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

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

Auger electrons have the potential for therapy mainly because of their short range and high level of toxicity. Biological effects of these electrons are critically dependent on the cellular and subcellular localization of their emitters. In this study 123I was used due to its relatively short half-life (13.2 h) and ideal y-ray energy (Ey=I59 keV). The short half-life is advantageous since it allows significant accumulation of disintegrations at normal physiological conditions without the need to freeze cells as required when long lived isotopes such as 1251 (T1/2=60.1 days) are used. The main objective of this study was to synthesize an organic compound that would deliver the radioactive 1231 into cellular DNA and to quantify the resulting damage from micronucleation. The higher degree of damage obtained in cell samples treated with 4-[123I]iodoantipyrine than with [123I]Nal is an indication of Auger electron effect. Also, micronuclei counts showed that 4-[123I]iodoantipyrine and [123I]Nal were prepared in a suitable manner and did not interfere with cellular kinetics. RBE values of 1.8 and 1.9 were obtained for CHO-Kl clone # 7 and # 16 cells, respectively, indicating lack of differential response for the two clones. When the current data is collectively analysed with that of other investigators using the same radiolabeled compounds, a reduction in Auger electron RBE is noticeable with radiosensitivity. This is consistent with the high-LET nature of Auger electrons. The extremely radiosensitive CHO-XRS 1 failed to give a dose-response as most cells underwent apoptosis. The significant radioprotection of CHO-K1 cells irradiated, in the presence of a chemical protector MEA, with 4-[ 123I]iodoantipyrine than with [123I]Nal showed that the mode by which Auger electrons cause damage is not the same as that of a-particles. Further studies involving the use of carriers that would specifically deliver radionuclides to nuclear DNA may be useful in understanding the mechanisms underlying the cellular responses to Auger electron exposure. / Thesis (M.Sc. (ARST) North-West University, Mafikeng Campus, 2005

Oxygen is known to sensitize X-irradiated cells to lethal radiation damage. At low ambient oxygen tensions, however, the molecular mechanisms of the sensitization process and the metabolic requirements of the cell may be forced to compete for the cellular oxygen supply. The effect of cell respiration on the availability of intracellular oxygen during irradiation was consequently investigated by comparing the radiosensitivities of respiring and non-respiring cells. Cultured mammalian cells were irradiated in single cell suspensions and thin film monolayers at respiration inhibiting (4°C) and at normal cell culturing (37°C) temperatures. Due to oxygen equilibration and radiolytic depletion problems, the results of the suspension culture experiments were inconclusive. By subsequently analyzing the diffusive mass transfer of oxygen in the suspension medium, the stirrer flask was determined to be an inappropriate culture vessel in which to irradiate cells at constant low oxygen concentrations. A thin film cell culture system in which the oxygen concentrations to which the cells were exposed during irradiation could be more accurately controlled was then developed. A comparison of the oxygen enhanced radiosensitivities of the respiring and non-respiring cells in thin film monolayers suggested that the metabolic depletion of oxygen at low oxygen tensions has a significant effect on the local and intracellular oxygen distribution. These effects are representative of those that would be produced if respiration induced oxygen gradients existed inside and immediately around respiring cells. The magnitude of the differential radiosensitivities was found to be dependent on cell shape and to have values that agreed very well with theoretical predictions based on the existence of such gradients. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The ability of mammalian cells to locomote is important in a variety of normal and pathological processes. Previous work has suggested that low doses of x-irradiation may perturb cell motility, a finding that may have important consequences in embryogenesis, cancer metastasis, and immune response. This thesis has sought to study in more detail the effect of radiation on mammalian cell motility. Work performed in other laboratories used the colloidal gold assay and time lapse cinemicroscopy to study x-irradiation induced changes to 3T3 fibroblast motility in tissue culture. These studies were repeated here, with qualitative results similar to those reported earlier. However, these methods were not amenable to a detailed quantitative analysis. For this, spatial and temporal information on the motility and dynamic morphology of a large number of cells is required. Such a task would be impossible to perform manually, thus an automated microscope system was developed that used a computer-driven precision stage and a solid state optical sensor to track individual cells in tissue culture. Information on motility and morphology was concurrently extracted from many cells. As part of the thesis, several techniques were developed to analyze and display these data, and to correlate motility and morphology observations. These techniques were directed at preserving the actual process of 3T3 cell motility, and parameters were measured to quantify the short term persistence of cell movement (on a time scale of 0.5 to 2 hours), and the long term persistence of cells in maintaining certain characteristic behaviour (on a time scale of 3 to 12 hours). The response of 3T3 fibroblasts to x-irradiation was characterized by a number of parameters. The population average cell speed was measured following treatment, and a dose response and time response was determined in the range of 1.5 Gy. Other motility parameters indicate that the normal process of cell motility, evidenced by a series of motile segments, was disrupted by x-rays. This was thought to reflect perturbation to the control mechanisms of cell motility. The morphology of 3T3 cells stained with Coomassie blue was examined in an effort to correlate the observed motility changes with changes in the fixed cell morphology. This stain is a general structural protein stain with higher affinity toward microfilaments. High doses of x-rays were required to produce significant perturbation to cell morphology, and in the dose regime of interest, the morphology of irradiated cells was not identifiably different from control. Of note is that it was the well spread, quiescent cells that seemed least perturbed by large doses of irradiation. In summary, x-rays apparently disrupt the normal process of cell motility. Several lines of evidence suggest that actively migrating cells are the most perturbed by irradiation. This work has developed techniques to quantify cell motility in a meaningful way, and to characterize the x-ray induced perturbations. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

