The study of the effects of damaging agents on living cells is of importance both for the assessment of the potential consequences of exposure, and for the advancement of medical technologies concerned with the treatment of cancer and other diseases. Such agents, however, are commonly studied at doses and levels of effect much higher than those seen in environmental or even medical exposures. Studies of cell survival after treatment with ionizing radiation, for instance, generally involve the measurement of dose-response over several decades of cell kill on a logarithmic scale. In contrast, cell survival rates for a typical clinical treatment dose are on the order of 50%. Survival measurements in this first decade of cell kill require that the exact fate of thousands of cells is determined in a single experiment. This is beyond human capabilities. Measurements at these levels of effect therefore require the development of a rapid, automated system of cell detection, characterization, and follow-up. The primary aim of this thesis was to test the hypothesis that the algorithms necessary for generating survival data with such an automated system can be developed to standards of accuracy comparable to those of an experienced human observer. Using an image cytometry device specifically designed for the detection and analysis of live, unstained cells, automated scanning procedures were optimized for selected cell lines, and means of maintaining appropriate focus levels during a scan were devised. Algorithms to distinguish cells from other objects detected in the flask were also developed. These performed with comparable accuracy, but at greater speed, than could be achieved by a human observer. Because the endpoint used in survival measurements is the ability of treated cells to proliferate to form colonies, the hypothesis that automated methods of assaying colony formation could be developed was also tested. Using rapidly collected, low resolution image data obtained at locations in the tissue culture vessel where individual cells had been detected on the day of treatment, it was found that the survival status at 70-90% of these locations could be determined automatically. Manual assessments were required at the remaining locations. The final objective of this thesis was to use the methods developed for automated sample selection and survival assessment to examine a question relevant to both radiotherapeutic applications and the understanding of mechanisms of radiation action. In particular, the Theory of Dual Radiation Action (Kellerer and Rossi, 1972), which predicts that the relative biological effectiveness (RBE) of different modalities of sparsely ionizing radiations may change dramatically with decreasing dose, was tested experimentally. Using two different mammalian cell lines, the experimental data in general rejected the predictions of this theory, although a modified theory, developed by the same authors, could accommodate the results. In summary, the data showed that there may be a slight dose dependence in the RBE of the radiations tested. Specifically, the RBE of low energy X-rays relative to ⁶⁰Co γ-rays was found to increase slightly with decreasing dose. The measured RBE's in the zero-dose limit were ~1.4 for 55 kVp X-rays and ~1.1-1.2 for 250 kV[subscript]p X-rays. High energy (11 MeV) electrons, on the other hand, showed a small decrease in RBE relative to ⁶⁰Co γ-rays as the dose approached zero, having a zero-dose limit of ~0.95. All of the aforementioned radiation modalities had an RBE of 1.0-1.1 at high doses (10 Gy or more). / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Criteria are developed for the design of an experiment to study steady radiation fronts in Cs vapor. According to these criteria, a pulsed light source of supposedly sufficient intensity (≃10²²photons/cm²-sec) and pulse length (≃90ys) was built, and an absorption tube for Cs vapor inside an oven was developed (through several models) which produced a maximum absorber density of ≃10¹⁸particles/cm³. Light and electrical probe measurements were carried out to detect ionizing radiation fronts in the cesium. While the light measurements were inconclusive due to stray light problems, the probe measurements showed that photoionization takes place but at a much lower rate than expected. A subsequent study of the light source confirmed that indeed the light source, a constricted capillary arc, driven by a 90μs square current pulse, has a much lower intensity in the test section of the absorption tube than is required to drive a front. The low intensity of this light pulse rendered the initial aim of the experiment namely the study of steady ionization fronts in cesium vapor, unattainable with the available apparatus. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The primary objective of this study was to investigate the radiosensitivity of Pseudotsuga menziesli (Mirb.) Franco, Douglas-fir, from two different provenances representing the coastal and interior regions in British Columbia, Picea sitchensls (Bong.) Carr., Sitka spruce, and Tsuga heterophylla (Raf.) Sarg., Western hemlock. A secondary objective was to correlate the radiosensitivity with some cytogenetical and biochemical characteristics. Induction of mutations and radiostimulation of seed germination and seedling growth were also sought. Filled seeds of the above mentioned species were given five different dosages of gamma-irradiation from a cobalt-60-source control, 500, 2,000, 5,000, and 10,000 R. Stratification for 14 days at 0°-2°C as post-irradiation treatment was tested. Germination values were evaluated and germinants were transplanted. Survival and growth under controlled environmental conditions were recorded for 182 days. The species exhibited differential responses to seed irradiation. All showed drastic reduction in germination and survival at the higher dosages, (5,000-10,000 R). There was some stimulation of Interior Douglas-fir seed germination and seedling survival at 500 and 2,000 R irradiation dosages over the control. The same exposures stimulated the height growth of Coastal Douglas-Fir. Stratification after irradiation reduced seed germination and seedling growth and survival in all the species. The tolerance to gamma-irradiation decreased in the following Order: Interior Douglas-fir, Coastal Douglas-fir, Western hemlock and Sitka spruce. Sitka spruce had a significantly larger nuclear volume than Western hemlock and Douglas-fir. No correlation was found between nuclear volume, or interphase chromosome volume, and LD₅₀ (germination). The amount of DNA per cell and per chromosome differed significantly among the three species with Sitka spruce having the highest DNA content followed by Western hemlock, Coastal Douglas-fir and Interior Douglas-fir. Significant negative correlations were found between DNA content per cell and per chromosome, and LD₅₀ (germination). This indicated that DNA content plays a more important role than nuclear volume in determining the radiosensitivity of the species. The differences in radiosensitivity, nuclear volume and DNA content between the Coastal and Interior forms of Douglas-fir are discussed in relation to their ecogeographical distribution and taxonomy. Chromosome breaks, micro-nuclei and chromosome erosion were detected in irradiated seeds of Douglas-fir. Intraspecific hybridization was carried out in Douglas-fir using gamma-irradiated pollen grains. Pollen irradiation up to 5,000 R increased the number of filled seeds/cone. Seedlings from pollen irradiated at 500 R, exhibited some increase in height growth. Similar effects were obtained for pollen germination in vitro. The possibilities of utilizing seed and pollen irradiation in forest tree improvement are discussed' and recommendations are made for future mutation breeding work. / Forestry, Faculty of / Graduate

Survival studies using cell sorting techniques together with data averaging on asynchronously dividing V79-WNRE cells reveal statistically significant evidence of substructure in the radiation survival response at low dose (2 to 3 Gy) that cannot be adequately characterized by a single linear-quadratic function, S = exp( -αD -βD²). The data suggest a two-component response that is particularly evident when plotted in the linear form of the linear-quadratic equation, -ln(S)/D = α + βD. Application of other survival models (Single Hit + Multi Target and Repair-Misrepair) does not eliminate the substructure. The substructure may result from subpopulations of cells at different stages of the cell cycle, which differ in their radiosensitivity, giving rise to a two-component survival response. In order to explore this hypothesis, studies were carried out with populations of partially synchronized cells. The mitotic selection technique was employed alone or in combination with 12 hours exposure to 1 mM hydroxyurea or 1 μg/ml aphidicolin to obtain large numbers of synchronized cells. G1/S phase populations obtained by mitotic selection followed by hydroxyurea or aphidicolin do not show the substructure found for asynchronous cells, thus supporting the hypothesis. G1 phase populations obtained by mitotic selection alone revealed substructure which could be characterized by a single fit to the RMR or SH+MT models. Structure was also present in the response of heterogeneous S/G2 phase populations synchronized by mitotic selection followed by hydroxyurea, but may not be present in the response of S phase populations synchronized by mitotic selection alone. The data are consistent with the hypothesis that the substructure is a result of sensitive (G1, G2 or M) and resistant (S) subpopulations of cells. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The microwave dielectric constant and conductivity of MEM(TCNQ)₂ were studied in the neighbourhood of the monomer to dimer transition at 61°C using cavity-perturbation techniques at 9 GHz. The conductivity was found to be in general agreement with four-probe d.c. results. Thus, doubts about the reliability of the d.c. measurements above the destructive transition have been removed. The complex dielectric constant of TTF-TCNQ at liquid helium temperatures was studied using dielectric resonance techniques. Some anomalies regarding the interpretation of the dielectric resonance mode plots were resolved. Values for Є’ of (3.0±0.4)X10³ and for Є’of greater than 9 were implied by the results. Finally, preliminary results and proposed directions for a bolometric absorption measurement in TTF-TCNQ in the microwave bands are presented. This technique may prove useful for the direct observation of the pinned charge density wave in TTF-TCNQ. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

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