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Faculty of Medicine and Health
School of Medicine
Leeds Institute of Biomedical and Clinical Sciences
Section of Translational Anaesthetic and Surgical Sciences
Research Fellow (based at St James’s University Hospital)
This full-time post is fixed term for 5 years and is available from 1st November 2016
Applications are invited for a Research Fellow post to be based within the group of Professor Paul Allen studying the genetics, molecular and cell biology of Malignant Hyperthermia. Understanding how mutations in RyR1 and other EC coupling proteins in skeletal muscle will not only allow us to facilitate diagnosis, but potentially will allow better understanding of abnormal responses to heat stress. The post is funded by a USA National Institutes of Health grant and will contribute to the execution, analysis and interpretation of the mechanisms that are responsible for MH, and the active site(s) of action for the only current therapy, dantrolene.
The project will require an excellent knowledge of modern techniques of electrophysiology (with a particular focus on intracellular cation concentrations) advanced imaging techniques (including fluorescence imaging and confocal microscopy). You will also evaluate susceptible muscles using functional assays, including the assessment of protein-protein interactions using affinity purification methods and post translational modifications (phosphorylation, nitrosylation and sulfonation) (Using Biotin switch techniques), so previous experience of these systems is desirable. A good knowledge of biochemical assays is essential.
Candidates must be enthusiastic, highly motivated and demonstrate a high level of relevant laboratory experience and technical competence. In addition, the post holder must show the ability to manage their time effectively and work under pressure, exhibiting effective interpersonal, communication, and team-working skills. You must have a PhD in molecular/cell biology or a relevant biomedical subject (or have a date set for examination) and an interest in electrophysiology and cell physiology. A background in electrophysiology is required. Candidates with MBBS/MD/DVM and appropriate basic science training will be considered.
The University of Leeds is committed to providing equal opportunities for all and offers a range of family friendly policies which can currently be found at(http://hr.leeds.ac.uk/homepage/4/policies). The University is a charter member of Athena SWAN and holds the Bronze award. The School of Medicine gained the Bronze award in 2013. We are committed to being an inclusive medical school that values all staff, and we are happy to consider job share applications and requests for flexible working arrangements from our employees.
University Grade 7 (£32,004 – £38,183 p.a) depending upon qualifications and experience.
Informal enquiries regarding the post should be directed to Professor Paul D Allen, Email: .
If you have any specific enquiries about your online application please contact Rosemary Sannaee, Email: or Sharon Collins, Email:
Job Ref: MHBCS1037
Closing Date: 28 November 2016
Interview Date: 8 December 2016
MAIN DUTIES AND RESPONSIBILITIES
· Performing experiments on murine models of Malignant Hyperthermia.
· Performing electrophysiologic measurements of intracellular Ca2+ and Na+ in mouse skeletal muscle in vivo and in vitro.
· Under the guidance of the principal investigator plan and manage your own research activity (in collaboration with others as appropriate), assisting in designing specific strategies towards achieving the research goals of the project and managing individual components of the study.
· Ensuring good day-to-day progress of the projects and contributing to strategic development.
· Designing experimental procedures and technical details.
· Molecular and cell biology techniques to be used will include:
- DNA/RNA extraction
- mammalian cell culture
- preparation of plasmid constructs
- preparation of lentivirus
- transfection with plasmids, lentivirus or siRNA/shRNA
- RT-PCR and quantitative (‘real-time’) RT-PCR
- biochemical assays of protein-protein interactions using affinity purification methods (TAP-tagging)
- analysis of post-translational modification (Phosphorylation, nitrosylation, sulfonation)
- confocal microscopy and live-cell imaging
- bioinformatic analyses of data-sets
· Generating research output (e.g. publications/presentations) and analysing and interpreting the results.
· Communicating and/or presenting research results within the Group and Section.
· Developing independent and original ideas, as appropriate.
· Continually evaluating your working practices and protocols and adapting/evolving these where necessary.
· Troubleshooting problems with optimisation, assay design, screening, data analysis and validation and seeking solutions via published literature, company websites and personal contacts.
· Maintaining accurate and up-to-date laboratory and other records.
