Leeds.
Dr Sikha Saha, Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine, University of Leeds.
Dr Ahamad Hassan, Consultant Stroke Physician, Leeds Teaching Hospital Trust, Leeds.
Key words: Stroke, biomarker, prognosis, diagnosis, stroke mimics, hemorrhagic stroke, insulin resistance, blood pressure
Summary: Stroke is the second most common cause of death worldwide. Globally, stroke occurrence is rising , and there are over 33 million stroke survivors . In the UK alone there are around 1.2 million stroke survivors, 30% of whom will experience a recurrent stroke .
The diagnosis of stroke remains largely clinical, yet widely used stroke scoring systems such as the FAST TEST or CINCINNATI STROKE SCALE do not allow distinction of patients with acute stroke from those with mimic presentations (e.g., migraine, seizures, infections and mass brain lesions) [4,5,6]. The proportion of patients with TIA as opposed to a mimic is even lower than that confirmed to be true stroke.
Diffusion-weighted magnetic resonance imaging (DW-MRI) can be helpful in distinguishing ischaemic stroke from mimic but this is not suitable as an initial point of care (POC) test in hyper-acute stroke and often patients with acute stroke do not tolerate scanning. It is also very costly, not available out of hospital and insufficiently sensitive in minor stroke . It is believed that biomarkers in body fluids may be one of the keys to address this problem [8,9]. The most pressing need for a diagnostic biomarker is in the ultra-acute phase when patients are assessed by paramedics out of hospital or by emergency physicians in the accident and emergency department (A&E), as this is where the most important time critical decisions have to be made.
As well as rapid diagnosis there is an unmet need to identify biomarkers which predict stroke outcome in the acute phase e.g. following reperfusion therapy, or in the long term. For example information obtained from biomarker signatures in the acute phase could be combined with clinical and imaging data to identify if there is salvageable tissue present or the likelihood of reperfusion10. Alternatively a candidate biomarker associated with cardiovascular events post stroke may influence intensity of long term secondary prevention11.
The list of biomarker studied will include but not be limited to Neuron-specific enolase (NSE), a protein present in neuronal cytoplasm, S100B, a calcium-binding protein mainly expressed in astrocytes, Myelin basic protein (MBP), a myelin membrane proteolipid, glial fibrillary acidic protein (GFAP), an astrocytes specific protein and occludin, a blood brain barrier junctional protein. Identification will be with different techniques (e.g., Mass Spectrometry, Nuclear magnetic resonance spectroscopy, Microarray and Laser spectrometry). Prognostic factors studied will be ‘C’ reactive protein, Insulin and insulin resistance, Matrix metalloproteinase-9, thrombomodulin, vascular cell adhesion molecule, and von Willebrand factor.
Why this study is important
Although stroke is a major cause of death and disability, it is treatable and preventable. This study is clinically important as if diagnostic and prognostic biomarkers can be identified for stroke patients it will improve access of stroke patients to the most appropriate therapies.
Aims and Objectives
The aim of the study is to quantify circulating biomarkers which
Distinguish between acute stroke and stroke mimics
Differentiate between ischaemic and haemorrhagic stroke
Accurately predict early and longer term stroke outcome
Patients with suspected stroke in A&E
Patients with confirmed stroke on the acute stroke unit
Subject has capacity and refuses consent
Subject lacks capacity and relative informed consent cannot be obtained
Patients with suspected acute stroke will be identified by the brain attack team in the emergency room at Leeds General Infirmary. The details of all such patients are routinely recorded in a log book and will be passed onto the research team.
Patients with a suspected acute stroke will be managed according to routine clinical care pathways. The population will include a mixture of confirmed strokes and also stroke mimics (commonly sepsis, seizures, migraine, syncope and sepsis).
As part of the acute management blood samples are taken and forwarded to the pathology lab to assess clotting (plasma) and serum (biochemical profile) and full blood count. Where possible, an extra 5ml sample of serum ad plasma will be collected at the same time and sent to the pathology lab along with the routine samples.
Identified patients will be approached for participation in the study on either the acute stroke unit or general medical wards. Patients with suspected stroke will be invited to participate in the main diagnostic study. Patients with clinically confirmed stroke will also be invited to participate in the follow up prognostic study.
Obtaining and Processing Samples
In subjects with confirmed stroke we will seek permission to use existing blood samples (serum/plasma) which were collected during routine care in A&E. Samples are usually kept by NHS pathology laboratories for 3 days after receipt before being routinely discarded. We will use the excess sample to undertake studies of diagnostic markers.
The samples will be pseudoanonymised and snap frozen in liquid nitrogen and stored at -70oC in the LIGHT laboratory freezer.
In subjects with clinically confirmed stroke, we will seek permission to obtain a fresh 5 ml serum and plasma sample solely for the purposes of research. This is necessary because certain biomarkers (e.g. insulin and clotting factors) have specific collection techniques e.g. transportation on ice or taken in a fasting state. Whenever possible we will try and time the research sample with routine clinical sampling usually undertaken on the morning round by ward phlebotomists.
All patient identifiable data will be stored on a password protected database in the NHS
Data taken offsite for analysis will be in pseudoanonymised format. The baseline data collected will include patient demographics, waist height ratio, blood pressures of arms and legs, final diagnosis and stroke characteristics. The follow up data will include discharge functional outcome, mortality and cardiovascular outcome events up to 3 years. This will be collated from the paper records and the electronic patient record (PPM+).
Assays will be performed on defrosted serum/plasma using commercially available ELISA kits in accordance with the manufacturer’s instructions and also with other techniques (e.g., Mass Spectrometry, Nuclear magnetic resonance spectroscopy, Microarray and Laser spectrometry).
The assays will be performed In the NHS or University of Leeds laboratories depending on the biomarker being studied.
Continuous variables will be analysed using the two independent samples, Mann-Whitney U test. Categorical data will be cross tabulated and a Chi-Square test will be performed to study differences in confirmed stroke patients and stroke mimics. Logistic regression analysis will be carried out to identify independent biomarkers predictive of stroke and stroke outcome with risk factors in the model. Hazards ratios will be calculated to determine the association between biomarkers and survival/ major adverse cardiovascular events. All statistical analysis will be performed with IBM SPSS statistics version 23.
Rationale for the Study Design and Methodology
All suspected stroke are assessed in A&E thus representing a complete population of confirmed strokes and mimics for study. However, we recognise that in the initial phase of stroke, treatment is time critical and this limits access to research procedures in the department. Therefore the design of the study is that it will not interfere in clinical acute stroke care. Blood samples from hyperacute stroke patients will only be obtained in A&E at the time of routine blood sampling and retrospective patient consent or relative informed consent will be obtained to use the left over sample once the patient has completed any treatment and is on the ward. Using existing samples will also maximise ascertainment and prevent any bias in determining the performance of a biomarker.
Further blood sampling will be necessary for patients in the prognostic study, as certain candidate biomarkers such as insulin and clotting factors require collection on ice, and it will not be possible to use the remnant sample.
Feigin, V.L., Forouzanfar, M.H., Krishnamurthi, R., Mensah, G.A., Connor, M., Bennett, D.A., Moran, A.E., Sacco, R.L., Anderson, L., Truelsen, T. and O'Donnell, M., 2014. Global and regional burden of stroke during 1990–2010: findings from the Global Burden of Disease Study 2010. The Lancet, 383(9913), pp.245-255.
Naghavi, M., Wang, H., Lozano, R., Davis, A., Liang, X., Zhou, M., Vollset, S.E., Ozgoren, A.A., Abdalla, S., Abd-Allah, F. and Aziz, M.I.A., 2015. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. 2016. Lancet, 385(9963), pp.117-171.
State of the Nation, Stroke Statistics January 2016 https://www.stroke.org.uk/sites/default/files/state_of_the_nation_2016_110116_0.pdf
Anathhanam SA and Hassan A. 2017 Mimics and chameleons in stroke Clinical Medicine 2017 Vol 17, No 2: 156–60
Vilela P Acute stroke differential diagnosis: Stroke mimics. Eur J Radiol. 2017 Nov;96:133-144. doi: 10.1016/j.ejrad.2017.05.008. Epub 2017
Liberman AL, Prabhakaran S. Stroke Chameleons and Stroke Mimics in the Emergency Department. Curr Neurol Neurosci Rep. 2017 Feb;17(2):15. doi: 10.1007/s11910-017-0727-0.
Brazzelli M Diffusion-Weighted Imaging and Diagnosis of Transient Ischemic Attack ANN NEUROL 2014;75:67–76
Jickling GC and Sharp FR. 2015 Biomarker panels in ischemic stroke Stroke. 2015 March ; 46(3): 915–920
Whiteley W, Sandercock P (2008) Blood Biomarkers in the Diagnosis of Ischemic Stroke:A Systematic Review Stroke. 2008 Oct;39 (10):2902-9.
Calleja AI, Garcia-Bermejo P, Cortijo E et al. Insulin Resistance Is Associated With a Poor Response to Intravenous Thrombolysis in Acute Ischemic Stroke. Diabetes Care. 2011; 34: 2413–2417
Di Napoli M, Papa F, Bocola V. C-Reactive Protein in Ischemic Stroke An Independent Prognostic Factor. Stroke. 2001;32:917-924
Cardiovascular risk in younger patients with double diabetes
Supervisor(s): RA Ajjan and S Plein
Key words: Double Diabetes, Thrombosis, Glycaemia
The term double diabetes has been used to describe individuals with type 1 diabetes (T1DM) who are overweight and display features of type 2 diabetes (T2DM) such as insulin resistance, hypertension and dysplipidaemia. Patients with double diabetes are at higher risk of diabetic vascular complications, thereby increasing diabetes-related morbidity and mortality. There are no established guidelines for the treatment of this group of patients, despite the documented increased risk of complications.
Recent work has shown that at least one member the sodium glucose transporter-2 inhibitors (SGLT-2i) reduces cardiovascular and all-cause mortality in patients with T2DM. Moreover, real life observational data have shown that all three agents in this class reduce heart failure in patients with diabetes The exact mechanisms for the beneficial cardiovascular effects are unclear but likely to be multifactorial involving different pathways including weight loss, a drop in blood pressure, improved insulin sensitivity and reduction in blood glucose. We hypothesise that the use of SGLT-2i in individuals with double diabetes reduces weight, increases insulin sensitivity and improves markers of cardiovascular health.
We propose to investigate whether the use of SGLT-2i in younger overweight T1DM diabetes patients, in addition to insulin therapy, results in weight reduction and improvement in glycaemic parameters, cardiovascular markers and quality of life measures.
This will be an open label, single centre, two arm study comparing standard insulin therapy, using multiple daily injections or an insulin pump, with the addition of SGLT2i to insulin in patients with double diabetes for a period 6 months.
Inclusion criteria are patients with a known diagnosis of T1DM for at least 2 years, who meet all the following criteria: aged 18 to 40 years, suboptimal glycaemic control (HbA1c >58 mmol/mol) and body mass index >27 kg/m2 or >25 kg/m2 in those of South Asian origin or a family history of T2DM.
A total of 48 consenting adults will be randomised 1:1 to:
Control group using insulin only for the treatment of glycaemia.
Intervention group, treated with SGLT-2i in addition to insulin.
Metabolic, cardiovascular and personal parameters will be analysed at baseline, 90, 180 and 365 days (the last time point at 6 months following cessation of intervention). The primary end point is reduction in weight and/or improvement in estimated glucose disposal rate (eGDR). Secondary and exploratory end points are:
Glycated haemoglobin (HbA1c)
Time spent in euglycaemia
Time spent in hypoglycaemia
Vascular inflammatory markers: C-reactive protein and complement C3
Vascular thrombotic markers: fibrin clot structure and lysis
Clinical vascular markers: blood pressure, microalbuminuria and cardiac function assessed by magnetic resonance imaging
Health-related quality of life
Hospital admissions for any cause
This study in patients with double diabetes will establish safety and efficacy of SGLT2i in reducing weight, modulating glycaemic, inflammatory and thrombotic markers and improving quality of life. Data generated have the potential to change clinical management of younger individuals with double diabetes, helping to reduce morbidity and mortality in this high risk population.
Pozzilli P, Guglielmi C. Double diabetes: a mixture of type 1 and type 2 diabetes in youth. Endocr.Dev. 2009;14:151-166.
Pozzilli P. Type 1 diabetes mellitus in 2011: Heterogeneity of T1DM raises questions for therapy. Nat.Rev.Endocrinol. 2011;8:78-80.
Erbey JR, Kuller LH, Becker DJ, Orchard TJ. The association between a family history of type 2 diabetes and coronary artery disease in a type 1 diabetes population. Diabetes Care 1998;21:610-614.
Merger SR, Kerner W, Stadler M et al. Prevalence and comorbidities of double diabetes. Diabetes Res.Clin.Pract.
Famulla S, Pieber TR, Eilbracht J et al. Glucose Exposure and Variability with Empagliflozin as Adjunct to Insulin in Patients with Type 1 Diabetes: Continuous Glucose Monitoring Data from a 4-Week, Randomized, Placebo-Controlled Trial (EASE-1). Diabetes Technol.Ther. 2017;19:49-60.
Pieber TR, Famulla S, Eilbracht J et al. Empagliflozin as adjunct to insulin in patients with type 1 diabetes: a 4-week, randomized, placebo-controlled trial (EASE-1). Diabetes Obes.Metab 2015;17:928-935.
Khunti K, Davies M, Majeed A et al. Hypoglycemia and risk of cardiovascular disease and all-cause mortality in insulin-treated people with type 1 and type 2 diabetes: a cohort study. Diabetes Care 2015;38:316-322.
Modulation of hypofibrinolysis in diabetes
Supervisor(s): RA Ajjan, D Tomlinson and V Ponnambalam
Key words: Diabetes, hypofibrinolysis, plasminogen activator inhibitor-1
Hypofibrinolysis is associated with increased risk of thrombotic disease. Diabetes is characterized by hypofibrinolysis and elevated levels of plasminogen-activator inhibitor (PAI)-1 is one of the mechanisms implicated. We hypothesise that inhibition of PAI-1 activity using a novel methodology improves hypofibrinolysis in high risk conditions, including diabetes, and consequently decreases the risk of thrombosis.
