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Mitochondrial bioamarkers in Huntington's disease
Vizelka, Vera ; Hansíková, Hana (advisor) ; Dračínská, Helena (referee)
Huntington' s disease (HD) is a severe hereditary neurodegenerative disorder. The cause of this disease is a mutation of the gene that codes the protein huntingtin (htt). The mutation manifests itself as an abnormal extension of CAG triplets. In this way, a polyglutamine chain is formed in the htt molecule, which causes its fragmentation and aggregation. The result is a misfolded protein and this results in damage of neurons, especially in the area of striatum. The biochemical basis of neuronal death has not yet been clarified. Today, there is no effective treatment for HD. Htt participates in many different cellular processes, for example it participates in the regulation of transport events in the cell, participates in the transcription of some genes and also controls apoptosis. Mutated htt can be the cause of mitochondrial dysfunction. The consequence of the action of mutated htt is the occurrence of oxidative stress and subsequently the onset of the neurodegeneration process, but the exact state of mitochondria during the development of the HD has not yet been carefully investigated. Previous studies have studied mitochondrial disorders not only in the brain, but also in other extraneuronal tissues. The aim of the bachelor's thesis was to optimize the preparation of a sample from buccal smear...
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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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Preparation and characterization of chicken antibodies against pathogenic yeast Candida albicans
Vodecký, Matúš ; Hodek, Petr (advisor) ; Dračínská, Helena (referee)
The increase of infections caused by yeast pathogens is an alarming global public health problem. People with compromised immune systems, such as after infection with human immunodeficiency virus or diabetes mellitus, are most at risk. Diagnosis is often inadequate, and treatment relies on a limited selection of antifungal drugs, to which resistance is also increasing. These aspects represent a major problem, as second-choice drugs, which may have toxic side effects (e.g., nephrotoxicity in the case of amphotericin B), must then be applied to resistant pathogens. Given the severity of yeast infections, scientists are trying to find new ways of protection against these pathogens. One option is passive immunotherapy. In this bachelor thesis, antibodies from hen's eggs were chosen for this purpose, as they have the potential for wide therapeutic use. The preparation of hen's egg yolk IgY antibodies is both ethically and economically acceptable. Moreover, egg yolk antibodies show relatively high stability. Polyclonal antibodies were isolated from eggs of hens immunized with either cell wall or whole C. albicans cells in a medium simulating the vaginal environment (VSF). Antibody reactivity was tested by ELISA. The cell walls of C. albicans in VSF medium or YPD medium were used as antigen. The results...
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Circulating tumor DNA in urine and its use as a potential biomarker of colorectal cancer
Surkovová, Veronika ; Ptáčková, Renata (advisor) ; Dračínská, Helena (referee)
Colorectal cancer (CRC) is a serious disease whose incidence has been steadily increasing. There is a high chance of cure and a good quality of life, if the treatment is started in the early stages of this disease. The so-called liquid biopsy, in which biomarkers from various body fluids are obtained and analyzed, could help with early detection of the disease, monitoring of the patient's condition and appropriate determination of the diagnosis. The aim of this study is to isolate and analyse circulating tumor DNA (ctDNA) from the urine of patients with advanced CRC. In these patients the ctDNA was detected in plasma. The method used for analysis is based on the principle of PCR with the formation of heteroduplexes, followed by the separation of "wild type" and mutated DNA fragments by denaturing capillary electrophoresis. Unlike plasma samples, the presence of KRAS mutations was not confirmed in urine samples. Neither the established procedure for plasma analysis, the attempt to enrich ctDNA by so-called purification, nor other tested isolation kits using the selected detection method allowed detection of the KRAS mutation in urine via ctDNA. Although urine ctDNA testing in general appears to be a promising method for improving the treatment level of (colorectal) cancer, and although its use could...