D.Tech. (Biomedical Technology) / Stem cells are defined as undifferentiated cells that can proliferate and have the capacity of both self-renewal and differentiation to one or more types of specialised cells (Bishop et al., 2002). The two types of stem cells are embryonic and adult stem cells. Adult stem cells have been isolated from adipose tissue in abundance and with ease (Mvula et al., 2010) and these cells have been differentiated into smooth muscle cells (SMCs) with the enhancement of low intensity laser irradiation and the growth factors (de Villiers et al., 2011). Smooth muscles play an important role in diseases like cancer, hypertension, asthma and others (Rodriguez et al., 2006). Studies have shown that low intensity laser irradiation (LILI) can increase proliferation of cells, cellular attachment, differentiation and production of transforming growth factor-beta 1 (TGF-β1) in cells indicating that in vitro LILI can modulate the activity of cells and tissues (Khadra et al., 2005). Further studies have also discovered that LILI enhances wound healing (Fiszerman and Markmann, 2000). LILI has been successfully used for pain attenuation and to induce wound healing in non-healing defects (Hawkins and Abrahamse, 2005). LILI has been shown to increase viability and proliferation of adipose derived stem cells (ADSCs) (Mvula et al., 2008 and Mvula et al., 2010). Growth factors such as retinoic acids (RA) have been shown to have major influences on cells. They are involved specifically in apoptosis, cell proliferation, differentiation and maturation (Duong and Rochette, 2011; Gudas and Wagner, 2011). Co-culturing is used to achieve several cellular processes including proliferation, differentiation and migration (Kim et al., 2012). When two types of cells are cultured together, they are exposed to a number of complex environmental factors such as cytokines, extracellular matrix components, cell interactions, mechanical stimuli, signalling transcriptional pathways and transcriptional factors such as growth factors. v These factors are able to affect migration, proliferation and differentiation of one cell type into another (Zhang et al., 2012). The aim of this study was to investigate the differentiation potential of ADSCs when co-cultured with (SMCs) and to determine the role of LILI on the co-cultured cells. Short and long term biological effects were monitored on these cells following exposure to LILI and addition of growth factors. The study used commercial and isolated human ADSCs and SMCs (SKUT-1) cells. After growing cells to semiconfluency for ADSCs and confluency for SMCs, they were co-cultured in a ratio of 1:1 using the established methods supplemented with and without growth factors (TGF-β1and RA) and then exposed to LILI. The cellular morphology, viability and proliferation activities of the irradiated cells were then assessed using direct inverted and differential interference contrast microscopy (DIC), trypan blue test, adenosine triphosphate luminescence, optical density analysis, and carboxyfluorescein diacetate succinimdyl ester (CFSE) methods. In particular the expression of the specific markers of both ADSCs, β1 Integrin (CD29) and Thy-1 (CD90) and SMCs, Myosin Heavy Chain (MHC) were investigated through immunoflourescent microscopy and flow cytometric analysis. Up and down regulation of genes involved in the human mesenchymal stem cell array were analysed through Reverse Transcriptase Polymerase Chain Reaction (RTPCR)...