We developed a phage display library of a scaffold protein that constrains two variable conformational 9AA peptides (>3x1010 random peptides), mimicking the configuration of antibodies, termed Affimers. These Affimers have a low molecular weight and are able to interact with proteins. We will use the library of high diversity random library to screen for PAI-1 binders employing a phage display system. The project will have three main objectives:
Isolate high affinity PAI-1 binding Affimers and characterise their role in modulating fibrinolysis.
Study binding affinities/specificity of Affimers of interests and pinpoint interaction sites.
Conduct preliminary in vivo studies to establish the role of Affimers in bleeding and thrombosis.
Objective 1. Isolate high affinity PAI-1 binding Affimers and characterise their role in modulating fibrinolysis.
Screening for PAI-1 binding Affimers. Screening the library of Affimers against immobilised PAI-1 will be undertaken and high affinity binders enriched. A total of 250 clones will be picked and sequenced. Affimers with distinct sequences will be subcloned for large scale protein production.
Functional analysis of Affimers. This will be undertaken using turbidimetric and thromboelastometric assays employing plasma and whole blood systems. Plasma systems will investigate the role of Affimers in modulating fibrinolysis in the presence of other plasma proteins, whereas whole blood will study the potential effects of cells. Initially, pooled plasma samples and the validated high-throughput turbidimetric assay will be employed to identify PAI-1 binding Affimers the modulate clot lysis. Subsequently, individual samples from healthy control and various clinical conditions (ischaemic heart disease, type 1 and type 2 diabetes plasma; n=40 in each group), will be tested to establish inter-individual variability in response in various conditions. Turbidimetric experiments will be complemented by confocal and electron microscopy to visualise the clot and ensure the structure remains physiological.
Having narrowed down the Affimers of interest, clot formation/lysis experiments on whole blood using rotational elastometry will be conducted on individual Affimers as well as a combination of Affimers as appropriate.
Testing combination of Affimers. In the unlikely event that none of the high affinity binding Affimers shows an effect on fibrinolysis, a number of Affimers will be used in combination to establish whether more than one Affimer is required to effectively block PAI-1 activity.
Objective 2. Study binding affinities/specificity of Affimers of interests and pinpoint interaction sites
Binding assays. These will be conducted using surface plasmon resonance to establish the binding affinity of Affimer to PAI-1 and study association/dissociation rates. PAI-1 will be coupled to a CM5 sensor chip using amine-coupling chemistry and Affimers will be passed over the surface of PAI-1 with binding constants calculated using BIAeval software. Experiments will be repeated by immobilizing Affimers of interests and flow PAI-1 over the protein.
Pull down assays. This will be performed to investigate interaction of Affimers of interest with PAI-1 in solution and to ensure that this interaction is specific and does not involve other plasma proteins. Optimisation will be carried out using purified proteins before using plasma samples.
Microarray analysis. Peptide chips of overlapping 15 AA peptides will be created for PAI-1. Affimers of interest will be added to these peptide chips and after extensive washing binding will be detected with biotinylated anti-Affimer antibody.
Crystallography. this will be used to study Affimers-PAI-1 complexes. Data generated will help to understand molecular interactions that underlie interactions sites of Affiemrs of interests with PAI-1.
Objective 3. Conduct preliminary in vivo studies to establish the role of Affimers in bleeding and thrombosis.
Our work so far has shown that >70% of Affimers that modulate coagulation protein activity, other than PAI-1, have a similar effects in human and mouse plasma. Affimers of interest with equal effect in human and mouse plasma will further undergo in vivo testing. Mice will be anaesthetised and cannulae placed in the jugular vein followed by injection of selected Affimers. Simultaneously, femoral vessels will be dissected and thrombosis induced by application of filter paper soaked in ferric chloride for 3 minutes with serial images taken to quantify thrombus size. In a separate set of experiments, tail bleeding time will be measured in anaesthetised animals after injecting Affimers of interest with scaffold only protein used as control in all the in vivo experiments.
The risk of atherothrombotic events in diabetes subjects is increased and the prognosis following vascular ischaemia remains poor despite advances in treatment. Hypofibrinolysis is one of the mechanisms implicated in increased thrombosis risk in diabetes, which is partially mediated by raised PAI-1 levels. Using both human and animal studies, the proposed work comprehensively investigates the role of inhibiting PAI-1, using a novel methodology, in modulating hypofibrinolysis, which may have future therapeutic implications in high vascular risk conditions.
Yarmolinsky J, Bordin Barbieri N, Weinmann T, Ziegelmann PK, Duncan BB, Inês Schmidt M. Plasminogen activator inhibitor-1 and type 2 diabetes: a systematic review and meta-analysis of observational studies. Sci Rep. 2016 Jan 27;6:17714.
Kearney K, Tomlinson D, Smith K, Ajjan R. Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk. Cardiovasc Diabetol. 2017 Mar 9;16(1):34.
Tiede et al. Affimer proteins are versatile and renewable affinity reagents. Elife. 2017, 27;6, pii: e24903.
Role of Neutrophils in Fibrin Structure and Function: How do Cells, Fibrin and Neutrophil Extracellular Traps (NETs) Integrate in Thrombi and Blood Clots?
Supervisor(s): Robert Ariëns (Primary, LICAMM), Simon Connell (Physics), Helen Philippou (LICAMM)
Key words: Clot structure, thrombosis, neutrophils, NETs, stroke, heart attack
Blood clots that cause heart attacks and strokes are composed of fibrin, platelets and red blood cells. However, white blood cells are also incorporated into the clot, and their role in clot architecture and stability is poorly understood. Neutrophils are a type of white cell involved in innate immunity and are able to secrete a number of proteases such as elastases that are known to degrade fibrin. Furthermore, recent studies have shown neutrophils are capable of extruding their DNA and histones, producing an extracellular fibrous network that traps bacteria and pathogens (Neutrophil Extracellular Traps or NETs). Platelets have been implicated in NETs formation and NETs have been found in arterial and venous thrombi (Fig 1). Currently it is not fully known how these NETs interact with the fibrin network and how NETs influence formation of the clot, its function and its stability. Moreover, recent studies have shown that fibrin interacts with red blood cells via a b3 integrin receptor, and with platelets via the a2bb3 and GPVI receptors. Fibrin further binds endothelial cells via integrin receptors. It is not known if fibrin also binds neutrophils, but fibrin is known to interact with NETs (Longstaff C, JBC 2013). This project will focus on the 1) Interaction of neutrophils with fibrin and the consequences of this interaction on clot formation and stability, 2) Role of NETs in regulating fibrin elastic properties and resistance to fibrinolysis, and 3) Investigation of how NETs incorporate into the clot network in vivo.
Text Box: Fig. 1. Neutrophils when activated by PMA undergo chromatin decondensation leading to the expulsion of DNA and histone (Neutrophil Extracellular Traps or NETs), which trap bacteria, and interact with platelets and fibrin, playing a role in immunothrombosis and deep vein thrombosis. The interaction of NETs with fibrin is poorly understood. Image from Figure from Fuchs TA et al. ATVB 2012;32:1777-1783.
Fig. 1. Neutrophils when activated by PMA undergo chromatin decondensation leading to the expulsion of DNA and histone (Neutrophil Extracellular Traps or NETs), which trap bacteria, and interact with platelets and fibrin, playing a role in immunothrombosis and deep vein thrombosis. The interaction of NETs with fibrin is poorly understood. Image from Figure from Fuchs TA et al. ATVB 2012;32:1777-1783.
The activation of neutrophils in the vicinity of fibrin formation leads to the incorporation of NETs into the fibrin network, which leads to a denser clot architecture, increases clot stiffness, reduces elastic properties of the clot, increases clot resistance to fibrinolysis and worsens thrombosis.
The role of neutrophils and NETs in thrombosis provides an important link between inflammation and blood coagulation but is hitherto poorly understood. This PhD project will elucidate the role of neutrophils and NETs in clot formation, and may lead to the identification of novel therapeutic targets for the treatment of thrombosis.
Plan of Investigation
The plan of investigation will focus on 3 main objectives: 1) Interaction of neutrophils with fibrin and the consequences of this interaction on clot formation and stability, 2) Role of NETs in regulating fibrin elastic properties and resistance to fibrinolysis, and 3) Investigation of how NETs incorporate into the clot network in vivo.
Neutrophils will be obtained from blood by standard density gradient centrifugation methods. Neutrophil interactions with fibrin(ogen) will be investigated at a single molecule interaction level by AFM force spectroscopy. Fibrinogen will be coupled to AFM tips using amine-coupling methods as previously described (Carvalho FA, ACS Nano 2010) and used to probe washed neutrophils. We will also probe neutrophil fibrinogen interactions with optical tweezers recently set up in our laboratory. In some of the experiments, fibrinogen will be converted to fibrin by bathing the tip in thrombin. In addition, neutrophils will be activated with C5a, LPS or PMA prior to probing of fibrin(ogen) binding. Binding kinetics will be investigated using plate-binding assays.
The role of NETs in regulating clot elastic properties (Young’s modulus, G’, and loss tangent, G’’) will be investigated using magnetic tweezers. Fibrin clots will be made from fibrinogen and from plasma in the presence of PMA stimulated neutrophils to induce NETs formation. Clot stiffness and viscous properties will be investigated using magnetic tweezers as previously described (Domingues M, Blood 2016; Allan P, JTH 2012). The candidate will also analyse the effects of NETs on fibrin intrafibrillar structure to investigate whether NETs influence protofibril packing of the fibres or whether NETs integrate with fibrin at the fibre level. Intrafibrillar structure will be investigated using turbidimetric analysis of protofibril packing and high-resolution cold-field scanning electron microscopy as previously described (Domingues M, Blood 2016). The effects on overall clot network structure will be investigated by confocal microscopy and permeation analysis. Finally, rates of fibrinolysis by tPA and plasminogen will be investigated using standard laser scanning confocal microscopy and turbidity measurements.
The role of NETs interaction with fibrin during in vivo thrombosis will be investigated using murine models of thrombosis (Duval C, ATVB 2016). The candidate will develop a novel restricted blood flow thrombosis model based on ligation of the murine vena cava, which previously has been shown to induce thrombosis and fibrin deposition accompanied by inflammation and NETosis (Brill A, JTH 2012). The murine vena cava will be exposed through dissection under general anaesthesia. Ligatures will be placed around the vena cava, with a small-diameter hypodermic needle as spacer. The spacer will be removed and the ensuing thrombosis occurs over 3-5 days post-intervention. The animals will be sacrificed and the thrombi obtained for analysis of fibrin and NETs using immunohistochemical analysis. We will also explore the use of light sheet microscopy to image the thrombi for NETs and fibrin.
The candidate will be trained in Leeds for in vitro methods of clot structure and function, including turbidity, permeation, laser scanning confocal microscopy and scanning electron microscopy. We have a number of research staff (post-doctoral research fellows and research technicians) who will provide the training in these methods. Furthermore, the candidate will be trained in AFM force spectroscopy, magnetic tweezers and turbidimetric analysis of protofibril packing. We have a biophysical post-doctoral research fellow (Dr Stephen Baker) and another PhD student who are well versed with these interdisciplinary methods and will provide training. All these methods have been set up and the expertise is available in house. For the in vivo skills, the candidate will be trained in Leeds, where we have set up our murine thrombosis models using FeCl3 and laser injury of the vessel wall, and in which thrombus formation is analysed by intravital microscopy. For this studentship, we will establish a new model for venous thrombosis that is based on restriction of blood flow in the vena cava by placing ligatures, which leads to the development of venous thrombus over the course of 3-5 days. NETs have been shown to be part of the ensuing thrombus in this model.
Fit with Current On-going Research
Thrombosis is a leading cause of death worldwide and recent studies show an important role for clot structure and function in determining thrombosis risk. Our group focuses on novel mechanisms regulating clot structure and stability with the aim to develop novel diagnostics and therapeutics (BHF programme led by Ariëns). This studentship will cement existing interactions and create a new link between in vivo studies (Ariëns, Philippou) and molecular physics (Connell). This studentship will combine the area of expertise in haemostasis and thrombosis (Ariëns, Philippou) with that in Nanoscale biophysics (Connell).
Fit with Overall Cardiovascular Research Strategy
Cardiovascular disease and thrombosis are one of the major causes of disability and mortality in the ageing population worldwide. Older people are particularly at risk of developing stroke, heart disease or venous thrombosis. However, with increases of the knowledge of the disease processes and advances in surgical procedures, repair and recovery from cardiovascular disease is improving. This research project is focused on elucidating the mechanisms that link inflammation with thrombosis and that determine the structure and function of the blood clot or thrombus. This knowledge and expertise will lead to advances in the early diagnosis and treatment of cardiovascular disease, and improve repair from this devastating disease. Treatments for thrombosis are available but have problems. There is a high risk of bleeding in patients on anticoagulation, even with the newer direct oral anticoagulant inhibitors. Furthermore, treatment of ischaemic stroke with tPA to lyse the fibrin clot is effective only in a proportion of the patients, for reasons that are unknown. A better understanding of the structure of the blood clot and the mechanisms that link inflammation with thrombosis may indicate new direct ways to treat thrombosis, increase efficacy and reduce unwanted side effects such as bleeding.
Fuchs TA, Brill A, Duerschmied D, Schatzberg D, Monestier M, Myers DD Jr, Wrobleski SK, Wakefield TW, Hartwig JH, Wagner DD. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci U S A. 2010 Sep 7;107(36):15880-5.
Longstaff C, Varjú I, Sótonyi P, Szabó L, Krumrey M, Hoell A, Bóta A, Varga Z, Komorowicz E, Kolev K. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem. 2013 Mar 8;288(10):6946-56.
Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012 Jan;10(1):136-44.
Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol. 2012 Aug;32(8):1777-83.
Carvalho FA1, Connell S, Miltenberger-Miltenyi G, Pereira SV, Tavares A, Ariëns RA, Santos NC. Atomic force microscopy-based molecular recognition of a fibrinogen receptor on human erythrocytes. ACS Nano. 2010 Aug 24;4(8):4609-20.
Domingues MM, Macrae FL, Duval C, McPherson HR, Bridge KI, Ajjan RA, Ridger VC, Connell SD, Philippou H, Ariëns RA. Thrombin and fibrinogen γ‘ impact clot structure by marked effects on intrafibrillar structure and protofibril packing. Blood. 2016 Jan 28;127(4):487-95.
Allan P, Uitte de Willige S, Abou-Saleh RH, Connell SD, Ariëns RA. Evidence that fibrinogen γ‘ directly interferes with protofibril growth: implications for fibrin structure and clot stiffness. J Thromb Haemost. 2012 Jun;10(6):1072-80.
Duval C, Ali M, Chaudhry WW, Ridger VC, Ariëns RA, Philippou H. Factor XIII A-subunit V34L variant affects thrombus cross-linking in a murine model of thrombosis. Arterioscler Thromb Vasc Biol. 2016 Feb;36(2):308-16.
Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA, Wagner DD. Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 2012 Jan;10(1):136-44.
Missing data methods for large and complex data - multiple imputation and latent variable approaches
Supervisor(s): Baxter P, Gilthorpe M
Key words: Missing data, multiple imputation, statistical epidemiology
Summary: Missing data undermines the potential of routinely recorded patient data to identify mechanisms of disease and predict outcomes1. Researchers currently rely on the use of multiple imputation2,3 (MI) to mitigate the problems caused by missing data. However, for MI to be effective, imputation models need to be specified correctly for every variable that is incomplete4. For large and complex datasets, this is difficult if not impossible to achieve given the large numbers of variables to select from, all with potential issues of nonlinearity, interaction and collinearity. Instead, we propose the novel approach of partitioning data into a number of well-populated subgroups where each subgroup incurs minimal non-linearity, collinearity and interaction and default MI can then be applied. Latent Class5 (LC) methods offer a partitioning approach that can be semiautomated and implicitly accounts for uncertainty in the overall partition construct. In this proposal we develop the LCMI method. This PhD project will evaluate robustness and computational efficiency of the process through the use of simulations and analysis of observational data for risk prediction scoring (e.g. of survival, recurrence or morbidity).
Sterne JA, White IR, Carlin JB, Spratt M, Royston P, Kenward MG et al. Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls. BMJ 2009; 338:b2393
White IR, Royston P, Wood AM. Multiple imputation using chained equations: Issues and guidance for practice. Stat Med 2011; 30(4):377-399
van Buuren S, Groothuis-Oudshoorn K. mice: Multivariate Imputation by Chained Equations in R. Journal of Statistical Software 2011; 45(3):1-67
von Hippel PT. How to Impute Interactions, Squares and Other Transformed Variables. Sociological Methodology 2009; 39(1):265-291
Mplus [Los Angeles, CA]: Muthén & Muthén; 2014
Structural insights into the pharmacological modulation of cardiovascular ion channels.
Supervisor(s): Dr Robin S. Bon, Prof. David J. Beech and Dr Stephen P. Muench
Key words: ion channels, pharmacology, chemical biology, structural biology, cryo-electron microscopy, mass spectrometry
Summary: Many human diseases result from abnormalities in the function of ion channels, and many successful therapeutic drugs work by activating or blocking ion channels. Our research focuses on understanding the roles in cardiovascular health and disease of ion channels formed by the proteins TRPC4 and TRPC5. In order to study specific TRPC4/5 channel isoforms in cells, tissues and animal models, specific small molecule modulators with known molecular mechanisms of action are required. Despite >15 years of research in the field, the development of such molecules is hindered by the lack of structural insights into TRPC4/5 small molecule binding sites. We previously discovered that the small molecule Pico145 potently inhibits different TRPC4/5 channels, and that it has some selectivity for heteromeric TRPC1/4 channels over other TRPC4/5 channels. In this project, we will study how Pico145 interacts with different TRPC4/5 channels in molecular detail.
We will use Pico145 analogues that can chemically react with TRPC4/5 channels, and use mass spectrometry to identify the sites of these reactions. In addition, we will use cryo-electron microscopy to determine the three-dimensional structures of TRPC4/5 channels and their complexes with Pico145.
These results will reveal how specific TRPC4/5 channels can be regulated by small molecules. This will enable future development of small molecules that target specific TRPC4 or TRPC5 channels, which will enable detailed studies of TRPC4/5 biology and future TRPC4/5 drug discovery projects.
Rubaiy HN, Ludlow MJ, Henrot M, Gaunt HJ, Miteva K, Cheung SY, Tanahashi Y, Hamzah N, Musialowski KE, Blythe NM, Appleby HL, Bailey MA, McKeown L, Taylor R, Foster R, Waldmann H, Nussbaumer P, Christmann M, Bon RS, Muraki K, Beech DJ. Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels. J Biol Chem. 2017 May 19;292(20):8158-8173.
Rubaiy HN, Ludlow MJ, Bon RS, Beech DJ. Pico145 - powerful new tool for TRPC1/4/5 channels. Channels 2017 Sep 3;11(5):362-364.
Naylor J, Minard A, Gaunt HJ, Amer MS, Wilson LA, Migliore M, Cheung SY, Rubaiy HN, Blythe NM, Musialowski KE, Ludlow MJ, Evans WD, Green BL, Yang H, You Y, Li J, Fishwick CW, Muraki K, Beech DJ, Bon RS. Natural and synthetic flavonoid modulation of TRPC5 channels. Br J Pharmacol. 2016 Feb;173(3):562-74.
Bon RS, Beech DJ. In pursuit of small molecule chemistry for calcium-permeable non-selective TRPC channels -- mirage or pot of gold? Br J Pharmacol. 2013 Oct;170(3):459-74.
Myocardial perfusion, interstitial fibrosis and cardiomyocyte volume in the development of cardiac hypertrophy measured with contrast-enhanced MRI
Supervisor(s): David Buckley, John Greenwood & John Biglands
Key words: magnetic resonance imaging, mathematical modelling, physics, computing.
To assess the sensitivity of contrast-enhanced MRI to changes in cardiomyocyte volume.
Recent pre-clinical studies have indicated that in addition to measures of myocardial perfusion and interstitial fibrosis, contrast-enhanced MRI may be sensitive to changes in cardiomyocyte volume due to limitations in the rate of water exchange across the cell wall . This is a potentially useful measure of early cardiac hypertrophy but the sensitivity and precision of such measurements has not yet been established.
Simulation studies will be undertaken to determine the sensitivity of dynamic contrast-enhanced MRI to changes in cardiomyocyte volume. The effect of multiple contrast agent injections and changes in the way in which the MRI data are acquired will be assessed. Once an optimized imaging protocol is designed, MRI studies will be performed in volunteers and subsequently in patients before and after transcatheter aortic valve implantation (TAVI). Estimates of myocardial perfusion, cardiomyocyte and interstitial volume will be obtained from those data.
The project will produce recommendations for optimized MR imaging protocols to measure cardiomyocyte volume and assess the effect of TAVI on the parameters measured.
Coelho-Filho OR, Mongeon FP, Mitchell R, Moreno H, Jr., Nadruz W, Jr., Kwong R, et al. Role of transcytolemmal water-exchange in magnetic resonance measurements of diffuse myocardial fibrosis in hypertensive heart disease. Circulation: Cardiovasc Imaging. 2013;6(1):134-41.
Assessment of the relationship between myocardial microstructure and microvascular perfusion using cardiac magnetic resonance: novel imaging biomarkers to risk stratify patients with cardiomyopathy
Supervisor(s): Erica Dall’Armellina, Irvin Teh, Sven Plein, Jurgen Schneider
Key words: HYPERTROPHIC CARDIOMYOPATHY, FIBRES DISARRAY, PERFUSION, DTI/IVIM
The spatial interaction between the vessels and the arrangement of the cardiomyocytes is crucial to maintain an efficient delivery of oxygen to the cells. Current imaging techniques allow for the assessment of myocardial blood flow as indirect measurement of perfusion but lack to quantify the actual efficiency of oxygen delivery in the context of the 3D myocardial architecture. Knowledge of this interrelationship is particularly crucial in cardiac disease such as hypertrophic cardiomyopathy (HCM), which is characterized by cardiomyocytes disarray and myocardial perfusion abnormalities due to dysfunction of the microcirculation. Recent technical developments in diffusion-weighted Magnetic Resonance Imaging could potentially allow for investigating the perfusion system in relation to cardiomyocyte architecture in more detail and provide new biomarkers of cardiac disease. This proposal aims to establish a novel diffusion weighted technique (i.e. Intra-Voxel Incoherent Motion – IVIM) to assess myocardial perfusion in relation to the myocardial micro-architecture. We propose to validate the technique on the isolated perfused rat heart; we will then establish baseline parameters and reproducibility in volunteers. We finally will investigate the clinical utility of the IVIM biomarkers in HCM patients, although novel IVIM biomarkers may also allow for more accurate risk stratification in other cardiac diseases.
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Identifying the risks of cardiovascular, metabolic and other health outcomes following treatment for childhood and young adult cancer and their care pathways: a population-based data linkage study.
Supervisor(s): Feltbower RG, Gale CP, Hall M, Glaser AW.
Key words: Epidemiology, long-term outcomes, data linkage, cancer registration.
Summary: Previous work has begun to describe the risks of cardiovascular disease appearing as long-term adverse health effects following treatment for childhood and young adult cancer in Yorkshire, UK (1). This has been undertaken through electronic linkage between the Yorkshire Specialist Register of Cancer in Children and Young People (2) and hospital episode statistics (HES) data (1). We propose to develop this analysis further by incorporating more up-to-date information on HES admissions for the Yorkshire cohort, examine the risks of cardiovascular and metabolic disease (3) and describe the clinical pathways leading up and beyond the cardiovascular diagnosis to understand the management of these conditions.
The supervisory team comprises cancer and cardiovascular epidemiologists, statisticians and clinicians alongside experts in data linkage through whom you will develop both your clinical, epidemiological and data analytic skills.
van Laar M, Feltbower RG, Gale CP, Bowen DT, Oliver SE, Glaser A. Cardiovascular sequelae in long-term survivors of young peoples‘ cancer: a linked cohort study.. Br J Cancer 2014; 110: 1338-1341.
Fairley L, Stark DP, Yeomanson D, Kinsey SE, Glaser AW, Picton SV, Evans L, Feltbower RG. Access to principal treatment centres and survival rates for children and young people with cancer in Yorkshire, UK. BMC Cancer 2017; 17: 168.
Bright CJ, Hawkins M, Guha J, Henson KE, Winter D, Kelly JS, Feltbower RG, Hall M, Cutter DJ, Edgar AB, Frobisher C, Reulen RC. Risk of Cerebrovascular Events in 178,962 5-Year Survivors of Cancer Diagnosed Aged 15-39 Years: The Teenage and Young Adults Cancer Survivors Study (TYACSS). Circulation 2017; 135: 1194-1210.
Computational phenotyping of cardiovascular disease following acute myocardial infarction in primary care: a large scale electronic healthcare record study
Supervisor(s): Dr Marlous Hall, Professor Chris Gale
Key words: acute myocardial infarction, long-term disease trajectories, electronic health record data, epidemiology
Summary: Cardiovascular disease remains the leading cause of death worldwide, contributing to 30% of all deaths globally. Presently, most patients with acute myocardial infarction (AMI; heart attacks) are elderly and more patients are living longer following AMI due to increased use of guideline recommended care. To date, there are no large scale population based studies providing high resolution insights into the healthcare burden following AMI. Such large scale population based studies are increasingly important as they provide insights into patterns of care and outcomes for ‘real world’ populations in clinical settings, unlike the strictly controlled environments of randomised clinical trials.
This project will apply advanced statistical techniques to large scale electronic health record data from primary care to discover the multimorbidity disease profiles of patients following AMI. Such disease profiles may result in the development of novel disease phenotypes (so called ‘data-driven computational phenotypes’) which can inform future clinical trial designs as well as lead to development of clinical guidelines in areas where representative trials spanning a number of multimorbid conditions are unlikely or impractical.
The student will work within a multidisciplinary team of statisticians, epidemiologists and clinicians under the overarching Cardiovascular Survivorship theme. The project offers the opportunity to lead on, and contribute to, high impact peer-reviewed publications (for example [1-3]), as well as the opportunity to further develop advanced applied epidemiological and data analytical skills for the effective, efficient and clinically relevant use of data to ultimately improve patient outcomes.
M Hall, TB Dondo, AT Yan, MA Mamas, AD Timmis, JE Deanfield, T Jernberg, H Hemingway, KAA Fox, CP Gale. Multimorbidity and survival for patients with acute myocardial infarction in England and Wales: Latent class analysis of a nationwide population-based cohort. Plos Medicine [In Press]
TB Dondo, M Hall, RM West et al. Beta-Blockers and mortality after acute myocardial infarction in patients without heart failure or ventricular dysfunction. Journal of the American College of Cardiology (2017) 69(22):2710-2720.
M. Hall, TB Dondo, A. Yan et al. Association of clinical factors and therapeutic strategies with improvements in survival following non ST-elevation myocardial infarction, 2003-2013. Journal of the American Medical Association (2016) 316(10):1073-1082.
The role of NLRP family proteins in the development of the oocyte and preimplantation embryo
Supervisor(s): John Huntriss (1st), Helen Picton (2nd).
Key words: Oocyte, preimplantation embryo, genomic imprinting, NLRP proteins (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing Proteins), NLRP5, DNA methylation, gene expression.
