Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: The assignment of radiation weighting factors to high energy neutron sources is important as there is reason to believe that neutron relative biological effectiveness (RBE) may be related to the inherent radiosensitivity of different individuals. A study was undertaken to quantify the inherent radiosensitivities of lymphocytes obtained from different donors to 60Co y-rays and p(66)/Be neutrons. For this a novel semi-automated image analysis process has been employed. In addition the responses of lymphocytes with different inherent radiosensitivities have also been tested using Auger electrons emitted by 123I. Methods: The RBE of neutrons was determined from dose-response curves for lymphocytes from different donors. Isolated T-lymphocytes irradiated in vitro were cultured to induce micronuclei in binucleated cells and micronuclei (MN) formations numerated using a semi-automated Metafer microscope system. The accuracy in obtaining dose response curves with this method has been tested by evaluating dispersion parameters of MN formations in the response to the different treatment modalities. Differences in the inherent radiosensitivities of cells from different donors were ascertained using 95 % confidence ellipses. [123I]Iododeoxyuridine was prepared in a formulation that allows incorporation of 123I into the DNA of lymphocytes. Micronucleus formations to this treatment were evaluated in lymphocytes with established differences in inherent radiosensitivities. Results: The image analysis system proved to be consistent in detecting micronuclei frequencies in binucleated lymphocytes. As a result, differences in the inherent radiosensitivities of different individuals were distinctive and could be stated at the 95% confidence level. The inter-individual radiosensitivity variations were considerably smaller for blood cells exposed to high energy neutrons compared to 60Co y-rays. Relative biological effectiveness (RBEM) values between 2 and 13 were determined that are highly correlated with the inherent radioresistance of lymphocytes obtained from different individuals. As such radiation weighting factors for high energy neutrons cannot be based on cytogenetic damage determined in lymphocytes from a single donor. Dispersion parameters for micronuclei formations proved to vary according to ionization density. The variation in RBE with neutron dose changed according to theoretical considerations and automated image analysis detection of MN is thus a suitable method to quantify radiation weighting factors. A clear reduction in the variation in radiosensitivity is noted for lymphocytes exposed to Auger electrons compared to 60Co y-rays. The effectiveness of Auger electrons from [123I]IUdR to induce biological damage is demonstrated as the number of disintegrations needed to yield micronuclei formations was found to be more than two orders of magnitude less than that of other compounds. An increase in the RBE of Auger electrons with radioresistance can be inferred from these findings and constitutes a basis for therapeutic gain in treating cells compared to using radioisotopes emitting low-LET radiation. / AFRIKAANSE OPSOMMING: Agtergrond: Die bepaling van straling gewigsfaktore vir hoë energie neutron bronne is belangrik, aangesien daar rede is om te glo dat die relatiewe biologiese effektiwiteit (RBE) kan verband hou met die inherente stralings sensitiwiteit van verskillende individue. Hierdie studie is onderneem om die inherente radiosensitiwiteit van limfosiete verkry vanaf verskillende skenkers te kwantifiseer na blootstelling aan 60Co y -strale en p(66)/Be neutrone. Vir hierdie doel is daar van 'n semi-outomatiese beeldontleding metode gebruik gemaak. Daarbenewens is die reaksie van limfosiete met vooraf bepaalde inherente radiosensitiwiteite ook getoets aan die hand van Auger elektrone wat uitgestraal word deur 123I. Metodiek: Die RBE van neutrone was bepaal uit dosis mikrokerne frekwensie verwantskappe verkry vir limfosiete. Geïsoleerde T-limfosiete was in vitro bestraal en gekweek om mikrokerne te vorm in dubbelkernige selle. Die mikrokerne was gekwantifiseer deur die gebruik van 'n semi-outomatiese Metafer mikroskoop stelsel. Die akkuraatheid in die verkryging van dosis-effek krommes met hierdie metode is getoets deur die ontleding van verspreidings parameters van MN vorming in reaksie op behandeling met die verskillende stralings modaliteite. Verskille in die inherente stralingsensitiwiteite van die selle van verskillende skenkers was vasgestel deur die konstruksie van 95 % betroubaarheidsinterval ellipse. [123I]Iododeoxyuridine was ook berei om 123I in die DNA van limfosiete in te bou. Die mikrokerne vorming op die behandeling is beoordeel in limfosiete met gevestigde verskille in inherent radiosensitiwiteite. Resultate: Die beeld analise stelsel bewys om