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 technique of single-cell analysis using time-resolved inductively coupled plasma-mass spectrometry has been characterized and optimized. Determination of the metal contents of individual cells provides data on the natural metal contents of the cells and the corresponding distributions in the population. The distribution is a useful indicator of the health and the state of development of the cells. The contents of sorbed metals of individual cells over a duration of time are required to understand the dynamics of metal-cell interactions. A green alga, Chlorella vulgaris, was used as a model biological cell in this study. The criteria and procedures for proper sampling of the cells into the ICP will be discussed. Ideally, each ICP-MS spike corresponds to one cell, but cell overlapping occurs because the cells enter the ICP randomly. Selection of cell number density and sample uptake rate to minimize spike overlapping will be discussed. A cell counting method based on the frequency of the spikes has been developed. The distribution of the metal contents of cells was determined by measuring large number of spikes. The minimum number of spikes required was determined by statistical analysis. The spike intensity distribution was correlated with the size distribution of the cells. The peak maximum of the spike intensity distribution was used for the determination of the average metal content of the cells. The use of the peak maximum reduces errors due to spike overlapping in the measurement. Quantitative determination of the metal contents was achieved using standard particles for calibration. Errors in calibration using standard solution nebulization were discussed. The technique was applied in the study of metal-cell interactions. Sorption of heavy metal ions (as environmental pollutants) by Chlorella vulgaris, and uptake of biometal (as nutrient) and metallodrug (as toxin) by Helicobacter pylori were studied. The technique requires simple sample preparation of removing the culture medium by filtration or centrifugation. The health state of the cells in the presence of toxic metals was related to the change in cell number density. The ratio of the FWHM of the spike intensity distributions of the sorbed metals to the natural metal contents of the cells is identified as a possible indicator of the location of the sorbed metals. The kinetics of metal sorption by the cells can be studied using a single cell culture. The method reduces errors due to uncertainties in cell number density and metal concentration in multiple samples that are required in conventional methods. The optimal ICP-MS sampling depth of 17 elements, introduced into the ICP by conventional solution nebulization of aqueous standard solutions, has been determined. The elements were selected to represent a wide range of boiling points and ionization potentials. Boiling point of the dried residues and ionization potential of the analyte element were identified as the major factors that determine the optimal sampling position. Since dried sample solution aerosols are effectively nanoparticles, the study provides useful insight on the optimization of the operation conditions and calibration strategies for single-particle analysis using ICP-MS. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

While single-cell RNA sequencing provides a remarkable window on pathophysiologic tissue biology and heterogeneity, its high gene-dropout rate and low signal-to-noise ratio challenge quantitative analyses and mechanistic understanding. This thesis addresses this issue by developing PISCES, a pipeline for regulatory network-based single-cell analysis of mammalian tissues. PISCES accurately estimates the mechanistic contribution of regulatory and signaling proteins to cell state implementation and maintenance based on the expression of their lineage-specific transcriptional targets, inferring protein activity for a putative set of transcriptional regulators and cell-state markers. Experimental validation assays – including technical analysis via downsampling of high depth data and biological analysis by assessing concordance with CITE-Seq-based measurements – show a significant improvement in the ability to identify rare subpopulations and to elucidate key lineage markers compared to gene expression analysis. The improved ability to identify biologically meaningful cellular subpopulations makes PISCES an ideal tool to deconvolute heterogeneity in a wide variety of biological contexts. A systematic analysis of single-cell gene expression profiles in the Human Protein Atlas (HPA) by PISCES generated tissue-specific clustering and master regulator analyses across 26 human tissues, as well as a publicly available repository of ready-to-use regulatory networks specific to cell-lineages in each tissue. This resource will allow researchers to access the algorithmic advantages of PISCES without requiring prohibitively expensive or technically challenging computational resources. Additionally, PISCES is able to unravel the heterogeneous stromal environment of Pancreatic Ductal Adenocarcinoma, a malignancy defined by a large and complicated stromal compartment. This analysis reveals several novel candidate subpopulations, including a fibroblast subtype that has never been observed in humans, a potential pro-metastatic population of endothelial cells, and a population of immune-suppressing stellate cells. PISCES is also able to deconvolute more continuous forms of heterogeneity, as demonstrated by an analysis of epithelial cells in the developing murine lung. Here, PISCES is able to computationally reconstruct a developmental trajectory between Sox9+ distal cells and Sox2+ proximal cells, which is then leveraged to identify several novel markers of the critical intermediate population. Subsequent analysis suggests that these transition zone cells may share programs similar to those seen in injury repair and identifies a candidate therapeutic target that can drive cells into or out of this transition state. Finally, protein activity measured by PISCES is used to refine faulty experimental labels through differential density analysis. This analysis lead to the development of a machine learning classifier that accurately predicted increased degrees of stemness in experimentally transduced populations. Additionally, the density analysis paradigm has been extended to unsupervised settings, allowing for the detection of stable cellular populations and transitory trajectories.

Cells have been found to have an inherent heterogeneity that has led to an increase in the development of single-cell analysis methods to characterize the extent of heterogeneity that can be found in seemingly identical cells. With an understanding of normal cellular variability, the identification of disease induced cellular changes, known as biomarkers, may become more apparent and readily detectable. Biomarker discovery in single-cells is challenging and needs to focus on molecules that are abundant in cells. Lipids are widely abundant in cells and play active roles in cellular signaling, energy metabolism, and are the main component of cellular membranes. The regulation of lipid metabolism is often disrupted or lost during disease progression, especially in cancer, making them ideal candidates as biomarkers. Challenges exist in the analysis of lipids beyond those of single-cell analysis. Lipid extraction solvents must be compatible with the lipid or lipids of interest. Many lipids are isobaric making mass spectrometry analysis difficult without separations. Single-cell extractions using nanomanipulation coupled to mass spectrometry has shown to be an excellent method for lipid analysis of tissues and cell cultures. Extraction solvents are tunable for specific lipid classes, nanomanipulation prevents damage to neighboring cells, and lipid separations are possible through phase dispersion. The most important aspect of single-cell analysis is that it uncovers the extent of cellular heterogeneity that exists among cellular populations that remains undetected during averaged sampling.

Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.

Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.