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).

Made available in DSpace on 2014-10-09T12:25:25Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:05Z (GMT). No. of bitstreams: 1 06048.pdf: 4286403 bytes, checksum: 125c0216eea47b9a749977ad0ec8b66a (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:38:33Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:05:10Z (GMT). No. of bitstreams: 1 06004.pdf: 4196576 bytes, checksum: 707f1ed9565795d26cc6aa938c5f4995 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:47:05Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:10:25Z (GMT). No. of bitstreams: 1 08287.pdf: 1642417 bytes, checksum: 48f844079650f71035ccb87b1dffe7a4 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:25:25Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:05Z (GMT). No. of bitstreams: 1 06048.pdf: 4286403 bytes, checksum: 125c0216eea47b9a749977ad0ec8b66a (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:38:33Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:05:10Z (GMT). No. of bitstreams: 1 06004.pdf: 4196576 bytes, checksum: 707f1ed9565795d26cc6aa938c5f4995 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:47:05Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:10:25Z (GMT). No. of bitstreams: 1 08287.pdf: 1642417 bytes, checksum: 48f844079650f71035ccb87b1dffe7a4 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Environ, 60 % des personnes atteintes d’un cancer auront au moins une séance de radiothérapie au cours de la prise en charge thérapeutique de leur maladie. Les doses de radiothérapie sont limitées en raison du risque important de fibrose séquellaire des tissus sains. Les rayonnements ionisants (RI) peuvent induire différents types de mort cellulaire y compris l'apoptose et la sénescence. Les cellules sénescentes ont une sensibilité réduite à l'apoptose et un phénotype sécrétoire inflammatoire. De plus, les RI peuvent induire la production d’espèces réactives de l’oxygène (ERO) qui provoquent des lésions de l'ADN dans les tissus non ciblés, et des effets systémiques associés à l'inflammation. Différentes équipes ont proposé des tests prédictifs de la radiosensibilité individuelle des patients basés sur l’évaluation du taux d'apoptose radio-induite des lymphocytes T CD4+/CD8+ (LT). Cependant, l’impact des différences de sensibilité à l’apoptose/sénescence des sous-populations de LT sur le taux d’apoptose n’a pas été étudié. Notre hypothèse est que la sensibilité à l’apoptose/sénescence radio-induite des LT circulants est associée à la sur/sous-représentation de sous-populations particulières de LT CD4+ dont les fonctions sont en rapport avec la survenue de fibrose. Nos résultats chez le donneur sain montrent que les LT CCR6+Th17 pro-fibrogéniques sont moins sensibles à l’apoptose et plus sensibles à la sénescence que les LT CCR6negTh et les Treg. Cette sénescence peut être préjudiciable car les lymphocytes CCR6+Th17 situés dans les tissus irradiés peuvent sécréter de l'IL-8 et du VEGF-A. La modulation des voies ERO/MAPK ou mTOR pourrait être une cible potentielle pour la prévention de la radiotoxicité induite par les CCR6+Th17 sénescents. Enfin, le ratio de cellules circulantes H2A.J+CCR6+Th17 sénescentes / CCR6+Treg pourrait être utilisé comme marqueur potentiel de la radiosensibilité individuelle. / On average, 60% of cancer patients have at least one radiation session during their care throughout the history of their disease. The doses of radiotherapy are limited because of the high risk of fibrosis-type side effects of healthy tissues. Ionizing radiation can induce a variety of cell death responses including apoptosis, but also senescence. Senescent cells have reduced sensitivity to apoptosis, and a pro-inflammatory secretory phenotype. In addition, ionizing radiations can induce the production of reactive oxygen species (ROS) that cause DNA damage in non-target tissues, and systemic effects associated with inflammation. In order to improve the personalization of radiotherapy, different teams proposed predictive tests of the individual radiosensitivity of patients by establishing a relationship between a low rate of radio-induced apoptosis of CD4+/CD8+ T lymphocytes (LT) and a high risk of secondary fibrosis. However, the impact of the differences in individual cell sensitivity to radiation-induced senescence on the ratio between LT cell subpopulations has not been studied. Our results on healthy donors show that pro-fibrogenic CCR6+ Th17 cells are less sensitive to apoptosis and more susceptible to senescence compared to CCR6neg LT. This senescence can be detrimental as irradiated CCR6+Th17 lymphocytes located in the irradiated tissue can secrete IL-8 and VEGF-A. Modulation of ROS/MAPK or mTOR signaling pathways could be potential targets for the prevention of this CCR6+Th17-induced radiotoxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CCR6+Treg cells may be used as a potential marker of individual radiosensitivity.

This thesis demonstrates the relevance of bystander effect mechanisms after exposure to two Synchrotron modalities – Microbeam Radiation Therapy and Pencilbeam – that are currently at the preclinical stage but aim to treat brain tumours. We elucidate the relationship between the hyper-radiosensitivity phenomenon and radiation-induced bystander effects by studying the dose response of three glioma cell lines. The relevance of these low-dose effects for both Synchrotron modalities is because the tissue exposed to low valley-doses is predicted to be where hyper-radiosensitivity and bystander effects might be expected to predominate. In vivo experiments were conducted in the European Synchrotron radiation Facility in Grenoble, France and also in the University of Freiburg’s Hospital in Freiburg, Germany. Experiments conducted in vitro were performed at McMaster University. The most relevant results of this thesis revealed that the low-dose hyper-radiosensitivity phenomenon can coexist with radiation-induced bystander effects and evidence points towards bystander signalling mechanisms as the primary cause of cell killing during hyper-radiosensitivity. Bystander and abscopal effects can occur in rats and even in immune-compromised nude mice after exposure to Synchrotron Microbeam Radiation and Pencilbeam. Bystander effects can be communicated from irradiated rats to healthy unirradiated cage mate rats and the presence of a tumour modulates both the bystander and abscopal responses. The γ-H2AX biomarker can successfully be used for the detection of DNA damage in the brain of rodents after Synchrotron Radiation. In conclusion, this thesis considerably expands the understanding of the role of bystander effects in cells lines, tissues, and animals exposed to Synchrotron radiation. It is suggested that further exploration of the role of bystander effects and hyper-radiosensitivity during Synchrotron treatments could identify new targets leading to better tumour control. / Thesis / Doctor of Science (PhD)

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.