MSc (Med), Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / INTRODUCTION: Ionising radiation can induce DNA damage, in the form of
DNA double strand breaks (DSBs), which the affected cell may or may not be able
to repair. Micronuclei are indicators of cytogenetic damage, which result from
aneugenic (spontaneous loss of chromosomes) or clastogenic (chromosomal
fragments) events. The micronuclei may be centromere-positive (CM+MN) for
aneugenic events or centromere-negative (CM–MN) for clastogenic events. A pancentromeric
marker would help differentiate between CM+MN and CM–MN,
especially important among exposures to very low doses of ionising radiation.
METHODOLOGY: Micronucleus assays were performed on blood samples
collected from healthy donors and HIV+ donors. The blood samples were
irradiated at various doses of ionising radiation. Two methods were used to create a
pan-centromeric probe. First, the p82H plasmid, which contains centromere
specific α repetitive human DNA sequences, was used. Second, human centromeric
sequences were amplified using polymerase chain reaction (PCR). In both cases,
the pan-centromeric probe was labelled and hybridised using fluorescent in situ
hybridisation (FISH) to micronucleus slide preparations from healthy and HIV+
donors. The slides were scored manually and on an automated system, MetaFer®.
RESULTS: The p82H probe did not hybridise to any centromeres when FISH was
performed, while the synthetic probe made by means of PCR bound to the
centromeres of all chromosomes. Henceforth, all experiments were performed with
the synthetic pan-centromeric probe. A dose response study was performed on
micronucleus slides from healthy donors, from which significant differences in the
number of micronuclei and the percentage of centromere-negative micronuclei
could be seen between doses. The HIV study involving HIV+ donors and HIV–
controls did not yield any significant differences between the two groups.
DISCUSSION & CONCLUSION: Combining the micronucleus assay with the
pan-centromeric probe greatly improves its sensitivity. The dose response study
corroborated previous work performed by Vral et al (1997). Contrary to what was
expected and published (Baeyens et al, 2010), no significant differences were
observed between HIV+ and HIV- individuals. Issues, improvements and possible
future work are discussed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/8829 |
| Date | 13 October 2010 |
| Creators | Swanson, Rosemary Veronica |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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