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Title: The role of Claudins in the increase in paracellular permeability induced TNF-α in CaCo-2 Cells
Authors: Darazi, Emale El
Advisors: Itany, Omar
Subjects: Cell junctions
Epithelial cells
Tight Junctions (cell biology)
Issue Date: 2013
As with all epithelial tissue, the intestinal epithelium is formed of sheets of contiguous cells joined tightly by junctional complexes; thus creating compartments of different molecular milieu. A critical component of the junctional complex is the tight junction, or zonula occludens. In addition to riveting adjacent cells together, tight junctions are dynamic and regulated structures located at the most apical end of the epithelia that are responsible for developing and maintaining the barrier and gate functions of the epithelia in a variety of ways. Claudins are the main structural component of the tight junction. Members from this family, interact with one another as well as with other proteins to shape and regulate paracellular pores. Inflammatory processes have been shown to perturb permeability properties in epithelia. TNF-α, an inflammatory cytokine, plays a fundamental role in the intestinal inflammation of a variety of inflammatory disorders, including Crohns disease. Studies have revealed that TNF-α increases the permeability of intestinal cells. This increase in permeability, manifested by a drop in transepithelial electrical resistance (TER), could be attributed to changes in claudin expression. The hypothesis proposed by this study is that TNF-α disrupts the permeability of intestinal epithelial cells by altering claudin expression and/or localization. Caco-2 cells were seeded on microporous filters and plastic wells for 5 days to allow the formation of tight junctions. TNF-α was then added at different concentrations (1, 10ng/ml) for 72 hrs. The effect of TNF-α on the viability of Caco-2 cells was first assessed first by trypand blue and propidium iodide. Changes in proliferation were determined using the MTT proliferation assay. The alterations in permeability were evaluated through the daily measurement of TER. Finally, expression and localization of claudin-1 and claudin-2 were determined on Day 8 by western blot analysis and immunofluorescence respectively. Our results suggest that TNF-α does not affect the viability of Caco-2 cells, confirming that a drop in TER is not due to cell death. TNF α was also shown to decrease TER in a time dependent manner. Accompanying this decrease in TER was an increase in claudin-2 expression, an acclaimed leaky component of the tight junction; with a slight upregulation in claudin-1 expression. The localization of both claudin proteins was not affected. Taken together, our results suggest a possible mechanism in which TNF-α increases the permeability in intestinal cells through modulation of claudin proteins.
Includes bibliographical references (p.80-114).

Supervised by Dr. Omar Itani.
Rights: This object is protected by copyright, and is made available here for research and educational purposes. Permission to reuse, publish, or reproduce the object beyond the personal and educational use exceptions must be obtained from the copyright holder
Ezproxy URL: Link to full text
Type: Thesis
Appears in Collections:UOB Theses and Projects

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