Cummins, AdrianHowarth, GordonDudhwala, Zenab Mustansir2022-12-202022-12-202022https://hdl.handle.net/2440/137094The small intestine is important for nutrition. It is the site for absorption and digestion. Intestinal failure is a state of malabsorption in which there is inadequate digestion necessary for health and growth. To improve nutrition, one of the major approaches is to enhance intestinal growth. Intestinal crypt fission contributes to intestinal growth but has been poorly studied. It is thought to be initiated by division of intestinal stem cells in the base of the crypts. Further, the Wnt--catenin signalling pathway has been well known to maintain homoeostasis of intestinal stem cells and peaks at the same stage of development as the peak of crypt fission. This thesis has concentrated on several key gaps in current scientific knowledge that required investigation to enable a complete understanding of the role of the Wnt-β-catenin signalling pathway in promoting crypt fission in both humans and rats. The different aspects of this thesis approached these fundamental knowledge gaps through a series of discrete research chapters, which when combined, provide evidence of the similarity by which the Wnt-β-catenin signalling pathway could initiate intestinal growth via crypt fission. Intestinal crypt fission has been mainly characterised in adult mice and rats. Morphometry analysis were performed to determine the three-dimensional structures of the small intestine. By conducting this analysis, this showed an increase in crypt width by counting bifid crypts per 100 crypts. This is the first study to demonstrate a peak in crypt fission at 6–12 months in humans and at day 14 of life in rats. There was also a decrease in apoptosis and an increase in mitotic cell counts. Therefore, this study confirms that growth of the small intestine is at its peak during infancy. Next, the role of the Wnt-β-catenin signalling pathway was analysed in rats and humans and its correlation with crypt fission. This is the first study to investigate the Wnt-β- catenin signalling pathway with its correlation to crypt fission. Furthermore, protein levels were investigated using immunohistochemistry in the small intestinal tissue (duodenum in humans and jejunum in rats) and showed an increase in the Wnt signalling pathway proteins: β-catenin, Axin-2, Wnt3, Wnt2b and R-spondin-1 at the same as the peak of crypt fission. Peaks of certain Wnts were demonstrated but only Wnt3 and Wnt2b that were present at the base of the crypts where activity of β-catenin is high also peaked. To further confirm the increase in Wnt-responsive genes, including the small intestinal stem cell marker, LGR5, was examined with real-time PCR on rat small intestinal tissue. This demonstrated that there was a peak of LGR5 genes at day 14 of life To further investigate if the expansion of intestinal stem cells that occurs through division of whole crypts, the expression of LGR5 intestinal markers was examined in small intestinal tissues of humans and rats by the in-situ method. LGR5 was shown to be more highly expressed in infants than in adults. Although the LGR5 expression was greater, there was no accumulation of stem cells readily observed. Thus, this is suggestive that the stem cells are distributed and differentiate to new crypts to increase intestinal growth. This thesis provides important insights into understanding the complex role of the Wnt-β-signalling pathway and provides tangible evidence to suggest crypt fission and initiate small intestinal growth. This is an important finding in developing and targeting appropriate strategies for better and faster recovery of infants with intestinal failure.enCrypt fissionintestinal stem cellsLGR5Wnt beta catenin and intestinal morphologyThesis