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The influence of cytochrome b5 on naphthalene oxidation catalyzed by human cytochrome P450 1A2
Stránský, Jaroslav ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochromes P450 (CYP) form a crucial group of oxidoreductase-class enzymes participating in the biotransformation of numerous endo- and exogenous compounds (xenobiotics). A negative aspect to the activity of this enzyme, however, can be the bioactivation, a process during which an originally only slightly toxic compound is transformed into a substance of much higher toxicity. Cytochrome P450 has therefore long been standing at the forefront of biochemical and medicinal interest. For the proper function of cytochrome P450 a supply of electrons into a catalytic cycle is necessary. This is usually taken care of by NADPH:cytochrome P450 oxidoreductase. Cytochrome b5, an enzyme present in the membrane of the endoplasmic reticulum of eukaryotic cells along with NADPH:cytochrome P450 oxidoreductase and cytochrome P450, however, can also act as an alternative electron donor. The participation of cytochrome b5 in the catalytic cycle of cytochrome P450 have a potential to modulate this cycle. Over the course of evolution, many isoforms of cytochrome P450 have developed, each possessing different substrate specifity, catalytic properties, distributions within various tissues etc. This bachelor thesis is focused on the isoform 1A2 (CYP1A2) which represents nearly 10 % of all human hepatic cytochrome P450s....
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Preparation of fusion domains of human immunoreceptors for their utilization in immunotherapy
Cmunt, Denis ; Vaněk, Ondřej (advisor) ; Moserová, Michaela (referee)
The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there's an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody...
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Study of metabolism carcinogenic and nephrotoxic natural compound aristolochic acid II
Martináková, Lenka ; Stiborová, Marie (advisor) ; Moserová, Michaela (referee)
Aristolochic acids (AA) have been considered as toxicants of plants which were found in plants of the family Aristolochiaceae. The most abundant acids in mentioned plants are aristolochic acid I (AAI) and aristolochic acid II (AAII). AA have been considered as causes kidney disease called Aristolochic acid nephropathy (AAN). AAN was initially discovered in patients of one Belgian clinic in Brussels specialized on treatment of patients leading to a decrease in their body weight. The first name of this disease was Chinese herb nephropathy (CHN). Later, it was discovered that one component of herbal preparation was changed by a mistake with the Aristolochiaceae plant. The second type of renal disease caused by AA was discovered in populations of countries along the Danube river, called as Balkan endemic nephropathy (BEN), which was probably caused by the contamination of grains with plants containing AA. These renal diseases (AAN and BEN) are often associated with development of upper urothelial cancer (UUC). AA (AAI + AAII) in organisms are subject to biotransformation leading to its reductive activation or oxidative detoxification. Both cytosolic enzymes [NAD(P)H:quinone oxidoreductase] and microsomal enzymes [cytochromes P450, NADPH:cytochrome P450 reductase] participate in their reduction. The...
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The inhibitors of tyrosine kinases and their effect on the expression of biotransformation enzymes
Dvořák, Josef ; Dračínská, Helena (advisor) ; Moserová, Michaela (referee)
Tumor diseases are one of the most common causes of death in the human population. One of the many possible causes of tumor growth is abnormal function of tyrosine kinases, which are involved in signal transfer and regulation of the most important cell processes. These processes include the control of cell growth, division and cell differentiation and apoptosis. For the therapy of tumor diseases caused by the abnormal function of tyrosine kinases, their specific inhibitors are developed. For the targeted treatment of thyroid tumors, the tyrosine kinases vandetanib and lenvatinib are newly used. In this bachelor thesis, the effect of vandetanib and lenvatinib on the gene expression of the cytochrome P450 family of 2 (CYP2A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1) biotransformation enzymes has been investigated as they are involved in the biotransformation reactions of a large portion of the available drugs on the market. To examine the relative gene expression of CYP2, quantitative PCR of samples of cDNA, synthesized from isolated RNA from rat liver and kidney exposed to the above-mentioned tyrosine kinase inhibitors, was used. The results suggest, that vandetanib and lenvatinib do not have a significant effect on the gene expression of cytochrome P450 family 2 in rat liver and kidney tissue. KEY WORDS:...