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Interaction of the anticancer drug lenvatinib with cytochrome P450 subfamily 2C
Srogoň, Jiří ; Dračínská, Helena (advisor) ; Hýsková, Veronika (referee)
Lenvatinib is a multi-targeted kinase inhibitor capable of inhibiting these kinases at nanomolar concentrations. For this reason, it is used as a drug in the treatment of various types of cancer. Like many other xenobiotics, lenvatinib is metabolized by cytochromes P450, which can cause occurrence of drug interactions with other substances. Identification of the most important and clinically relevant drug interactions is essential to ensure the safety of patients already suffering from reduced quality of life due to cancer. The main aim of this bachelor thesis was to investigate the effect of lenvatinib on cytochromes P450, specifically on isoforms of subfamily 2C in humans and rats. The inhibitory effect was measured in vitro using marker rections on rat liver microsome samples for the CYP2C6 isoform and on human recombinant CYP2C9 isoform samples expressed in SuperosomesTM or Bactosomes® . Furthermore, the effect of lenvatinib on CYP2C11 and 2C6 expression in the liver of rats exposed to lenvatinib was investigated. Lenvatinib caused a decrease in the activity of the human recombinant CYP2C9 isoform, whereas no effect was observed on the activity of the CYP2C6 isoform in rat liver microsomes. One of the other objectives was to determine the IC50 for CYP2C9, but the objective was not met. When...
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Production and characterisation of therapeutical antibody Farletuzumab - Thiomab E154C S378C
Mochánová, Michaela ; Brynda, Jiří (advisor) ; Dračínská, Helena (referee)
5 Abstract The human folate receptor α (FRα) is a receptor that binds folic acid, which as one of the vitamins is required for basic cellular processes, cellular growth, and differentiation. FRα's expression is strictly regulated in healthy tissue but is highly overexpressed in some tumor types. A humanized monoclonal antibody against folate receptor α called Farletuzumab has been developed. Binding of Farletuzumab to FRα leads to the activation of the immune system via antibody-dependent cytotoxicity and complement-dependent cytotoxicity. To enhance the antitumor effect, selected amino acids in the Farletuzumab antibody molecule were mutated to cysteines, allowing the subsequent use of THIOMAB technology. These cysteines are used to specifically conjugate the antibody to an artificial effector molecule that triggers the cGAS-STING (cyclic GMP-AMP synthase - stimulator of interferon genes) signaling pathway. The combination of the antibody's own anti-tumor effect and the triggering of the immune response to the cGAS-STING signaling pathway maximizes the immune response against cancer cells. Therefore, the aim of my work was to express the antibody in a suitable eukaryotic cell system and then to do a basic characterization of the protein. The Farletuzumab Thiomab antibody was prepared by heterologous...
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Effect of the anticancer drug cabozantinib on cytochrome P450 activity
Slobodníková, Eva ; Dračínská, Helena (advisor) ; Václavíková, Radka (referee)
Cabozantinib is an anti-cancer drug used mainly for the treatment of thyroid and renal cell carcinoma. It is classified as a low molecular weight selective tyrosine kinase inhibitor. Tyrosine kinases play a key role in signal transduction and regulation of many cellular processes such as growth, differentiation, and proliferation. The changes in the tyrosine kinase pathways are associated with the formation and progression of tumors where their growth is uncontrolled. Tyrosine kinase inhibitors act on tyrosine kinase receptors, thereby preventing the spread of cancer cells and slowing down the progression of cancer. Because cabozantinib, like other clinically used drugs, is metabolized by cytochromes P450, adverse drug interactions may occur that result in altered pharmacokinetics of the administered drugs and a consequent decrease in the efficacy of these drugs. In this diploma thesis, the effect of cabozantinib on the activity of the main enzymes of phase I biotransformation of xenobiotics, cytochromes P450, was investigated in vitro. The effect on the activity of both rat and human cytochrome P450 isoforms involved in xenobiotic metabolism was studied. CYP isoforms were predominantly incubated with cabozantinib at two concentrations; 10 µM and a concentration corresponding to the substrate...
