O presente estudo teve como objetivo avaliar o efeito da inibição das cadeias inflamatórias reguladas pela ação do interferon-gama (IFN-γ) e da enzima óxido nítrico sintase indutível (iNOS) no dano radioinduzido após exposição a dose letal (8 Gy) ou moderada a severa (4 Gy) nos tecidos hematopoiéticos (baço e medula) de modelos experimentais irradiados nestas doses. Grupos de camundongos isogênicos C57Bl/6j foram expostos à radiação correspondente a 4 ou 8 Gy em exposições de corpo inteiro em fonte panorâmica de 60Co. Da mesma forma, foram irradiados camundongos cuja expressão de IFN-γ ou iNOS é ausente ou indetectável. Outros grupos receberam via oral por toda a duração do experimento um inibidor atividade de iNOS, aminoguanidina, ou via intraperitoneal uma quimiocina primordial promotora da hematopoiese, CXCL12, até o quarto dia após a exposição. Outra divisão experimental recebeu os dois agentes concomitantemente. Os animais foram sacrificados nos dias 2º, 4º e 8º após a irradiação, e fragmentos dos baços e fêmures foram preservados para histologia. Os esplenócitos e células não aderentes da medula óssea femoral foram removidos e divididos, fornecendo alíquotas para posterior RT-PCR e suspensões celulares apropriadas para ensaios de citometria de fluxo específicos para a detecção da freqüência de populações CD34+. Nestes mesmos dias de experimento, alíquotas de sangue caudal foram coletadas para contagem de hemácias e plaquetas periféricas. Os resultados mostraram que a ausência da produção de interferon-gama no local irradiado aumenta a sobrevivência e a quantidade de células progenitoras hematopoiéticas e que a ausência de iNOS ou seu bloqueio funcional diminuem a extensão do dano radioinduzido nos tecidos hematopoiéticos. Além disso, foi possível observar que a suplementação com CXCL12 sintética aumenta a freqüência do fenótipo CD34+ P.chave: radiação ionizante; medula óssea; óxido nítrico; aminoguanidina nos baços dos modelos testados, e que seu efeito parece antagonizar com a inibição da produção de NO pela aminoguanidina. / This study aimed to evaluate the effect of inhibition of the inflammatory chains governed by the action of interferon-gamma (IFN-γ) and the enzyme inducible nitric oxide synthase (iNOS) in damage after radiation exposure to lethal dose (8 Gy) or moderate /severe dose (4 Gy) in hematopoietic tissues (spleen and bone marrow) of experimental models irradiated at these doses. Groups of isogenic C57Bl/6j mice were exposed to radiation (4 or 8 Gy) in whole-body exposures in a 60Co panoramic source. Similarly, were irradiated mice whose iNOS or IFN-γ expression was absent or undetectable. Other groups received orally in all days of experiments an inhibitor of iNOS activity, aminoguanidine, or CXCL12, a primordial chemokine known as an hematopoeisis promoter intraperitonially by days 0 to 4th after radiation events Another groups received the two agents concomitantly. The animals were sacrificed on days 2nd, 4th and 8th after irradiation, and fragments of the spleens and femurs were preserved for histology. Splenocytes and non-adherent cells from femoral bone marrow were removed and divided, providing aliquots for subsequent RT-PCR and cell suspensions suitable for flow cytometry experiments specific to the detection of the frequency of CD34+ cell populations. In same days of experiment, tail blood samples were collected for counting of peripheral red blood cells and platelets. The results showed that the absence of the production of interferon-gamma at the irradiated sites increases the survival and the amount of hematopoietic progenitor cells and that the absence of iNOS or its functional blockade reduces the extent of radioinduced damage in tested hematopoietic tissues. Furthermore, it was possible to observe that supplementing with synthetic CXCL12 increases the frequency of CD34+ Keywords: ionizing radiation, bone marrow, nitric oxide, aminoguanidine phenotype in the spleens of tested models, and that its effect seems to antagonize the inhibition of the production of NO by aminoguanidine.

