Visualization and quantification of autophagy in primary gastrointestinal epithelia cell cultures.
type: CreativeWork
description: Autophagy is an intracellular bulk degradation pathway which can be used during cellular maintenance to encapsulate and target intracellular content such as organelles and proteins to be degraded by the lysosome. The autophagosome is a double membrane vesicle which is used to encapsulate this cellular content. During the cellular response to starvation, autophagy can degrade cytoplasmic organelles and long-lived proteins in lysosomes, to recover amino acids within the cell and form new proteins. Stimulation of autophagy during infection can enhance immune responses by degrading pathogens and increasing presentation of microbial components to the immune system. Genome wide association studies (GWAS) have identified mutations in the autophagy related (Atg) protein Atg16L1, and NOD2 as risk alleles for Crohn’s disease. Atg16L1 is essential for the formation of autophagosomes and autophagosome formation is stimulated following recognition of bacterial cell wall peptide muramyl dipeptide (MDP) by NOD2. Crohn’s disease results from an autoimmune reaction to gut epithelial cells, and this raises the possibility that the risk alleles alter autophagy and/or NOD2-mediated microbial sensing in gut epithelial cells. The aim of this thesis has been to develop methods to quantifying autophagy in primary epithelial cells cultured from human intestinal biopsy material. Atg8/LC3 is the major structural protein of the autophagosome. Images of GFP-Atg8/LC3 puncta generated in response to starvation, or NOD2 signalling, were taken by fluorescence microscopy, and pixel densities were rendered to generate digital datasets that were amenable to statistical analysis. Rendered puncta were displayed graphically to indicate autophagosome diameters, spatial location within the cell, lifetime, expansion and movement. The methods were validated in tissue culture cell lines and then extended to study human colon crypts cultures transduced by adenoviral vectors expressing GFP-LC3. In comparison to…
abstract: Autophagy is an intracellular bulk degradation pathway which can be used during cellular maintenance to encapsulate and target intracellular content such as organelles and proteins to be degraded by the lysosome. The autophagosome is a double membrane vesicle which is used to encapsulate this cellular content. During the cellular response to starvation, autophagy can degrade cytoplasmic organelles and long-lived proteins in lysosomes, to recover amino acids within the cell and form new proteins. Stimulation of autophagy during infection can enhance immune responses by degrading pathogens and increasing presentation of microbial components to the immune system. Genome wide association studies (GWAS) have identified mutations in the autophagy related (Atg) protein Atg16L1, and NOD2 as risk alleles for Crohn’s disease. Atg16L1 is essential for the formation of autophagosomes and autophagosome formation is stimulated following recognition of bacterial cell wall peptide muramyl dipeptide (MDP) by NOD2. Crohn’s disease results from an autoimmune reaction to gut epithelial cells, and this raises the possibility that the risk alleles alter autophagy and/or NOD2-mediated microbial sensing in gut epithelial cells. The aim of this thesis has been to develop methods to quantifying autophagy in primary epithelial cells cultured from human intestinal biopsy material. Atg8/LC3 is the major structural protein of the autophagosome. Images of GFP-Atg8/LC3 puncta generated in response to starvation, or NOD2 signalling, were taken by fluorescence microscopy, and pixel densities were rendered to generate digital datasets that were amenable to statistical analysis. Rendered puncta were displayed graphically to indicate autophagosome diameters, spatial location within the cell, lifetime, expansion and movement. The methods were validated in tissue culture cell lines and then extended to study human colon crypts cultures transduced by adenoviral vectors expressing GFP-LC3. In comparison to…
author: Whelband, Matthew
language: en
date published: 2013-09
date modified: 2026-07-24T02:12:01Z
provider: University of East Anglia
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of East Anglia