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(Construction of deletion mutants of human cytochrome b5 using gene synthesis)
Kotlánová, Iveta ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochrome b5 is a small amphipathic protein. The human form is anchored to the outer membrane of the endoplasmic reticulum and mitochondria, a free form is located in red blood cells. It consists of two domains: a large hydrophilic domain binds heme, a small hydrophobic domain anchors cytochrome b5 to the microsomal membrane. Both domains are connected by linker chain of about 15 amino acids, which gives a flexibility to the protein. Its length plays an important role in transferring electrons to cytochrome P450. If the linker domain is too short, cytochrome b5 is not able to tranfer electrons to cytochrome P450 and not participates in the reactions of MFO system. Other functions are preserved. The aim of this study was to design and build 4 deletion mutants of cytochrome b5 using gene synthesis. The linker domain contains long and short deletions, which are expected to have distortion interaction with cytochrome P450. Part of this thesis was the expression of heterologous proteins by cells of Escherichia coli strain XL10-Gold and DH5α. As expression vectors for the transformation were used plasmids pET- 30a(+) and pET-22b. DNA from cells was isolated and the accuracy of the genetic code was verified using the sequencing. Keywords: cytochrome b5, heterologous expression, gene synthesis (In Czech)
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Metabolism carcinogens and drugs by the system of monooxygenases
Moserová, Michaela ; Stiborová, Marie (advisor) ; Entlicher, Gustav (referee) ; Čeřovská, Noemi (referee)
Ellipticine, an alkaloid isolated from Apocynaceae plants, exhibits significant antitumor and HIV activities. Ellipticine is a pro-drug, whose pharmacological and genotoxic effects depend on activation by cytochromes P450 (CYP) and peroxidases (Px) to a reactive species generating DNA adducts. To elucidate contribution of CYPs (and which of them) and Px to ellipticine activation, we used rat and mouse models, mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in the liver (HRNTM ) and a control mouse line (WT), rats treated with ellipticine, and microsomal systems isolated from the liver of mouse lines and from the liver, kidney and lung of rats. The purified enzymes, CYP1A1 and 3A4, reconstituted with NADPH:CYP reductase were also used. The effect of cytochrome b5, a facultative component of the mixed function monooxygenase system, on ellipticine oxidation by CYP1A1 and 3A4 was also investigated. Carcinogenic benzo(a)pyrene (BaP), known to covalently bind to DNA after its activation with CYPs, was investigated for its potential to generate DNA adducts and to induce CYP and NADPH:CYP reductase enzymes in mouse livers. We investigated an influence of each of components of the mixed function oxidases (MFO) system on metabolism of BaP. CYP1A1 is widely accepted to be the...
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Transport of charged and neutral particles across the model biomembranes
Parisová, Martina ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....
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Development of the new method for in vitro selection of DNA aptamers
Bláhová, Kamila ; Míšek, Jiří (advisor) ; Moserová, Michaela (referee)
Aptamers are short sequences of single-stranded DNA or RNA that are able to specifically bind various molecules (drugs, lipids, sugars, proteins, etc.). These aptamers are isolated from large libraries of random oligonucleotide sequences by SELEX (Systematic Evolution of Ligands by Component Enrichment) or in vitro selection. Despite the success of this method, in vitro selection often requires more than ten rounds of affinity selection as well as optimization of selection conditions. To increase the efficiency of aptamer selection, several methods have been developed that use an increase in the number of secondary structures in random oligonucleotide libraries. These methods, based on increasing the possibility of canonical base pairing in single-stranded oligonucleotides, increased the efficiency of the in vitro selection method. In this work, it was tested whether increasing the probability of occurrence of G-quadruplexes, as structural motifs in random sequences, will lead to increased efficiency of aptamer selection. Four single-stranded DNA libraries with different numbers of guanine (25 %, 35 %, 45 %, 55 %) in a random sequence were used. Streptavidin was chosen as the model molecule for selection, against which several aptamers that are not rich in guanine (G) have previously been selected....
