| 1. |
- Aasa, Jenny, et al.
(författare)
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Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions
- 2016
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier BV. - 1383-5718 .- 1879-3592. ; 805, s. 38-45
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Tidskriftsartikel (refereegranskat)abstract
- Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration x cell treatment time. Gly induced mutations in the hprt-gene at ante of 0.08 +/- 0:01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5 rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.
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| 2. |
- Al-Minawi, Ali Z., et al.
(författare)
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The ERCC1/XPF endonuclease is required for completion of homologous recombination at DNA replication forks stalled by inter-strand cross-links
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 37:19, s. 6400-6413
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Tidskriftsartikel (refereegranskat)abstract
- Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.
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| 3. |
- Biverstal, Anna, et al.
(författare)
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Cyclobutane pyrimidine dimers do not fully explain the mutagenicity induced by UVA in Chinese hamster cells
- 2008
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Ingår i: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 648:02-jan, s. 32-39
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Tidskriftsartikel (refereegranskat)abstract
- UVA generates low levels of cyclobutane pyrimidine dimers (CPDs). Here we asked the question whether CPDs could fully explain the level of mutations induced by UVA. Relative mutagenicities of UVA and UVC were calculated at equal levels of CPDs in cell lines, deficient in different aspects of repair. Survival and gene mutations in the hprt locus were analyzed in a set of Chinese hamster ovary (CHO) cell lines, i.e., wild-type, Cockayne syndrome B protein-deficient (CSB), XRCC3-deficient and XRCC1-deficient adjusted to the same level of CPDs which was analyzed as strand breaks as a result of DNA cleavage by T4 endonuclease V at CPD sites. Induced mutagenicity of UVA was approximately 2 times higher than the mutagenicity of UVC in both wild-type and XRCC1-deficient cells when calculated at equal level of CPDs. Since this discrepancy could be explained by the fact that the TT-dimers, induced by UVA, might be more mutagenic than C-containing CPDs induced by UVC, we applied acetophenone, a photosensitizer previously shown to generate enhanced levels of TT-CPDs upon UVB exposure. The results suggested that the TT-CPDs were actually less mutagenic than the C-containing CPDs. We also found that the mutagenic effect of UVA was not significantly enhanced in a cell line deficient in the repair of CPDs. Altogether this suggests that neither base excision- nor nucleotide excision-repair was involved. We further challenge the possibility that the lesion responsible for the mutations induced by UVA was of a more complex nature and which possibly is repaired by homologous recombination (HR). The results indicated that UVA was more recombinogenic than UVC at equal levels of CPDs. We therefore suggest that UVA induces a complex type of lesion, which might be an obstruction during replication fork progression that requires HR repair to be further processed.
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| 4. |
- Dahle, Dag Olav, et al.
(författare)
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Uric acid and clinical correlates of endothelial function in kidney transplant recipients
- 2014
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Ingår i: Clinical Transplantation. - : Wiley. - 0902-0063 .- 1399-0012. ; 28:10, s. 1167-1176
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Tidskriftsartikel (refereegranskat)abstract
- Uric acid is associated with increased mortality in kidney transplant recipients (KTRs), but it is uncertain if this involves endothelial dysfunction. We hypothesized, first, that there was an association between uric acid and endothelial function, and second, that there were associations between endothelial function and cardiac and mortality risk scores.METHODS: One hundred and fifty-two patients were examined 10 wk after kidney transplantation by two measures of endothelial function, the brachial artery flow-mediated dilatation (FMD) expressed as percent dilatation (FMD%), and fingertip peripheral arterial tone (PAT) expressed as log-reactive hyperemia index (LnRHI). Risk scores were calculated from a recently validated formula. Other clinical correlates of endothelial function were described in stepwise linear regression models.RESULTS: Uric acid was associated negatively with FMD% in an age- and gender-adjusted model, while not in the multivariable model. No association was shown between uric acid and LnRHI. FMD% was associated negatively with risk scores in both crude and age- and gender-adjusted models (p < 0.01). LnRHI was associated negatively with risk scores in the latter model only (p < 0.05).CONCLUSIONS: Uric acid was neither associated with FMD% nor LnRHI in KTRs. There were significant associations between endothelial function indices and cardiac and mortality risk scores.
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| 5. |
- Fotouhi, Asal, et al.
