Date of Award

8-11-2023

Publication Type

Thesis

Degree Name

M.Sc.

Department

Biological Sciences

Supervisor

Phillip Karpowicz

Rights

info:eu-repo/semantics/openAccess

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Abstract

The circadian clock is a transcription-translation feedback loop that controls our body’s daily rhythms. Previous work in our lab has shown that the circadian clock controls regeneration in the small intestinal epithelium and that the Hippo signaling pathway may serve as a link between regeneration and the circadian clock. In order to elucidate if the circadian clock directly controls the Hippo pathway as well as the Wnt pathway, both vital for intestinal homeostasis, intestinal stem cell-derived organoids, a 3D culture system that is able to recapitulate the cell biology of the intestinal epithelium in vitro, were used to examine the temporal pattern of gene expression between epithelia that were circadian clock wildtype (Bmal1+/+) vs clock dead (Bmal1-/-). The expression of a core clock gene, Per2, was tracked with a luciferase reporter and organoids were collected for RNA analysis and staining every 6 hours over the course of 24 hours. RT-qPCR was used to confirm rhythmic expression of Per2 as well as determine differences in patterns of expression of Wnt target gene Axin2 and Hippo target Ereg. Mitosis, visualized by phosphorylated histone H3 staining, was used to compare circadian patterns of cell division in the Bmal1+/+ versus Bma1-/- organoids. Clock functional organoids showed rhythmic patterns of Per2, Ereg, and mitosis which were absent in the clock-dead organoids, suggesting that regeneration in the intestinal epithelium may be coordinated with the circadian clock via the Hippo pathway. I also piloted methods to transfect human intestinal epithelial organoids for future translational work.

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