Student Number : 9906382M
MSc (Med) dissertation -
Faculty of Health Sciences / The G1862T mutation in the bulge of the RNA encapsidation signal, in the precore
region of hepatitis B virus, results in reduced expression of HBeAg and accumulation of
the HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell.
This accumulation can disturb the functioning of the ER and lead to the ER stress
response that can affect various cellular pathways, in turn affecting cell viability. The aim
of this study was to determine whether apoptosis or necrosis occurred when cultured
Huh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmid
constructs, with and without the G1862T mutation, were used to transfect cells. To
differentiate between necrosis and apoptosis cells were stained with propidium iodide or
YO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry and
qualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti-
HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a result
of the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling was
carried out using a fluorogenic-based assay. When cells were transfected with wild-type
plasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cells
were transfected with the G1862T mutant plasmid. The highest levels of apoptosis
occurred at 72 hours post-transfection. Confocal microscopy revealed the co-localization
of aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection with
G1862T mutant plasmids resulted in significant differences in the expression of caspase
3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. The
accumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment,
leads to apoptosis and affects the levels of caspase expression.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/1879 |
| Date | 17 November 2006 |
| Creators | Viana, Raquel Valongo |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 6651825 bytes, 49932 bytes, application/pdf, application/pdf, application/pdf, application/pdf |
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