konsekwent te wees in die opsporing van mikrokerne wat vorm in dubbelkernige limfosiete. Verskille in die inherente radiosensitiwiteite van verskillende skenkers kon vasgestel word op die 95 % betroubaarheidsvlak. Die skommeling in inter-individuele stralings sensitiwiteite was kleiner vir bloed selle blootgestel aan hoë-energie neutrone in vergelyking met 60Co y-strale. Relatiewe biologiese effektiwiteit (RBEM) waardes tussen 2 en 13 is bepaal wat sterk verband hou met die inherente radioweerstandbiedendheid van limfosiete verkry vanaf verskillende persone. As sodanig kan straling gewigsfaktore vir hoë energie neutrone nie gebaseer word op sitogenetiese skade in limfosiete van 'n enkele skenker nie. Verspreidings parameters vir mikrokern vorming het gewissel as ‘n funksie van ionisasiedigtheid van die straling. Die verandering in RBE met neutron dosis verloop volgens teoretiese oorwegings en die semi-outomatiese beeldontledings metode om mikrokerne op te spoor is dus geskik om stralings gewigsfaktore te kwantifiseer. 'n Duidelike afname in die verandering in die stralingsensitiwiteite is waargeneem vir limfosiete blootgestel aan Auger elektrone in vergelyking met 60Co y-strale. Die hoë doeltreffendheid van Auger elektrone afkomstig van [123I]IUdR om biologiese skade te veroorsaak, word weerspieël deur die feit dat die getal disintegrasies wat nodig is om mikrokerne te vorm meer as twee ordes grootte minder is as dié van ander verbindings. 'n Toename in die RBE van Auger elektrone in selle wat radioweerstandbiedend is kan afgelei word uit hierdie bevindinge. Dit vorm 'n basis vir terapeutiese wins in die behandeling van selle in vergelyking met die gebruik van radio-isotope wat lae ionisasie digthede tot stand bring.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

Les bactéries du genre Deinococcus sont extrêmement tolérantes à de fortes doses de radiations. Des études antérieures ont montré que IrrE est nécessaire à la radiotolérance et à l'induction des gènes de réparation de l'ADN après exposition des cellules à l'irradiation. Pendant des années il est resté inconnu comment IrrE active l'expression de ces gènes. L'objectif de ma thèse était la caractérisation de la voie de signalisation dépendent de IrrE chez Deinococcus deserti. Pour cela, des approches biochimiques et génétiques ont été utilisées. Les premiers résultats ont fortement suggéré que IrrE agit indirectement sur l'activation de l'expression des gènes. En utilisant des expériences in vitro et in vivo, nous avons montré que IrrE de Deinococcus deserti interagit avec DdrO, un régulateur potentiel qui est codé par un gène radio-induit et qui est, comme IrrE, conservé chez les Deinococcus. De plus, IrrE clive DdrO in vitro mais aussi in vivo lorsque les deux protéines sont co-exprimées chez Escherichia coli. Ce clivage est abolit en présence d'un agent chélateur de métaux, l'EDTA. Chez D. deserti, le clivage de DdrO dépendent de IrrE a été observé mais seulement après exposition à l'irradiation. En parallèle, nous avons montré que la répression du promoteur d'un gène radio-inductible est dépendante de DdrO. Nos résultats montrent donc que IrrE est une métalloprotéase et nous proposons que le répresseur DdrO soit désactivé après clivage par IrrE conduisant à l'induction de différents gènes indispensables pour la réparation de l'ADN et la survie des cellules après exposition de Deinococcus à l'irradiation. / Deinococcus bacteria are famous for their extreme tolerance to high doses of radiation. Earlier studies have shown that IrrE protein is required for radiation tolerance and for induction of DNA repair genes after exposure of cells to radiation. However, for years it has remained unknown how IrrE activates gene expression. The aim of my thesis was to characterize the IrrE-dependent regulation pathway in Deinococcus deserti. For this, biochemical and genetic approaches were used. The first results strongly suggested that IrrE activates gene expression in an indirect manner. Then, using other in vivo and in vitro experiments, IrrE from Deinococcus deserti was found to interact with DdrO, a predicted regulator encoded by a radiation-induced gene that is, like irrE, highly conserved in Deinococcus. Moreover, IrrE was found to cleave DdrO in vitro and also in vivo when the proteins were co-expressed in Escherichia coli. This cleavage was not observed in the presence of the metal chelator EDTA. In D. deserti, IrrE-dependent cleavage of DdrO was observed only after exposure to radiation. Furthermore, DdrO-dependent repression of the promoter of a radiation-induced gene was shown. Our results demonstrate that IrrE is a metalloprotease and we propose that IrrE-mediated cleavage inactivates repressor protein DdrO, leading to transcriptional induction of various genes required for DNA repair and cell survival after exposure of Deinococcus to radiation.

