It generally accepted that the critical target of ionizing radiation in cellular reproductive death is DNA. What is less known is what types of lesions are involved, how they are repaired and how various agents enhance, protect or modify them. One of the main reasons for these difficulties has been the lack of assays for molecular damage sensitive enough to measure yields at doses where significant numbers of cells survive. Recently however, various techniques have appeared that exhibit this sensitivity. One of these, Rydbergs’ double-label unwinding method, can routinely detect damage in the DNA of mammalian cells from doses as low as 0.1 Gy. We have used this technique to measure initial yields, repair kinetics and residual yields of damage in the dose range 1-6 Gy. We have found, in substantial agreement with measurements at higher doses, the existence of at least two distinct repair processes with half-lives of 7 minutes and 250 minutes respectively. We have also found that the distribution of breaks between the fast and slow processes can be altered by various conditions; for example: whether or not the irradiation was done in the presence of oxygen or nitrogen, post-treatment in hypertonic solution, incubation with ARA-A, (an inhibitor of DNA synthesis) or post-treatment in dimethyl sulphoxide. We have also found that the yield of unrepaired lesions (as defined by damage remaining after two hours incubation at 37°C) seems to include a quadratic variation with initial dose suggesting that among the slowly repairing breaks, there exist complex, combination-type lesions (which may include DNA double-strand breaks). Such studies, combined with results from other assays should help in understanding the nature of the lesions in DNA and the repair processes acting on them and will eventually aid in identifying the critical lesion(s) responsible for cell death. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

No description available.

During the last two decades there has been growing interest in the application of organic dielectric liquids for dosimetry of ionizing radiations. The main problem associated with the liquid application in radiation detectors has been the difficulty in securing saturation charge collection and controlled charge multiplication to permit operation in the ionization chamber and proportional counter modes. In an attempt to understand better the fundamental mechanisms involved in the limitation to charge collection an extensive review has been made of the published theoretical and experimental research. The theoretical work attributes the unattainability of saturation charge collection to losses caused by different types of recombination depending on the initial separation of ions liberated and on the magnitude of the applied electric field. None of the presented theories is found to be fully consistent with the reported experimental results obtained for a range of different di-electric liquids, especially in high field regions. Liquid hydrocarbons, especially those characterised by high charge mobility and high yield of ions, have been widely investigated experimentally to explore the mechanisms responsible. The experimental measurements are found to be strongly dependent on: the purity of the liquids, their chemical structure, the type of materials used for the electrodes in contact with the liquid and on the temperature. These conclusions reflect inadequacies both in the theoretical knowledge of charge transport in liquids and in the practical difficulties of measurement which indicate the need for more detailed experimental investigation. The origin of the natural intrinsic dark current in liquids is found to be due to the presence of impurities; the effect of cosmic-ray interactions; and the presence of radioactivity in the construction materials of the detector. Upon application of high electric fields other factors such as electron emission, molecular dissociation and field ionization become significant. The extensive range of results reported on transport properties (mobility, free ion-yield, conduction band energy, di-electric strength, and theoretical W-values) of charge earners in liquid hydrocarbons and liquified rare-gases, and their dependence on the electric field and temperature have been compiled into tabulated form in appendix B to provide a ready reference. New experimental work, aimed at assessing the role and the key factors involved, was conducted with two separate ionization chambers filled with liquid tetramethylsilane (TMS). Information was obtained on the dependence of the current-field characteristic, for the dark and ionization currents, on various parameters such as purity, electrode separation, surface asperities, electrode construction material, and the charge collection area. For the ionization current, the dependence on the radiation intensity, produced with a 4 mCi source of 57Co of y-rays, was also measured. Liquid purity was confirmed to be very important. Chemical and electrical purification, could lead to orders of magnitude reduction in the background dark current. Tests, made to assess the efficiency of ion collection in liquid TMS, indicated the need for much larger, and more uniform, electric fields. These were achieved by fitting electrodes made from tissue-equivalent plastic. The improved surface smoothness of the latter was found to improve the current-to-noise ratio by a factor of 2-3 orders of magnitude. From the results of the present investigation at fields ? 500 kV/cm it was concluded that the saturation collection of charge was attainable. Limitation to achieving saturation is discussed in terms of charge multiplication produced inside localised gas bubbles on the electrode surfaces. Field induced polarization of liquid molecules could be a contributing factor at high fields. There appears to be realistic prospects of achieving saturation collection of charge, and possibly proportional multiplication, by appropriate design using advanced technology to ensure ultra-smooth surfaces and uniform electric fields.

No description available.