Summary: NLRP proteins (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing Proteins) are members of the NLR (Nod-like receptors) protein family. NLRPs are expressed abundantly in mammalian oocytes. For example, NLRP5 (also known as MATER-maternal antigen that embryos require) is a key protein within the mammalian subcortical maternal complex (SCMC), a multiprotein complex uniquely expressed in mammalian oocytes and early embryos that has multiple functions including spindle formation, positioning of the meiotic spindle, the regulation of translation, and epigenetic reprogramming of early embryos . Notably, NLRP5 is essential for epigenetic regulation, since in humans, mutations in the NLRP5 gene have been associated with reproductive wastage and Multilocus Imprinting Disturbance (MLID) . NLRP5 is also essential for early embryo development in other mammals [3,4] and appears to be involved in a number of important processes in the oocyte, including a potential role in the formation of the cytoplasmic lattice (CPL), an essential structure in the oocyte .
This PhD studentship aims to establish the precise role(s) of at least one member of the NLRP protein familyin the regulation of mammalian oogenesis and embryogenesis. The bovine reproductive model will be used as a model for understanding their role in epigenetic processes in humans. The favoured target is the NLRP5 gene. The functional impact of NLRP5 knock-down on epigenetic programming will be studied in the bovine oocyte and preimplantation embryo by knocking the gene down in oocytes using DsiRNAs. Changes in the oocyte and embryonic transcriptome and the embryonic DNA methylome will be assessed to reveal gene pathways and regions of the genome that are affected upon NLRP5 depletion. Embryonic development after knockdown will be assessed using for example, time-lapse techniques and/or other established metrics of embryo development. The project will also map the expression patterns of NLRP familytranscripts and proteins in bovine tissues, ovarian follicles, oocytes and preimplantation embryos.
The student will receive training in a range of cellular and molecular techniques that are applicable across many themes in biomedical research. These include molecular biology, including transcriptome analysis (for example: real time PCR, cDNA library generation from single oocytes/embryos and single cells, RNA sequencing), DNA methylation analysis (for example: pyrosequencing, reduced representation bisulfite sequencing). In addition the student will be trained in techniques in reproductive biology, (for example: oocyte in vitro maturation (IVM), microinjection, in vitro fertilization (IVF) and preimplantation embryo culture). In summary, these experiments will attempt to dissect the role of NLRP proteins in mammalian oogenesis and preimplantation development.
Bebbere D, Masala L, Albertini DF, Ledda S. The subcortical maternal complex: multiple functions for one biological structure? J Assist Reprod Genet. 2016 33(11):1431-1438.
Docherty LE, Rezwan FI, Poole RL et al. Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans. Nat Commun. 2015 1;6:8086.
Peng H, Liu F, Li W, Zhang W. Knockdown of NLRP5 arrests early embryogenesis in sows. Anim Reprod Sci. 2015 163:151-6.
Maternal depletion of NLRP5 blocks early embryogenesis in rhesus macaque monkeys (Macaca mulatta). Wu X. Hum Reprod. 2009 24(2):415-24.
Kim B, Kan R, Anguish L, Nelson LM, Coonrod SA. Potential role for MATER in cytoplasmic lattice formation in murine oocytes. PLoS One 2010 7;5(9):e12587.
The role of RNA methylation in the development of the oocyte and preimplantation embryo.
Supervisor(s): John Huntriss (1st), Helen Picton (2nd)
Key words: RNA methylation, N6-methyladenosine (m6A) RNA methylation, epitranscriptomics, oocytes, preimplantation embryo, gene expression.
Summary: RNA methylation is a form of epitranscriptomic modification that modifies several RNA properties including stability, translation, splicing, transport and localization . N6-methyladenosine (m6A) RNA methylation is the most prominent mark on messenger RNA (mRNA) and is known to regulate mRNA stability , making it a good candidate mark for the regulation of mRNA transcripts during the oocyte to embryo transition. A number of players in the m6A RNA methylation machinery have been identified. Thus, N6-methyladenosine (m6A) RNA methylation is mediated by the ‘writers’ METTL3, METTL14, WTAP, which are RNA methyltransferases that methylate adenosine residues within RNA molecules to form m6A. Further, m6A is ‘erased’ by RNA demethylases FTO and ALKBH5 and m6A marks are ‘read’ by YTHDF1, YTHDF2 and YTHDF3. Collectively, these factors control gene expression by virtue of their involvement in m6A RNA methylation . There is growing evidence of an essential role of RNA methylation in reproduction, during gametogenesis and early development. For example, mice deficient in the RNA demethylases ALKBH5 have increased m(6)A in their mRNA and consequently have impaired fertility and altered expression of transcripts regulating spermatogenesis . Very recently, the RNA m6A reader YTHDF2 has been shown to be essential for oocyte competence and the post-transcriptional regulation of the oocyte transcriptome .
This PhD studentship aims to establish the precise role(s) of at least one member of the RNA methylation machinery and will hence explore the role of epitranscriptomic processes in the regulation of mammalian oogenesis and embryogenesis. The bovine reproductive model will be used as a model for human reproduction. The favoured targets for knockdown are currently METTL14, WTAP, and YTHDF3. The functional impact of gene knock-down will be studied in the bovine oocyte and preimplantation embryo by knocking the gene(s) down in oocytes using DsiRNAs. Changes in the oocyte and embryonic transcriptome and, if possible, the m6A RNA methylome will be assessed to reveal gene pathways that are affected to hopefully give valuable insight into the role of RNA methylation in reproduction. Embryonic development after knockdown will be assessed using for example, time-lapse techniques and/or other established metrics of embryo development. The project will also map the expression patterns of m6A RNA methylation machinery gene transcripts and proteins in bovine tissues, ovarian follicles, oocytes and preimplantation embryos.
The student will receive training in a range of cellular and molecular techniques that are applicable across many themes in biomedical research. These include molecular biology, including transcriptome analysis (for example: real time PCR, cDNA library generation from single oocytes/embryos and single cells, RNA sequencing). In addition the student will be trained in techniques in reproductive biology (for example: oocyte in vitro maturation (IVM), in vitro fertilization (IVF), preimplantation embryo culture and microinjection). In summary, these experiments will attempt to dissect the role of (m6A) RNA methylation machinery in mammalian oogenesis and preimplantation development.
Liebers R, Rassoulzadegan M, Lyko F. Epigenetic regulation by heritable RNA. PLoS Genet. 2014 10(4):e1004296.
N6-methyladenosine-dependent regulation of messenger RNA stability. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C. Nature. 2014 505(7481):117-20.
Meyer KD, Jaffrey SR. The dynamic epitranscriptome: N6-methyladenosine and gene expression control. Nat Rev Mol Cell Biol. 2014 15(5):313-26.
Zheng G, Dahl JA, Niu Y, Fedorcsak P, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013 49(1):18-29.
Ivanova I, Much C, Di Giacomo M, Azzi C. The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence. Mol Cell. 2017 67(6):1059-106.
Dissecting the role of endothelial insulin-like growth factor-1 signalling in the vascular and metabolic complications of obesity-induced type 2 diabetes mellitus.
Supervisor(s): Mark Kearney Katherine Paradine
Key words: Diabetes, obesity, IGF-1, angiogenesis
Summary: In extensive pilot studies we have discovered a previously unrecognised role for endothelial cell (EC) insulin-like growth factor-1 (IGF-1) in the pathophysiology of obesity-induced type 2 diabetes. We have shown that reducing expression of IGF-1 receptors (IGF-1R) in EC leads to favourable changes in white and brown adipose tissue (WAT and BAT) in the response to nutritional obesity. We show that reducing EC IGF-1R enhances WAT angiogenesis, increases expression of BAT genes in WAT, and also leads to a favourable potentially anti-atherosclerotic metabolic profile. Consistent with this, we have shown that increasing EC IGF-1R has a potentially disadvantageous effect on metabolic profile. We now plan to examine: 1) the effects of long-term deletion of EC IGF-1R on the metabolic and vascular response to nutritional obesity. 2) the effects of increasing EC IGF-1R in the vascular and adipose tissue response to nutritional obesity.
Cubbon RM et. al. Endothelial IGF-1 Receptor signalling in Diabetes and insulin resistance. Trends Endocrinol. Metab. 2016; 27: 96-104.
Abbas A et. al. The Insulin-Like Growth Factor-1 Receptor Is a Negative Regulator of Nitric Oxide Bioavailability and Insulin Sensitivity in the Endothelium. Diabetes 2011;60, 2169-2178.
Yuldasheva NY et. al. Haploinsufficiency of the insulin-like growth factor-1 receptor enhances endothelial repair and favourably modifies angiogenic progenitor cell phenotype. Arterioscler. Thromb. Vasc. Biol. 2014; 34, 2051-2058.
Imrie H et. al. Novel role of the IGF-1 receptor in endothelial function and repair: studies in endothelium-targeted IGF-1 receptor transgenic mice. Diabetes 2012; 61: 2359-2368.
Small molecules to manipulate insulin and IGF-1 signaling
Supervisor(s): Prof MT Kearney and Dr K J Simmons
Key words: Insulin Signalling, Insulin Receptor, IGF1 Receptor, homology model, virtual high-throughput screening, Bioluminescence Resonance Energy Transfer Assay, Electron Microscopy
Summary: Type 2 diabetes mellitus- where the body produces insufficient amounts of insulin or the insulin that is produced does not function properly to control blood glucose-is an increasingly common disorder. Around 2.9 million people in the UK are affected by diabetes and there are estimates of another 850 000 undiagnosed cases. Insulin resistance, which precedes the development of type 2 diabetes by many years, is also an independent risk factor for the development of premature cardiovascular disease.
It is well established that IGF-1R and IR are able to form hybrid receptors consisting of an IR αβ heterodimer and an IGF1R αβ heterodimer. IR: IGF1R stoichiometry is a critical determinant in endothelial cell insulin sensitivity, nitric oxide bioavailability and vascular repair, the mechanism of which being a negative effect of the IGF-1R on insulin signaling by forming insulin resistant hybrid receptors with IR. (1)
Using a homology model of the IR:IGF-1R hybrid structure(2, 3) and hotspot prediction software to predict the key regions involved in hybrid dimer formation together with virtual high-throughput screening tools(4, 5), you will identify small molecules which are predicted to inhibit formation of the IR:IGF-1R hybrids. You will screen these using a Bioluminescence Resonance Energy Transfer assay(6) to measure the interaction of the IR and IGF-1R monomers and thereby quantify the action of small molecule probes on selectively inhibiting dimer formation.
Structural studies to characterize the IR:IGF1R dimer structure and its perturbation by small molecules, will also be carried out using Electron Microscopy(7).
Gatenby, V.K. Imrie, H. and Kearney, M. The IGF-1 receptor and regulation of nitric oxide bioavailability and insulin signalling in the endothelium. Pflugers Archiv : European journal of physiology. 2013, 465(8), pp.1065-74.
Croll, T.I. Smith, B.J. Margetts, M.B. Whittaker, J. Weiss, M.A. Ward, C.W. and Lawrence, M.C. Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain. Structure. 2016, 24(3), pp.469-76.
Yang, J. Yan, R. Roy, A. Xu, D. Poisson, J. and Zhang, Y. The I-TASSER Suite: protein structure and function prediction. Nature methods. 2015, 12(1), pp.7-8.
Sterling, T. and Irwin, J.J. ZINC 15-Ligand Discovery for Everyone. J Chem Inf Model. 2015, 55(11), pp.2324-2337.
Halgren, T.A. Murphy, R.B. Friesner, R.A. Beard, H.S. Frye, L.L. Pollard, W.T. and Banks, J.L. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem. 2004, 47(7), pp.1750-1759.
Blanquart, C. Gonzalez-Yanes, C. and Issad, T. Monitoring the activation state of insulin/insulin-like growth factor-1 hybrid receptors using bioluminescence resonance energy transfer. Molecular pharmacology. 2006, 70(5), pp.1802-11.
Smith, M.T. and Rubinstein, J.L. Structural biology. Beyond blob-ology. Science. 2014, 345(6197), pp.617-9.
The role of matricellular proteins in notch signalling and cell survival after myocardial infarction.
Supervisor(s): Azhar Maqbool, Neil Turner and Mark Drinkhill
Key words: Notch, cardiac fibroblasts, myocardial infarction, Tenascin C
Summary: Studies using genetic ablation or activation of Notch signalling have demonstrated that Notch signalling, plays a critical role in cardiac repair and regeneration after myocardial injury. The activation of Notch signalling suppresses cardiomyocytes apoptosis, minimizes fibrosis, increases neovascularization and improves cardiac function and outcome after myocardial infarction (MI). Hence, modulation of Notch signalling may serve as a tool to limit ventricular remodelling and dysfunction after injury. The matricellular protein, tenascin C (TNC) is upregulated following cardiac injury where it plays a role in tissue remodelling. TNC can activate Notch signalling in tumour tissue promoting both survival and growth, however, its role in Notch-mediated cardio-protection in the injured heart remains unexplored. This study aims to investigate the effect of TNC on Notch mediated protective signalling in the heart following injury. Studies will be conducted on isolated cardiac fibroblasts and myocytes as well as mouse models of myocardial injury.
Natalie Gude, and Mark Sussman. Notch signaling and cardiac repair. J Mol Cell Cardiol. 2012 Jun; 52(6): 1226–1232.
Yuxin Li, Yukio Hiroi and James K. Liao. Notch Signaling as an Important Mediator of Cardiac Repair and Regeneration after Myocardial Infarction. Trends Cardiovasc Med. 2010 Oct; 20(7): 228–231.
Imanaka-Yoshida K. Tenascin-C in cardiovascular tissue remodeling: from development to inflammation and repair. Circ J. 2012; 76(11):2513-20.
Maqbool A et al. Interleukin-1 has opposing effects on connective tissue growth factor and tenascin-C expression in human cardiac fibroblasts. Matrix Biol. 2013; 32: 208-14.
Maqbool A et al. Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4. World J Cardiol. 2016; 26; 8: 340–50.
Turner NA. Inflammatory and fibrotic responses of cardiac fibroblasts to myocardial damage associated molecular patterns (DAMPS). J Mol. Cell. Card 2016; 94:189-200
Turner NA, Porter KE. Function and fate of myofibroblasts after myocardial infarction. Fibrogenesis & Tissue Repair 2013; 6:5.