O crescente aumento do interesse no desenvolvimento em técnicas cirúrgicas menos invasivas, como nas reconstruções tendíneas e ligamentares, tem levado ao aumento das pesquisas referentes ao uso de aloenxertos esterilizados por radiação ionizante. O processamento por radiação ionizante é um método seguro e não deixa resíduos, sendo utilizado como esterilização final. O presente estudo teve como proposta avaliar os efeitos da aplicação de radiação ionizante, produzida por fonte de 60Co, em amostras de tendões humanos pré processados de doadores multiorgãos obtidas por meio de colaboração com Bancos de Tecidos. O pré-processamento das amostras deu-se por métodos químicos e preservação por congelamento em -80 °C. As doses aplicadas no processamento por radiação foram de 12,5 kGy, 15,0 kGy e 25,0 kGy, cada uma com seu respectivo controle não irradiado. As amostras foram avaliadas por meio de testes histológicos, ópticos e biomecânicos, com o objetivo de analisar possíveis modificações morfológicas e estruturais. Os resultados apresentados demonstraram que o processamento por ultrassom e peróxido de hidrogênio causaram alterações na morfologia dos tecidos, o que ocasionou danos à sua estrutura, inviabilizando as amostras. Nas amostras processadas por álcool e antibiótico não foram observados danos na rede de colágeno pela aplicação da radiação. Os resultados dos testes biomecânicos apresentaram diferenças significativas entre os métodos aplicados. As amostras processadas com álcool e antibiótico apresentaram perda pouco significativa no módulo de elasticidade, comparadas às amostras processadas por ultrassom e peróxido de hidrogênio que mantiveram a propriedade viscoelástica. Contudo na dose de 12,5 kGy foi observado um aumento no módulo elástico e na viscoelasticidade. Com base nas análises, podemos concluir que o método de processamento com álcool, antibiótico e irradiação demonstrou menor dano, tanto na biomecânica quanto na esterilização, sendo que as amostras irradiadas a 15,0 e 25,0 kGy apresentaram características semelhantes ao controle não irradiado. / The increasing interest of development in less invasive surgical techniques, such as reconstructions of ligament tendon, has led to the increase of the research concerning the use of Allografts sterilized by ionizing radiation. Processing by ionizing radiation is a safe method and leaves no residues, being used as final sterilization. The present study was to evaluate the effects of proposed application of ionizing radiation, produced by 60Co source in human tendon pre-samples processed multiorgans donors obtained through collaboration with tissue banks. The pre-processing of samples given by chemical methods and preserved by freezing at -80 °C. The doses applied in radiation processing were 12.5, 15.0 and 25.0 kGy, each with your corresponding non-irradiated control. The samples were evaluated by means of histological and biomechanical testing, with the purpose of analyzing possible structural and morphological changes. The results showed that the ultrasound processing and hydrogen peroxide caused changes in the morphology of the tissues, which caused damage to the structure, making your samples. In the samples processed by alcohol and antibiotics were not observed damage on the network of collagen by the application of radiation. The results of biomechanical tests showed significant differences between the methods used. The samples processed with alcohol and antibiotics showed negligible loss in modulus of elasticity compared with the samples processed by ultrasound and hydrogen peroxide which kept the viscoelastic property, however in 12.5 kGy dose was observed an increase in elastic modulus and viscoelasticity. Based on the analysis we can conclude that the method of processing with alcohol, antibiotics and irradiation showed less damage, both in biomechanics and sterilization, in the samples irradiated with 15.0 and 25.0 kGy, showing results similar to the non-irradiated control.

