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The use of cell-free nucleic acids in maternal plasma for non-invasive prenatal diagnosis of monogenic diseases, placental insufficiency-related complications and Down syndrome
Veselovská, Lenka ; Hromadníková, Ilona (advisor) ; Schierová, Michaela (referee)
Since the discovery of cell-free fetal DNA in peripheral blood of pregnant women, cell-free nucleic acids in maternal plasma are explored in relation to non-invasive prenatal diagnosis of various fetal conditions and pregnancy complications. Non-invasive prenatal diagnosis of monogenic diseases represented by TSC1-linked tuberous sclerosis could be achieved by detection of paternally-inherited mutant allele in the pool of maternal alleles in plasma. Reliability of detection of mutant allele could be improved by simultaneous mutation haplotype analysis or detection of universal fetal marker. None of the 3 methods (allele- specific real-time PCR, SNaPshot minisequencing and quantitative fluorescent PCR) evaluated using artificial mixtures and maternal plasma samples reliably and accurately detected low-frequency allele distinguished by point mutation, SNP or microsatellite in TSC1 gene or in its close proximity. We developed a strategy for prediction of proportion of informative couples for panel of SNPs of interest that can be applied to any monogenic disease. Exploiting differential methylation of promoters of genes RASSF1A, HLCS and OLIG2 in maternal and fetal genome, we failed to establish functional fetal marker. MicroRNAs of placental origin released into plasma could serve as biomarkers of...
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Mechanisms of resistance and iron metabolism in cancer stem cells
Lettlová, Sandra
(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....
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Mechanisms of resistance and iron metabolism in cancer stem cells
Lettlová, Sandra
(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....
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Mechanisms of resistance and iron metabolism in cancer stem cells
Lettlová, Sandra ; Truksa, Jaroslav (advisor) ; Kovář, Jan (referee) ; Brábek, Jan (referee)
(EN) Analogously to normal stem cells within the tissues, cancer stem cells (CSCs) have been proposed to be responsible for maintenance and growth of tumours. CSCs represent a small fraction of cells within the tumour, which is characterised by self-renewal capacity and ability to give rise to a tumour when grafted into immunocompromised mice. Cells with increased stemness properties are believed to be responsible for tumour resistance, metastases formation and relapse after tumour treatment. The first part of this work concentrates on resistance of the tumours, which is often associated with increased expression of ATP-binding cassete (ABC) transporters pumping chemotherapeutics out of the cells. For the purposes of this study, we utilized an in vitro model of CSCs, based on cultivation of cells as 3D "spheres". Expression profiling demonstrates that our model of CSCs derived from breast and prostate cancer cell lines express higher mRNA level of ABC transporters, particularly ABCA1, ABCA3, ABCA5, ABCA12, ABCA13, ABCB7, ABCB9, ABCB10, ABCC1, ABCC2, ABCC3, ABCC5, ABCC8, ABCC10, ABCC11 and ABCG2 among the cell lines tested. The protein level of ABC transporters tested in breast CSCs showed higher expression of ABCB8, ABCC1, ABCC2, ABCC10 and ABCG2 but downregulation of ABCB10 and ABCF2 proteins....
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The use of cell-free nucleic acids in maternal plasma for non-invasive prenatal diagnosis of monogenic diseases, placental insufficiency-related complications and Down syndrome
Veselovská, Lenka ; Hromadníková, Ilona (advisor) ; Schierová, Michaela (referee)
Since the discovery of cell-free fetal DNA in peripheral blood of pregnant women, cell-free nucleic acids in maternal plasma are explored in relation to non-invasive prenatal diagnosis of various fetal conditions and pregnancy complications. Non-invasive prenatal diagnosis of monogenic diseases represented by TSC1-linked tuberous sclerosis could be achieved by detection of paternally-inherited mutant allele in the pool of maternal alleles in plasma. Reliability of detection of mutant allele could be improved by simultaneous mutation haplotype analysis or detection of universal fetal marker. None of the 3 methods (allele- specific real-time PCR, SNaPshot minisequencing and quantitative fluorescent PCR) evaluated using artificial mixtures and maternal plasma samples reliably and accurately detected low-frequency allele distinguished by point mutation, SNP or microsatellite in TSC1 gene or in its close proximity. We developed a strategy for prediction of proportion of informative couples for panel of SNPs of interest that can be applied to any monogenic disease. Exploiting differential methylation of promoters of genes RASSF1A, HLCS and OLIG2 in maternal and fetal genome, we failed to establish functional fetal marker. MicroRNAs of placental origin released into plasma could serve as biomarkers of...
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