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- Kotova, Natalia, 1975-
(author)
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Biomarkers for DNA damage in human biomonitoring
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Genomic DNA in humans is constantly exposed to different kinds of damage. Therefore, it is desirable to implement methods for detecting and measuring of inflicted body burden. Human biomonitoring (HBM) can here be a useful tool as a link between environmental exposure and disease outcome. The present thesis aims to monitor DNA damage in humans by studies on: 1) urinary thymidine dimer (T=T) as a novel biomarker (BM) of human exposure to UV-light; 2) enumeration of variants in HPRT gene in human peripheral blood lymphocytes by developing a sensitive flow cytometric (FCM) analysis; 3) the impact of dietary habits on genomic stability in vegetarians and omnivores in terms of micronuclei (MN) induction detected by FCM. Urinary T=T was quantified by a 32P-postlabeling technique, the kinetics of T=T excretion was studied and the method was validated by delivering controlled UV-doses. The major conclusion was that the amount of urinary T=T was determined by the UV dose, and hence T=T can be used as a BM of UV exposure. Moreover, a new approach for rapid and sensitive enumeration of HPRT-variants by FCM was developed. The obtained HPRT-frequencies were comparable to those previously published by others. Finally, the FCM assay for MN enumeration was applied to study effects of dietary habits in vegetarians and omnivores. The main finding was that vegetarians had significantly lower MN frequencies compared to omnivores. In summary, the applied BMs and respective methods have high sensitivity and/or throughput possibility which are important factors considered in HBM.
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| 2. |
- Lagerqvist, Anne, 1979-
(author)
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Factors Influencing the Yield of Mutations Induced by Polycyclic Aromatic Hydrocarbons in Mammalian Cells
- 2008
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Doctoral thesis (other academic/artistic)abstract
- Environmental contaminants are ubiquitously present in the urban environment, this has the implication that humans are exposed to toxic and carcinogenic chemicals. Polycyclic aromatic hydrocarbons (PAH) are one type of environmental contaminants, which are produced by combustion of organic compounds. A wide variety of different PAH areformed of which most need metabolic activation to be transformed into the ultimate carcinogenic metabolite, a reactive diol epoxide (PAH-DE) that binds to DNA. PAH induced DNA damage is occasionally removed by different repair processes.This thesis focuses on four PAH-DE(benzo(a)pyrene-diol-epoxide (BPDE), dibenzo(a,l)pyrene-diol-epoxide (DBPDE), dibenzo(a,h)-anthracene-diol-epoxide (DBADE) and benzo(c)phenanthrene-diol-epoxide (BPhDE)) and the role of repair of the induced adducts and their efficiency to induce mutations. The highest level of adducts per µMh was found for the two PAH-DE with fjord region conformation (DBPDE and BPhDE). The highest mutation frequency was exerted by DBPDE followed by BPDE, DBADE and BPhDE explained by differences in both nucleotide excision repair (NER) and replication fidelity. When investigating the repair efficiencies and the effect on replication fork (RF) progression we found that NER enhanced the RF progression whereas HR delayed this process. Inhibition of translesion synthesis was found to delay the RF progression in both wild-type, NER and HR deficient cells. BPDE-induced adducts were most efficiently repaired by NER, whereas DBPDE adducts were not repaired. Antioxidants were tested against PAH-DE mutagenicity and their effects were not dependent on the fjord or bay region structures but on some other property of in the individual compounds.All together, the results indicate that it is not possible to categorize the mutagenic potency of PAH-DE according to common structural features (bay/fjord), why these compounds need to be evaluated individually.
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| 3. |
- Vare, Daniel, 1982-
(author)
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Interstrand Crosslinks - Induction and repair
- 2012
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Doctoral thesis (other academic/artistic)abstract
- DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process.
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