La détection de rayonnements ionisants - notamment les neutrons de basse énergie qui sont la signature de matériaux fissibles - est un enjeu majeur pour des applications duales civiles et militaires. La raréfaction de l’hélium 3 indispensable à la production de détecteur nécessite la découverte et la mise au point d’alternative fiable. Le développement de prototypes de détecteurs pour la caractérisation de neutrons permet de répondre aux risques d’accidents technologiques et de terrorisme. Mais ils permettent également l’amélioration de l’analyse de radiations ionisantes en recherche fondamentale. Une approche convergente de la synthèse des matériaux salins luminescents permet d’adresser à la fois la détection et la discrimination de rayonnements ionisants mais également de développer de nouveaux matériaux pour la détection de petites molécules en phase gazeuse. Les matériaux ioniques à base de sels d’imidazolium présentent des propriétés physiques remarquable tout en ayant une structure chimique malléable à façon. Ce projet vise à explorer de nouvelles méthodes pour la synthèse de sels d’imidazolium luminescents et leurs propriétés en détection. / Detector of ionizing radiation is a key challenge for both civil and military applications. New prototype of ionizing radiation discrimination helps us against technological accident and terrorism act. For instance, neutron of low energy is sign of fissile material. However current detectors require helium 3 which is scarce resource. Thus, a sustainable alternative has to be found. A convergent approach to synthesis active luminescent salt materials cope both. It identities and discriminate ionizing radiation. Especially for small cell in gas phase, this approach allows to develop new material to detect them. Based on imidazolium salt, ionic material has remarkable property with the benefit of being scalable.This project aims to explore new synthesis methods of luminescent imidazolium and their properties to identify ionizing radiation characteristic.

Liver cancer is the fourth leading cause of cancer-associated deaths globally, and among primary liver cancers, hepatocellular carcinoma (HCC) encompasses 75-85% of all cases. HCC is a highly lethal disease due to limited treatment options – only a small subset of patients qualify for surgical resection or transplantation; the remaining patients often display resistance to radiation therapy or chemotherapy. Overexpression of the oncogene astrocyte elevated gene-1 (AEG-1) is associated with poorer survival and increased tumor recurrence in HCC, and numerous studies show its role in initiation of hepatocarcinogenesis. A prior study also demonstrated AEG-1 expression inhibits senescence by diminishing the ATM/Chk1/Chk2/p53/p21 DNA damage response (DDR) pathway. The aim of this study is to understand if AEG-1 expression promotes radioresistance in HCC. A CRISPR/Cas9 plasmid system was used to delete AEG-1 in the QGY-7703, HuH7 and DihXY cell lines, which model HCC. The cell lines were then treated with ionizing radiation (IR). We find that knockout of AEG-1 in these cell lines induces sensitivity to IR at 2.5 Gy. In response to radiation, AEG-1 wildtype cells more profoundly upregulate ATR, Chk1, and Chk2 signaling; and also more rapidly induce γH2AX, ATM, and BRCA1 signaling, which sense dsDNA breaks to initiate homologous recombination repair. We conclude that AEG-1 expression protects HCC cells from IR through two mechanisms: 1) rapidly initiating the DNA damage response; and 2) increasing replication fork stabilization. These findings indicate AEG-1 can be a therapeutic target in combination with radiation treatment to improve outcomes for HCC patients who demonstrate radioresistance.

<p>Radiation quality is described by the RBE (relative biological effectiveness) that varies with the ionizing ability of the radiation. Microdosimetric quantities describe distributions of energy imparted to small volumes and can be related to RBE. This has made microdosimetry a powerful tool for radiation quality determinations in both radiation protection and radiation therapy. The variance-covariance method determines the dose-average of the distributions and has traditionally been used with two detectors to correct for beam intensity variations. Methods to separate dose components in mixed radiation fields and to correct for beam variations using only one detector have been developed in this thesis. Quality factor relations have been optimized for different neutron energies, and a new algorithm that takes single energy deposition events from densely ionizing radiation into account has been formulated. The variance-covariance technique and the new methodology have been shown to work well in the cosmic radiation field onboard aircraft, in the mixed photon and neutron fields in the nuclear industry and in pulsed fields around accelerators.</p><p>The method has also been used for radiation quality characterization in therapy beams. The biological damage is related to track-structure and ionization clusters and requires descriptions of the energy depositions in nanometre sized volumes. It was shown that both measurements and Monte Carlo simulation (condensed history and track-structure) are needed for a reliable nanodosimetric beam characterization. The combined experimental and simulated results indicate that the dose-mean of the energy imparted to an object in the nanometre region is related to the clinical RBE in neutron, proton and photon beams. The results suggest that the variance-covariance technique and the dose-average of the microdosimetric quantities could be well suited for describing radiation quality also in therapy beams.</p>

