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<p>Radiation induced bystander effects have given the cancer risk analysis a whole new paradigm. However the actual mechanism involved in producing the effects is still not clear. The basic bystander signal is assumed to be a biological signal. In this study we proposed and tried to quantify the presence of a physical signal in the form of electromagnetic radiation that can trigger a biological response in the bystander cells. In bystander effect studies where the cells are exposed to very low fluence of charged particles there could be several regions that can produce electromagnetic radiation due to the process of atomic/molecular excitations and relaxations. We focused on quantifying the number of ultraviolet photons emitted when charged particles pass through different media that have relevance to radiation biology experiments. The choice of UV photons was made due to the reason that its effects on living cells are very well documented. For this purpose we developed a system which employed the technique of single photon counting to measure the light emitted from samples irradiated under vacuum by a charged particle beam. Photon counting was done using a fast photomultiplier tube (Hamamatsu R7400p) with a peak cathode response at 420 nm wavelength.</p> <p>In the early set of “proof of principle experiments” we tested polystyrene targets for ion beam induced luminescence. Polystyrene is one of the materials that are used as a cell substrate for radiation biology experiments. The luminescence yield from polystyrene was measured in terms of absolute value i.e. number of photons per second per unit solid angle. The output appeared to have a non-linear behavior with the incident Ion fluence: it rose exponentially to an asymptotic value. We irradiated the samples with beam energies varying from 1 MeV to 2.0 MeV and showed saturation at or before an incident fluence rate of 3×10¹³ H⁺/cm²s. The average saturation value for the photon output was found to be 40 × 10⁶ cps. Some measurements were performed using filters to study the emission at specific wavelengths. In the case of filtered light measurements, the photon output was found to saturate at 28×10³, 10×10⁶, and 35×10⁶ cps for wavelengths of 280±5 nm, 320±5 nm and 340±5 nm respectively. Using the IBIL signal evolution characteristics with the ion fluence we determined the ions produce a damage having a cross section of the order of 10^-14 cm² in polystyrene. The average radiant intensity was found to increase at wavelengths of 280 nm and 320 nm when the proton energy was increased. Having found an evidence of a significant production of UV in ion irradiated, biologically relevant, material we extended this study further into the measurements from other relevant materials in radiation biology.</p> <p>Here charged particle irradiation was performed using positively charged protons (H⁺) ranging in energy from 1.2 MeV to 2.2 MeV at a fluence rate of 2.7×10¹⁰ protons mm^-2s^-1.The materials chosen for this study were polypropylene, Mylar, Teflon, and Cellophane as they are all materials commonly used in radiation biology experiments as cell substrates or containers. In addition, we performed measurements of two NIST standard materials derived from living cells: oyster tissue and citrus leaves. These materials were measured as a powder.</p> <p>All the container materials were found to emit UV frequency photons at emission levels that are significant enough to warrant further investigation of the potential biological consequences. In addition, the NIST standard reference materials oyster tissue and citrus leaves also emitted UV when irradiated. This suggested that biological materials may themselves emit UV at significant levels when irradiated with charged particles.</p> <p>We established this further by irradiated cells with β-particles. Cells were plated in Petri-dishes of two different sizes, having different thicknesses of polystyrene (PS) substrate. Exposure of the cell substrates (polystyrene) only resulted in the production of 1035 photons per unit activity in μCi of ⁹⁰Y which was equivalent to an exposure of 840 <em>β</em>-particles/cm² to the substrate. For a collimated electron beam exposure, we observed 158-167 photons per unit μCi (18 β-particles per cm² on the substrate) for different thicknessesof the substrate. Upon irradiating HPV-G cells plated on the PS dishes we determined that the luminescence gradually increased with the increasing exposure of β-particles; reaching up to 250 % of that of the luminescence without any cells for an activity of 180 μCi. For general irradiation conditions we found statistically significant difference in luminescence output for varying cellular densities with cells only and with the application of medium on top of the cells. The colourless medium increased the total luminescence yield while the coloured medium decreased it. When the cells were irradiated using a collimated beam of electrons it was found that the luminescence decreases with the increasing cellular density thus providing an evidence of re-absorption of photons within the surroundings.</p> <p>After establishing the fact that charged particles induce light emission from the materials that have a relevance to the radiation biology experiments. We extended our study further to find out other sources of radiation that could affect the dose distribution in radiation biology experiments. In radiation biology experiments the low doses of radiation are usually delivered usingamicrobeam charged particle accelerator. Microbeams delivers a highly localized and small dose to the biological medium by using a set of collimators that confine the charged particle beam to a very narrow (micron level) region. Since the collimation block a significant proportion of the beam therefore there is a chance of the production of low energy x-rays and secondary electrons. We used Monte Carlo simulations to investigate the production of particle induced x-rays and secondary electrons in the collimation system and its possible effects on the final dose delivery to the biological medium. We found no evidence of the escape of x-rays or secondary electrons from the collimation system for proton energies of up to 3 MeV. The thickness of the collimators was sufficient to reabsorb all the generated low energy x-rays and secondary electrons. However if the proton energy exceeds 3 MeV then a significant proportion of 10 keV and 59 keV (K-α) x-rays can be emitted by the collimator. Further it was established that due to the phase space distribution of particles in various orientations with the beam axis there are significant chances of hitting non-targetted cells in microbeams that employ a collimator to confine the beam.This may happen due to the beam particles travelling obliquely with the beam axis thus passing the collimator edge and hitting the non-targetted cells. Another reason could be the scatter of beam particles inside the collimator.</p> <p>The evidence of the production of UV in materials relevant to the radiation biology experiments suggest that the conclusions and hypotheses derived from some radiation bystander experiments need to be re-thought, as charged particle irradiation leads to some level of UV emission in experimental materials which may be the cause of some “non-targeted” effects.</p> / Doctor of Philosophy (PhD)