· Treating data with confidentiality and in accordance with current policies.
· Building internal contacts and participating in internal networks for the exchange of information and for future collaboration.
· Working collaboratively with other staff/students within the Group, and other research groups as appropriate, including external collaborators.
· Actively engaging with the national and international MH community, ensuring research outputs are shared via existing networks and providing specialist knowledge to others.
· Encouraging and promoting good working practices within the team and within the Institute.
· Providing guidance, support, training and assistance in relevant techniques to staff and students within the Group and Section.
· Publishing outcomes from the project in appropriate high quality journals and presenting at national or international conferences as required.
· Working with the Principal Investigator to identify future opportunities for research and preparing proposals and funding applications.
· Attending and contributing to relevant meetings.
· Being aware of the risks in the laboratory environment and working in accordance with the GM and COSSH assessments.
· Contributing to GM, RA and COSSH assessments when appropriate.
· Actively supporting and adhering to the Faculty’s commitment to Equality and Diversity in line with the requirements of the Faculty Diversity Action Plan.
Please note, you may be required to work flexibly outside of normal working hours according to the needs of the project.
Job descriptions cannot be exhaustive and you may be required to undertake other duties that are broadly in line with the above key responsibilities.
RELATIONSHIPS
The post holder will be responsible on a day-to-day basis to the Principal Investigator, Professor Paul D. Allen, through whom s/he will be accountable to the Director of the Institute (Professor Philip Hopkins), and to the Dean of the Faculty of Medicine and Health. S/he will be expected to interact with other members of the Institute, comprising post-doctoral research fellows, PhD students and research support staff, and to present data at Institute meetings.
UNIVERSITY VALUES
All staff are expected to operate in line with the university’s values and standards, which work as an integral part of our strategy and set out the principles of how we work together. More information about the university’s strategy and values is currently available at http://www.leeds.ac.uk/comms/strategy/
PERSON SPECIFICATION
Essential
1. A PhD in molecular/cell biology or a relevant biomedical subject (or pending with a date set for examination)
2. A BSc degree (or equivalent) in one of the biomedical sciences or related subject or an MBBS/MD/DVM with previous basic science training
3. A demonstrable interest in cell biology
4. Extensive experience in electrophysiology
5. Extensive experience in biochemical assays of protein-protein interactions
6. Extensive experience in cell culture, including culturing primary cells
7. Practical experience of immunofluoresence microscopy
8. Experience in molecular cloning techniques and generation of vectors
9. A demonstrable ability to analyse and interpret research results
10. A demonstrable ability to work unsupervised, with the direction from the Principal Investigator and as part of the wider research team
11. Effective communication and interpersonal skills
12. A developing publication record in high impact peer reviewed journals
13. A demonstrable understanding of health and safety issues within the laboratory setting
14. A demonstrable understanding of translational research
15. Strong IT skills, including the use of Microsoft Office and image processing applications
16. A willingness to work flexibly, when necessary, to fulfil the needs of the research project
Desirable
1. A demonstrable research background in human molecular genetics or developmental biology (or a familiarity with their concepts)
2. Demonstrable experience in mouse primary cell culture
3. Demonstrable experience in intracellular cation measurements with ion-selective microelectrodes
FURTHER INFORMATION
Research Background
The long-term goal of this research is to determine the mechanisms by which mutations that alter intracellular Ca2+ homeostasis at rest cause fulminant MH after exposure to triggering agents. To achieve our objectives we will use the RyR1-R163C mouse which phenocopies human MH and CSQ1 nulls, which have an MH like phenotype. We hypothesize that because the amount of Ca2+ in internal stores is limited compared to the extracellular pool, after MH is triggered sarcolemmal channels are critical to its progression and that a primary mechanism for this is increased ROS production promoted by increased [Ca2+]i causing activation of TRPCs, increasing RCaE and supporting the MH syndrome. In addition to using in vitro mechanistic analyses and in vivo pharmacological approaches to reduce RyR1 leak, ROS production via NOX2 and sarcolemmal Ca2+ entry to abrogate the MH phenotype, because TRPCs exist as heteromultimers, we will molecularly suppress function of all TRPC3/6/7 family members by expression of a dominant negative non-conducting TRPC6 channel in MH muscles. Finally we will use similar approaches to discover the mechanism(s) by which the syndrome is both prevented and aborted by dantrolene, which has heretofore been thought to act only through inhibition of RyR1 Ca2+ release. Within this framework we will investigate the transformative concept that while RyR1 leak is a common convergent pathway leading to all MHS, the RyR1 leak is the proximate driver of a mitochondrial Ca2+ overload and augmented ROS and NO production, which in turn activates TRPC dependent sarcolemmal Ca2+ and Na+ entry that initiates and sustains the fulminant MH episode. Such a mechanism can not only explain responses to MH triggering agents through dysregulated EC coupling in MH, but also more chronic adaptations and progression of muscle damage ascribed to a vicious cycle of increased RyR1 Ca2+ leak, sarcolemmal Ca2+ and Na+ entry, ROS, RNS, TRPC activity and mitochondrial impairments in other muscle diseases.