Este trabalho consiste em um estudo acerca dos efeitos da radiação em circuitos programáveis e sistemas em chip, do inglês System-on-Chip (SoC), baseados em FPGAs (Field-Programmable Gate Array). Dentre os diversos efeitos que podem ensejar falhas nos circuitos integrados, destacam-se a ocorrência de Single Event Effects (SEEs), Efeitos Transitórios em tradução livre, e a Dose Total Ionizante, do inglês Total Ionizing Dose (TID). SEEs podem ocorrer em razão da incidência de nêutrons originários de interações de raios cósmicos com a atmosfera terrestre, íons pesados provenientes do espaço e prótons originários do Sol (vento solar) e dos cinturões de Van Allen. A Dose Total Ionizante diz respeito à exposição prolongada de um circuito integrado à radiação ionizante e cuja consequência é a alteração das características elétricas de partes do dispositivo em razão das cargas elétricas induzidas pela radiação e acumuladas nas interfaces dos semicondutores. Dentro desse contexto, este trabalho descreve em detalhes a caracterização do SoC-FPGA baseado em memória FLASH e de sinais mistos SmartFusion A2F200-FG484, da empresa Microsemi, quando exposto à radiação (SEEs e TID) através do uso da técnica de Redundância Diversificada visando a detecção de erros. Também, uma arquitetura que utiliza um esquema baseado em Redundância Modular Tripla e Diversificada é testada através da sua implementação no FPGA baseado em memória SRAM da família Spartan-6, modelo LX45, da empresa Xilinx, visando a detecção e correção de erros causados pela radiação (SEEs). Os resultados obtidos mostram que os diversos blocos funcionais que compõe SoC SmartFusion apresentam diferentes níveis de tolerância à radiação e que o uso das técnicas de Redundância Modular Tripla e Redundância Diversificada em conjunto mostrou-se extremamente eficiente no que se refere a tolerância a SEEs. / This work consists in a study about the radiation effects in programmable circuits and System-on-Chips (SoCs) based on FPGAs (Field-Programmable Gate Arrays). Single Event Effects (SEEs) and Total Ionizing Dose (TID) are the two main effects caused by the radiation incidence, and both can imply in the occurrence of failures in integrated circuits. SEEs are due to the incidence of neutrons derived from the interaction of the cosmic rays with the terrestrial atmosphere, as well as heavy ions coming from the space and protons provided from the solar wind and the Van Allen belts. Total Ionizing Dose regards the prolonged exposure of an integrated circuit to the ionizing radiation, which deviates the standard electrical characteristics of the device due to radiation-induced electrical charges accumulated in the semiconductors’ interfaces. In this context, this work aims to describe in details the characterization of Microsemi’s mixed-signal SoC-FPGA SmartFusion A2F200-FG484 when exposed to radiation (SEEs and TID), using a Diverse Redundancy approach for error detection. As well, an architecture using a Diversified Triple Modular Redundancy scheme was tested (SEEs) through its implementation in a Xilinx’s Spartan-6 LX45 FPGA, aiming error detection and correction. The results obtained show that several functional blocks from SmartFusion have different radiation tolerance levels and that the use of the Triple Modular Redundancy together with Diversified Redundancy proved to be extremely efficient in terms of SEEs tolerance.