The role of Rab46 in cardiovascular disease
Supervisor(s): Dr Lynn Mckeown; Dr Bernadette Moore; Dr Mar Pujades Rodriguez
Key words: endothelial, Rab GTPases, calcium, Weibel Palade body, inflammation
Endothelial cells line the blood vessels and play a central role in vascular function. Weibel Palade bodies (WPBs) are specialized storage vesicles found specifically in endothelial cells (1). These vesicles contain a pool of readily made cargo that, upon release, control vascular events such as haemostasis, inflammation, angiogenesis and vascular tone. Inappropriate release of WPB cargo into the blood vessel lumen results in the pro-inflammatory and pro-thrombotic environment associated with cardiovascular disease. My research involves understanding the mechanisms underlying the release of WPB cargo and how these become perturbed in cardiovascular disease. Recently, we described a novel Rab GTPase (Rab46) in endothelial cells that has GTPase and Ca2+ binding activities and is located on WPBs (2)(2). Thereby, this research project involves a range of techniques including;- cell culture, siRNA transfection, calcium imaging, biochemistry, MRI and confocal microscopy, to address the role of Rab46 in WPB trafficking and endothelial cell secretion. In addition, we will investigate how mutations in this protein may contribute to cardiovascular diseases and explore the association of Rab46 with other inflammatory diseases using data from the UK Biobank.
Weibel, E. R. (2012) Fifty years of Weibel-Palade bodies: the discovery and early history of an enigmatic organelle of endothelial cells. Journal of thrombosis and haemostasis : JTH 10, 979-984
Wilson, L. A., McKeown, L., Tumova, S., Li, J., and Beech, D. J. (2015) Expression of a long variant of CRACR2A that belongs to the Rab GTPase protein family in endothelial cells. Biochemical and biophysical research communications 456, 398-402
Non-mendelian effects on offspring phenotype: origins and characterisation of small RNAs of sperm from mice.
Supervisor(s): David Miller, Karen Forbes, Julie Aspden (FBS)
Key words: Sperm RNA, inter (trans) generational inheritance, epigenetics, non-mendelian.
Recent evidence points to small non-coding RNAs carried by the fertilising sperm, conveying epigenetic information to offspring that can affect phenotype. While this mode of transmission is likely, we do not know whether the epigenetic information is introduced to the sperm from the testis, epididymis or accessory glands (prostate and seminal vesicles). This project will initially look for differences in small (s) non (n) coding (c) sncRNA content in the proximal (caput), intermediate and distal (cauda) sperm of experimental mice that will tell us whether their RNA content is influenced by transport across the epididymis. This will be done with one or more experimental models in current use on unrelated projects, hence minimising the use and waste of animals. Sperm will also be recovered from the reproductive tracts of dames after removal of post-coital plugs, giving us samples exposed to all male reproductive secretions. In parallel, sncRNA from the epididymides, seminal vesicles and prostate will be isolated and characterised. Differences in sncRNA content between caput, intermediate and cauda sperm will tell us whether some RNAs are introduced via the epididymis (the most likely conduit for soma to germline transmission). Furthermore, mapping the sncRNAs of epididymides (washed free of sperm), prostate and seminal vesicles will help refine identification of the main signalling RNAs and their likely sources. We anticipate screening males from several experimental studies making use of interventions (dietary or injection) that can affect offspring phenotype.
Yuan S, Schuster A, Tang C, Yu T, Ortogero N, Bao J, Zheng H, Yan W. Sperm-borne miRNAs and endo-siRNAs are important for fertilization and preimplantation embryonic development. Development 2016;143:635-647.
Chen Q, Yan M, Cao Z, Li X, Zhang Y, Shi J, Feng GH, Peng H, Zhang X, Zhang Y et al. Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 2016;351:397-400.
Gapp K, Jawaid A, Sarkies P, Bohacek J, Pelczar P, Prados J, Farinelli L, Miska E, Mansuy IM. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Nature neuroscience 2014;17:667-669.
Rassoulzadegan M, Grandjean V, Gounon P, Vincent S, Gillot I, Cuzin F. RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Nature 2006;441:469-474.
The role of platelet dysfunction in promoting diabetic thrombo-inflammation.
Supervisor(s): Khalid Naseem, Ramzi Ajjan
Key words: diabetes, insulin resistance, platelets, Inflammation, interleukin 1b
Insulin resistance (IR), defined as resistance to insulin-stimulated glucose uptake, is a common precursor to both diabetes and associated vascular disease. IR is a multi-system disorder that is characterised by multiple metabolic and cellular alterations including atherogenic dyslipidemia, glucose intolerance and inflammation. In subjects with Type 2 diabetes (T2D) immune cells are transformed into a proinflammatory state that is associated with increased production of the cytokine interleukin 1b (IL-1b). 1 This proinflammatory cytokine is deposited in the vascular wall of atherosclerotic coronary arteries in humans and promotes several atheroclertoic processes in mice 2. These observations are the basis for a large scale clinical trial (CANTOS) targeting IL-1b with a human monoclonal antibody to reduce of the incidence of myocardial infarction.
Subjects with type 2 diabetes (T2D) have increased circulating levels of platelet-monocyte complexes that are known to drive vascular inflammation. 3 However, the precise role of platelet-mediated inflammation in T2D is unclear. Recent evidence suggests platelet driven inflammation proceeds through the ability to modify their proteome. Experiments performed in vitro demonstrate that platelets produce proteins that potentially influence the thromboinflammatory process including IL-1b. 4 In immune cells IL-1bis generated through the activation of inflammasomes, multimeric protein signaling complexes that trigger the activation of caspase-1 and maturation of IL-1b. The most widely characterised of these is the NOD-like receptor (NLR)P3 inflammasome and is proposed to be expressed in human platelets 5.
In this project you will establish the molecular mechanisms that facilitate platelet IL-1b production and determine if the capacity for platelet IL-1bsynthesis is elevated in patients with T2D. The project provides excellent opportunity to gain training in a wide range of cell and molecular biology techniques, including immunoblotting, immunoprecipitation, flow cytometry, immunofluorescence, super-resolution microscopy and in vivo thrombosis assays.
Lee, H.-M. et al. Upregulated NLRP3 Inflammasome Activation in Patients With Type 2 Diabetes. Diabetes 62, 194–204 (2013).
Kaptoge, S. et al. Inflammatory cytokines and risk of coronary heart disease: new prospective study and updated meta-analysis. Eur Heart J 35, 578–589 (2014).
Ferroni, P., Basili, S., Falco, A. & Davì, G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2, 1282–1291 (2004).
Lindemann, S. et al. Activated platelets mediate inflammatory signaling by regulated interleukin 1β synthesis. J Cell Biology 154, 485–490 (2001).
Abderrazak, A. et al. NLRP3 inflammasome: From a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases. Redox Biology 4, 296–307 (2015).
Unravelling the unusual secretory pathway of blood clotting factor XIII-A
Supervisor(s): Richard Pease, Paul Cordell
Key words: Transfection, mutagenesis, super-resolution microscopy, primary cell isolation.
Clotting factor XIII-A is a member of the transglutaminase family of enzymes. Transglutaminases introduce covalent cross-links between other proteins, and in the case of FXIII-A, it cross-links the chains of fibrinogen to one another to stabilize blood clots. Transglutaminases are unusual in that, while they can be secreted from cells, they lack the signals required to direct them to the ER-Golgi pathway. Thus, they are among a small group of proteins that undergo unconventional secretion. Recent studies have begun to clarify the pathways by which a few other unconventionally secreted proteins are released from cells, and we intend to apply the approaches that have emerged to understand the secretion of FXIII-A. Our recent studies have suggested that vascular macrophages secrete FXIII-A into the blood-stream and we will concentrate on isolating these cells, and establishing in vitro culture models under which cells adopt the characteristics of vascular macrophages.
Association of coagulation factor XIII-A with Golgi proteins within monocyte-macrophages: implications for subcellular trafficking and secretion. Cordell PA et al (2010); Blood. 115 2674-81.
Cre/lox Studies Identify Resident Macrophages as the Major Source of Circulating Coagulation Factor XIII- A Beckers CML et al (2017);, Arteriosclerosis, Thrombosis, and Vascular Biology. 37 1494-1502
Bioengineering and evaluation of artificial ovaries for fertility preservation
Supervisor(s): Prof Helen Picton, Dr John Huntriss, and Dr Virginia Pensebene
Key words: ovary, follicle, oocyte, fertility preservation, nanotechnology, microfluidics, metabolomics, imprinting
Summary: In recent years the improvement in the diagnosis, management and treatment of a range of solid and haematological malignancies has lead to a marked increase in the chances of long-term survival for a significant number of children and adolescents. Unfortunately germ cells, like the cancer cells that are their intended targets, are highly susceptible to damage by radiation and by the alkylating agents and platinum compounds that are commonly used in chemotherapy formulations. Thus the radiotherapy and chemotherapies used to cure many cancers can render patients of either sex or any age temporarily or even permanently infertile. This adverse side effect of cancer treatment is particularly relevant to young girls who, in the event of ovarian failure, are faced with the prospects of life long hormone replacement therapy- a regime that is not, it, risk free. Health care professionals are therefore faced with the challenge of not only developing and improving the technologies that will treat the cancer and protect the individual’s wellbeing, but they must also implement strategies that will conserve the fertility of these young patients.
The current options available to preserve the fertility of young female cancer patients range from no medical intervention at all to the use of invasive procedures to harvest and freeze-store tissues or isolated cells. Assisted reproductive techniques can be used for the collection and storage of mature oocytes (eggs) and embryos. However these approaches are only suitable for patients of reproductive age and it is clearly not an option for most adolescents or for any prepubertal girls. An alternative approach that is suitable for both children and adults is the cryopreservation of ovarian tissue. Ovarian tissue cryopreservation involves freezing the earliest staged primordial oocytes in situ either through freezing small pieces of the surface of the ovary or by freezing the whole organ. The tissue fragments or ovary can then be transplanted back to the patient when she is in remission and wishes to start her family. Unfortunately, ovarian autografting is not suitable for patients with blood bourn diseases such as leukemia or steroid related cancers as these diseases carry a high chance of ovarian metastasis and there is therefore a high risk of reintroducing the cancer to the body through the transplanted tissue. Where any such risk exists a safer alternative for the restoration of fertility is to grow the immature oocytes in the ovarian tissue to maturity in the laboratory through a process known as the in vitro growth (IVG) of follicles. Once the eggs are fully grown they can then be matured by in vitro maturation (IVM) and fertilised using in vitro fertilisation (IVF)technologies so that only embryos that are free from cancer cells are transferred back to the patient to establish a pregnancy.
The development of technologies for the in vitro derivation of fertile oocytes from the most abundant primordial follicles holds many attractions for assisted conception, in humans and for animal production technology. Any culture strategy designed to support the complete IVG of oocytes must mimic the developmental sequence of events and cellular checkpoints seen in vivo in terms of follicle and oocyte growth rates, gene expression patterns and molecular milestones and metabolic requirements. During their extended growth phase oocytes progressively synthesise and accumulate the payload of proteins and RNAs that are required to support both nuclear and cytoplasmic maturation of the fully-grown oocytes and early post-fertilisation development of the zygote. Finally, there are stage specific changes in DNA methylation of the genes associated with genomic imprinting during oocyte growth and early embryo development. The team at Leeds has recently developed a physiologically relevant, 3D culture strategy for follicle that supports the complete in vitro development of oocytes over extended periods of up to 3-4 months. While it is possible to grow oocytes from primordial stages using our multiphase 3D approach, the system is inefficient, extended open culture systems are at risk of contamination, and the health and genetic normality of the oocytes so derived remain to be tested. The complexity of the tissue and the need for a tightly controlled culture environment during IVG may be resolved using a microfluidic centred culture approach. Using Organ on chip (OoC) microfluidic devices, cells are perfused in μm-sized chambers to more closely replicate organ physiology in vivo than conventional disaggregation cultures in vitro. The stage is therefore set to test whether an ovary OoC microfluidic device can be developed and used to enhance the health and developmental competence of in vitro-derived oocytes from ovarian tissues.
This Ph.D. project will therefore develop and test an OoC model of the ovary as a vehicle to support the complete in vitro production and fertilisation of oocytes. The Ph.D. will address the hypothesis that “Organ on Chip models can be used to support the in vitro production and fertilisation of healthy oocytes from primordial follicles in vitro”.
The Reproduction and Early Development Research Group in the LICAMM Institute and have been instrumental in establishing the technology to grow oocytes from large animals and humans to maturity in the laboratory (Prof Picton) but also to test their health and genetic normality (Dr Huntriss). Dr Pensebene has a background in nanotechnology she has developed ultrathin membranes to support in vitro cell growth and to repair fetal membrane in utero. She has developed the OoC model of the endometrium for studying the effect of environmental exposure to bacteria and dioxin on the establishment and development of pregnancy.
This project aims to combine the recent advances in nanotechnology, microfluidics, and follicle and oocyte culture to test the health and efficiency of complete IVG, IVM and IVF of immature eggs derived from fresh ovarian tissues. The proposed experiments will ultimately test the developmental capacity of IVG/IVM oocytes derived using either: (a) an in-house, open and highly defined multi-step, physiological, follicle culture system which takes 40-60 days to complete; or (b) a novel, closed microfluidic ovary OoC co-culture strategy. A series of end point assays will be used to evaluate the health and developmental competence of oocytes derived using both approaches. Follicle and oocyte size will be measured and growth rates recorded. Follicle development and culture stress will be assessed by measuring follicle and oocyte metabolism and waste product production in samples of spent culture media. The capacity of in vitro-derived oocytes to complete meiotic maturation and produce fertile gametes will ultimately be assessed using methods for IVM and IVF. Embryo developmental potential will be quantified by culturing embryos for 7 days and recording embryo cleavage rates, blastocyst production and blastocyst cell number. Tissues will be archived throughout the cultures for later molecular analysis. All of the technologies for IVG, IVM and IVF of sheep oocytes, together with analytical techniques used to assess growth and fertility are in routine use in the Leeds laboratory. Due to the limited availability of human ovarian tissue for research, sheep ovaries and oocytes will be used as a model for human ovary/oocytes through the course of this Ph.D. The novel ovary OoC microfluidic device will be fabricated in Polydimethylsiloxane (PDMS) and biocompatible polymers using soft lithography and spin coating deposition techniques. Fabrication and assembly of the devices will be carried out at the School of Electronic and Electrical Engineering in a state of the art class 100 clean room.
