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New tools for investigating intestinal barrier function in-vitro

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  • Full or part time
    Dr C Duckworth
    Prof M Pritchard
    Dr Cath Booth
    Dr Jim Wilson
  • Application Deadline
    No more applications being accepted
  • Competition Funded PhD Project (European/UK Students Only)
    Competition Funded PhD Project (European/UK Students Only)

Project Description

The intestine has important and unique functions that are absolutely essential for maintaining whole organism health. Around 40% of the body’s energy expenditure can be attributed to regulation of the intestinal barrier between luminal contents and the body. This is exquisitely controlled to prevent malnutrition or systemic infection by commensal or pathogenic bacteria. Daily absorption of fluids (dietary and secretions) from the intestinal lumen into the body is in the region of 8 litres. To achieve efficient absorption, the intestine has a surface area of around 400m2 and an epithelial layer that is only one cell thick. This process provides a huge challenge to prevent invasion by the luminal microbiome. Intestinal permeability and epithelial cell health status are particularly important regulators of intestinal barrier function and if compromised can lead to a multitude of diseases (intestinal and systemic) and inflammation. The physiological mechanisms that maintain this tightly regulated barrier remain incompletely understood and are difficult to explore owing to the lack of suitable in-vitro models.

Intestinal permeability is difficult to investigate due to a lack of suitable in-vitro assays. Cell line monolayers derived from human colon cancers on transwell filters are often used to assess transepithelial electrical resistance and flux of substances across the epithelium, but these culture systems do not recapitulate normal gut growth dynamics. Other studies consist of oral dosing of whole animals or tissue explants that are maintained in short-term culture.

Ground-breaking research by Hans Clevers’ group has determined culture conditions for maintaining intestinal stem cell growth permitting the generation of long-lived three-dimensional organoid “mini-gut” cultures. Organoids maintain all differentiated cell types and growth dynamics observed in the intestinal epithelium in-vivo and grow with a central lumen. The apical membrane on the inside and basolateral membrane on the outer surface makes the apical pole difficult to access. Application of dietary components and bacterial products/bacteria to the apical pole of epithelial cells is important as apical and basolateral membranes act very differently to the same stimuli. Several groups including our own have developed microinjection techniques to access the lumen and apply dietary/microbiome/mycobiome components and permeability markers to assess intestinal permeability, however, these assays are time consuming and do not lend themselves to high-throughput studies.

This year, techniques have been developed to reverse the polarity of intestinal organoid culture resulting in the apical membrane on the outside and basolateral membrane on the inside. We now want to develop high-throughput assays utilising apical-out and basal-out organoid systems to investigate how diet/genetics/age and microbiome interact to modulate intestinal permeability and barrier function.

This four-year CASE studentship is based at the University of Liverpool under the supervision of Dr Carrie Duckworth and Prof Mark Pritchard, and includes an industrial placement based at Epistem Ltd in Manchester, UK (www.epistem.co.uk). Co-supervisors are Dr Cath Booth (Epistem Ltd) and Dr Jim Wilson (Epistem Ltd).

HOW TO APPLY
Applications should be made by emailing [Email Address Removed] with a CV (including contact details of at least two academic (or other relevant) referees), and a covering letter – clearly stating your first choice project, and optionally 2nd and 3rd ranked projects, as well as including whatever additional information you feel is pertinent to your application; you may wish to indicate, for example, why you are particularly interested in the selected project(s) and at the selected University. Applications not meeting these criteria will be rejected.
In addition to the CV and covering letter, please email a completed copy of the Additional Details Form (Word document) to [Email Address Removed]. A blank copy of this form can be found at: https://www.nld-dtp.org.uk/how-apply.
Informal enquiries may be made to [Email Address Removed]

Funding Notes

This is a 4 year BBSRC CASE studentship under the Newcastle-Liverpool-Durham DTP. The successful applicant will receive research costs, tuition fees and stipend (£15,009 for 2019-20). The PhD will start in October 2020. Applicants should have, or be expecting to receive, a 2.1 Hons degree (or equivalent) in a relevant subject. EU candidates must have been resident in the UK for 3 years in order to receive full support. Please note, there are 2 stages to the application process.

References

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells. Cell Death Dis. (2019); in press

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium. Front Cell Infect Microbiol. (2019); 28;9:300

Developing a 3D intestinal epithelium model for livestock species. Cell Tissue Res. (2018); 375(2):409-424.

Oral iron exacerbates colitis and influences the intestinal microbiome. PLoS One. (2018);13(10):e0202460.

Progastrin-induced secretion of insulin-like growth factor 2 from colonic myofibroblasts stimulates colonic epithelial proliferation in mice. Gastroenterology (2013) vol 145 pp 197-208

A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Disease Models & Mechanisms; (2013) vol 6 pp 1388-99

Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease. Gut (2012) vol 61 pp 1146-53

The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding. Gastroenterology (2011) vol 140 issue 4 pp 1208-1218

Suppression of apoptosis, crypt hyperplasia, and altered differentiation in the colonic epithelia of bak-null mice. Gastroenterology (2009) vol 136 issue 3 pp 943-52

Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium. Exp Cell Res. (1999);249(2):359-66



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