TRF2 is an essential telomeric protein involved in preventing the telomere ends from being recognized as DNA breaks. I have shown that TRF2 does not appear to play a major role in the UV -induced DNA damage response in IMR90, Cockayne syndrome or XPC deficient cells. TRF2 binds telomeric DNA via its Myb domain and also contains an N-terminal basic domain. Expression of TRF2MMM causes telomere fusions, whereas TRF2^(ΔB) causes rapid deletion of telomeric DNA, as both phenotypes result in senescence. These phenotypes are dependant upon recombination events. Thus, the basic domain of TRF2 may be essential to suppress recombination events at telomeres. However, it is not fully understood what amino acid residues in the basic domain of TRF2 are indispensable to maintain its function. By creating mutations in the arginine residues in the basic domain of TRF2, I have shown that the positive charge of the basic domain alone is not sufficient to maintain its protective function. By expressing these TRF2 mutants in the presence or absence of the Myb domain in HT1080 and BJ/hTERT cells, I have been able to recapitulate the TRF2^(ΔB) and TRF2^(ΔBΔM) decreased proliferation and senescence phenotypes. Furthermore, by analyzing anaphase and metaphase chromosomes and performing Southern blotting, I have shed light on the molecular mechanisms responsible for the deleterious phenotypes observed in the TRF2 mutants. Amino acid changes from arginines to lysines introduced into the basic domain of TRF2 results in a significant increase in telomere doublets. However, when these TRF2 mutants are expressed in the absence of the Myb domain, a significant increase in telomere fusions events occur. Collectively, my results indicate that more than one arginine residue in the basic domain is essential to maintain the protective function of TRF2, as these arginine residues may act as substrates for protein arginine methyltransferases. / Thesis / Master of Science (MSc)

Efficient optical-to-electrical conversion is a fundamental requirement of a range of silicon devices such as those which employ photodetection, solid-state-imaging and photovoltaic power generation. This thesis investigates the effects of using indium, a deep-level acceptor in silicon, as a dopant for thin film single crystalline silicon solar cells and UV-A detectors. Indium acts as a p-type dopant in silicon and has been proposed previously as a substitutional lattice defect that would enable sub-band gap transitions as described by the so-called impurity photovoltaic (IPV) effect. The physical mechanisms responsible for operation of the devices presented in this work are described. Models for electrical performance, optical absorbance and device fabrication are used as methods to interpret data and optimize device parameters. Specifically, a two-diode model is used to account for the electrical loss mechanisms within a device, while modeling optical absorption by a multilayer structure consisting of Silicon-On-Insulator (SOI) is approached using a novel multi-wavelength numerical model that describes the reflections and transmissions at each of the device’s layers. Additionally, Technology Computer Aided Design (TCAD) simulations were used to optimize the critical fabrication parameters associated with the ion implantation and thermal annealing techniques used during the device fabrication process. Selected from multiple devices fabricated during the course of this work, the most efficient solar cells in SOI (2.5 μm thick active layer) exhibited a maximum conversion efficiency of 4.74 % for indium-doped and 4.16 % for boron-doped layers. The most efficient UV-A detector fabricated in SOI (100 nm thick) exhibited a maximum responsivity to 365 nm light of 20 mA/W for indium-doped and 16 mA/W for boron-doped devices. In both types of devices, indium doping consistently resulted in a relative increase in efficiency when compared to equivalently fabricated, boron doped devices, despite experimental carrier decay measurements confirming the action of the indium as a recombination centre. External and internal quantum efficiency measurements confirm a relative enhancement in absorption, for solar cells and detectors doped with indium, which is correlated with the p-type dopant concentration and the ratio of n-type to p-type concentrations. The origin of the enhancement is postulated to be caused by a relaxation of the momentum-space restrictions associated with undoped silicon, a postulate supported by previously reported absorption data. This thesis presents the first comprehensive data from indium doped silicon devices designed for optical-to-electrical conversion. The implications for a range of widely deployed devices may be significant. / Thesis / Doctor of Philosophy (PhD)

In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)

The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1. / Thesis / Master of Science (MS)

Host cell reactivation (HCR) of UV-irradiated (UV'd) herpes simplex virus type 2 (HSV-2), capacity of UV'd cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV'd HSV-2 were examined in human fibroblasts. The cells were derived from four Cockayne's Syndrome (CS) patients, 5 xeroderma pigmentosum (XP) patients and 5 normal patients. Survival curves for HCR of HSV-2 plaque formation showed 2- components. HCR was not significantly different in the CS strains and an XP variant strain compared to normal, whereas all excision deficient strains showed a significant reduction in HCR. The o37 values for the delayed capacity curves were in the range 8.6-12.4 J/m2 for the normal strains, 3.1-5.1 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.40-1.98 J/m2 for the XP excision deficient strains examined. UVER was also examined for HSV-2 UV-irradiated to survival levels of 10-2 and 10-3 in unirradiated cells. Maximum delayed UVER was observed in normal strains at a UV dose of 15 J/m2 to the virus. Maximum UVER in CS cells was detected at a UV dose of 5 J/m2 to the cells, in XP excision deficient cells maximum UVER occurred at doses ranging from 0.5-2.5 J/m2 to the cells, and in XP variant maximum UVER occurred at 10 J/m2 to the cells. In all cell strains the level of UVER increased with increasing UV dose to the virus. Results are discussed in terms of the repair defects of CS and XP cells and their relationship to possible viral repair functions. In addition, the possible existence of an inducible DNA repair response is discussed in terms of the results of this study. / Thesis / Master of Science (MSc)