<p>Cancer therapy operates by inflicting damage in malignant cells. The most lethal target is the genomic DNA. As a single double strand DNA break has the potential to kill the cell, mechanisms have evolved to detect and block propagation of the damage. Genes and their products function in a highly connected network-structure with ample cross-talk between different pathways. This interplay can be studied by genome-wide experiments, such as expression profiling. The aim of this thesis is to study the cellular effects of DNA damaging agents.</p><p>A theoretical framework is explored to improve understanding of expression profiling results. To analyse large datasets, computational methods were developed to model the data. Further, the response to DNA damage was investigated in different cellular systems. As late radiation toxicity is a severe limitation of radiotherapy of cancer patients, patients were enrolled in a study to search for a molecular signature to identify high-risk patients. Ex vivo irradiation of lymphocytes revealed a signature of functionally related gene sets that were capable to separate patients with regard to toxicity status. </p><p>The gene set analysis was also applied to a dataset where mouse embryonic stem cells had been exposed to various doses of cisplatin. At several time-points after administration of the drug, expression profiles were determined. In addition to the expected increase of genes related to apoptosis and cell cycle progression, damaged cells also seemed to have embarked upon a p53-dependent differentiation programme. Finally, in a study of cardiac rodent cells, the genotoxic treatment with irradiation was compared to the mechanical stress induced in heart tissue.</p><p>In conclusion, this thesis presents evidence for the advantage of using functionally related sets of genes in analysis and interpretation of genome-wide experiments. This strategy may improve clinical understanding of the effects of DNA damaging agents used for cancer therapeutics.</p>

Electronic components, based on current semiconductor technologies and operating in radiation rich environments, suffer degradation of their performance as a result of radiation exposure. Silicon carbide (SiC) provides an alternate solution as a radiation hard material, because of its wide bandgap and higher atomic displacement energies, for devices intended for radiation environment applications. However, the radiation tolerance and reliability of SiC-based devices needs to be understood by testing devices  under controlled radiation environments. These kinds of studies have been previously performed on diodes and MESFETs, but multilayer devices such as bipolar junction transistors (BJT) have not yet been studied. In this thesis, SiC material, BJTs fabricated from SiC, and various dielectrics for SiC passivation are studied by exposure to high energy ion beams with selected energies and fluences. The studies reveal that the implantation induced crystal damage in SiC material can be partly recovered at relatively low temperatures, for damag elevels much lower than needed for amorphization. The implantation experiments performed on BJTs in the bulk of devices show that the degradation in deviceperformance produced by low dose ion implantations can be recovered at 420 oC, however, higher doses produce more resistant damage. Ion induced damage at the interface of passivation layer and SiC in BJT has also been examined in this thesis. It is found that damaging of the interface by ionizing radiation reduces the current gain as well. However, for this type of damage, annealing at low temperatures further reduces the gain. Silicon dioxide (SiO2) is today the dielectric material most often used for gate dielectric or passivation layers, also for SiC. However, in this thesis several alternate passivation materials are investigated, such as, AlN, Al2O3 and Ta2O5. These materials are deposited by atomic layer deposition (ALD) both as single layers and in stacks, combining several different layers. Al2O3 is further investigated with respect to thermalstability and radiation hardness. It is observed that high temperature treatment of Al2O3 can substantially improve the performance of the dielectric film. A radiation hardness study furthermore reveals that Al2O3 is more resistant to ionizing radiation than currently used SiO2 and it is a suitable candidate for devices in radiation rich applications. / QC 20120117

