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Recently excavated burial grounds of the Únětice culture near Kolín (Central Bohemia)
Král, Vlastimil ; Bláhová, Zuzana (advisor) ; Limburský, Petr (referee)
Theses is focused on analysis of early bronze age burial sites excavated during the rescue excavation caused by highway construction around the town of Kolín. Two burial sites in a range of 70 and 13 burials were examined. Information from both of these burial sites were analyzed on the basis of fieldwork research from years 2008-2009. The larger burial site was ascribed to late Únětice Culture by material finds, the smaller burial site was recognized as early Únětice Culture. Burial rite had a same standard characteristic as in other Únětice regions. Local differences in burial rite dwelled in a specific finds, which are not common in an eastern part of Middle Bohemia (amber, animal blade bone). Secondary burial disturbances was traced in all kind of burials of men, women and children. The reason why was secondary burial disturbance not connected to only one group of burials, but to all of them is not clear. Types of ceramic shapes support connection with Moravian region. Thanks to new knowledge about Únětice culture in the eastern part of Middle Bohemia, we can observe, that settlement concentration was in late Únětice culture same as in other Únětice regions. Key words: early bronze age, Únětice Culture, burial sites, burial rite, Kolín region, burial disturbance
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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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Recombinant Fragments of Antibodies
Král, Vlastimil ; Sedláček, Juraj (advisor) ; Pěknicová, Jana (referee) ; Hašek, Jindřich (referee)
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Recombinant Fragments of Antibodies
Král, Vlastimil
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Recombinant Fragments of Antibodies
Král, Vlastimil
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Recombinant Fragments of Antibodies
Král, Vlastimil ; Sedláček, Juraj (advisor) ; Pěknicová, Jana (referee) ; Hašek, Jindřich (referee)
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Functional analysis of hPrp8 mutations linked to retinitis pigmentosa.
Matějů, Daniel ; Cvačková, Zuzana (advisor) ; Král, Vlastimil (referee)
hPrp8 is an essential pre-mRNA splicing factor. This highly conserved protein is a component of the U5 small ribonucleoprotein particle (U5 snRNP), which constitutes one of the building blocks of the spliceosome. hPrp8 acts as a key regulator of spliceosome activation and interacts directly with U5 snRNA and with the regions of pre-mRNA that are involved in the transesterification reactions during splicing. Mutations in hPrp8 have been shown to cause an autosomal dominant form of retinitis pigmentosa (RP), an inherited disease leading to progressive degeneration of retina. In this study, we analyzed the effects of the RP-associated mutations on the function of hPrp8. Using BAC recombineering, we created mutant variants of hPrp8-GFP construct and we generated stable cell lines expressing the recombinant proteins. The mutant proteins were expressed and localized to the nucleus. However, one of the missense mutations affected the localization and stability of hPrp8. Further experiments suggested that RP-associated mutations affect the ability of hPrp8 to interact with other components of the U5 snRNP and with pre-mRNA. We further studied the biogenesis of U5 snRNP. We depleted hPrp8 by siRNA to interfere with U5 snRNP assembly and we observed that the incompletely assembled U5 snRNPs accumulate in...
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Recently excavated burial grounds of the Únětice culture near Kolín (Central Bohemia)
Král, Vlastimil ; Bláhová, Zuzana (advisor) ; Limburský, Petr (referee)
Theses is focused on analysis of early bronze age burial sites excavated during the rescue excavation caused by highway construction around the town of Kolín. Two burial sites in a range of 70 and 13 burials were examined. Information from both of these burial sites were analyzed on the basis of fieldwork research from years 2008-2009. The larger burial site was ascribed to late Únětice Culture by material finds, the smaller burial site was recognized as early Únětice Culture. Burial rite had a same standard characteristic as in other Únětice regions. Local differences in burial rite dwelled in a specific finds, which are not common in an eastern part of Middle Bohemia (amber, animal blade bone). Secondary burial disturbances was traced in all kind of burials of men, women and children. The reason why was secondary burial disturbance not connected to only one group of burials, but to all of them is not clear. Types of ceramic shapes support connection with Moravian region. Thanks to new knowledge about Únětice culture in the eastern part of Middle Bohemia, we can observe, that settlement concentration was in late Únětice culture same as in other Únětice regions. Key words: early bronze age, Únětice Culture, burial sites, burial rite, Kolín region, burial disturbance
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Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
Král, Vlastimil ; Fábry, Milan ; Hořejší, Magdalena ; Závada, Jan ; Sedláček, Juraj
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former scFv is derived from a monoclonal antibody M75 specific for a cell surface protein MN/CA IX, strongly associated with many types of human carcinomas. The latter scFv is derived from a monoclonal antibody TU-20 specific for neuronal beta-III-tubulin.
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