The response of mammalian cells to ionizing radiation has been extensively studied with single cells exposed to acute doses. Little information is, however, available for cells growing in tissues, especially for cells subjected to multiple exposures. Our aim in this thesis was therefore to use a more complex in vitro system, three dimensional spheroids grown from V79-171b Chinese hamster lung cells, to determine the role of repair, redistribution and repopulation during multifraction irradiation. Repair and redistribution effects were isolated by using spheroids under normal culture conditions of 37°C, or at 22°C where repair occurs but cell proliferation is markedly inhibited. As expected, we found that cells surviving an initial 8 Gy dose showed cell cycle dependent fluctuations in radiosensitivity when allowed to progress at 37°C before exposure to a second 8 Gy dose. Sublethal radiation damage was repaired more rapidly at 37°C than at 22°C, and was also affected by proliferation. Due, however, to the small proliferating population in the spheroid system, a large initial dose was required to produce a population with enough synchrony for the expected split dose survival fluctuations throughout the cell cycle to be observed. When two doses of 6 Gy separated by 4 hours were administered to spheroids, the subsequent cellular radiosensitivity to a third dose remained quite constant for at least 10 hours, indicating a more extended mitotic delay than observed in the two dose experiments. Mitotic delay consequently was not linear with dose, a result apparently dependent upon the fractionation scheme used, and the complexity of the multicell system. In multifraction schedules where doses of 6 Gy or 8 Gy were administered daily for 6 days, we found, as expected, that repair, redistribution and repopulation all affected cell viability. However, each effect dominated at different times throughout the experiments. The overall cytotoxicity for each 6 Gy fraction decreased with increasing fraction number, while the 8 Gy fraction survival remained fairly constant. A novel feature of our experimental design, administering each 6 Gy or 8 Gy fraction in 1-2 Gy increments, also allowed evaluation of successive responses to the clinically relevant dose of 2 Gy. Cell survival at that level fluctuated greatly due to a decreasing repair capacity, 'and an increasing effect of repopulation with fraction number. Using two radioactive Iridium sources of different activities, high dose rate fractionated exposure was compared to continuous low dose rate irradiation. Also, the linear quadratic model was used to predict the equivalent doses. We found that the model did not provide a good prediction; more repair of radiation induced damage was observed at the lower dose rates than the higher dose rates, an effect which could not be incorporated into this theoretical model. We conclude that, with fractionated radiation exposures to the spheroid system, repair, redistribution, repopulation and cell killing all contribute to the multifraction responses. Each has varying significance on each fraction. An equal effect per fraction, often implicit in radiotherapy regimens, is therefore only achieved in the fortuitous situation where repair, redistribution, repopulation and cell killing combine in different proportions to result in the same overall survival. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The behavior of bimodal suspensions of oblate spheroids undergoing simple shearing flow has been described by analyzing the frequency spectra of orientation-dependent macroscopic properties. In particular, the frequency spectra of rheo-optical transients in turbidity and angular light scattering obtained experimentally from mono- and bimodal suspensions of human and frog erythrocytes showed peaks corresponding to the dominant frequencies of the particles in the flow. The method can, in principle, be applied to multimodal dispersions to obtain information on particle geometry. / The effect of high polydispersity on the rheo-optical transients of sheared suspensions have been studied. The time-dependent behavior of such systems is neither determined by the period of rotation defined by the mean axis ratio nor by the mean period of rotation amongst particles, but by the most probable period of rotation (i.e. the value corresponding to the maximum in the distribution of periods of rotations). Rheo-optical transients in polydisperse kaolin (clay) suspensions have been experimentally obtained. Here the oscillations, in contrast to the slightly polydisperse suspensions studied previously, are highly damped. Frequency spectra obtained from these experiments have peaks near 2/T(,m) , where T(,m) is the most probable period in the ensemble. / Finally, the influence of the rotary Brownian motion on the rheo-optical transients of sheared spheroidal suspensions is considered by using simulation procedures such as those employed previously to analyze rheological properties and memory loss. The rotary Brownian motion induced two effects: damping of oscillations and a shift in the extrema of the oscillations resulting in variation in the apparent mean period of rotation. Experiments with human erythrocytes at low Brenner number confirmed such behavior. Also a system with strong rotary Brownian motion was observed for which the rheo-optical transients showed monotonic changes. / The observations made in this work contribute to a better understanding of the transient rheo-optical properties of flowing suspensions.

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Traditional Chinese medicine (TCM) plays an important role in the Chinese healthcare system for over five thousand years. It includes the use of herbal medicine, acupuncture, Tui Na (推拿), and diet therapy. TCM helps to maintain a balance of Yin-Yang (阴阳), Five Phases (五行), Meridians (经络) and Qi (气) inside the body. In practise, pregnant women take tocolytic drugs to tonify the blood and qi to provide a continuous supply of nutrients for baby. Traditional Chinese herbal medicines usually prescribed as a complex formula to produce synergistic or agonistic effect to maintain a well balance of the above components in human bodies. Moreover, TCM usually cannot produce immediate effect on patients, therefore, the efficacy of individual component remains largely unknown. This study aims to investigate whether Chinese tocolytic drug components could modulate fertility by affecting the in vitro spheroid (blastocyte surrogate) attachment process by using trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells to mimic the embryo-endometrial implantation process. Nine Chinese herbal medicinal compounds (Atractylenolide I(白术内酯), Atractylenolide II(白术内酯II), Atractylenolide III(白术内酯III), Paeoniflorin(芍药苷), Albiflorin(芍药内酯苷), Nuzhenide(女贞子甙), Ecliptasaponin A(旱莲甙A), Wedelolactone(蟛蜞菊内酯) and Columbianadin(二氢欧山芹醇当归酸酯)) which are commonly found in traditional Chinese tocolytic drug formula were selected to study (1) the toxicity of the drugs on trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells growth, (2) the effect of three tocolytic drugs (Atractylenolide I, Atractylenolide II and Atractylenolide III) on spheroid attachment, and (3) their effect of the expression of Wingless (Wnt) signaling molecules (Active-β-Catenin, Axin-2, β-catenin, E-cadherin, GSK-3β, and Mucin-1). It was found that the nine compounds, Atractylenolide I, Atractylenolide II, Atractylenolide III, Paeoniflorin, Albiflorin, Nuzhenide, Ecliptasaponin A, Wedelolactone and Columbianadin did not affect cell viability at 25μM, 25μM, 5μM, 0.2μM, 125μM, 125μM, 125μM, 5μM and 25μM, respectively, by cell proliferation assay. However, at these concentrations, the spheroid attachment was not significantly increased by Atractylenolide I, Atractylenolide II and Atractylenolide III. Interestingly, the protein expression of GSK-3β and Active-β-catenin were up-regulated by the three compounds in both cells and JEG-3 cells respectively. The expressions of Axin-2 and E-cadherin were up-regulated by Atractylenolide III in Ishikawa cells and Atractylenolide II in JEG-3 cells. Atractylenolide I and Atractylenolide III increase the Ishikawa cells expression of Active-β-catenin and β-catenin respectively and together suppress the JEG-3 cells Mucin-1 and β-catenin expression. In conclusion, the nine tocolytic compounds have different effect on cell proliferation. Atractylenolide I, Atractylenolide II and Atractylenolide III did not enhance the attachment rate of JEG-3 spheroid onto Ishikawa monolayer. However, they affected Wnt-signaling molecules expression, suggesting that they may modulate endometrial receptivity. Further experiments are needed to study their combined effect on co-culture and expression of Wnt-signaling molecules. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences