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Laboratory diagnostics of hepatitis E - experience from routine laboratory
Tužáková, Andrea ; Janďourek, Ondřej (advisor) ; Diepoltová, Adéla (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Andrea Tužáková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title thesis: Laboratory diagnosis of hepatitis E - personal experience in a routine laboratory This thesis takes a comprehensive review of viral hepatitis E (VHE), including theoretical and practical aspects. In the theoretical part, the VHE is presented in detail, including epidemiology, genotypic variants, clinical symptoms, diagnostic methods, prevention and treatment. The epidemiological trends and prevalence of VHE are analysed, along with genotypic variants and their correlation with clinical manifestations. Various diagnostic modalities and therapeutic options in the context of VHE are discussed. Hepatitis E is a liver infection caused by the hepatitis E virus. It is one of the five main types of hepatitis viruses: A, B, C, D, and E. Hepatitis E virus is transmitted mainly through the faecal-oral route, often via contaminated water or food. It can cause acute illness characterized by jaundice (yellowing of the skin and eyes), dark urine, fatigue, abdominal pain, and loss of appetite. Most cases of hepatitis E resolve on their own within a few weeks to months without causing...
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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously uncharacterized protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential...
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Isolation and characterization of antibodies specific to the DNA quadruplex structure
Oršel, Jiří ; Stříž, Radim (referee) ; Brázda, Václav (advisor)
The aim of present bachelor thesis has been to isolate guanine quadruplex (G-quadruplex) binding antibodies and characterise them by the means of SDS-PAGE, western blot and immunostaining with a subsequent evaluation of their binding capabilities using an electrophoretic mobility shift assay. G quadruplexes are non-canonical secondary DNA structures formed by guanine rich sequences. They are commonly found in telomeres or oncogene promoters where they fulfil important regulatory functions and contribute to the onset of some tumours. The first part of this thesis is dedicated to the isolation process of BG4, SG4 and SG4 R105A antibodies from the E. coli BL21 DE3 expression system, their determination using the Bradford protein assay followed by the aforementioned western blot and immunostaining, confirming their purity. Their binding capabilities have been assessed using the electrophoretic mobility shift assay in the second part. On the one hand, SG4 has been found to bind G-quadruplexes with high specificity especially in lower DNA:protein molar ratios (1:5), which is consistent with available literature. BG4 on the other hand, has shown comparable affinity to both guanine quadruplexes and double stranded DNA. A surprising finding has been a relatively large affinity of SG4 R105A, a mutant of SG4 engineered to have a low affinity to G-quadruplexes, towards single stranded DNA. Furthermore, it’s measured affinity to G-quadruplexes has been comparable to that of BG4 in lower DNA:protein ratios.
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Preparation of mouse monoclonal antobodies against cyclin-dependent kinase
Šupák, Marek ; Ševčíková,, Sabina (referee) ; Kohoutek,, Jiří (advisor)
The aim of this master‘s thesis is to prepare a monoclonal antibody against cyclin-dependent kinase 13 (CDK13). The theoretical part focuses on antigen-antibody binding, which is essential for the use of monoclonal antibodies in the determination of CDK13 as well as the transcription that this kinase affects. This section is also devoted to Western blot and ELISA methods for detection of newly generated antibodies. Furthermore, the antibodies and the antigen definition are stated, which are later on discussed. The practical part is devoted to the preparation of antigen - its isolation and purification on a peristaltic pump. It also addresses immunization, its course, and the amount of antigen used to immunize mice. After immunization, the work focuses on fusion of sp-2 cells and splenocytes, which were first removed from the immunized mouse and purified. After the fusion alone, selection of hybridomas on HAT selection medium is mentioned, followed by detection first by ELISA and later by Western blotting. The resulting hybridomas with positive ELISA response are frozen for further testing at the Veterinary Research Institute in Brno, where the entire practical part of this thesis was carried out. These frozen hybridomas are further tested by immunoprecipitation to conclude this thesis.
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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously undescribed protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential function in...
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Current possibillities of laboratory diagnostics of EBV
Janů, Denisa ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Charles university Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study programme: Bioanalytical laboratory diagnostics in health care Author: Bc. Denisa Janů Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: Current possibillities of laboratory diagnostics of EBV Background: The aim of this diploma thesis was to get acquainted with the issue of Epstein-Barr virus infection and its laboratory diagnostics. Other aim was to divide the patients into groups according to the serological results, then evaluate the prevalence of EBV and evaluate the incidence of primary EBV infection in the tested group of patients for the years 2019-2022. Methods: Chemiluminescence immunoassay and indirect immunofluorescence were used to determine specific antibodies against EBV. In some cases, the examination was completed by determining the avidity of anti-VCA IgG antibodies and the microblot array method. Real-time PCR was used to detect EBV DNA. Microsoft Excel 2016 was used to process data and to create tables and graphs. Results: For the years 2019-2022, 14,736 patients were tested for the presence of specific antibodies against EBV in the infectious serology and virology laboratory of Vidia-Diagnostika, 263 patients for the presence of heterophile antibodies...
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Determination of opioid receptors in cerebral cortex of rats exposed to multifunctional opioid peptides
Kusová, Pavla ; Svoboda, Petr (advisor) ; Novotný, Jiří (referee)
The subject of this bachelor thesis was to study the effects of multifunctional peptides LYS739 and LYS744 on the amount of µ-, δ-, ĸ-opioid receptors (µ-OR, δ-OR, ĸ-OR) in rat cortex. Peptides were administered by the dose of 10 mg/kg for seven days. The reference group was administered a dose of 10 mg/kg morphine for the same period of the time. The effect of morphine is mainly through µ-OR. Long-term exposure to this opioid agonist results in a reduction in the function and quantity of these receptors in the development of tolerance and dependence. Multifunctional opioid peptides have begun to be investigated for their therapeutic potential to reduce adverse effects and increase efficacy. Their potential lies in their ability to interact with multiple opioid receptors. Peptides LYS739 and LYS744 act as agonists of µ-OR, δ-OR and simultaneously as antagonists of ĸ-OR. The amount of opioid receptors was determined by SDS-PAGE followed by Western blot. The results were compared with a control group that was administered by saline and a reference group that was administered by morphine. There was almost no change in the δ-OR, ĸ-OR receptors. In the case of µ-OR, there was a decrease in morphine and LYS739, but this change was not assessed as statistically significant.
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NMDA receptors outside CNS and their subunit composition
Hotovec, Matěj ; Kolcheva, Marharyta (advisor) ; Horáková, Olga (referee)
N-Methyl-D-Aspartate receptors (NMDARs) are a type of ionotropic glutamate receptor that is widely present in the central nervous system and in lesser numbers in other parts of the body, including the gastrointestinal tract. NMDARs play a crucial role in various physiological and pathological processes in the CNS, including synaptic plasticity, learning and memory, and excitotoxic damage. The role of NMDARs outside the CNS is still under investigation. This thesis aims to confirm the presence of NMDARs in the gastrointestinal tract and their subunit composition. Subunit specific NMDAR modulators showed effect on spontaneous phasic activity of ileum, indirectly confirming the presence of GluN1, GluN2A-D and GluN3 subunits. Attempt at direct evidence of protein expression by immunolabeling with monoclonal antibodies against NMDAR subunits was unsuccessful.
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Cathepsin L of Sphaerospora molnari - localisation, function and diagnostic tools
NECHVILE, Rudolf Lukas
The aim of this thesis was to develop diagnostic and quantitative assays/tools based on western blotting, confocal microscopy and immunogold electron microscopy, as well as flow cytometry, to determine the localisation and potential function of the highly expressed cathepsin L of the myxozoan parasite Sphaerospora molnari (SmolCL) in its fish host, the common carp. Furthermore, a recombinant SmolCL was used in a vaccine trial to estimate its potential for raising antibodies in carp and test their immunoprotective potential towards S. molnari. This thesis provides new methodological tools for research and allows a greater understanding of myxozoan parasite-fish host interactions based on proteolytic enzymes.
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