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Preparation of cDNA of selected gene and cloning.
Bogdanovič, Igor ; Miletín, Miroslav (advisor) ; Bunček, Martin (referee)
ABC drug efflux transporters are transmembrane proteins which are known to play a crucial role in drug resistance. The ability of these proteins to actively transport a wide variety of compounds out of the cell is one of the key reasons behind the resistance of cancer cells to chemotherapeutic agents. To study the activity of efflux transporters and identify their substrates or inhibitors a number of in vitro models have been developed. These models are based on the mammalian cells genetically modified in order to express the protein of interest. For genetic manipulations of the cells various expression plasmids are used. In our study we attempted to prepare a universal expression plasmid, containing a short sequence of V5 tag. Such plasmid enables further cloning of coding sequences of selected efflux transporters so that the V5 tag is localized on their 3'-end. Once constructed, the plasmid can further be used to transfect the desired cell line, thus developing an in vitro cell model. Since the transporter is produced as a fusion protein with the V5 tag localized on their C-end, the tag could be used as a universal marker of the transporter expression detectable by immunohistochemical methods. To construct the V5 tag sequence two approaches were employed both leading to the product of the desired...
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Pharmacological approach to the results of the estrogen and progesterone receptors in the breast tumours.
Bařinková, Vlasta ; Hochmann, Jiří (advisor) ; Bunček, Martin (referee)
In the part devoted to discussion of different authors about the importance of the progesterone receptor (PR) value we concentrated primarily to the works of foreign authors published in professional journals. We divided the opinions of this experts to such, which show that PR testing is needless and to the part about profitability of this testing. In the chapter about importance PR testing some authors critize, that some patients - negative in estrogen receptors (ER) (such of them that are simultaneously PRpositive) could be deprived in the possibility of hormonal therapy, because PR testing wouldn't be made. And in the addition, the other - (in this case) the less effective treatment would be given them preferentially. PR testing is important also for detection of the group of ER-positive tumors with PR negativity. At this group is the smaller possibility that the tumors would respond to the hormonal treatment (in contrast to ER+/PR+). In the part devoted to gene therapy we described first the information about learning from the textbooks. Futher we describe different modes of classification of the gene therapy of the breast cancer in the foreign literature. We modified their classification further according to our necessity to these subheads: (1) mutation compensation of differentiation genes,...
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β-tubulin paralogs in Aspergillus: taxonomical importance and molecular tools for distinguishing
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Bunček, Martin (referee)
A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...
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β-tubulin paralogs in Aspergillus: taxonomical importance and molecular tools for distinguishing
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Bunček, Martin (referee)
A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...
|
|
|
Preparation of cDNA of selected gene and cloning.
Bogdanovič, Igor ; Miletín, Miroslav (advisor) ; Bunček, Martin (referee)
ABC drug efflux transporters are transmembrane proteins which are known to play a crucial role in drug resistance. The ability of these proteins to actively transport a wide variety of compounds out of the cell is one of the key reasons behind the resistance of cancer cells to chemotherapeutic agents. To study the activity of efflux transporters and identify their substrates or inhibitors a number of in vitro models have been developed. These models are based on the mammalian cells genetically modified in order to express the protein of interest. For genetic manipulations of the cells various expression plasmids are used. In our study we attempted to prepare a universal expression plasmid, containing a short sequence of V5 tag. Such plasmid enables further cloning of coding sequences of selected efflux transporters so that the V5 tag is localized on their 3'-end. Once constructed, the plasmid can further be used to transfect the desired cell line, thus developing an in vitro cell model. Since the transporter is produced as a fusion protein with the V5 tag localized on their C-end, the tag could be used as a universal marker of the transporter expression detectable by immunohistochemical methods. To construct the V5 tag sequence two approaches were employed both leading to the product of the desired...
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|
|
Pharmacological approach to the results of the estrogen and progesterone receptors in the breast tumours.
Bařinková, Vlasta ; Hochmann, Jiří (advisor) ; Bunček, Martin (referee)
In the part devoted to discussion of different authors about the importance of the progesterone receptor (PR) value we concentrated primarily to the works of foreign authors published in professional journals. We divided the opinions of this experts to such, which show that PR testing is needless and to the part about profitability of this testing. In the chapter about importance PR testing some authors critize, that some patients - negative in estrogen receptors (ER) (such of them that are simultaneously PRpositive) could be deprived in the possibility of hormonal therapy, because PR testing wouldn't be made. And in the addition, the other - (in this case) the less effective treatment would be given them preferentially. PR testing is important also for detection of the group of ER-positive tumors with PR negativity. At this group is the smaller possibility that the tumors would respond to the hormonal treatment (in contrast to ER+/PR+). In the part devoted to gene therapy we described first the information about learning from the textbooks. Futher we describe different modes of classification of the gene therapy of the breast cancer in the foreign literature. We modified their classification further according to our necessity to these subheads: (1) mutation compensation of differentiation genes,...
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