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Specific heme interaction modulates the conformational dynamics and function of p53
Sergunin, Artur ; Martínková, Markéta (advisor) ; Dračínská, Helena (referee)
Tumor suppressor p53 is one of the most studied proteins in terms of cancer and the mechanism of its formation. The general function of p53 is based on the transcriptional regulation of various genes, which can differently influence numerous cellular processes. Recent studies revealed a relationship between p53 and iron homeostasis within the cell. In particular, p53 was shown to interact with a molecule of heme, and this interaction ultimately disrupts the DNA-binding ability of p53 and promotes its proteasomal degra- dation. This work focuses on a detailed description of heme binding to the p53. For this purpose, we isolated two forms of p53, heme-free and heme-bound. We discovered that conformational dynamics of heme-free and heme-bound p53 differ, with the latter exhibi- ting a higher degree of flexibility. We also confirmed previous reports that heme indeed interacts with a cysteine residue in a specific manner. However, heme binding does not disrupt the oligomeric state of p53 or its native zinc binding ability. Finally, we showed that heme-bound p53 exhibits severely impaired DNA-binding ability as opposed to the heme-free form. Keywords: heme, sensor proteins, p53 protein, transcription factor, intrinsically disor- dered proteins
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Identification of expression profile changes of resistance-associated genes in experimental in vivo model of ovarian carcinoma
Bryndová, Barbora ; Václavíková, Radka (advisor) ; Dračínská, Helena (referee)
Ovarian carcinoma is one of the most serious gynecological malignancies in the world. High mortality of this cancer is mainly due to the development of multiple drug resistance to conventional treatments involving taxanes. Therefore, current research is focused on the study of new taxane-based derivatives capable to overcome known mechanisms of drug resistance. At present, there are several promising Stony Brook taxane derivatives (SB-T-taxanes), investigated in frame of efficacy, toxicity and mechanism of action. The aim of this bachelor thesis was to study changes in the expression of genes involved in the development of resistance in tissues and tumor obtained from in vivo mo- dels after the administration of derivatives SB-T-121605 compared to conventional taxane paclitaxel and non-treated models. Expression of selected ABC transporter genes (ABCB1, ABCC1 and ABCG2), taxane metabolizing enzyme genes (CYP3A11, CYP3A13 and CYP2C29) and their mouse orthologs was estimated in liver, intestinal and tumor tissue samples taken from in vivo mouse xenograft models. Tumor tissue was derivated from the highly resistant NCI/ADR-RES ovarian cancer cell line. Gene expression was determined by real-time quantitative polymerase chain reaction. Administration of the SB-T-121605 derivative led to several...
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Optimization of a loop-mediated isothermal amplification method for testing Streptococcus pneumoniae infection
Friček, Matúš ; Jeřábek, Petr (advisor) ; Dračínská, Helena (referee)
Streptococcus pneumoniae is grampositive pathogen bacteria. According to World Health Organisation, diseases like pnemoniae or meningitidis are most commonly caused by S. pneumoniae infection. Gold standard for pathogen detection is currently polymerase chain reaction based on specific DNA amplification. During the last decades, many DNA amplification methods have been developed. One of them is loop mediated isothermal amplification (LAMP). Thanks to low instrumental requirements and simple interpretation, this method should be appropriate alternative for pathogen detection. Putting LAMP into clinical practise is complicated mainly because of common false positive results occurance in non DNA containing samples. Based on that, one of the aims of the thesis was to identificate and eliminate main sources of DNA contamination. According to the results, the main source of contamination seems to be the products from previous reactions. Nowdays, lytA and piaB genes are the most commonly used as targets for PCR detecion of S. pneumoniae. In available publications, there is not any mention about targetting LAMP method at piaB gene for detection of S. pneumoniae. That is why the aim of this bachelor thesis was to design optimal LAMP "primers" set for piaB gene detection. Within the optimization of the...
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