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Preparation of tumour ligand B7-H6 with coiled-coil tag and verifying of its binding to receptor NKp30
Krejčová, Kateřina ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
Natural killer cells (NK cells) are part of innate antitumor immunity. These cells have the capacity to prevent viral infection or malignant transformation without prior antigen sensitization. Activation of NK cells consist of different recognition strategies. Mechanism of activation is based on down-regulated expression of MHC gp I molecules on the cell surface. NK cells possess both activation and inhibitory receptors that transmit activation or inhibitory signals which determine if NK cells are activated or not, and thus whether the target cell will be lysed or spared. NKp30 is a type I transmembrane receptor which recognize the stress-induced cell surface ligand B7-H6. Interaction of these two proteins leads to the initiation of immune response. The main aim of this thesis is the preparation of cell ligand B7-H6 with coiled-coil peptide tag in human embryonic kidney cell lines HEK293 GnTI- and HEK293T. Successful preparation of B7-H6 with coiled-coil tag on its C-terminus was verified by mass spectrometry. Its interaction with natural cytotoxicity receptor NKp30 was also proven by sedimentation analysis. Key words: NK cells, recombinant expression, B7-H6, HEK293, coiled-coil (This thesis is written in Czech)
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Enzymatic fucosylation of oligosaccharides
Horáčková, Karolína ; Bojarová, Pavla (advisor) ; Moserová, Michaela (referee)
Fucosylated oligosaccharides occur naturally in the mammalian body, for example in the form of blood group antigens or human milk oligosaccharides. Human milk oligosaccharides act as inhibitors of pathogen binding to infantile gastrointestinal epithelial cells, thereby preventing diarrheal diseases. The most abundant human milk oligosaccharides are 2' fucosylgalactose and 2' fucosyllactose using mutant α α 2' Key words carbohydrate; enzymatic synthesis; fucosidase; fucosylation; human milk
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Preparation of reporter cell line for validation of recombinant protein biological activity
Kozojedová, Petra ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
Natural killer cells belong to immune cells mediating non-specific immune response. On their surface, natural killer cells express many ligand-specific activation or inhibition receptors, among which belongs natural cytotoxicity activation receptor NKp30, which is able to trigger cytotoxic immune response upon interaction with its ligands. Natural cytotoxicity receptor NKp30 comprises of one immunoglobulin-like ligand-binding extracellular domain with short stalk domain, allowing the extracellular domain of NKp30 receptor to form oligomers, which leads to strengthening of ligand-receptor interaction. Furthermore, signalization leading to cell activation is affected by oligomerization of the extracellular domain of NKp30 receptor. This thesis focuses on preparation of constructs inspired by chimeric antigen receptor theory that carry extracellular domain of NKp30 receptor with stalk domain or only with ligand- binding domain and on validation of biological activity of prepared constructs after expression on the cell membrane. Two vectors for expression of the constructs inspired by chimeric antigen receptors carrying genes for extracellular domain of NKp30 receptor were prepared and to validate their biological activity, they were transfected into Jurkat cells. Key words: NK cells, NKp30, reporter...
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V-ATPase expression in hypoxic conditions in neuroblastoma cells
Kittlerová, Kateřina ; Eckschlager, Tomáš (advisor) ; Moserová, Michaela (referee)
Tumor diseases belong to one of the most common death causes all around the world these days, therefore scientists still work on new therapeutic procedures. Tumor diseases are the second most common death causes among kids and juvenile. One of the freqeuently diagnosed tumor among kids and juvenile is neuroblastoma. Neuroblastoma is malignant embryonic tumor of the peripheral nervous system. Chemotherapy is used as tumor treatment by therapeutic procedures such as surgical removal and tumor irradiation. Cisplatin is one of the most applicated cytostatics, however it's efficiency it's lowered despite of creating resistance during the treatment. Vacuolar ATPase (V-ATPase) acidifies some of the cell organelles including lysosomes, which can lead to lysosomal sequestration of some of the substances including cytostatics and therefore the cure can't get to the therapeutic target. Key role in lysosomal activity regulation performes transcription factor EB (TFEB). This study deals with expression of d subunit of vacuolar ATPase (ATP6V0D1) and TFEB in neuroblastoma sensitive cell line UFK-NB-4 and resistant to cisplatin UKF-NB-4CDDP in normoxia and hypoxia. After exposure of neuroblastoma resistant cells to cisplatin the expression of ATP6V0D1 gene raised compared to normoxia. In case of TFEB the...
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