Este trabalho consiste em um estudo acerca dos efeitos da radiação em circuitos programáveis e sistemas em chip, do inglês System-on-Chip (SoC), baseados em FPGAs (Field-Programmable Gate Array). Dentre os diversos efeitos que podem ensejar falhas nos circuitos integrados, destacam-se a ocorrência de Single Event Effects (SEEs), Efeitos Transitórios em tradução livre, e a Dose Total Ionizante, do inglês Total Ionizing Dose (TID). SEEs podem ocorrer em razão da incidência de nêutrons originários de interações de raios cósmicos com a atmosfera terrestre, íons pesados provenientes do espaço e prótons originários do Sol (vento solar) e dos cinturões de Van Allen. A Dose Total Ionizante diz respeito à exposição prolongada de um circuito integrado à radiação ionizante e cuja consequência é a alteração das características elétricas de partes do dispositivo em razão das cargas elétricas induzidas pela radiação e acumuladas nas interfaces dos semicondutores. Dentro desse contexto, este trabalho descreve em detalhes a caracterização do SoC-FPGA baseado em memória FLASH e de sinais mistos SmartFusion A2F200-FG484, da empresa Microsemi, quando exposto à radiação (SEEs e TID) através do uso da técnica de Redundância Diversificada visando a detecção de erros. Também, uma arquitetura que utiliza um esquema baseado em Redundância Modular Tripla e Diversificada é testada através da sua implementação no FPGA baseado em memória SRAM da família Spartan-6, modelo LX45, da empresa Xilinx, visando a detecção e correção de erros causados pela radiação (SEEs). Os resultados obtidos mostram que os diversos blocos funcionais que compõe SoC SmartFusion apresentam diferentes níveis de tolerância à radiação e que o uso das técnicas de Redundância Modular Tripla e Redundância Diversificada em conjunto mostrou-se extremamente eficiente no que se refere a tolerância a SEEs. / This work consists in a study about the radiation effects in programmable circuits and System-on-Chips (SoCs) based on FPGAs (Field-Programmable Gate Arrays). Single Event Effects (SEEs) and Total Ionizing Dose (TID) are the two main effects caused by the radiation incidence, and both can imply in the occurrence of failures in integrated circuits. SEEs are due to the incidence of neutrons derived from the interaction of the cosmic rays with the terrestrial atmosphere, as well as heavy ions coming from the space and protons provided from the solar wind and the Van Allen belts. Total Ionizing Dose regards the prolonged exposure of an integrated circuit to the ionizing radiation, which deviates the standard electrical characteristics of the device due to radiation-induced electrical charges accumulated in the semiconductors’ interfaces. In this context, this work aims to describe in details the characterization of Microsemi’s mixed-signal SoC-FPGA SmartFusion A2F200-FG484 when exposed to radiation (SEEs and TID), using a Diverse Redundancy approach for error detection. As well, an architecture using a Diversified Triple Modular Redundancy scheme was tested (SEEs) through its implementation in a Xilinx’s Spartan-6 LX45 FPGA, aiming error detection and correction. The results obtained show that several functional blocks from SmartFusion have different radiation tolerance levels and that the use of the Triple Modular Redundancy together with Diversified Redundancy proved to be extremely efficient in terms of SEEs tolerance.