Molecular evaluation of the health and normality of in vitro-derived oocytes. Samples of oocytes, embryos and follicular somatic cells will be archived from Experiment 1. These samples will be used for quantification of the expression of key gene markers of oocyte (e.g. Gdf-9, Bmp15) and follicle development (e.g. Fshr, Lhr, Amh, Has2, Egfr, Amphiregulin, Epiregulin, Ptx2) and embryo health and imprinting status (e.g. Oct4, H19, Igf2r, Snrpn). All of the techniques required for single egg and embryo RNA and DNA extraction, amplification and molecular analysis are fully validated and are in routine use in the Leeds laboratory.
Fabrication and testing of the novel ovary OoC microfluidic device. Cultures will be conducted to test the utility of our new microfluidic device as a vehicle to co-culture (i) ovarian stroma; (ii) ovarian cortex; (iii) preantral ovarian follicles and (iv) cumulus oocyte complexes over extended periods. Replicate cultures will be conducted for each combination of cell types. Viable cell number and parameters of follicle and oocyte growth and maturity will be recorded. Follicle and oocytes morphology will be assessed histologically at the end of culture in fixed tissues.
Comparison of the efficacy of the open, multi-phase 3D culture system and the ovary OoC for the in vitro production of oocytes. This experimental series will directly compare the 2 culture strategies as vehicles for the complete IVG of follicles and oocytes. Follicle growth dynamics, the capacity for oocytes from both sources to undergo IVM, IVF and embryo development will be used to test the efficacy of both culture approaches. Oocytes and embryos will be archived for analysis using the range of molecular markers of oocyte development and normality as defined during Experiment 2.
This multidisciplinary project will predominantly be conducted in the Light laboratories. The ovary OoC will be fabricated at the School of Electronic and Electrical Engineering, assembled and characterized in the class 100 clean room. Cell loading and testing will be carried out in the LIGHT laboratories. In addition to the acquisition of generic skills in data analysis, presentation and thesis writing etc, the student will receive training in: (a) tissue culture methods (aseptic cortex harvest, follicle and oocyte isolation and culture, IVM, IVF and embryo culture; (b) metabolism assays; (c) molecular techniques (RNA isolation, real time PCR, cDNA generation, cloning and sequencing) and (d) microfluidic device fabrication and testing.
Anckaert E, De Rycke M, Smitz J.(2013) Culture of oocytes and risk of imprinting defects. Hum Reprod Update 19(1):52-66.
Chambers EL, Gosden RG, Yap C, Picton HM (2010). In situ identification of follicles in ovarian cortex as a tool for quantifying follicle density, viability and developmental potential in strategies to preserve female fertility. Hum Reprod. 2010 Oct; 25(10): 2559-68
Gnecco JS, Pensabene V, Li DJ, Ding T, Hui EE, Bruner-Tran KL, Osteen KG (2017) Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium. Ann Biomed Eng. 45(7):1758-1769
Huntriss J, Lu J, Hemmings K, Bayne R, Anderson R, Rutherford A, Balen A, Elder K, Picton HM (2017). Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos. J Assist Reprod Genet. 34(1):23-31
Huntriss J, Woodfine K, Huddleston JE, Murrell A, Picton HM (2015). Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®. Methods Mol Biol. 2015;1315:259-70. doi: 10.1007/978-1-4939-2715-9_19.
Labrecque R, Sirard MA (2014). The study of mammalian oocyte competence by transcriptome analysis: progress and challenges. Mol Hum Reprod 20(2):103-16.
Newton HL, Glaser AW, Picton HM (2017). Fertility preservation options in prepubertal females: feasibility, safety and outcomes. Minerva Ginecol. 69(6):568-586
Pensabene V, Costa L, Terekhov AY, Gnecco JS, Wikswo JP, Hofmeister WH (2016). Ultrathin Polymer Membranes with Patterned, Micrometric Pores for Organs-on-Chips. ACS Appl Mater Interfaces. 31;8(34):22629-36
Picton HM, Harris SE, Muruvi W, Chambers EL (2008). The in vitro growth and maturation of follicles. Reproduction. 136(6):703-15
Smitz J, Dolmans MM, Donnez J, Fortune JE, Hovatta O, Jewgenow K, Picton HM, Plancha C, Shea LD, Stouffer RL, Telfer EE, Woodruff TK, Zelinski MB (2010). Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation. Hum Reprod Update. 16(4):395-41
Stewart KR, Veselovska L, Kelsey G (2016) Establishment and functions of DNA methylation in the germline. Epigenomics. 8(10):1399-1413
The effect of inorganic nitrate on systemic energy homeostasis in a setting of perturbed energy balance: implications for diabetes and obesity.
Supervisor(s): Dr Lee Roberts, Dr Scott Bowen (FBS)
Key words: Diabetes, Obesity, Metabolism, Physiology, Imaging
The World Health Organization estimates 500 million people worldwide are obese and over 340 million suffer from type 2 diabetes (T2D). The quality of life of these individuals and the burden to healthcare make the discovery of novel therapeutics or interventions imperative.
Inorganic nitrate is found in many dietary foodstuffs, with a high concentration observed in green leafy vegetables. Nitrate was considered a non-bioactive metabolite of NO and a potentially toxic dietary constituent1. However, nitrate has since been shown to reduce blood pressure and the oxygen demand of exercise; indicating that this anion may be beneficial for metabolic health2,3. Recent complementary studies demonstrated that nitrate treatment led to anti-obesity and anti-diabetic effects in endothelial NOS-deficient mice, a strain prone to a metabolic syndrome–like phenotype4. Moreover, studies conducted by the Dr Roberts and co-workers have identified that nitrate has distinct metabolic effects on both skeletal muscle and adipose tissue; nitrate increased mitochondrial biogenesis and fatty acid oxidation in skeletal muscle (SKM), activated the thermogenic programme in brown adipose tissue (BAT), and induced the “browning response”, a switch from a white adipose like-phenotype to a brown adipose-like phenotype in subcutaneous white adipose tissue (WAT)5-7. The metabolic effects of nitrate in adipose and skeletal muscle indicate increased energy expenditure within these independent tissues. Increasing energy metabolism may be a viable therapeutic strategy for treating obesity and T2D, however the systemic effect of nitrate on whole body energy metabolism in a setting of perturbed energy balance, as seen in obesity is, as yet, unknown.
Aim: This PhD project will examine the physiological effects of inorganic nitrate on whole-body systemic energy balance and metabolism in dietary induced models of obesity and type 2 diabetes.
Project :A multidisciplinary approach, employing metabolic and in vivo imaging techniques, will be utilized to phenotype the effects of inorganic nitrate on systemic energy homeostasis in dietary-induced mouse models of obesity and type 2 diabetes. Employing the state-of-the-art preclinical imaging facility (which includes high-field MRI, optical imaging, CT and PET/CT) within the Leeds Institute of Cardiovascular and Metabolic Medicine, in vivo longitudinal measures of body composition and BAT, WAT and SKM metabolic activity in the mouse models will be made (MRI, PET/CT). Metabolic phenotyping will include determination of whole body metabolic phenotypes using an advanced Comprehensive Lab Animal Monitoring System (CLAMS), which enables the accurate measurement of weight, food and water intake, activity, indirect calorimetry and respiratory quotient and exchange ratios using specialized metabolic cages. Glucose and insulin tolerance tests will be employed to monitor glucose homeostasis.
PhD training opportunities
The project will suit a student with a background in physiology, biochemistry or related discipline with a strong interest in diabetes and metabolism. The multidisciplinary project provides training in cutting-edge physiological / metabolic phenotyping and imaging techniques.
Moncada, S. The L-arginine: nitric oxide pathway, cellular transduction and immunological roles. Adv Second Messenger Phosphoprotein Res 28, 97-9 (1993).
Larsen, F.J., Ekblom, B., Sahlin, K., Lundberg, J.O. & Weitzberg, E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med 355, 2792-3 (2006).
Larsen, F.J., Weitzberg, E., Lundberg, J.O. & Ekblom, B. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol (Oxf) 191, 59-66 (2007).
Carlstrom, M. et al. Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice. Proc Natl Acad Sci U S A 107, 17716-20 (2010).
Roberts, L.D. et al. Inorganic nitrate promotes the browning of white adipose tissue through the nitrate-nitrite-nitric oxide pathway. Diabetes 64, 471-84 (2015).
Roberts, L.D. et al. Inorganic Nitrate Mimics Exercise-Stimulated Muscular Fiber-Type Switching and Myokine and gamma-Aminobutyric Acid Release. Diabetes 66, 674-688 (2017).
Ashmore, T. et al. Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism. BMC Biol 13, 110 (2015).
Role of clotting changes in cardiovascular events induced by air pollution
Supervisor(s): Dr Michael Routledge & Prof Robert Ariens
Key words: Airborne particulate matter, PM2.5, traffic emissions, coagulation, vascular endothelial cells, acute myocardial infarction
Summary: There is increasing evidence that exposure to fine particulate matter (PM2.5) present in air pollution can trigger acute events such as myocardial infarction and deep vein thrombosis in susceptible individuals. We have previously shown that PM2.5 can induce changes in blood clot structure and function (1,2) and induce pro-coagulant response in vascular endothelial cells (3). The aims of this project will be to further explore these changes, determine the mechanisms by which they are induced and use a molecular epidemiology approach to studying changes in susceptible individuals exposed to ambient levels of PM2.5.
Metassan S, Charlton AJ, Routledge MN, Scott DJA and Ariens RAS (2010) Alteration of fibrin clot properties by ultrafine particulate matter. Thrombosis and Haemostasis 103, 103-113.
Pan X, Gong YY, Martinelli I, Angelici L, Favero C, Bertazzi PA, Mannucci PM, Ariens RAS and Routledge MN (2016) Fibrin clot structure is affected by levels of particulate air pollution exposure in patients with venous thrombosis. Environment International 92-93, 70-76.
Pan X, Gong YY, Xu Y, Ariens RS and Routledge MN (2017) Urban Particulate matter induces changes in gene expression in vascular endothelial cells that are associated with altered clot structure in vitro. Thrombosis and Haemostasis, in press.
Development of novel techniques for detection and quantification of stroke biomarkers
Supervisor(s): Dr Sikha Saha, Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine, University of Leeds.
Prof Gin Jose, School of Chemical and Process Engineering, Faculty of Engineering, University of Leeds.
Prof Paul Millner, School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds.
Key words: Stroke, Biomarker, Laser spectroscopy, Rare earth ions, Nanoparticles
Summary: Stroke is the leading cause of death worldwide, with high medical costs and rates of long-term disability. Although brain imaging remains the gold standard for diagnosis of stroke, computerised tomography (CT) and magnetic resonance imaging (MRI) scan are expensive and time consuming. Rapid identification of ischemic stroke allows clinicians to treat patients with the only available thrombolytic drug, tissue plasminogen activator (tPA). A number of biomarker panels have been evaluated for the diagnosis of stroke in human patients with variable results. Among these markers neuron-specific enolase (NSE), protein S100B, and glial fibrillary acidic protein (GFAP) have been shown promising.
The main aim of this PhD project is to develop a single step optical biosensor platform for rapid analysis of diagnostic and prognostic biomarkers panel which will enable specific quantification within a few minutes.
This will involve the bioreceptors modified to chelate lanthanide ions (e.g. europium) where analyte binding causes fluorescence quenching or bioreceptor-functionalised nanoparticles that show fluorescent up-conversion where near infra-red excitation gives visible light emission and analyte binding enhances emission. The sensors can be used for multi-analyte detection with a simple add and measure protocol. The student will be based in the Division of Cardiovascular and Diabetic Research, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds and will get training in laser spectroscopy, bioconjugation and functionalization of lanthanides / nanoparticles, cell culture, electron microscopy, ELISA and other relevant techniques. The student will have advantage in working in a multidisciplinary environment including Biologist, Biochemist and Engineer.
Maas MB and Furie KL Molecular biomarkers in stroke diagnosis and prognosis Biomark Med. 2009 3(4): 363–383.
Jickling GC. & Sharp FR. Blood Biomarkers of Ischemic Stroke Neurotherapeutics (2011) 8:349–360
Development and characterisation of a novel human dynamic in vitro Blood Brain Barrier model to explore the effects of amyloid β-peptide on the barrier integrity
Supervisor(s): Dr Sikha Saha, Leeds Institute of Cardiovascular and Metabolic Medicine, Faculty of Medicine, University of Leeds.
Dr Virginia Pensabene, School of Electronic & Electrical Engineering, Faculty of Engineering, University of Leeds.
Key words: Blood brain barrier, endothelial cells, astrocytes, pericytes, Alzheimer disease, amyloid β-peptide
Alzheimer‘s disease (AD), which is defined by memory deficits and cognitive impairment, is the most common form of dementia in the elderly with more than 26 million people worldwide living with the disease. AD is characterized, among other pathological features by the accumulation of beta-amyloid peptides (Aβ peptides) in the brain. Aβ peptides are thought to contribute to synaptic dysfunction and neuro-inflammation in the brain and play a significant role in the blood brain barrier (BBB) dysfunction during the course of AD. At present, there are not proper human in vitro blood brain barrier models, which mimic physiological human conditions, and the effects of each Aβ fragment on the individual BBB cells as well as BBB integrity remains unknown.
