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Made available in DSpace on 2014-10-09T12:36:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:17Z (GMT). No. of bitstreams: 1 03966.pdf: 1375579 bytes, checksum: 57621423a9d166633d51a36fc9c8a997 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:36:17Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:59:17Z (GMT). No. of bitstreams: 1 03966.pdf: 1375579 bytes, checksum: 57621423a9d166633d51a36fc9c8a997 (MD5) / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Targeted therapy for prostate cancer may offer potential improvement over current conventional therapies because of its specificity. Although conventional treatments are effective, they are not curative and have several limitations. In prostate cancer, activation of both the epidermal growth factor receptor (EGFR) and the phosphatidylinositol 3 – kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway have been implicated in tumorigenesis and resistance to both conventional and targeted anticancer therapies. Having a better understanding of the molecular mechanisms involved in PCa development, progression and resistance to therapy, could assist in the design of novel therapeutic strategies. The objective of this study was to inhibit key molecular targets of the human epidermal growth factor receptor signalling pathway and expose prostate cell lines to doses of radiation, so as to establish potential therapeutic targets that may be amenable to combined modality therapy, and formulate a cocktail of inhibitors to evaluate its radiosensitising capability. The EGFR/PI3K/mTOR pathway plays an important role in the radiosensitivity of the human prostate carcinoma cell line (DU145) and the normal cell line (1542N). In our study we have shown that AG-1478, an EGFR inhibitor, and BEZ-235, a dual inhibitor of the PI3K/mTOR pathway, singly or in combination, at low and relatively high radiation doses, resulted in radiosensitisation of DU145 cells. Radio-protection was achieved in 1542N cells. AG-1478 had no effect on radiosensitivity. / AFRIKAANSE OPSOMMING: Geteikende terapies wens hul spesifisiteit teenoor konvensionele terapies vir prostaat kanker, mag potensieel verbetering offer. Konvensionele behandeling is wel effektief maar nie genesend nie wens ‘n aantal beperkings, sowel as die toksisiteit vir normale selle. In prostaat kanker is die aktivering van beide die epidermiese groei faktor reseptor (EGFR) en fosfatidielinositol 3-kinase/Akt/soogdier teiken vir rapamisien (mTOR) seingewing baan sterk betrek by tumor groeisel en weerstand teen konvensionele en geteikende anti-kanker terapies. Beter begrip van die molekulêre meganismes betrokke by prostaat kanker ontwikkeling, bevordering en weerstand teen terapie, kan die ontwerp van nuwe terapeutiese strategies ondersteun. Die doelwit van hierdie studie was om sleutel molekulêre teikens van die epidermiese groei faktor reseptor seingewing baan te inhibeer en om prostaat selle bloot te stel aan dosisse bestraling, om potensiële terapeutiese teikens te vestig wat vatbaar is vir gekombineerde modaliteit terapie, om ‘n mengsel van stremmiddels te formuleer, en om die straling gevoeligmaking bekwaamheid daarvan te evalueer. Die EGFR/PI3K/mTOR seingewingbaan speel ‘n belangrike rol in the radiosensitiwiteit van die menslike prostaat kanker sellyn (DU145) en die normale prostaat sellyn (1542N). Die studie bevind dat AG-1478, ‘n EGFR stremmer, en BEZ-235, ‘n tweevoudige beperker van die fosforinositied 3-kinase (PI3K) en soogdier teiken vir rapamisien (mTOR) seingewingbaan, enkel of in kombinasie die DU145 selle radiosensitiseer vir straling dosisse van 2 en 6 Gy. Stralings beskerming was verkry met die 1542N sellyn. AG-1478 het geen effek getoon op radiosensitiwiteit nie.