Este trabalho consiste em um estudo acerca dos efeitos da radiação em circuitos programáveis e sistemas em chip, do inglês System-on-Chip (SoC), baseados em FPGAs (Field-Programmable Gate Array). Dentre os diversos efeitos que podem ensejar falhas nos circuitos integrados, destacam-se a ocorrência de Single Event Effects (SEEs), Efeitos Transitórios em tradução livre, e a Dose Total Ionizante, do inglês Total Ionizing Dose (TID). SEEs podem ocorrer em razão da incidência de nêutrons originários de interações de raios cósmicos com a atmosfera terrestre, íons pesados provenientes do espaço e prótons originários do Sol (vento solar) e dos cinturões de Van Allen. A Dose Total Ionizante diz respeito à exposição prolongada de um circuito integrado à radiação ionizante e cuja consequência é a alteração das características elétricas de partes do dispositivo em razão das cargas elétricas induzidas pela radiação e acumuladas nas interfaces dos semicondutores. Dentro desse contexto, este trabalho descreve em detalhes a caracterização do SoC-FPGA baseado em memória FLASH e de sinais mistos SmartFusion A2F200-FG484, da empresa Microsemi, quando exposto à radiação (SEEs e TID) através do uso da técnica de Redundância Diversificada visando a detecção de erros. Também, uma arquitetura que utiliza um esquema baseado em Redundância Modular Tripla e Diversificada é testada através da sua implementação no FPGA baseado em memória SRAM da família Spartan-6, modelo LX45, da empresa Xilinx, visando a detecção e correção de erros causados pela radiação (SEEs). Os resultados obtidos mostram que os diversos blocos funcionais que compõe SoC SmartFusion apresentam diferentes níveis de tolerância à radiação e que o uso das técnicas de Redundância Modular Tripla e Redundância Diversificada em conjunto mostrou-se extremamente eficiente no que se refere a tolerância a SEEs. / This work consists in a study about the radiation effects in programmable circuits and System-on-Chips (SoCs) based on FPGAs (Field-Programmable Gate Arrays). Single Event Effects (SEEs) and Total Ionizing Dose (TID) are the two main effects caused by the radiation incidence, and both can imply in the occurrence of failures in integrated circuits. SEEs are due to the incidence of neutrons derived from the interaction of the cosmic rays with the terrestrial atmosphere, as well as heavy ions coming from the space and protons provided from the solar wind and the Van Allen belts. Total Ionizing Dose regards the prolonged exposure of an integrated circuit to the ionizing radiation, which deviates the standard electrical characteristics of the device due to radiation-induced electrical charges accumulated in the semiconductors’ interfaces. In this context, this work aims to describe in details the characterization of Microsemi’s mixed-signal SoC-FPGA SmartFusion A2F200-FG484 when exposed to radiation (SEEs and TID), using a Diverse Redundancy approach for error detection. As well, an architecture using a Diversified Triple Modular Redundancy scheme was tested (SEEs) through its implementation in a Xilinx’s Spartan-6 LX45 FPGA, aiming error detection and correction. The results obtained show that several functional blocks from SmartFusion have different radiation tolerance levels and that the use of the Triple Modular Redundancy together with Diversified Redundancy proved to be extremely efficient in terms of SEEs tolerance.

The objective of this work is to provide a comprehensive analysis of the small-signal and broadband noise performance of highly scaled silicon-based field-effect transistors (FETs), and develop high-frequency noise models for robust radio frequency (RF) circuit design. An analytical RF noise model is developed and implemented for scaled Si-CMOS devices, using a direct extraction procedure based on the linear two-port noise theory. This research also focuses on investigating the applicability of modern CMOS technologies for extreme environment electronics. A thorough analysis of the DC, small-signal AC, and broadband noise performance of 0.18 um and 130 nm Si-CMOS devices operating at cryogenic temperatures is presented. The room temperature RF noise model is extended to model the high-frequency noise performance of scaled MOSFETs at temperatures down to 77 K and 10 K. Significant performance enhancement at cryogenic temperatures is demonstrated, indicating the suitability of scaled CMOS technologies for low temperature electronics. The hot-carrier reliability of MOSFETs at cryogenic temperatures is investigated and the worst-case gate voltage stress condition is determined. The degradation due to hot-carrier-induced interface-state creation is identified as the dominant degradation mechanism at room temperature down to 77 K. The effect of high-energy proton radiation on the DC, AC, and RF noise performance of 130 nm CMOS devices is studied. The performance degradation is investigated up to an equivalent total dose of 1 Mrad, which represents the worst case condition for many earth-orbiting and planetary missions. The geometric scaling of MOSFETs has been augmented by the introduction of novel FET designs, such as the Si/SiGe MODFETs. A comprehensive characterization and modeling of the small-signal and high-frequency noise performance of highly scaled Si/SiGe n-MODFETs is presented. The effect of gate shot noise is incorporated in the broadband noise model. SiGe MODFETs offer the potential for high-speed and low-voltage operation at high frequencies and hence are attractive devices for future RF and mixed-signal applications. This work advances the state-of-the-art in the understanding and analysis of the RF performance of highly scaled Si-CMOS devices as well as emerging technologies, such as Si/SiGe MODFETs. The key contribution of this dissertation is to provide a robust framework for the systematic characterization, analysis and modeling of the small-signal and RF noise performance of scaled Si-MOSFETs and Si/SiGe MODFETs both for mainstream and extreme-environment applications.