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Modulation of the ankyrin TRPA1 receptor by divalent cations
Hynková, Anna ; Hudeček, Jiří (advisor) ; Hodek, Petr (referee)
TRPA1 is the only mammalian member of the ankyrin type subfamily of transient receptor potential (TRP) ion channels. This channel is widely expressed in many cell types and tissues, including sensory neurones, heart, brain, and lung, where it detects stimuli of different sensory modalities, including a broad spectrum of potentially harmful irritants and pungent compounds, such as isothiocyanates, thiosulfinates, acrolein, cinnamaldehyde derivatives, eugenol, nicotine, as well as multiple products of oxidative stress. In addition to these chemicals, TRPA1 channel can be also activated and, concurrently, inhibited by intracellular calcium, however, the exact mechanisms of this regulation are still unclear. TRPA1 is a nonselective cation channel, permeable for calcium ions, however, relative scattered data on TRPA1 channel selectivity provide divergent estimates ranging from 0,84 to 7,9; in addition, these values strongly depend on the activation mode of the channel. It appears that the extent to which calcium ions dynamically modulate TRPA1 depends on the total amount of cations permeating the ion channel pore. This diploma thesis contributes to the understanding of this topic by (i) investigating the effects of extracellular calcium ions on chemically and voltage- induced activity of human TRPA1...
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Chicken antibodies as a tool of cytochrome P450 immunodetection
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
Cytochrome P450 is an important enzyme which catalyzes a large amount of reactions of endogenous and exogenous metabolism. There are compounds which increase expression of this enzyme and it leads up to the carcinogen activation and cancer formation. It includes much propagated chemopreventive compounds which are consumed abundantly in dietary supplements. It is therefore important to have the methods for determination of cytochrome P450 induction. One of these methods is the cytochrome P450 immunodetection by Western blotting. The detection of proteins on the membrane is done by the specific antibodies mostly gained from mammals. In this bachelor thesis there is the evidence that the antibodies from hen egg serve as well as the mammalian antibodies. The cytochromes P450 1A1 and 1A2 was detected by these egg antibodies after rat exposure to the model chemopreventive compounds β-naphthoflavone.
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Stimulation of pancreatic β-cell proliferation by synthetic mRNA
Volejníková, Anna ; Hodek, Petr (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a severe metabolic disease associated either with loss or functional impairment of insulin-producing β-cells. A major goal in diabetes research is to develop effective ways of pancreatic β-cells restoration. One of the possible approaches is the stimulation of β-cell proliferation. Transcription factor c-Myc is one of the promising targets for stimulation of β-cell proliferation. However, the adenovirus-mediated long-term overexpression of this oncoprotein is associated with risk of cellular dedifferentiation, apoptosis and development of cancer. Therefore, the applicability of an alternative approach for the c-Myc ectopic expression, based on the in-vitro transcribed synthetic mRNA, was verified in this thesis. Use of in vitro transcribed c-Myc-mRNA allows achieving short-term overexpression of transcription factor c-Myc in host cells. High level of c-Myc expression by the β-cells, was detected following the transfection with c-Myc-mRNA. However, within the 48 hours the expression level of c-Myc protein declined to the basic level. The physiological degradation of c-Myc-mRNA by the host cells is a key factor that reduces the risk of cancer development. Transfection of rat islet cells with c-Myc-mRNA resulted in a significantly increased β-cell proliferation. Under the...
