Role of cutaneous circadian clocks in mediating skin repair and regeneration

Background: Biological clocks play a key role in mediating the function of organisms, tissues and cells. These cell autonomous circadian clocks are responsible for generating 24 hour rhythms, governing cell division, DNA damage response, cell metabolism and tissue repair/regeneration. In liver tissue, the expression of at least ~10% of proteins is rhythmically regulated by clock genes. A functional clock ensures temporal segregation of different events to different part of the 24 hr day, maintaining tissue homeostasis and promoting effective repair. Disruptions to clock rhythms (e.g. during ageing) have been linked to increased risks of various human diseases. Whilst it is clear that skin also possess these peripheral clocks, the role which they play in mediating susceptibility to damage (by ultraviolet radiation [UVR] exposure for example) or the efficacy of repair strategies (such as day or night creams) remains unknown. This PhD project aims to characterise the influence of peripheral clocks on: i) the physiology of skin ii) the morphology and cellular phenotype of skin and that of progenitor cells cultured on compliant and stiff environments, iii) the extent of UVR-induced damage and iv) the efficacy of topical anti-ageing treatments with the ultimate goal of optimising strategies for chronotherapy (timing the delivery of medication) for skin repair.

Main questions to be answered: The project with address three objectives:
1. Influence of chronic UVR exposure on circadian clocks and the physiology of skin. The student will recruit young and old volunteers and collect punch biopsies from photo-protected and photo-exposed sites at 0, 6, 12 and 18 hour time points (9AM-3AM). Bisected biopsies will be processed for immunohistochemistry (to localise clock protein expression in skin cells and progenitor cells) and for RNASeq and mass spectrometry to characterise the influence of peripheral clocks on the transcriptome and proteome, and their changes in response to UVR and ageing (Janich, Meng, Benitah. Curr Opin Cell Bio 2014; Dudek et al J Clin Invest 2016).
2. Dynamic clock gene expression and skin cell biology in response to UVR. Primary dermal fibroblasts, keratinocytes and progenitor cells will be cultured from human skin biopsies (young /old /photo-protected and photo-exposed) on substrates of varying compressive stiffness (characterised by atomic force microscopy) and transfected with bioluminescent lentivirus reporters for clock genes in order to characterise the influence of age (young/ old volunteers), chronic UVR exposure (photo-protected / photo-exposed sites) and acute UVR exposure on the dynamic expression of clock genes (using real-time photon counting/imaging) and cellular morphology (by optical microscopy and atomic force microscopy).
3. Optimising chronotherapy. The student will characterise the response of clock synchronised cells (cultured on both compliant and stiff matrices) to therapeutic agents administered: i) at differing points in the circadian cycle and ii) at points in the circadian cycle which mimic likely damage and therapeutic scenarios (i.e. UVR exposure in the afternoon followed by afternoon or evening treatment).

This project will follow the training elements of the CDT Regenerative Medicine programme. PLease see the link below for further details:

http://www.regenmedcdt.manchester.ac.uk/programme/#.WY2_A2PqmIQ