Hypothesis 1: MH susceptibility is the result of a conformational change in RyR1 caused either by a RyR1 mutation, or induced indirectly by mutations in other proteins closely associated with RyR1 which results in increased RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry.
Specific Aim 1: To determine the filling state of the SR and rate of SR Ca2+ leak in MH muscle fibers.
Specific Aim 2: To determine the role of TRPCs in causing abnormalities in sarcolemmal Na+ and Ca2+ entry RyR1-R163C and CSQ1 null muscles and then to determine if skeletal muscle specific over-expression of a dominant negative non-conducting TRPC6 channel, overexpressing SERCA1A and KO of TRPV1 can modify RyR1-R163C's MH phenotype.
Hypothesis 2: In addition to blocking RyR1 SR Ca2+ release, dantrolene abrogates the MH phenotype by modulating RyR1 SR Ca2+ leak and sarcolemmal Na+ and Ca2+ entry.
Specific Aim 3: To determine the mechanisms by which dantrolene diminishes aberrant Ca2+signaling.
Successful completion of these specific aims will provide a more comprehensive understanding of MH.
The project will require measurements of intracellular Ca2+ and Na+ with ion-selective microelectrodes, biochemical assays of protein-protein and drug-protein interactions and post translational protein modifications. The project will involve cell culture, including derivation of mouse myoblasts, advanced microscopy techniques, recombinant DNA technology, and will have a significant component of bioinformatic and image analysis.
For background on methodologies, see the following publications:
Allen PD, Lopez JR, Sanchez V, Alamo L, Ryan JF, Sreter FA: EU 4093 decreases intracellular [Ca2+]i in skeletal muscle fibers from control and malignant hyperthermia - susceptible swine. Anesthesiology 1992; 76:132-138.
Feng, W., Barrientos, G., Cherednichenko, G., Yang, T. Padilla, I., Truong, K. Allen, P. D., Lopez, J. R., Pessah, I. N. (2011) Functional and biochemical properties of RyR1 channels from heterozygous R163C malignant hyperthermia susceptible mice. Molec Pharmacol 79, 420-31. PMC3061367
Yuen, B. J., Boncompagni, S., Feng, W., Yang, T., Lopez, J. R., Matthaei, K. I., Goth, S. R., Protasi, F., Franzini-Armstrong, C., Allen, P. D., Pessah, I. N. (2012) Mice expressing T4826I-RYR1 are viable but exhibit sex- and genotype dependent susceptibility to malignant hyperthermia and muscle damage FASEB J 26, 1311-22
Eltit JM, Bannister RA, Moua O, Altamirano F, Hopkins PM, Pessah IN, Molinski TF, López JR, Beam KG, Allen PD. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor. Proceedings of The National Academy of Science USA. 2012 109:7923-7928
José M. Eltit, Xudong Ding, Isaac N. Pessah, Paul D. Allen, José R. Lopez Non-specific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia. FASEB J. 2013 27:991-1000
Altamirano F., Eltit J.M., Robin G., Linares N., Ding X., Pessah I.N., Allen P.D., López J.R. Ca2+ influx via the Na+/ Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle J Biol Chem. 2014 289:19180-90
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