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How does ageing-related loss of basement membrane collagen regulate epidermal barrier homeostasis?
Closing date
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Supervisors
Professor Edel O’Toole, e.a.otoole@qmul.ac.uk, Edel's interests are genetic skin disease (palmoplantar keratodermas and ichthyoses), paediatric dermatology and medical dermatology. , Queen Mary University of London
Project Details

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Background:  During ageing, skin atrophy or thinning is associated with decreased basement membrane collagens (BMC) and an impaired epidermal barrier with associated keratinocyte  innate (intrinsic) immune response.   Preliminary data from the O’Toole group suggest that loss of BMC changes the cytoskeleton and nuclear organisation with type VII  (Col7) and XVII (Col17) collagen having opposing effects.    Our hypothesis is that decreased BMC results in altered nuclear signalling and organisation with resulting increased inflammation, oxidative stress and an impaired barrier.  The student will use keratinocytes with knockdown of Col7 or Col17 in 3D skin equivalents to study these processes with a goal of reducing inflammation and restoring the skin barrier in ageing.

Importance:  Skin ageing is a universal problem.  The impaired skin barrier in ageing results in impaired wound healing and increased skin infections.  The link between the basement membrane and the nucleocytoskeleton is novel and the student will be exploring a new area which we believe will be of broad interest.

Aims and objectives:

Aim 1  Establish 3D skin equivalents incorporating immortalised keratinocytes with shRNA knockdown of Col7 or Col17.   Integrin receptors, actin, nucleocytoskeleton and differentiation proteins will be examined using immunofluorescence.   Existing RNAseq data from keratinocytes with knockdown (KD) of Col7 and Col17 will be mined to direct further analyses.  The skin barrier will be characterized using dye exclusion, Nile Red staining and transglutaminase activity assays.  Experiments will be replicated using primary  keratinocytes with KD of Col7/Col17.

Aim 2  Apply mechanical/UV stress to the Aim 1 3D skin equivalents.   The student will develop the 3D models on a stretchable porous membrane (Julien Gautrot lab) to apply mechanical stretch.   An alternative stressor is UV irradiation.  In parallel, glycolysis and oxidative phosphorylation will be measured using Seahorse technology in monolayer cultures with KD of Col7/Col17 . Immunostaining will be performed for oxidative stress, proliferation and DNA damage markers.   Changes in nuclear morphology and chromatin remodelling will be analysed by immunostaining of histone marks and high-resolution confocal microscopy.

Aim 3 Use epigenomic methods to identify regulators of the identified transcriptional response  from BM loss.  Keratinocytes from Aim 2 will be subjected to chromatin immunoprecipitation (ChIP) using antibodies for selected transcriptional regulators followed by Next Generation Sequencing (NGS).  The student will integrate these data with the existing RNAseq data to identify how the BM is regulating epidermal homeostasis.  Findings can be confirmed in young and aged skin by immunostaining.

Aim 4  Identify and test compounds to mitigate effects of BM alterations on epidermal homeostasis

At Unilever, bioinformatic and chemi-informatic tools will be used to identify compounds via a combination of RNA-based signature analysis, pathway-driven and target/lead ID approaches. Proof of principle options will be tested in aged keratinocyte models to examine if the barrier and basement membrane can be improved.

 

Research Environment- QMUL

The Centre for Cell Biology and Cutaneous Research (CBCR) is one of the largest hubs of Cutaneous Research within the UK and is home to >60 staff and students. There is a weekly seminar series at which PhD students and Post Docs present their work creating a vibrant and highly collegiate student environment. The CBCR is embedded within the Blizard Institute and our 400 researchers enjoy an active programme of external seminars, journal clubs and research meetings, and excellent core support, including Advanced Microscopy training, which will be utilised in this project.

Research Environment- Unilever

Unilever is a world leading supplier of fast moving consumer goods with products sold in over 190 countries and used by 2 billion consumers every day. It employs >6,000 R&D professionals globally, located in six key sites, including Colworth (UK) where the placement will take place.  The Unilever Human Biology team  is part of a global research group for bioscience and is extremely well equipped to conduct research in human cell biology and houses a state of the art Genomics Facility at Unilever Colworth. Unilever’s expertise in sophisticated in vitro skin models and genomics/bioinformatics/chemi-informatics are central to this proposal. The site is partner for >20 BBSRC PhD awards and 10 engineering apprentices, and is an active participant in national and international collaborative government funded projects providing a rich network of applied researcher contacts.

References

3D model of harlequin ichthyosis reveals inflammatory therapeutic targets. 

Enjalbert F, Dewan P, Caley MP, Jones EM, Morse MA, Kelsell DP, Enright AJ, O'Toole EA.

J Clin Invest. 2020 Sep 1;130(9):4798-4810. doi: 10.1172/JCI132987.

PMID: 32544098

 

 Loss of Laminin α3 induces cell invasion and macrophage infiltration in cutaneous squamous cell

 carcinoma.  Caley MP, Martins VL, Moore K, Lashari M, Nissinen L, Kähäri VM, Alexander S, Jones E,

 Harwood CA, Jones J, Donaldson M, Marshall JF, O'Toole EABr J Dermatol. 2020 Aug 7. doi: 10.1111/bjd.19471. Online ahead of print.  PMID: 32767748

 

RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation. 

Jones M, Dry IR, Frampton D, Singh M, Kanda RK, Yee MB, Kellam P, Hollinshead M, Kinchington PR, O'Toole EA, Breuer J.  PLoS Pathog. 2014 Jan 30;10(1):e1003896. doi: 10.1371/journal.ppat.1003896. eCollection 2014 Jan.  PMID: 24497829 

 

Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen.  Martins VL, Vyas JJ, Chen M, Purdie K, Mein CA, South AP, Storey A, McGrath JA, O'Toole EA.

J Cell Sci. 2009 Jun 1;122(Pt 11):1788-99. doi: 10.1242/jcs.042895. Epub 2009 May 12.

PMID: 19435799 

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