(författare)
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Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA
- 2011
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Ingår i: Mutation research. - : Elsevier BV. - 0027-5107 .- 1873-135X. ; 715:1-2, s. 13-18
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Tidskriftsartikel (refereegranskat)abstract
- UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.
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| 6. |
- Gradecka-Meesters, Dobroslawa, et al.
(författare)
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Assessment of the protective effects of selected dietary anticarcinogens against DNA damage and cytogenetic effects induced by benzo[a]pyrene in C57BL/6J mice
- 2011
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Ingår i: Food and Chemical Toxicology. - : Elsevier BV. - 0278-6915 .- 1873-6351. ; 49:8, s. 1674-1683
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Tidskriftsartikel (refereegranskat)abstract
- The protective action in C57BL/6J mice from orally administered ellagic acid (EA), benzyl isothiocyanate (BITC), an extract of epigallocatechins (Tegreen (R)) as well as chlorophyllin (CHL) against benzo[a]pyrene (B[a]P)-induced DNA damage and cytogenetic effects was investigated. In pilot experiment the comet assay indicated protective effects for all compounds, while such activity was confined to EA and CH with respect to B[a]P-DNA adducts and micronuclei. EA and CH were chosen for the main study where the levels of DNA adducts in liver after injection of 30 mg B[a]P/kg b.w. did not differ from those found for animals exposed to B[a]P and treated with the protective substances. In leukocytes no significant protective effect of CHL was detected while a 2-fold increase of adduct concentrations was observed after co-administration of EA. In the comet assay CHL or EA caused a 3-fold decrease of SSB, and a 2-fold decrease of FPG sites in comparison to animals treated with B[a]P. CHL or EA showed a significant protective effect against B[a]P-induced MN in polychromatic erythrocytes in bone marrow. In contrast, flow cytometry measurements in peripheral blood indicated the MN frequency after treatment with CHL or EA almost twice as high as that recorded for B[a]P alone.
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| 7. |
- Harms-Ringdahl, Mats, et al.
(författare)
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Tomato juice intake suppressed serum concentration of 8-oxodG after extensive physical activity
- 2012
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Ingår i: Nutrition Journal. - : Springer Science and Business Media LLC. - 1475-2891. ; 11, s. 29-
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Tidskriftsartikel (refereegranskat)abstract
- Background: DNA is constantly exposed to reactive oxygen species (ROS), spontaneously arising during the normal oxygen metabolism. ROS may result in temporary as well as permanent modifications in various cellular components such as lipids, proteins and DNA, which may have deleterious consequences. Demonstrating that a dietary supplementation of antioxidants can reduce oxidative DNA damage may provide evidence for the value of such supplementation in prevention of cancer and age related diseases. Findings: The present study was conducted to address whether tomato juice protects against ROS induced by extensive physical exercise in untrained individuals. As a marker of oxidative stress, serum levels of 8-oxodG were monitored using a modified ELISA. An intervention was performed involving 15 untrained healthy subjects who performed a 20 min physical exercise at 80% of maximum pulse using an ergometer bicycle. Blood samples were taken before and one hour after the exercise. The procedure was repeated after 5 weeks with a daily intake of 150 ml tomato juice and followed by a 5 weeks wash-out period and another 5 weeks with a daily intake of tomato juice. The results indicated that a daily intake of tomato juice, equal to 15 mg lycopene per day, for 5 weeks significantly reduced the serum levels of 8-oxodG after an extensive physical exercise. Conclusion: These data strongly suggest that tomato juice has a potential antioxidant effect and may reduce the elevated level of ROS induced by oxidative stress.
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| 8. |
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| 9. |
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| 10. |
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| 11. |
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| 12. |
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| 13. |
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| 14. |
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| 15. |
- Johansson, Fredrik, 1970-
(författare)
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Interplay between DNA repair pathways at replication forks in mammalian cells
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular DNA is continuously being damaged by various agents of both endogenous and exogenous origin including a wide range of chemical compounds, UV-light and ionizing radiation. In order to remove lesions from the DNA and maintain genomic stability, different repair processes have evolved. If not repaired prior to DNA replication, such lesions may block the replication machinery and the capacity to rescue replication stalled at the site of a DNA lesion is thus also important for cellular survival and the maintenance of genome integrity. To date, the interplay between pathways of DNA repair and mechanisms of replication bypass in mammalian cells has not been fully elucidated.The present thesis focuses on the roles played by base- and nucleotide-excision repair and by homologous recombination in reducing errors in connection with replication of damaged DNA in mammalian cells. We were also interested in the effects of caffeine on cellular mechanisms that facilitate replication bypass. Furthermore, we have investigated the mutagenicity of glycidamide and attempted to identify the DNA repair pathway responsible for removing lesions induced by this genotoxic metabolite of acrylamide. In all of these studies Chinese hamster cell lines harbouring mutations in genes encoding proteins associated with DNA repair were employed as the model system. Moreover, we explored the possibility of using these cell lines to develop a system for high-throughput, rapid detection of genotoxic agents.Our results indicates that both nucleotide excision repair and homologous recombination are involved in maintaining replication fork progression on DNA damaged by UV-light or benzo(a)pyrene-7,8-diol-9,10-epoxide. In this connection homologous recombination appears to be a time-consuming process. We also found that the replication fork delay observed following exposure to UV-light is prolonged even further by subsequent treatment with caffeine, both in the case of wild-type cells and cells deficient in homologous recombination or nucleotide-excision repair. In addition, the frequency of mutations induced by UV-irradiation was attenuated, while the level of recombination was enhanced by caffeine. These results indicate that caffeine inhibits translesion DNA synthesis, thereby favouring the use of homologous recombination to bypass lesions that stall replication.On the basis of our findings with methyl methanesulfonate (MMS) and N-methyl-N´-nitro-N-nitrosoguanidine (MNNG), we propose that O6-methylguanine is the major substrate for homologous recombination following treatment with alkylating agents. Both of these agents also stall replication forks, probably due to the presence of unrepaired intermediates in base-excision repair. Moreover, we discuss the possibility of a Rad51-mediated pathway for homologous recombination that is dependent on XRCC1.Furthermore, glycidamide was shown to cause mutations in the hprt gene of wild-type Chinese hamster ovary cells. Our results also suggest that the DNA lesions induced by this compound are repaired by short-patch BER. However, we were unable to identify the lesion responsible for the mutations.Finally, the Chinese hamster ovary cell lines employed here provide a useful tool for the screening of genotoxic compounds and, in addition, for obtaining mechanistic information concerning the pathways involved in the repair of DNA lesions induced by various agents.
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| 16. |
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| 17. |
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| 18. |
- Kotova, Natalia, et al.
(författare)
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A novel micronucleus in vitro assay utilizing human hematopoietic stem cells
- 2015
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 29:7, s. 1897-1905
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Tidskriftsartikel (refereegranskat)abstract
- The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed.
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| 19. |
- Kotova, Natalia, 1975-
(författare)
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Biomarkers for DNA damage in human biomonitoring
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 20. |
- Kotova, Natalia, et al.
(författare)
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Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians
- 2015
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Ingår i: European Journal of Nutrition. - : Springer Science and Business Media LLC. - 1436-6207 .- 1436-6215. ; 54:7, s. 1181-1190
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Tidskriftsartikel (refereegranskat)abstract
- Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p < 0.01, Student's t test). A multivariate analysis demonstrated that there was no association between the fMN and factors such as age, sex, intake of vitamins/minerals, serum folic acid and vitamin B12 levels, physical activity, and body mass index. The mean Hb adduct levels of acrylamide and glycidamide showed no significant differences between vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p < 0.04) between the two groups. These data suggest that the vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the metabolic rates of acrylamide was indicated. In addition, the observed significant difference in the background fMN in the two groups demonstrated that the MN analysis method has a sensitivity applicable to the biomonitoring of human lifestyle factors.
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| 21. |
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| 22. |
- Kotova, Natalia, et al.
(författare)
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Genotoxicity of alcohol is linked to DNA replication-associated damage and homologous recombination repair
- 2013
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Ingår i: Carcinogenesis. - : Oxford University Press (OUP). - 0143-3334 .- 1460-2180. ; 34:2, s. 325-330
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Tidskriftsartikel (refereegranskat)abstract
- Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.
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| 23. |
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| 24. |
- Lagerqvist, Anne, et al.
(författare)
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Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo(a)pyrene-diol-epoxide and dibenzo(a,l)-pyrene-diol-epoxide.
