Publicação
Contribution to fundamental aspects of biophysics, radiobiology, and modeling of cellular response to low radiation doses
| Resumo: | This thesis aims at studying the direct and bystander effects (observations in which effects of ionizing radiation arise in non-irradiated cells) induced by alpha-particle irradiation, using a Po-210 source, in a human lung adenocarcinoma cells (A549), and at investigating and providing a contribution to the clarification of the protective or potentially detrimental effects of low-dose exposures (< 100 mGy). Besides addressing and analyzing the toxic effects of α-radiation on the A549 cell line the studies undertaken may contribute toward the goal of improving the effectiveness of targeted therapies, with alpha-radiation, such as radioimmunotherapy or Boron Neutron Capture Therapy (BNCT). The work performed consisted, at first, of a dosimetric study in order to determine the average local dose in a cell monolayer, encompassed by experimental and computational studies, followed by a biological study of the direct and bystander effects of α-radiation exposure. The averaged dose rate at the cell monolayer was calculated; through the Linear-Energy-Transfer (LET) value measured using a Passivated Implanted Planar Silicon (PIPS) detector, to be 25 mGy/min. To study the cellular response at low-dose exposures, several studies were conducted using the A549 cell line and a 210Po α-source. Initially, the effects of directly irradiated A549 cells were analyzed through the micronucleus and clonogenic assay. Then, the time and dose dependence of directly and bystander effects in the region of very low doses, was investigated through micronucleus and clonogenic assay. The study encompassed three different cell culture conditions: i) a culture of irradiated cells, ii) a medium transfer culture with non-irradiated cells and iii) a culture with irradiated cells after centrifugation. A dependence on dose and time was observed in both directly irradiated and bystander cells. Taking the advantage of the in situ γ-H2AX technique, the way how the bystander signals released after low-doses of α-radiation are influenced by the number of irradiated cells was studied. Evidence that bystander signals can easily spread through the culture medium was observed. Moreover, the neighboring of irradiated cells differ with the number of irradiated cells, i.e. with a higher number of cells being irradiated, the cellular damage in bystander areas seems to increase. Finally, the gene expression, in GADD45A, BCL2A1 and PTGS2 genes, was assessed with the qRT-PCR technique. This study revealed that all genes are expressed both in directly irradiated and bystander cells, except PTGS2. Moreover, the differences between gene expression levels in both irradiated and bystander cells, for the same dose value, suggest that the mechanisms underlying the response of both types of cells to radiation are different from each other. Summarizing, our results emphasize that the risks attributable to low doses encompass a complex cellular response. The observed hyper-radiosensitivity, time and dose-dependence of the bystander effects and gene expression profiles and the easily spread of bystander signals over the cell culture, raise important questions about the potentially detrimental effects resulting from low dose exposures, which are not included in a simple linear extrapolation from higher dose effects. |
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| Autores principais: | Belchior, Ana Lúcia Vital, 1979- |
| Assunto: | Engenharia biomédica Radiação Raios alfa Pulmão Tumor Teses de doutoramento - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | This thesis aims at studying the direct and bystander effects (observations in which effects of ionizing radiation arise in non-irradiated cells) induced by alpha-particle irradiation, using a Po-210 source, in a human lung adenocarcinoma cells (A549), and at investigating and providing a contribution to the clarification of the protective or potentially detrimental effects of low-dose exposures (< 100 mGy). Besides addressing and analyzing the toxic effects of α-radiation on the A549 cell line the studies undertaken may contribute toward the goal of improving the effectiveness of targeted therapies, with alpha-radiation, such as radioimmunotherapy or Boron Neutron Capture Therapy (BNCT). The work performed consisted, at first, of a dosimetric study in order to determine the average local dose in a cell monolayer, encompassed by experimental and computational studies, followed by a biological study of the direct and bystander effects of α-radiation exposure. The averaged dose rate at the cell monolayer was calculated; through the Linear-Energy-Transfer (LET) value measured using a Passivated Implanted Planar Silicon (PIPS) detector, to be 25 mGy/min. To study the cellular response at low-dose exposures, several studies were conducted using the A549 cell line and a 210Po α-source. Initially, the effects of directly irradiated A549 cells were analyzed through the micronucleus and clonogenic assay. Then, the time and dose dependence of directly and bystander effects in the region of very low doses, was investigated through micronucleus and clonogenic assay. The study encompassed three different cell culture conditions: i) a culture of irradiated cells, ii) a medium transfer culture with non-irradiated cells and iii) a culture with irradiated cells after centrifugation. A dependence on dose and time was observed in both directly irradiated and bystander cells. Taking the advantage of the in situ γ-H2AX technique, the way how the bystander signals released after low-doses of α-radiation are influenced by the number of irradiated cells was studied. Evidence that bystander signals can easily spread through the culture medium was observed. Moreover, the neighboring of irradiated cells differ with the number of irradiated cells, i.e. with a higher number of cells being irradiated, the cellular damage in bystander areas seems to increase. Finally, the gene expression, in GADD45A, BCL2A1 and PTGS2 genes, was assessed with the qRT-PCR technique. This study revealed that all genes are expressed both in directly irradiated and bystander cells, except PTGS2. Moreover, the differences between gene expression levels in both irradiated and bystander cells, for the same dose value, suggest that the mechanisms underlying the response of both types of cells to radiation are different from each other. Summarizing, our results emphasize that the risks attributable to low doses encompass a complex cellular response. The observed hyper-radiosensitivity, time and dose-dependence of the bystander effects and gene expression profiles and the easily spread of bystander signals over the cell culture, raise important questions about the potentially detrimental effects resulting from low dose exposures, which are not included in a simple linear extrapolation from higher dose effects. |
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