Philosophiae Doctor - PhD / Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy. However, concerns regarding the carcinogenic potential of secondary neutrons produced during PBT, especially in terms of their effect on HSPCs harboured in the cranial bone marrow of paediatric patients, remain. In this study, the radiobiological differences between 60Co γ-rays and p(66)/Be(40) neutron exposure was investigated to resolve the underlying mechanisms for the high radiosensitivity of HSPCs (CD34+ cells) isolated from umbilical cord blood (UCB). For both radiation qualities, an apparent dose-dependent increase in the frequency of radiation-induced MN was observed in CD34+ cells. Furthermore, increased cytogenetic damage was observed with the CBMN assay after neutron irradiation, which highlights its leukaemogenic potential. In addition, no difference was observed in the nuclear division index of the CD34+ cells post-irradiation between both radiation qualities. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX foci, 2 and 18 hours after radiation exposure. A significant higher number of DNA DSBs were observed 2 hours after neutron irradiation with 0.5 Gy, but decreased to similar levels for both radiation qualities after 18 hours. Different stages of apoptosis in CD34+ cells were studied at 18 and 42 hours numerous time points post-irradiation by flow cytometry using the Annexin/PI assay. In contrast to the γ-H2AX foci results, a significant difference in late apoptosis was observed at 18 hours and 42 hours between the two radiation qualities. The results point towards a fast error-prone DNA repair in HSPCs after neutron irradiation, which might contribute to genomic instability and leukemogenesis. In the second phase of the PhD project, the impact of age on radiosensitivity was investigated by comparing newborn T-lymphocytes with adult peripheral blood (APB) T-lymphocytes. The major difference between UCB and APB T-lymphocytes, is their immunophenotypic profile. Since it is known that different T-lymphocyte subsets have a difference in radiosensitivity, the fraction of CD4+, CD8+, naïve (CD45RA+) and memory (CD45RO+) T-lymphocytes was determined via flow cytometry in the two groups. The cytokinesis-block micronucleus (CBMN) assay was used to determine the extent to which age influences the frequency of cytogenic damage in response to 60Co γ-rays radiation. For both APB and UCB, an outspoken dose-dependent increase in the frequency of radiation-induced MN was observed at 0.5, 1, 3 and 4 Gy. However, no significant difference was observed at 4 Gy when comparing MN yields of APB and UCB. An increased radiosensitivity of newborn to adult donors of 34%, 42%, 29%, 26% and 16% was observed based on the MN scoring at doses of 0.5, 1, 2, 3 and 4 Gy, respectively. The lowest radiosensitivity was identified at the highest dose, which might explain the non-significant difference at 4 Gy. In addition, there was a clear trend that females were more sensitive to 60Co γ-rays radiation than males in both adults and newborns, even though the difference was not significant. The immunophenotypic study revealed that that both the CD4+ and CD8+ T-lymphocytes of newborns are mainly naïve. This is illustrated by the co-expression of CD45RA+ on 90.70% (range: 80.80% – 98.40%) and 95.90% (range: 89.60% – 98.80%) of CD4+ and CD8+ cells respectively. The composition of adult T-lymphocytes, in contrast, is clearly different with a more equal distribution between CD45RA+ and CD45RO+ subpopulations. This finding demonstrates that there are differences in the radiosensitivity between newborn and adult T-lymphocytes which might be linked to the immunophenotypic change of T-lymphocytes with age.

<p> The ɣ-radiosensitivity of haploid and diploid Oedogonium cardiacum cells was measured and compared to other cell lines. With the doubling of the chromosome complement, the Do value doubled, but the extrapolation number decreased four-fold. A general conclusion was drawn from the results that at all doses of ɣ-radiation, the diploids were more resistant than the haploids. A new radiation technique was used and compared to that used routinely in the laboratory. The further use of the technique was not recommended since the data obtained with the diploid line only was not as reliable as one would like.</p> <p> The degree of synchrony of Oe. cardiacum zoospore cultures was measured using cell division as the biological end-point and a mathematical expression, the percent phasing, as the index of synchrony. It was intended that this research problem be secondary to the radiation studies. The percent phasing values were determined for cells growing in two inorganic media and in the presence of an inhibitor, hydroxyurea. However the degree of synchrony was not improved beyond that of the routine laboratory procedure. Attempts to improve the size of the synchronous populations collected also proved unsuccessful.</p> / Thesis / Master of Science (MSc)

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