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Study of inflammation using epithelial cells
Majerová, Barbora ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The human respiratory system is in constant contact with heterogeneous agents from the environment. There must be effective mechanical lung barriers and sufficient immune protection due to continuous deposition of various substances in the respiratory system. The mutual balance between the mechanisms of natural and adaptive immunity of the lungs is essential for destruction of infectious agents without initiation of inflammatory response. Overreaction of the immune system of the lungs may lead to the production of various inflammatory mediators and cytokines such as interleukins IL-1, IL-6 and IL-18. When determining the immunogenicity of a substance, it has to be exposed to lung epithelial cells, and then the concentration of cytokines produced is measured. To determine the immunogenicity of mammalian immunoglobulin G and chicken immunoglobulin Y the subsequent twenty-four hour exposure to A549 lung cancer cell line was made. Concentration measurement of cytokines IL-1 and IL-6 was performed using Luminex method, which pointed out the immunogenicity of goat immunoglobulin G and certain chicken immunoglobulins Y.
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Interaction of food additives with xenobiotic metabolising enzymes
Jandová, Eliška ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
Recently the use of various food supplements as a part of a healthy lifestyle has been very popular. Although most of them are natural products, their excessive consumption may not always be beneficial for health. Dietary supplements are usually of a flavonoid character. Flavonoid compounds are found in plants and they have beneficial effects on human health. For their antioxidant, anti-allergy and chemopreventive effects they are extensively studied. However, in recent years the negative impacts of flavonoids have been described, often caused by their excessive consumption. It has been shown that they interact with cytochrome P450, which play an important role in the biotransformation of xenobiotics. The change in the metabolism of xenobiotics (whether drugs or carcinogens) can cause serious health problems, including a tumor growth. Beside cytochrome P450, there is another possible points of intervention, cytochrome b5 (or NADH:cytochrome b5 reductase), which effects the catalytic cycle of cytochrome P450. Another point of potential danger is the elimination of xenobiotics out of the organism. There is a complex system of transporters, in which P-glycoprotein plays a very significant role. P-glycoprotein is involved in transmembrane efflux of xenobiotics, preventing the aggregation of these...
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Preparation and enzymatic incorporation of deoxyribonucleic triphosphates derived from pyrimido[4,5-b]indol to DNA using selected polymerases.
Bosáková, Andrea ; Konvalinka, Jan (advisor) ; Hodek, Petr (referee)
This bachelor thesis deals with the synthesis of pyrimido[4,5-b]indole 2'- deoxyribonucleosides and their following trifosforylation. Overall there three new analogues 2'-deoxyadenosine triphosphate with benzen ring were prepared. Furthermore, the ability of DNA polymerase to incorporate in total four modified 2'-deoxyribonucleosides into the oligonucleotide strand by primer extension (PEX) method was observed. All the modified 2'-deoxyribonucleotides were incorporated into the oligonucleotide strand, however the success of the subsequent elongation was different accoring to the DNA polymerase that was used and according to the substitution in position 6 in the structure of substrate. Key words nucleosides, modified nucleotides, DNA polymerase, PEX reaction
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Heterologous expression and isolation of human cytochromes P450 1A1/2
Milichovský, Jan ; Martínek, Václav (advisor) ; Hodek, Petr (referee)
Cytochromes P450 form a large family of hemoproteins. Some of them are responsible for the metabolism of endogenous substrates, but their major role is in detoxification of exogenous substrates (xenobiotics), some of them are activated to reactive species forming covalent adducts with DNA and increasing intracellular oxidative stress. Cytochrome P450 are considered by very promiscuous in terms of their substrate specificity, thus one enzyme can typically oxidize many substrates. Cytochrome P450 1A1 prefers a planar aromatic compounds (e.g. polycyclic aromatic hydrocarbons, azo dyes, etc.). Cytochrome P450 1A2 elicits similar substrate specificity, but prefers slightly basic aromatic derivatives, for example caffeine. This work focuses on (i) the preparation of expression vectors containing genes encoding human cytochromes P450 1A1 and 1A2, (ii) their consequent expression in heterologous system followed by (iii) isolation of corresponding proteins. The genes coding both proteins were modified and transferred from older vectors to the more efficient to expression plasmids pET-22b. However, the new constructs did not produce stable native proteins. The modified genes were therefore transferred to the original expression plasmids pCW. The problem with the incorporation of native human form of...
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