The role of bronchoconstriction induced oxidative damage, signalling and circadian clocks in smooth muscle remodelling in asthma and COPD

Supervisors: Dr Lesley Iwanejko, Dr Vanja Pekovic-Vaughan and Dr Brian McDonagh

Overview
This studentship will use human pulmonary smooth muscle to test the hypotheses that mechanical stress induced bronchoconstriction:
a) Causes disruption of the circadian clock and altered redox response.
b) Decreases mitochondrial turnover and alters the cytoskeleton
c) That these processes ultimately lead to the airway remodelling seen in respiratory disorders such as asthma and chronic obstructive pulmonary disease (COPD).
Bronchoconstriction i.e. constriction of airway smooth muscle, results in symptoms such as wheezing and dyspnea in both asthma and COPD and ultimately lead to worsening symptoms due to airway remodelling. Bronchoconstricting agents such as pollutants, viral infections, allergens and mechanical stimuli, stimulate a cascade of events resulting in an increase in reactive oxygen species (ROS), disrupted redox signalling, DNA damage signalling, release of inflammatory cytokines and circadian clock dysfunction, all of which are interdependent and instrinsically linked. For example, circadian clocks in peripheral tissues e.g. lung, are sustained by external redox signals. Recent reports indicate ROS regulated airway remodelling (regulated by the redox environment and peripheral clock) results in an increased mitochondrial content with subsequent downstream effects on redox- and DNA repair-initiated signalling dependent processes such as autophagy, apoptosis, and cytoskeleton remodeling. Further understanding of the roles theses process play in regulating airway remodelling and the involvement of the circadian clock will a) identify new therapeutic targets to prevent or reduce airway remodelling and b) optimise correct timing of therapies.

Research Plan
Studies will be performed using commercially available human pulmonary smooth muscle cell lines initially from healthy donors, but in later stages using cells from asthmatic and COPD donors. The student will assess the effects of mechanical stretching of these cells (simulating bronchoconstriction) on redox-and DNA repair- signalling pathways and the Circadian Clock. The Flexcell system will be used to generate mechanical strain (17-18% stretch) to mimic the increased mechanical strain on airways induced by bronchospasm and hyperinflation. Lentiviral delivery of clock gene::luciferase reporters and real-time bioluminescent imaging will be used to track human cellular clock gene rhythms. Specific markers of the downstream effects of mechanical stress induced ROS, and consequently potential therapeutic target processes, such as mitochondrial size, oxidative DNA damage, DNA-repair, autophagic activation, further ROS generation and cytoskeletal remodelling will be analysed using a combination of redox proteomics, qPCR, blotting, comet assay and immunofluorescence. Finally, the study will then investigate the role of the Circadian Clock in the timing effectiveness of delivering inhibitors of the possible therapeutic targets processes e.g. the autophagy inhibitor Bafilomycin.

Training
The student will gain laboratory skills in a number of highly specialised techniques e.g. circadian biology and redox proteomics. The student will also be trained in a range of standard techniques and approaches such as cell culture, comet assays, immunofluorescence, lentiviral transformations, qPCR, analyses of high throughput data, statistical and imaging analyses and data storage. In addition to joining the local research themes they will become a member of the North West and UK DNA Repair Networks and will be encouraged to join and take an active role in learned societies such as the Physiology and Biochemical Society. All three supervisors are involved in delivering employability and researcher development skills to MRes students and are particularly keen for their PhD students to discuss their career ambitions with the supervisory team and to take advantage of the skills and careers workshops provided by the University, as well as gaining other skills by undertaking undergraduate practical demonstrating. They will also be encouraged to take advantage of networking opportunities such as attendance at seminars within and external to the Institute, to become a mentee in a mentoring scheme and active participation on the Postgraduate Society.

Equality Statement
The Institute of Ageing and Chronic Disease is fully committed to promoting gender equality in all activities. We offer a supportive working environment with flexible family support for all our staff and students and applications for part-time study are encouraged. The Institute holds a silver Athena SWAN award in recognition of on-going commitment to ensuring that the Athena SWAN principles are embedded in its activities and strategic initiatives