Cancer therapy operates by inflicting damage in malignant cells. The most lethal target is the genomic DNA. As a single double strand DNA break has the potential to kill the cell, mechanisms have evolved to detect and block propagation of the damage. Genes and their products function in a highly connected network-structure with ample cross-talk between different pathways. This interplay can be studied by genome-wide experiments, such as expression profiling. The aim of this thesis is to study the cellular effects of DNA damaging agents. A theoretical framework is explored to improve understanding of expression profiling results. To analyse large datasets, computational methods were developed to model the data. Further, the response to DNA damage was investigated in different cellular systems. As late radiation toxicity is a severe limitation of radiotherapy of cancer patients, patients were enrolled in a study to search for a molecular signature to identify high-risk patients. Ex vivo irradiation of lymphocytes revealed a signature of functionally related gene sets that were capable to separate patients with regard to toxicity status. The gene set analysis was also applied to a dataset where mouse embryonic stem cells had been exposed to various doses of cisplatin. At several time-points after administration of the drug, expression profiles were determined. In addition to the expected increase of genes related to apoptosis and cell cycle progression, damaged cells also seemed to have embarked upon a p53-dependent differentiation programme. Finally, in a study of cardiac rodent cells, the genotoxic treatment with irradiation was compared to the mechanical stress induced in heart tissue. In conclusion, this thesis presents evidence for the advantage of using functionally related sets of genes in analysis and interpretation of genome-wide experiments. This strategy may improve clinical understanding of the effects of DNA damaging agents used for cancer therapeutics.

Radiation quality is described by the RBE (relative biological effectiveness) that varies with the ionizing ability of the radiation. Microdosimetric quantities describe distributions of energy imparted to small volumes and can be related to RBE. This has made microdosimetry a powerful tool for radiation quality determinations in both radiation protection and radiation therapy. The variance-covariance method determines the dose-average of the distributions and has traditionally been used with two detectors to correct for beam intensity variations. Methods to separate dose components in mixed radiation fields and to correct for beam variations using only one detector have been developed in this thesis. Quality factor relations have been optimized for different neutron energies, and a new algorithm that takes single energy deposition events from densely ionizing radiation into account has been formulated. The variance-covariance technique and the new methodology have been shown to work well in the cosmic radiation field onboard aircraft, in the mixed photon and neutron fields in the nuclear industry and in pulsed fields around accelerators. The method has also been used for radiation quality characterization in therapy beams. The biological damage is related to track-structure and ionization clusters and requires descriptions of the energy depositions in nanometre sized volumes. It was shown that both measurements and Monte Carlo simulation (condensed history and track-structure) are needed for a reliable nanodosimetric beam characterization. The combined experimental and simulated results indicate that the dose-mean of the energy imparted to an object in the nanometre region is related to the clinical RBE in neutron, proton and photon beams. The results suggest that the variance-covariance technique and the dose-average of the microdosimetric quantities could be well suited for describing radiation quality also in therapy beams.

The tumour suppressor p53-binding protein 1 (53BP1) is phosphorylated following DNA double strand breaks (DSBs); however, little is understood about the upstream signaling pathways that control this phosphorylation. Additionally, it is not known how these processes combine with 53BP1 to control the survival of cells following DNA damage such as that imparted by ionizing radiation (IR), which is the basis of radiotherapy. In this thesis, I have shown that 53BP1 is phosphorylated specifically in S-phase cells, but not relocalized to intranuclear foci, in response to severe oxygen stress. This occurs with only partial dependence on the ATM kinase (Chapter 2). Following IR, I find that both ATM and DNA-PKcs contribute to intranuclear phosphorylated 53BP1 foci, but that this phosphorylation is independent of proximal signaling molecules that control the localization of 53BP1 to initial DSBs (Chapter 3). Furthermore, I show that 53BP1 loss confers sensitivity to IR and this can be further augmented by inhibition of ATM and DNA-PKcs kinases suggesting that there are both 53BP1-dependent and -independent pathways of survival from IR (Chapter 4). These findings may have important implications for molecular pathology and personalized medicine as 53BP1 has recently been found to be activated or lost in subsets of human tumours. I have collaborated to initiate the development of a novel system to interrogate the implications of 53BP1 loss as traditional siRNA approaches in human cancer cells were not feasible (Chapter 5 and Appendix 2). This system can be used in vivo as tumour xenografts to further understand how 53BP1 and the tumour microenvironment interact endogenously and in response to IR. I also present the possibility and proof of concept for the use of 53BP1 as a biomarker in primary human prostate cancer tissue where little is known about 53BP1 biology (Chapter 5).