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The Study of metabolism of dihydromyricetin
Blecha, Tomáš ; Moserová, Michaela (advisor) ; Koblihová, Jitka (referee)
More attention has recently been paid to the chemopreventive compounds contained in food of plant origin. A large group of chemopreventive compounds are represented by flavonoids contained in many dietary supplements. One of the representatives of this group is dihydromyricetin, which is attributed to positive impact on human organism, in particular by its antioxidant, anti-inflammatory and hepatoprotective effects. Some flavonoids can act as modulators of enzymes of phase I and phase II metabolism of xenobiotics biotransformation by their inhibition or induction which can causean interference with endogenous metabolism. In first part of this present work, the effect of selected flavonoid compounds, such as biochanin A, dihydromyricetin, myricetin and their combinations on the gene expression of cytochrome P450 2E1 (CYP2E1) and sulfotransferases 1A1 (SULT1A1) in liver and small intestine of laboratory rats was studied. The small intestine was divided into three parts namely a proximal, middle and distal. At first, isolation of total RNA was made followed by conversion into cDNA using random hexamers as primers. By real-time PCR a significant decrease in gene expression of both enzymes in liver was found. The highest induction of gene expression was observed for both enzymes in proximal part of...
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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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Interaction of gut microflora with myricetin
Hušková, Andrea ; Hodek, Petr (advisor) ; Moserová, Michaela (referee)
Intestinal microflora could be considered a "forgotten" organ of the human body. In the gastrointestinal tract, xenobiotics (including drugs) can be converted into their active or inactive forms by intestinal microorganisms via various metabolic pathways. This, in turn, affects their biological activity. The variability of microorganisms in the hosts intestines leads to diverse metabolic reactions of xenobiotics. Thus, the resulting metabolites may vary across host organisms. It is almost imperative to keep this fact in mind while studying and testing new drugs, as well as dietary supplements, because many microbial metabolites can have serious implications for host organisms, especially when administered orally. In this work, mutual interaction between myricetin and faecal bacteria originating from human intestines was studied under various conditions. For the experiments, a high performance liquid chromatography (HPLC) method was used to determine the rate of myricetin degradation and the potential formation of dihydromyricetin as a reductive metabolite. The effect of myricetin on the growth of faecal bacteria was studied by PCR-DGGE. From the isolated DNA of the bacteria, the DNA sequence corresponding to 16S rRNA was amplified by the PCR method. The PCR products were then separated on a...
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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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Study of effect of flavonoids on biotransformation enzymes expression
Koucká, Kamila ; Moserová, Michaela (advisor) ; Mrízová, Iveta (referee)
Flavonoids belong to the group of phenolic compounds included in the secondary metabolites of plants, which are represented mainly in fruits and vegetables. These compounds have antioxidant and anti-carcinogenic effects. Some of them can affect biotransformation enzymes, which include the cytochrome P450 and can interfere with the metabolism of xenobiotics. In the present work we studied effect of dihydromyricetine on the gene expression of cytochrome P450 1A1, 1A2 and cytochrome b5 in livers, lungs, kidneys, colon and small intestine of laboratory rats. The small intestine was divided into three parts namely a proximal, middle and distal. At first, isolation of total RNA was made using commercial reagents and isolated RNA was converted to cDNA by reverse transcription. Finally, using polymerase chain reaction in real time (RT-PCR), the relatively gene expression of genes observed in the organs of laboratory rats pretreated dihydromyricetine and control laboratory rats (untreated) to a reference gene β-actin was determined. It was found that dihydromyricetine did not significantly affect the gene expression of studied genes in most organs. However, a significant decrease of gene expression of CYP1A1 and CYP1A2 was observed in the lungs. On the contrary, an increase of gene expression of CYP1A2 was...
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