Bhartiya, Archana; (2022) Radiation effects on the structure of chromosomes. Doctoral thesis (Eng.D), UCL (University College London).

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Abstract

The study aims to investigate the effect of ionising radiation on the structure of the chromosomes after lose-dose X-ray irradiation. The first aim was to investigate the changes that occurred in the molecular environment of the chromosomes after radiation exposure using Fluorescence Lifetime Imaging Microscopy (FLIM). The study’s second aim was to measure the mass of the human chromosomes and produce ‘X-ray karyotypes’ of each spread, plotting the measured mass against the number of base pairs using the X-ray ptychography technique. The purpose was to observe the changes in the mass of the X-ray induced chromosomes. A complete set of 46 human metaphase chromosomes in one frame is known as ‘spread’. Exponentially growing cells were irradiated with X-ray doses at 0.1 Gy (Gray), 0.5 Gy and 1 Gy, followed by chromosome preparation from the irradiated as well as non-irradiated cells. DNA specific stain, 4’,6-diamidino-2-phenylindole (DAPI), was used to stain the chromosomes during the analysis. Using FLIM, a significant reduction in the DAPI lifetime values (2.8 ± 0.03 ns) was observed in the heterochromatin region of heteromorphic chromosomes 1, 9, 15, and 16 compared to the non-heterochromatin region of the chromosomes (2.88 ± 0.02 ns) present in the non-irradiated chromosome spreads. Furthermore, in the case of irradiated chromosomes, the heterochromatic regions of chromosomes showed statistically significant reduced lifetime values, with the difference ranging between ~260 picoseconds (ps) to ~290 ps compared to the non-irradiated chromosomes. This indicates that chromosomes might have undergone chemical perturbations. However, DAPI lifetime values were not significantly different between low-dose radiations. Therefore, low-dose irradiation is thought to induce an adaptive or hermetic response under certain conditions. However, the sensitivity of the FLIM could assist in the early detection of epigenetic variations that occur in the structure of chromosomes due to radiation/diseases. Key results were obtained from the Diamond Light Source (Harwell), I-13-coherence beamline by imaging metal-stained and unstained human chromosome spreads at room temperature to determine the gain or loss of genetic material upon low-level X-ray irradiation doses due to radiation damage. The obtained better resolution phase-contrast images of chromosome spreads gave the distribution of electron density which correlates with the mass of the chromosomes. The masses of non-irradiated and irradiated chromosomes were karyotyped to observe the difference in the masses. Therefore, the mass of the human genome can be determined using phase-contrast X-ray ptychography. The X-ray ptychography results are fascinating. The importance of this research lies in developing a new and improved technique to image and weigh individual chromosomes that will benefit medicine in diagnosing cancer and other health problems from patient samples.

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