- 2008
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Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 7:8, s. 1202-1012
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Tidskriftsartikel (refereegranskat)abstract
- Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the 32P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.
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| 25. |
- Lagerqvist, Anne, et al.
(författare)
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DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells
- 2011
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 10:8, s. 877-886
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Tidskriftsartikel (refereegranskat)abstract
- Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and ()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides ()-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (+/-)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.
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| 26. |
- Lagerqvist, Anne, 1979-
(författare)
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Factors Influencing the Yield of Mutations Induced by Polycyclic Aromatic Hydrocarbons in Mammalian Cells
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 27. |
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| 28. |
- Lagerqvist, Anne, et al.
(författare)
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Structural requirements for mutation formation from polycyclic aromatic hydrocarbon dihydrodiol epoxides in their interaction with food chemopreventive compounds
- 2011
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Ingår i: Food and Chemical Toxicology. - : Elsevier BV. - 0278-6915 .- 1873-6351. ; 49:4, s. 879-886
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Tidskriftsartikel (refereegranskat)abstract
- Chinese hamster V79 cells were used to investigate the protective effect of four known antimutagens present in food, chlorophyllin (CHL), ellagic acid (EA), epigallocathechingallate (EGCG) and benzylisothiocyanate (BITC), against potent mutagenic polycyclic aromatic hydrocarbon dial epoxides (PAH-DE) derived from benzo[a]pyrene (BP), dibenzo[a,h]anthracene (DBA), dibenzo[a,l]pyrene (DBP), and benzo[c]phenanthrene (BPh) known to be deposited on crops from polluted ambient air or formed during food processing. As fjord-region PAH-DE are more toxic and mutagenic than bay-region PAH-DE, we adjusted the concentrations of PAH-DE to induce approximately the same levels of adducts. The studies were performed using an assay indicating toxicity in terms of reduced cell proliferation together with the V79 Hprt assay for monitoring mutant frequencies. CHL significantly increased the survival and showed a protective effect against the mutagenicity of all PAH-DE. A significant protective effect of EA was found towards the mutagenicity of BPDE, DBPDE and BPhDE and with EGCG for BPDE and BPhDE. BITC had a slight positive effect on the mutagenicity of DBADE and BPhDE. Taken together, a novel and unexpected finding was that the antimutagenic activity could differ as much as by a factor of 7 towards four carcinogenic PAH metabolites being relatively similar in structure and genotoxic activity.
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| 29. |
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| 30. |
- Lundin, Cecilia, et al.
(författare)
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Different Roles for Nonhomologous End Joining and Homologous Recombination following Replication Arrest in Mammalian Cells
- 2002
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Ingår i: Molecular and Cellular Biology. - : American Society of Microbiology. - 0270-7306. ; 22:16, s. 5869-78
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Tidskriftsartikel (refereegranskat)abstract
- Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.
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| 33. |
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| 34. |
- Matsuoka, Atsuko, et al.
(författare)
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Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition.
- 2004
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Ingår i: Mutat Res. - 0027-5107. ; 547:1-2, s. 101-7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- We conducted the recombination and sister chromatid exchange (SCE) assays with five chemicals (hydroxyurea (HU), resveratrol, 4-hydroxy-trans-stilbene, 3-hydroxy-trans-stilbene, and mitomycin C) in Chinese hamster cell line SPD8/V79 to confirm directly that SCE is a result of homologous recombination (HR). SPD8 has a partial duplication in exon 7 of the endogenous hprt gene and can revert to wild type by homologous recombination. All chemicals were positive in both assays except for 3-hydroxy-trans-stilbene, which was negative in both. HU, resveratrol, and 4-hydroxy-trans-stilbene were scavengers of the tyrosyl free radical of the R2 subunit of mammalian ribonucleotide reductase. Tyrosyl free radical scavengers disturb normal DNA replication, causing replication fork arrest. Mitomycin C is a DNA cross-linking agent that also causes replication fork arrest. The present study suggests that replication fork arrest, which is similar to the early phases of HR, leads to a high frequency of recombination, resulting in SCEs. The findings show that SCE may be mediated by HR.
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| 35. |
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| 36. |
- Vare, Daniel, et al.
(författare)
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DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells
- 2012
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 11:12, s. 976-985
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Tidskriftsartikel (refereegranskat)abstract
- DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.