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Pentoxifylline enhances the toxicity of radiation and has emerged as an effective modulator of the radiation response of tumour cells. The molecular mechanisms involved in the enhancement of radiotoxicity by pentoxifylline have not yet been elucidated. Cell cycle blocks, DNA repair and programmed cell death (apoptosis) are all pert of the cellular response to DNA damage and as such must be considered as targets of the drug. In this study, the influence of pentoxifylline on radiosensitisation, G2 block abrogation, DNA repair inhibition and the induction of apoptosis have been investigated in 8e11 and MeWo melanoma and 4197 and 4451 squamous cell carcinoma (SCC) cell lines. The influence of pentoxifylline on radiation-induced apoptosis in Jurkat J5 T-lymphocytic leukemia cells has also been assessed. Hela cervical carcinoma cells were used to investigate the molecular events involved in the abrogation of the G2 block by pentoxifylline. It is shown that pentoxifylline preferentially sensitises the TP53 mutant MeWo and 4451 cell lines and enhances radiotoxicity by factors of up to 14.5. In the MeWo melanoma, but not in the 4451 SCC cell line, radiosensitisation is accompanied by inhibition of DNA repair. No significant enhancement of radiation-induced apoptosis was observed in MeWo melanoma and 4451 SCC cells. However, Jurkat J5 cells showed an increase in apoptosis after irradiation in the presence of the drug. In irradiated Hela cervical carcinoma cells, pentoxifylline affects the expression of the two components of the mitosis promoting factor (MPF), namely cyclin 81 and p34cdC2, and rapidly restores cyclin 81/p34cdC2 ratios to control levels. Analysis of cyclin 81 expression in whole cells and isolated nuclei furthermore reveals an influence of the drug on the subcellular translocation of the MPF. It is concluded that G2 block abrogation is not the only mechanism involved in the radiosensitisation of tumour cells by pentoxifylline, but that DNA repair inhibition plays a role in certain cell types. Although pentoxifylline induces apoptosis in Jurkat J5 thymocytes, radiation-induced apoptosis plays no role in the radiosensitisation of the two TP53 mutant melanoma and sec cell lines. Abrogation of the G2 block by pentoxifylline, which sensitises tumour cells to a second irradiation or chemotherapeutic challenge, involves a modulation of the levels of cyclin 81 and p34cdC2, and the subcellular location of the MPF. These results are of utmost importance for the clinical potential of pentoxifylline as a dose modifier in cancer therapy. / AFRIKAANSE OPSOMMING: Pentoxifylline verhoog die toksisiteit van bestraling en het dus na vore getree as 'n effektiewe modulator van die sellulêre stralingsrespons in kankerselle. Die molekulêre meganismes betrokke by die verhoging van stralingstoksisiteit deur pentoxifylline is egter nog nie duidelik nie. Blokkering van die selsiklus, die herstel van ONS skade en geprogrammeerde seldood (apoptose) vorm almal deel van die sellulêre respons ná bestraling en word as sulks beskou as potensiële teikens van die middel. In hierdie studie is die invloed van pentoxifylline op stralings-sensitiwiteit, G2 blok verwydering, die vertraging van ONS herstel en die indusering van apoptose ondersoek in die Be11 en MeWo melanoom en 4197 en 4451 plaveisel-sel karsinoom sellyne. Die invloed van pentoxifylline op stralings-geïnduseerde apoptose in Jurkat J5 T-limfosiete is ook bestudeer. Hela servikale karsinoom selle is gebruik om die molekulêre gebeurtenisse rondom die verwydering van die G2 blok deur pentoxifylline te ondersoek. Dit word aangetoon dat pentoxifylline by voorkeur die radiosensitiwiteit van die TP53 mutante MeWo en 4451 sellyne verhoog, en stralingstoksisiteits verhogingsfaktore van tot 14.5 genereer. Hierdie effek gaan gepaard met die vertraging van ONS herstel in die MeWo melanoom, maar nie in die 4451 plaveisel-sel karsinoom sellyn nie. Die meting van apoptose toon geen noemenswaardige verhoging van stralings-geïnduseerde apoptose in MeWo melanoom óf in 4451 plaveisel-sel karsinoom selle nie. Jurkat J5 T-limfosiete toon egter wel 'n verhoging in apoptose wanneer bestraling in die teenwoordigheid van pentoxifylline gedoen word. In Hela servikale karsinoom selle affekteer pentoxifylline die uitdrukking van die twee komponente van die mitose promoverings faktor (MPF), naamlik siklien B1 en p34CdC2 , en restoreer die siklien 81/p34cdC2 verhoudings vinnig na normale vlakke. Ontleding van die siklien 81 uitdrukking in heel selle en in geïsoleerde selkerne toon verder dat die middelook die sub-sellulêre ligging van die MPF affekteer. Die gevolgtrekking word gemaak dat G2 blok verwydering nie die enigste meganisme is waardeur pentoxifylline radiosensitiwiteit verhoog nie, maar dat die vertraging van ONS herstel in sommige seltipes 'n rol speel. Alhoewel pentoxifylline apoptose verhoog in T-limfosiete, speel dit nie 'n rol in die verhoogde radiotoksisiteit wat waargeneem is in die TP53 mutante melanoom en plaveisel-sel karsinoom sellyne nie. Verwydering van die G2 blok deur pentoxifylline, wat selle meer sensitief kan maak vir 'n tweede stralings- of chemoterapie aanslag, behels die modulasie van siklien 81 en p34cdc2 vlakke en die sub-sellulêre ligging van die MPF. Hierdie resultate is van uiterste belang vir die kliniese aanwending van pentoxifylline as 'n dosis-modifiseerder in kankerterapie.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.