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Role of Next Generation Sequencing in Diagnostics and Prognosis of X-linked Lysosomal Storage Disorders
Řeboun, Martin ; Dvořáková, Lenka (advisor) ; Fajkusová, Lenka (referee) ; Krejčí, Jan (referee)
The characterisation of the molecular-genetic etiology of monogenic diseases includes not only the identification of the pathogenic variant, but also the description of its effect on the RNA structure and stability. Additionally, the X-inactivation plays an important role in X-linked diseases. In the presented thesis, we applied the methods of next generation sequencing to study three lysosomal disorders (mukopolysacharidosis type II, MPS II; Danon disease, DD; Fabry disease, FD). Two methodological approaches have been used: 1) panel sequencing with hybridization probes for identification of single nucleotide variants, small deletions/duplications and structure variants (CNVs) 2) amplicon sequencing for analysis of somatic mosaicism and allele specific expression. The panel sequencing enabled us to confirm the molecular-genetic basis of DD in two patients with the identification of two exons duplication and five exons deletion in LAMP2, respectively. The somatic mosaicism was being analyzed by the amplicon sequencing in families with DD, MPS II. We could identify the first case of somatic mosaicism in a patient with DD. The allele specific expression has enlarged the group of methods used in X-inactivation analysis. Its impact has been proved particularly in minimizing misinterpretation of XCI...
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Connection between the solubility and the mutagenicity of nitrobenzanthrones in water and organic solvents
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans. Nitronbezanthrones can form covalent adducts with purine bases in DNA after their metabolic activation. Genotoxic properties of 2-NBA are lover by 3-4 orders of magnitudes in comparison to 2-NBA, large difference in the solubility, and therefore also the difference in their bioavailability, of these isomers can be among the factors explaining this phenomenon. One of our goals was to determine the solubility of 3-NBA and 2-NBA in water and in methanol. The second objective was to determine the extinction coefficients for their solutions in water and methanol. Two different methods were employed to determine the solubility of 3-NBA and 2-NBA. The first method was based on stepwise adding of the stock solution of 3-/2-NBA in dimethylsulfoxide to excessive volume of water and simultaneous spectrophotometric verification of the Lambert-Beer law. Additional experiments confirmed the solubility of 3-/2- NBA utilizing spectrophotometric determination of 3-/2-NBA concentration in...
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One goal was to determine solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). Another objective was to evaluate the effect of model proteins (bovine serum albumin, lysozyme) on solubility of Sudan I and ellipticine. The last aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method measuring concentration of a compound in saturated solution (In Czech) Key...
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One of our goals was to determine the solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). The second aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method utilizing determination of concentration of a compound in equilibrium with solid phase. (In Czech) Key words: solubility, UV-VIS spectroscopy, 3-nitrobenzanthrone, 2-nitrobenzanthrone, ellipticine, Sudan I
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Solubility of lipophilic model carcinogens in water environment and effect of biomolecules
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven strong mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans, while 2- NBA shows genotoxic properties lower by 3-4 orders of magnitude. Here we consider the possibility that large difference in the solubility, and consequently also the difference in bioavailability of these isomers could be the factor partially explaining this phenomenon. One goal was to determine solubility of 2-NBA in water and compare it with 3-NBA and also with other carcinogens studied in our laboratory (Sudan I, ellipticin). Another objective was to evaluate the effect of model proteins (bovine serum albumin, lysozyme) on solubility of Sudan I and ellipticine. The last aim was to determine extinction coefficients of these compounds in water and in methanol. Two different methods were employed to determine the solubility of the model compounds. The first method was based on spectrophotometric verification of the Lambert- Beer law. The results were than compared with other method measuring concentration of a compound in saturated solution (In Czech) Key...
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Connection between the solubility and the mutagenicity of nitrobenzanthrones in water and organic solvents
Řeboun, Martin ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
2-Nitrobenzanthron (2-NBA) and 3-nitrobenzanthron (3-NBA) are pollutants widely occurring in the environment. The main sources of benzanthrones are combustion products (i.e. diesel exhausts, wood and cigarette smoke ...). 3-NBA is proven mutagen and carcinogen for bacteria and mammals and it is probably mutagenic also to humans. Nitronbezanthrones can form covalent adducts with purine bases in DNA after their metabolic activation. Genotoxic properties of 2-NBA are lover by 3-4 orders of magnitudes in comparison to 2-NBA, large difference in the solubility, and therefore also the difference in their bioavailability, of these isomers can be among the factors explaining this phenomenon. One of our goals was to determine the solubility of 3-NBA and 2-NBA in water and in methanol. The second objective was to determine the extinction coefficients for their solutions in water and methanol. Two different methods were employed to determine the solubility of 3-NBA and 2-NBA. The first method was based on stepwise adding of the stock solution of 3-/2-NBA in dimethylsulfoxide to excessive volume of water and simultaneous spectrophotometric verification of the Lambert-Beer law. Additional experiments confirmed the solubility of 3-/2- NBA utilizing spectrophotometric determination of 3-/2-NBA concentration in...
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