The main aim of this pilot project is to improve a novel human in vitro dynamic BBB model to examine the differential effects of specific Aβ peptide fragments (e.g., Aβ 1-40 and Aβ25-35) in both static and dynamic conditions. We will focus on the effect of each fragment over each cell type implicated in the BBB formation and transport. This will help us to design further experiments to study the role of BBB implication in AD in a more realistic way, which could lead to the development of new therapies for this devastating disease. The student will get training on cell culture, microfluidic system, immunohistochemistry, Western Blotting, and other relevant techniques. The student will participate to weekly lab meeting and journal club to share their work with the group. The student will have advantage in working in a multidisciplinary environment and gain experience in presentation and publication of research work.
Pflanzner T, Kuhlmann CR, Pietrzik CU. Blood-brain-barrier models for the investigation of transporter- and receptor-mediated amyloid-beta clearance in Alzheimer‘s disease. Curr Alzheimer Res 2010 Nov;7(7):578-590.
Zlokovic BV. Clearing amyloid through the blood-brain barrier. J Neurochem 2004 May;89(4):807-811.
Gosselet F, Saint-Pol J, Candela P, Fenart L. Amyloid-beta peptides, Alzheimer‘s disease and the blood-brain barrier. Curr Alzheimer Res 2013 Dec;10(10):1015-1033.
Endothelial Piezo1 channels of human placenta and their physiological roles
Supervisor(s): Shi J, Morley L, Ludlow M, Simpson N, Beech D
Key words: Ion channel, Physiology, cardiovascular research
Summary: Piezo1 is an intriguing newly discovered membrane protein. Three Piezo1s assemble to form a trimeric Ca2+-permeable non-selective cationic channel which senses membrane tension. We showed that Piezo1 channels are critical mechanical force sensors of cardiovascular biology with special roles in vascular development and long-term implications for cardiovascular health and disease. In a seminal article we found that disruption of endothelial Piezo1 in mice caused growth retardation from embryonic day 9.5. Moreover, endothelial cells of Piezo1-disrupted mice failed to respond to the frictional mechanical force of shear stress and reconstitution of Piezo1 channels in otherwise resistant cells conferred shear stress sensing (1, 2). We concluded that Piezo1 channels are critical sensors of shear stress driven by the newly beating heart, transducing this force into vascular maturation and organ perfusion. Furthermore we found that in adult physiology Piezo1 channels sense physical activity to advantageously reset vascular control (3). It has since been found that PIEZO1 mutations are linked to abnormalities in human lymphatic vascular development and non-immune hydrops fetalis. Therefore endothelial Piezo1 channels are important in human as well as mouse vascular development.
The aim of this project is to determine the properties of single Piezo1 channels in endothelial cells freshly-isolated from human placentas of women who delivered babies of normal or low birth weight. The studies will test the hypothesis that functional abnormalities in Piezo1 channels are a factor underlying intrauterine growth restriction.
The main techniques used in the studies will be methods for isolation of fresh endothelial cells, patch-clamp single channel and membrane potential recordings, methods for application of mechanical force, complementary biochemical and immunofluorescence approaches, and isobaric contraction recordings. Single channel recording is a specialized technique which yields highly quantitative and precise data about the properties of channels – it will be the primary focus of the main 3-year project.
The project will involve the student in an exciting collaborative project between basic scientists at the university (Jian Shi, Melanie Ludlow and David Beech) and medical doctors at the adjacent teaching hospital (Lara Morley and Nigel Simpson).
Li J et al., Piezo1 integration of vascular architecture with physiological force. Nature. 2014, 515(7526):279-82.
Li J et al., Endothelial Piezo1: life depends on it. Channels (Austin). 2015;9(1):1-2
Rode B et al., Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat Commun. 2017: 8:350
Development of 3D bioprinted tissue samples to validate quantitative magnetic resonance imaging biomarkers.
Supervisor(s): Steven Sourbron, Susmita Basak, Kerrie Smith
Key words: 3D bioprinting, Magnetic Resonance Imaging, Imaging Biomarkers, Relaxometry, Diffusion, Microstructure Imaging, Tissue function
Background: Advanced Magnetic Resonance Imaging (MRI) methods provide an opportunity for non-invasive measurement of biological tissue properties such as microvessel size, fibrosis, oxygenation, inflammation, perfusion, cell transporter expression, fat fraction and many others [1,2]. However the measurement relies on the complex interaction between magnetic fields and protons in biological tissue, and determining the accuracy and specificity of these so-called imaging biomarkers is a challenge. As a result there is currently a real risk of misinterpretation, and this can have significant consequences if patient management strategies are based on these data. The common approach to validating MRI biomarkers uses experimental correlations against invasive measurements in animal models of disease . However, a thorough understanding cannot be derived in this way due to the difficulty of isolating individual variables and controlling the precise microstructure of the samples.
Objectives:The purpose of this project is to develop and characterize 3D bioprinted materials  that can serve as reference objects to aid in the interpretation and validation of MRI biomarkers. A range of materials with various degrees of complexity will be created and extensively characterized using MRI, microscopy and digital pathology. The scalability and reproducibility of the approach will be tested by repeated measurements on independent samples. MRI biomarkers will be validated by comparison against microscopic reference values and theoretical predictions for a range of tissue parameters.
Workplan: We will start with simple materials and increase the complexity stepwise. All MRI scanning will be performed on a state-of-the-art 7T preclinical MRI scanner. Initially we will work with simple homogeneous cell suspensions in an extracellular matrix with varying properties, including for instance different cell types, different ratios of extracellular fluid, and changes in the extracellular composition using for instance MRI contrast agents added to the bio-ink. These samples will be extensively characterized with MR relaxometry (T1, T2, T2* including multi-compartmental analyses to determine water exchange across the cell walls), diffusion MRI with varying diffusion times and waveforms tor probe microstructure. In a second phase we will extend this to more tissue-like structures including blood vessels and perfused tissues which will include the possibility to selectively modify intrinsic tissue properties such as oxygenation and blood flow. In a final phase we will also look at specialized functional tissue types, in particular renal and liver tissue, and aim to extract functional measurements such hepatocellular uptake and excretion rates or renal tubular reabsorption fractions.
This is a largely experimental project suitable for a student with a strong technical background and an interest in biophysics or medical engineering.
J.P.B. O’Connor et al. Imaging Biomarker Roadmap for Cancer studies. Nat Rev Clin Oncol 2017; 14: 169-186.
A. Stadlbauer et al. Magnetic resonance imaging biomarkers for clinical routine assessment of microvascular architecture in glioma. J Cereb Blood Flow Metab 2017; 37(2): 632-43.
I. Friedli et al. New Magnetic Resonance Imaging Index for Renal Fibrosis Assessment: A Comparison between Diffusion-Weighted Imaging and T1 Mapping with Histological Validation. Scientific Reports 2016; 6: 30088.
S.V. Murphy and A. Atala. 3D bioprinting of tissues and organs. Nature Biotechnology 2014; 32: 773-85.
Developing a virtual MRI biopsy of the liver through multiscale computational modelling of MRI signal formation.
Supervisor(s): Steven Sourbron, Susmita Basak, Darren Treanor
Key words: Magnetic Resonance Imaging, Imaging Biomarkers, Computational Modelling, Digital Histopathology, Chronic Liver Disease
Liver disease is the only major cause of mortality and morbidity which is on the increase in England. Appropriate management can significantly improve patient outcomes, but choosing the right management is difficult due to a lack of non-invasive biomarkers that identify progression early. A biopsy provides a detailed assessment but this is a highly invasive procedure that cannot be repeated all too often. Magnetic Resonance Imaging (MRI) biomarkers have a great potential to fill this gap as they are highly sensitive to pathophysiological changes, are fully non-invasive and avoid sampling errors inherent to biopsy [1,2,3,4]. However, a key limitation is that they are also inherently unspecific. For instance it is currently impossible to differentiate between effects of inflammation and fibrosis, which is essential to inform management decisions. It is likely that multiple MRI biomarkers used in combination will be able to provide a higher specificity, but progress in this area is hampered by our poor understanding of the complex relationship between tissue microstructure and MRI signals.
The overall aim of this PhD project is to develop and validate a “virtual biopsy” method for liver disease using multiparametric MRI. Specifically, we will develop computational models that create a direct mechanistic link between tissue microstructure as seen on digital pathology [5,6] and MRI signals measured on the same samples. These models will then be used to identify and validate an inverse model that links selected MRI biomarkers to biological tissue characteristics. Ultimately this may remove the need for biopsy in some patients and improve the clinical management of liver disease.
This project will benefit from a powerful training- and test data set in patients with paired imaging-biopsy correlations. The data will be taken from a large ongoing MRC-funded project HEPARIM (Hepatectomy Risk Assessment with functional Magnetic Resonance Imaging) which aims to improve the outcomes of major liver resection through preoperative MRI. The study will collect data in >200 patients and paired histology data will be available from the resected liver segments.
Biopsy slides from a set of training data will be digitised, segmented and values of relevant tissue properties (T1, T2, proton density, water diffusion, susceptibility) will be assigned to the component structures (tubular fluid, blood, cells, interstitium, fibrosis, …). Then Monte-Carlo simulations of proton magnetisation will be performed on these structures that predict the measured macroscopic properties of the tissue (T1, T2, T2*, ADC, …). The model will be optimized and validated by comparison against the measured MRI properties of the same tissues. In a second step a hidden-variable model of the tissue microstructure will be developed that “interpolates” between the samples to model a continuum of tissue types and corresponding MRI parameters. The hidden-variable model will then be used to identify and validate a descriptive model that can predict histological fibrosis and inflammation scores from a selected combination of MRI biomarkers. The score will then be applied to a set of previously unseen test data and validated by comparison against the paired histopathology results.
Profile & Opportunities
The student will work in the area of computational physics or -biology with a multi-disciplinary team of MRI scientists, hepatologists, computer scientists and histopathologists. They will develop novel methods which are scientifically challenging but at the same time have a direct clinical relevance with potential for impact on practice in the short to medium term. The project is embedded in a prestigious MRC-funded project that will guarantee a wide visibility on the national and international stage. This will involve academic and industrial partners and thereby form a perfect stepping-stone for a future career in private- or public sector research.
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K Juluru, et al. Diagnostic accuracy of intracellular uptake rates calculated using dynamic Gd-EOB-DTPA-enhanced MRI for hepatic fibrosis stage. J Magn Reson Imaging 2017; 45(4): 1177-85.
R. Banerjee, et al. Multiparametric magnetic resonance for the non-invasive diagnosis of liver disease. J Hepatol 2014; 60(1): 69-77.
S. Sourbron, et al. Combined Quantification of Liver Perfusion and Function with Dynamic Gadoxetic Acid-enhanced MR Imaging. Radiology 2012; 263: 874-833.
N. Roberts, et al. Toward Routine Use of 3D Histopathology as a Research Tool. Am J Pathol 2012; 180(5): 1835-42.
Cardiovascular consequences of single nuclear polymorphisms in novel mechanosensing ion channel Piezo1.
Supervisor(s): Piruthivi Sukumar, Jian Shi, David Beech
Key words: Piezo1, endothelium, genetic mutations, UK Biobank, cardiovascular diseases
Mechanotransduction in biological systems is the conversion of mechanical stimuli into biological signals and is involved in the modulation of diverse cellular functions such as migration, proliferation, differentiation and apoptosis. Evidently, mechanotransduction is essential from embryo- and organo- genesis to growth and homeostasis. A newly discovered mechanosensitive transmembrane protein, Piezo1 is reported to be primarily responsible for mechanotransduction in diverse cells. We and others have identified that Piezo1 is essential for vascular development and health, blood pressure regulation and exercise related vascular contraction/relaxation. Recently, many gain and loss of function mutations in Piezo1 gene have been identified in a range of pathophysiological conditions including lymphatic dysplasia, anaemia and malarial resistance. While Piezo1 expression and importance is clearly shown by many investigators, Piezo1 mutations that can lead to cardiovascular consequences are not yet identified. The primary aim of this PhD study will be to identify single nucleotide polymorphisms (SNPs) in Piezo1 gene that are associated with cardiovascular diseases. About 500,000 imputed genomic data stored in the UK Biobank will be analysed using appropriate bioinformatics tools, to identify the prevalent SNPs. Furthermore, using in silico methods, SNPs will be characterised and suitable candidates will be selected for laboratory studies. Then the patho-physiological consequences of such mutations on cardiovascular health and disease will be explored using over expression system, CRISPR mediated introduction of mutations in in vitro cell culture and in vivo animal models.
With obesity reaching epidemic proportions worldwide, the resultant increase in diabetes, hypertension, myocardial infarction and stroke are the major causes of morbidity and mortality. Hence the search for better therapeutic targets for these complex, multifactorial diseases is never ending. All living organisms are subjected to mechanical forces from their environment and rely on mechanotransduction for their survival. The inner lining cells of all blood and lymphatic vessels, endothelial cells are constantly exposed to physical force i.e. shear stress produced by the beating heart and flowing blood/lymph. Hence they have pronounced mechanosensitivity which aid in development and maintenance of healthy vasculature. Although it is long known that multiple patho-physiological signalling pathways are regulated by physical forces in endothelial cells, our current knowledge is limited in understanding the sensory pathways of mechanical stimuli and their roles in cardiovascular diseases.
A recently discovered mechanosensitive transmembrane protein, Piezo1, expressed widely in various cells, is reported to be the primary mechanosensor protein in these cells too. Functional Piezo proteins were discovered in 2010, in a targeted short interfering RNA (siRNA) knock down analysis of candidate transmembrane proteins against mechanically activated ionic currents in the murine N2A neuroblastoma cell line. Its discovery has created huge opportunities in exploiting mechanotransduction induced signalling as diseases targets. Human Piezo1 encoded by the PIEZO1/FAM38A is a highly polymorphic gene with many coding variants reported in the 1000 Genomes Project database. Piezo1 mutations have been linked to multiple human disorders. Six of them are known to be gain-of-function mutations that slow the inactivation rate and are associated with dehydrated hereditary xerocytosis. Mechanistically, the mutant channels allow excess calcium influx into RBC leading to osmotically-driven dehydration. In contrast, several loss-of-function Piezo1 mutations occur in patients with generalised lymphatic dysplasia. Piezo1 channels are widely expressed throughout the cardiovascular system and have important roles in angiogenesis in developing embryos, blood pressure control and exercise mediated changes in blood flow distribution and hence can potentially have long-term implications for cardiovascular health and disease. Therefore, mutations in the Piezo1 gene affecting the protein function might play an important role in causation/progression of cardiovascular diseases (CVD). No such mutations are described so far in CVD.
