The analysis of Telomere maintenanace in the process of cataractogenesis

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University London.Telomeres are structures found at the end of chromosomes, involved in protecting the ends from being recognised as DNA damage. In normal somatic cells telomeres shorten as cells replicate due mainly to the end replication problem, however other mechanisms such as the exonucleolytic activity and oxidative stress are involved. Telomere length regulation can be affected by the activation of the DNA damage response pathway. These are activated when DNA damaging agents are subjected to DNA. One such agent is ionising radiation (IR). IR has shown to affect the length of telomeres adversely, whereby they shorten at more rapid rates. One interesting area of research is the link between radiation and formation of cataracts. Cataracts are basically the opacity of the lens, which is caused by the changes in the lens cells, and is the leading cause of blindness worldwide. Scientists have found that radiation causes early onset of cataracts. Given that telomere length regulation is effected due to radiation induction and that radiation causes early onset of cataracts, this study was designed to further understand the link between telomeres and cataractogenesis before and after radiation exposure. To do this Human Lens Epithelial (HLE) cells and Mouse Bone Marrow cells were used. Telomere length was measured in these cells before and after exposure to radiation, to see the changes in telomere length over a period of time. Furthermore, telomerase activity was measured in HLE cells to see if radiation caused a difference in activity. Alongside this, DNA damage response using the H2AX and TIF assay was examined to see if the cells repaired the damage caused by radiation effectively Furthermore, telomere length was measured in bone marrow cells from cataract model mice. The telomere length was measured over the course 24 months in this life-time study (24months). Our study in the HLE cells has shown an increase in telomere length as the cells age both before and after irradiation. Higher doses of radiation were found to increase telomere length more in these cells. Telomerase activity in HLE cells was shown to decrease as the passage increased. The activity was also found to decrease dose dependently. The DNA damage response mechanisms in HLE cells were found to be normal and repair took place effectively for up to 2Gy of radiation exposure. DNA damage foci were found to go back to normal background amounts after 72hours in both H2AX and TIF analysis. For the Mouse Bone Marrow cells derived from the life-time study two strains of mice were used; Ercc2 heterozygous mutant and wild-type. The telomere length in both the mutant and wild-type mice was found to decrease over a period of 24months. Telomere length was found to be longer in the wild-type samples when compared to the heterozygous mutant samples. Finally, telomere length in the female samples of the mouse bone marrow was found to be longer than the male, in both the mutant and wild-type samples