The efforts addressed in this thesis refer to assaying the degradations in modern solar cells used in space-borne and/or nuclear environment applications. This study is motivated to address the following: 1. Modeling degradations in Si pn-junction solar cells (devices-under-test or DUTs) under different ionizing radiation dosages 2. Preemptive and predictive testing to determine the aforesaid degradations that decide eventual reliability of the DUTs; and 3. Using electrical overstressing (EOS) to emulate the fluence of ionizing radiation dosage on the DUT. Relevant analytical methods, computational efforts and experimental studies are described. Forward/reverse characteristics as well as ac impedance performance of a set of DUTs under pre- and post- electrical overstressings are evaluated. Change in observed DUT characteristics are correlated to equivalent ionizing-radiation dosages. The results are compiled and cause-effect considerations are discussed. Conclusions are enumerated and inferences are made with direction for future studies. / by George A. Thengum Pallil. / Thesis (M.S.C.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Many experiments are being conducted to find compounds that offer radioprotection against radiation damage and that are also non-toxic. It is hopeful that in the future, research for this technology will benefit patients undergoing cancer treatment by reducing radiation damage to normal cells and therefore reducing short and long term side effects experienced from treatments. Hamster cells were irradiated at doses of 60 and 120 rad, with and without Sphingosine-1-Phosphate mixed in with their growth medium. Post irradiation, it was observed that the S1P molecule seemed to have a radioprotective effect by decreasing the amount of cell death compared to the amount of cell death that occurred with the absence of the molecule. The results of this experiment will sent to Dr. Jon Tilly at Massachusetts General Hospital. Dr. Tilly is currently researching S1P as a possible radioprotector. / Graduation date: 2003

A new nanodosimetry-based cell survival model for mixed high- and low-LET radiations has been developed. The new model employs three dosimetry quantities and three biological quantities. The three dosimetry quantities are related to energy depositions at two nanometer scales, 5nm and 25nm. The three biological quantities are related to lesion production and interaction probabilities, and lesion repair rate. The model assumes that the lesions created at the two nanometer scales are directly or indirectly responsible for cell death depending on the lesions interaction and repair rate. The cell survival fraction derived from the new model can be expressed by the familiar dose-dependent linear quadratic formula. The coefficients alpha and beta are based on the three nanodosimetry quantities and the three biological quantities. Validation of the new model has been performed both by using published data and by the experimental data obtained. Published cell survival curves for V-79 Chinese hamster cells irradiated with various LET of radiations were used for validation. The new model was applied to radiation therapy by irradiating V-79 cells with mixed fission neutron and gamma-rays. The results show that the new model has been successfully used in a mixed n+g field to predict the synergistic effect between neutron and gamma-ray lesions and the RBE for fission neutrons.

Thesis (MTech Biomedical Technology)--Cape Peninsula University of Cape Town, 2005 / In clinical radiotherapy, experiments are performed to determine optimal conditions of the radiation prior to radiotherapy. These experiments focus on the relative biological effectivness(RBE) determination and are predominantly applied in high linear energy transfer (LET) radiations i.e. fast neutrons, as the RBE values for such radiations vary greatly. In general, the RBE of a certain radiation relative to a given reference radiation flCo gamma) varies widely with the energy, dose, dose rate, fractionation, type of tissue and end-point used. Experience with neutron therapy at iThemba LABS has shown that treatment with more fractions and lower doses per fraction may be beneficial for some patients. To calculate the iso-effective treatment dose needed, an appropriate alp ratio for early effects is needed. In this study, the repair of mouse jejunum was measured for split-dose irradiations to determine if a suitable alP ratio for neutrons could be estimated using the known value for gamma rays and the applicable RBE.. Crypt stem cell survival was measured 3.5 days after split-dose exposures to p(66)/Be neutrons and 6OCo gamma rays. Dose response curves for both treatment modalities and for both acute and fractionated exposures were constructed by counting crypts of Leiberkhiin at the base of the villi in haematoxylin and Eosin-stained sections of mouse jejunum. Using a RBE value of 1.64 and an alP ratio of 7Gy noted for tbe fractionated photon exposures, an alP ratio of 11.5 IV could be estimated for neutrons.