The UK Biobank (http://www.ukbiobank.ac.uk/), a health research resource, recruited over 500,000 people aged 40 to 69 years in 2006–2010 across the UK. In July 2017, the genetic information from 501,708 samples was released which combined with the extensive information on medical history and lifestyle choices recorded gives an enormous opportunity to investigate how genetics and other factors impact the onset and development of diseases. This study aims to access the data from UK Biobank, screen for the Piezo1 gene variants/mutations which have associations with a range of CVD and then characterise the functional properties of these mutations, which may aid in the development of therapeutic agents targeting the mutants.
Mutations in mechanosensing nonselective cation permeable ion channel Piezo1 can lead to cardiovascular disease development/progression.
Identifying SNPs associated with cardiovascular diseases and further characterisation of candidate SNPs using in silico methods and, in vitro and in vivo models.
Objectives and Experimental Plans
Identifying and characterising SNPs associated with cardiovascular diseases in UK Biobank
The genetic data from UK Biobank will be analysed using genomic data analysis software packages (e.g. PLINK) using whole genome association analysis toolset that is designed to perform a range of basic, large-scale analyses in a computationally efficient manner. The focus will be purely on analysis of genotype/phenotype data. SNP index files will be procured from UK Biobank. Using an appropriate confidence interval, SNPs occurring in the locus of Piezo1 gene will be extracted and further downstream analysis will be performed. Then genetic data will be filtered on the basis of whether the subjects are suffering from any CVD or if they acquire any CVD later. Further, the Piezo1 SNPs in CVD patients with ischemic heart disease and hypertension and age matched controls will be analysed and the most frequent Piezo1 mutations or variants in CVD patients will be identified.
In the next stage, clinical and life style data such as blood pressure, biochemical markers, diet and exercise habits, will be analysed to find any associations between particular genetic variants and a wide range of CVD. Next, a variety of bioinformatics tools and approaches will be employed to predict functional effects of the identified SNPs. Bioinformatics tools include - PROVEAN (Protein Variation Effect Analyzer), PhD-SNP (Predictor of human Deleterious Single Nucleotide Polymorphisms), MutPred2 (for predicting pathogenicity of amino acid substitution), I-mutant (to estimate changes in stability arising due to single point mutation) and structural modelling methods. They will be utilised to predict and select the SNPs that would be of potential functional consequences for further investigation.
Elucidating the functional impact of selected SNPs in vitro
Using the above mentioned methods, 3-5 SNPs will be selected for further characterisation. By site directed mutagenesis, we will create those SNPs individually in human Piezo1 construct. HEK 293 cells in which native Piezo1 is knocked out stably will be used for over-expressing mutant Piezo1 and study the functional effects. Primarily, calcium influx using radiometric calcium indicator fluorescent dye and single channel current recordings using path-clamp technique will be used for functional studies. Based on those results, protein expression and trafficking studies will be conducted. Taking further the study into cardiovascular context, the selected mutations will be introduced into endothelial cells too by double transfection method (knocking out native Piezo1 by siRNA/shRNA and transfecting the mutant construct temporarily) or mutating the native Piezo1 using CRISPR technology. After checking the knockout and over-expression or mutations by mRNA/protein expression/sequence analysis, calcium response and current measurement, functional characteristics of endothelial cells such as proliferation, migration, tube formation and secretory activity will be studied.
Elucidating the functional impact of selected SNPs in vivo
Based on the results from the above objectives, one point mutation will be selected for in vivo studies. Using CRISPR technology, a transgenic line will be generated in which the mutation is introduced. Basic phenotyping and cardiovascular system specific phenotyping of the transgenic line will be performed to elucidate the functional impact of the mutation on whole body cardiovascular physiology and diseases development. In/ex vivo experiments will include blood pressure measurement, exercise testing, analysing propensity for atherosclerosis and vascular contraction studies. Depending on the findings, further studies on downstream signalling will be performed.
Short Methodology for non-routine experiments:
Fluorescence based measurement of calcium
For determining the Piezo1 mediated calcium entry in transfected HEK 293/endothelial cells, the raise in intracellular calcium ([Ca2+]i) level upon addition of Piezo1 modulators (e.g. Yoda1-activator; Dooku1-inhibitor) will be measured using calcium sensitive fluorescent indicator dye Fura-2 AM and state of the art automated plate reader called Flex Station (www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/flexstation-3-multi-mode-microplate-reader). Cells will be loaded with 2 M Fura-2 AM for 1 hour at 37°C and then washed in standard bath solution for 30 minutes at 37°C. Cells will be imaged in Flex Station with appropriate filters. Fura-2 is excitable with 340/380 nm light and emits at 510 nm and the ratio between its emission intensity at the two excitation wavelengths is directly proportional to [Ca2+]i. Flex station will be programmed to apply Piezo1 modulators as per the experimental plan at appropriate concentrations.
Patch-clamp recordings of Piezo1 channels in HEK 293 cells and endothelial cells
To determine the characteristics of Piezo1 channels overexpressed in HEK293 cells or endogenously expressed in endothelial cells, whole-cell and single-channel patch-clamp recordings will be used. Piezo1 plasmids will be transfected into the HEK 293 cells with lipofectamine transfection reagent. Endothelial cells will be freshly isolated from mouse second-order mesenteric arteries. The arteries will be enzymatically digested in dissociation solution containing collagenase (1 mg/ml) and then be pipetted gently to release the endothelial cells. For the patch-clamp recordings on these cells, heat-polished patch pipettes with tip resistances between 3 and 5 MΩ will be used and high resistance seals (≥5 GΩ) will be achieved through slight suction. The Piezo1-mediated non-selective cation currents on these cells will be recorded with Axopatch-200B amplifier equipped with Digidata 1550B and pCLAMP 10.6 software using whole-cell and single-channel configurations. The currents will be evoked by either the channel agonist Yoda1 or the mechanical force such as negative pressure. Then the current amplitude/channel kinetics and channel open probability will be analysed.
CRISPR for in vitro mutation studies
For introducing point mutation in endothelial cells, CRISPR technology will be employed. Either commercially obtained appropriate gRNA with Cas9 protein or plasmids with relevant inserts will be used to transfect the cells. The clonal selection and expansion will be done to get a stable cell line with required mutation.
Li, J., Hou, B., Tumova, S., Muraki, K., Bruns, A., Ludlow, M.J., Sedo, A., Hyman, A.J., McKeown, L., Young, R.S., et al. (2014). Piezo1 integration of vascular architecture with physiological force. Nature 515, 279–282.
Rode, B., Shi, J., Endesh, N., Drinkhill, M.J., Webster, P.J., Lotteau, S.J., Bailey, M.A., Yuldasheva, N.Y., Ludlow, M.J., Cubbon, R.M., et al. (2017). Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat. Commun. 8, 350.
Beech, D.J., and Xiao, B. (2018). Piezo channel mechanisms in health and disease: Editorial. J. Physiol. 596, 965–967.
Hyman, A.J., Tumova, S., and Beech, D.J. (2017). Piezo1 Channels in Vascular Development and the Sensing of Shear Stress. Curr. Top. Membr. 79, 37–57.
Computational approaches to understanding the mechanosensitive Piezo1 channel activation mechanism.
Supervisor(s): Antreas Kalli, Jian Shi, David Beech
Key words: Piezo1, mechanosensitive channels, molecular dynamics simulations, molecular modelling, cell membrane, shear stress
Mechanical forces play a very important role in the cardiovascular system as they regulate vascular functions such as endothelial cell alignment to blood flow, endothelium-dependent vasodilation and cardiac remodeling. For this reason, mechanical forces are important in a number of cardiovascular diseases. Despite some recent advancements, our understanding of how force is sensed at the molecular level by proteins in the cardiovascular system is limited. This project will focus on Piezo1 that is a critical mechanical sensor in endothelial cells, red blood cells and other cell types such as cardiac fibroblasts.
Piezo1 is a large protein and its function is to permeate ions in response to mechanical stimuli. Three recent electron microscopy (EM) structures showed that Piezo1 exists in a trimeric form in the membrane. The three Piezo1 subunits form a pore region with a C-terminal extracellular domain between the last two transmembrane helices. Whilst a number of recent structural and functional data increased our understanding of Piezo1 function, fundamental aspects of Piezo1 activation and ion transport remain largely unknown party due to their dynamic nature. Molecular dynamics simulations are a well-established technique that enables us to follow the dynamics of membrane proteins in a membrane environment. Therefore, it can provide detailed molecular and dynamic understanding of the function of membrane proteins. In this project, the student will use molecular dynamics simulations and molecular modelling to simulate Piezo1 in model membranes that resemble native membranes in which Piezo1 functions e.g. endothelia membrane and red blood cell membrane. The student will use these simulations to examine the Piezo1 activation and inactivation mechanisms and to investigate the role of the membrane environment in regulating Piezo1 function. The student will also use computational methodologies that were developed in the Kalli group to examine how disease-causing mutations may change Piezo1 activation. The models derived from the computer simulations will be evaluated/refined experimentally, using cell-based assays and molecular biology techniques in Beech/Shi groups.
The student will be a part of a multidisciplinary team that already studies Piezo1 and has extensive experience on how to study Piezo1 channel using both advanced computational and lab-based approaches. This project will suit anyone wishing to learn a range of complementary skills, including both computational and lab-based skills.
Kalli, A. C.; Reithmeier, R. A. F. Interaction of the Human Erythrocyte Band 3 Anion Exchanger 1 (AE1, SLC4A1) with Lipids and Glycophorin A: Molecular Organization of the Wright (Wr) Blood Group Antigen. PLoS Comput. Biol. 2018, 14 (7), e1006284.
Kalli, A. C.; Sansom, M. S. P.; Reithmeier, R. A. F. Molecular Dynamics Simulations of the Bacterial UraA H+-Uracil Symporter in Lipid Bilayers Reveal a Closed State and a Selective Interaction with Cardiolipin. PLoS Comput. Biol. 2015, 11 (3), e1004123.
Li, J.; Hou, B.; Tumova, S.; Muraki, K.; Bruns, A.; Ludlow, M. J.; Sedo, A.; Hyman, A. J.; McKeown, L.; Young, R. S.; et al. Piezo1 Integration of Vascular Architecture with Physiological Force. Nature 2014, 515 (7526), 279–282.
Li, J.; Hou, B.; Beech, D. J. Endothelial Piezo1: Life Depends on It. Channels 2015, 9 (1), 1–2.
Liver imaging – a novel imaging approach to assess early-stage cardiovascular disease.
Supervisor(s): Laeticia Lichtenstein, Marc Bailey, Khalid Naseem, Jurchen Schneider, David Beech
Key words: Ultrasound imaging, magnetic resonance imaging, intravital microscopy, light sheet microscopy, liver, Non-alcoholic fatty liver diseases, metabolic syndrome.
Summary: Cardiovascular disease (CAD) is responsible of 31% of the global death in the world (WHO data), and metabolic syndrome (MetS) is strongly associated with the risk of developing CAD. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and appears to be the liver expression of MetS1. Its prevalence is constantly increasing2 and associated with the key cardiovascular disease problem of atherosclerosis2. A cross-sectional Framingham Heart study revealed a strong association between markers of vascular endothelial dysfunction and NAFLD3.
Remarkably, NAFLD can evolve in 20% of the case into a more severe disease named non-alcoholic steatohepatitis (NASH) that is characterized by inflammation and that can later lead to development of hepatic fibrosis. The project aims to develop new pre-clinical imaging methodology for early detection of liver fibrosis. The applicant will study the kinetics of events in liver fibrosis using our state-of-the-art Experimental and Preclinical Imaging Centre (ePIC) in parallel with histological characterisation. Ultimately we expect the project will pave the way to new clinical imaging approaches for the detection of early-stage NAFLD. Unique opportunities for learning cutting-edge imaging and preclinical cardiovascular biology will be available.
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Regulation of human cardiac fibroblast function by specific microRNAs.
Supervisor(s): Dr Neil Turner, Dr Karen Forbes
Key words: heart, cardiac fibroblasts, microRNA, cell culture
The heart is made up of several different cell types with the majority being either muscle cells (cardiomyocytes) or cells that produce the structural scaffold of the heart (cardiac fibroblasts) [1,2]. Like all cells, cardiac fibroblasts express a class of recently discovered regulatory molecules called “microRNAs”. In fibroblasts, microRNAs are thought to regulate the structural remodelling of the heart following injury or stress (e.g. after a heart attack or in response to high blood pressure) [2,3]. Cardiac fibroblasts contribute to cardiac remodelling through altered proliferation, migration, differentiation, extracellular matrix turnover and secretion of paracrine signalling factors [1-3]. We have recently identified a handful of microRNAs expressed by cardiac fibroblasts that we think are of particular interest in regulating cardiac remodelling . This project will use a combination of molecular and cellular methods to explore the role of these microRNAs in regulating human cardiac fibroblast function. The project will involve a variety of cell and molecular biology techniques including primary human cell culture, proliferation & migration assays, signalling pathway analysis, SILAC proteomics, luciferase reporter assays, transfection, real-time RT-PCR, Western blotting, ELISA and immunocytochemistry.
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Turner NA, Porter KE. Function and fate of myofibroblasts after myocardial infarction. Fibrogenesis & Tissue Repair 2013;6:5.
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Bageghni SA, Hemmings KE, Porter KE, Denton C, Ainscough JFX, Drinkhill MJ, Turner NA. Cardiac fibroblast-specific p38 MAPK promotes cardiac hypertrophy via a putative paracrine IL-6 signalling mechanism. FASEB Journal 2018;32:4941-4954.