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| 37. |
- Vare, Daniel, 1982-
(författare)
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Interstrand Crosslinks - Induction and repair
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 38. |
- Vare, Daniel, et al.
(författare)
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Quantification and repair of psoralen-induced interstrand crosslinks in human cells
- 2014
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Ingår i: Toxicology Letters. - : Elsevier BV. - 0378-4274 .- 1879-3169. ; 226:3, s. 343-350
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Tidskriftsartikel (refereegranskat)abstract
- Bi-functional alkylating agents that cause crosslinks are commonly used in chemotherapy. However, there is no conclusive knowledge for human cells regarding the number of induced interstrand crosslinks (ICLs) and the unhooking rate when the lesion is removed from one of the DNA strand. Using a newly developed method, we quantified the number of induced ICLs for the five furocoumarins; psoralen, 5-methoxypsoralen, 8-methoxypsoralen, tri-methoxypsoralen and angelicin. In quantitative terms, the results were in agreement with the values found by others. In kinetic studies using mammalian cells, we found that half of the psoralen-induced ICLs were unhooked within 2.5 h. The rate in normal human diploid fibroblasts was found to be 20,000 ICLs/h/cell. In comparison to survival, 2500 ICLs per cell led to 50% toxicity, indicating that the unhooking of the ICLs is not the crucial step for ICL tolerance. Surprisingly, only 3500 ICLs per cell corresponded to a significant delay in the replication fork elongation. The results indicate involvements of additional pathway(s) for the delay since the effect on replication elongation could be monitored when only 10% of the replication forks encounter an ICL.
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| 39. |
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| 40. |
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| 41. |
- Wold, Dag Erik, et al.
(författare)
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Hva finnes av forskning på/om friluftsliv for/i høyere utdanning i Norge? - En oversiktsstudie over forskning(slitteratur) på friluftsliv innen høyere utdanning
- 2023
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Konferensbidrag (refereegranskat)abstract
- Friluftslivets rolle som historisk kulturbærer og identitetsmarkør i Norge har bidratt til at friluftsliv i dag har en sterk posisjon i det norske utdanningssystemet. I høyere utdanning kommer dette til uttrykk ikke bare i (kroppsøvings)lærerutdanningen, men også i bachelor utdanninger i idrett og friluftsliv, i naturguideutdanninger og i veglednings og eventyrspedagogikk. Ulike idéer og teorier om pedagogikk, lederskap og gruppedynamikk har blitt utviklet innenfor høyere utdanning i friluftsliv i Norge. En del av disse ideene har også handlet om hvorvidt relasjonen mellom mennesker og natur er økologisk bærekraftig. Per i dag savnes det, etter vår kjennskap, en oversikt over forskning på friluftsliv innen høyere utdanning. Hovedmålet med studien er derfor å kartlegge hvilke tema forskningslitteraturen på/om friluftsliv for/i høyere utdanning i Norge belyser.Vi finner det hensiktsmessig å kombinere en scoping review gjennom elektronisk søking i databaser med en manuell søking av kurslitteratur og pensum i emner og kurs i friluftsliv på norske universitet og høyskoler. Temaene og søkeordene som inngår i studien er friluftsliv (OR Outdoor education OR Outdoor* OR Adventure* OR Wilderness*) AND høyere utdanning (OR Higher Education OR Universit* OR Høyskole OR Høgskole OR Høgskule OR College OR School (folkehøgskole) OR Physical Education Teacher Education OR Teacher training*) AND Norge (OR Norwegian OR Norway). Den elektroniske søkingen kommer til å gjennomføres i databasene EBSCO(ERIC), Google Scholar, BASE, Norart og Idunn. Den manuelle søkingen kommer til å gjennomføres på x norske læresteder der litteraturlister for kurs med ordet friluftsliv i tittelen inkluderes. Målet med det manuelle søket er å inkludere vitenskapelige bokkapittel (antologier) og annen litteratur som er basert på forskning, men som ikke finnes tilgjengelig elektronisk. Ettersom studien nylig er påbegynt, har vi per i dag ingen resultater å legge frem. Preliminære funn vil bli presentert på konferansen. Vi vil ha søkelys på den historiske utviklingen, endringer og diskurser i friluftslivsundervisningen i høyere utdanning, samt diskutere danningspotensialer og tema som ikke blir belyst i forskningen.
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