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Application of novel genomic techniques in studies of pathogenesis of selected rare inherited disorders
Nosková, Lenka ; Kmoch, Stanislav (advisor) ; Sedláček, Zdeněk (referee) ; Živný, Jan (referee)
Rare diseases are a heterogeneous group of disorders. Knowledge of their molecular basis is poor and till recently there were no appropriate methodical approaches due to a limited number of patients. Novel genomic techniques, especially the DNA array technology and the next generation sequencing emerging in last few years, enabled studies of these diseases even in small families and sporadic cases. This PhD thesis focuses on application of novel genomic techniques in studies of rare inherited diseases. It describes a use of DNA array technology in linkage analysis, analysis of differential gene expression, analysis of copy number variations and homozygous mapping, and a use of next generation sequencing technology. Combination of these methods was used for identification of molecular basis of adult neuronal ceroid lipofuscinosis, Rotor syndrome, isolated defect of ATP synthase and mucopolysaccharidosis type IIIC.
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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry
Soukup, Jakub ; Holada, Karel (advisor) ; Šebestová Janoušková, Olga (referee) ; Živný, Jan (referee)
Prions (PrP) are the main cause of neurodegenerative diseases such as Scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. Although the cellular PrP (PrPC ) is involved in many cellular processes, its precise function still needs to be discovered. The disease is caused by the accumulation of a pathological form of PrP (PrPTSE ), which is caused by direct contact of PrPTSE and PrPC . PrP is anchored in the membrane by GPI and can be transmitted by cell-to-cell contact, tunnelling nanotubes, or extracellular vesicles (EVs). EV factions are divided by different biogenesis into exosomes, microvesicles, and apoptotic bodies. PrPTSE was found in exosomes and microvesicles, but these fractions were never compared to each other. The first aim of the doctoral thesis is a comparison of PrP content, prion-converting activity and infectivity in these fractions on CAD5 and N2a-PK1 cellular models of infection. We isolated a fraction of large EVs (20,000× g) and small EVs (110,000× g) by centrifugation from a conditioned medium. We characterised EVs by cryo-electron microscopy and western blot with Alix, TSG-101, CD63, CD9, and HSP70 markers. The contamination from other cellular compartments was checked by calnexin. EV fractions differed...
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The physiological impact of digestive changes in geriatric patients and their impact on dietary and fluid intake
Krotilová, Martina ; Maruna, Pavel (advisor) ; Živný, Jan (referee)
Introduction: This bachelor thesis focuses on the aging process, physiological changes in the digestive tract, and the potential severe problems associated with advanced age that may impact diet and fluid intake. In the theoretical part, it summarizes the most important factors discussing old age and its related aspects. The practical part compares the theoretical research with the collected results from a questionnaire survey and laboratory blood tests. Aims: The main general aim of this thesis was to conduct a search of literature and scientific studies, and then to compare these findings with the data collected in the practical part. It was necessary to obtain the specific changes in the given sections of the digestive tract from the literature and research. The practical part included a questionnaire survey and the results of laboratory blood tests of geriatric patients to compare the diet of current versus younger age and, if necessary, to find out the reasons for the change. Methods: The quantitative questionnaire survey was realized between December 2022 and February 2023 in the Home for the Elderly, Prague 4 Háje and was completely anonymous. The target group were individuals from the elderly population. The questionnaire was based on a professional article by Professor Hana Kubesova. The...
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Nutrition and movement among patients with dementia in > nursing homes
Mesl, Jan ; Matoulek, Martin (advisor) ; Živný, Jan (referee)
Not only the ageing population, but also the pitfalls of the current lifestyle represented by high caloric intake and negligible physical activity lead to a significant increase in civilisation diseases, the most prominent of which are obesity, type 2 diabetes and hypertension. All of these, but not exclusively, contribute to the rise in neurodegenerative diseases such as dementia. This bachelor's thesis deals with the nutritional status and physical fitness of clients with dementia syndrome in a residential care home (one of the types of residential social care facility). The aim of the thesis was to perform nutritional screening, obtain body mass composition values and assess the nutritional status of this group of people. Another aim was to assess the physical fitness status and muscle volume. The research was conducted in the beginning of 2023 (from January to April) among the clients of the residential facility in the Home Velké Březno near Ústí nad Labem. Self- managing clients with different degrees of dementia syndrome were included in the research. A total of 31 people participated in the research, 25 women and 6 men with an average age of 79 years. Each participant underwent a standardized nutritional MNA screening, body mass composition was measured using the InBody S10 device, and...
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Danio rerio as a model of serious human diseases
Hason, Martina ; Bartůněk, Petr (advisor) ; Živný, Jan (referee) ; Divoký, Vladimír (referee)
(ENGLISH) Over the last five decades, zebrafish (Danio rerio) has become a useful vertebrate model organism for the field of developmental biology and disease control. Using zebrafish in xenotransplantation studies is becoming more popular and progressed towards drug screening of anti-cancer drugs. Zebrafish are particularly suitable for high-throughput pre-clinical drug screening, due to the small size of embryos and the striking evolutionary conservation of cancer- related pathways between human and zebrafish. The fast, large-scale evaluation of the cancer- drug response in vivo could facilitate progress in personalized cancer therapy. Nevertheless, there is still a lack of methods which would allow for rapid and sensitive evaluation of tumor cell growth to facilitate high-throughput screening of drugs in vivo. In our bioluminescent zebrafish transplantation model, we proposed and validated a new screening platform for pre-clinical drug discovery in zebrafish embryos. In our experiments we used the NanoLuc luciferase, which enabled us to rapidly screen inhibitors of cancer growth in a sensitive and quantitative way with very low background compared to the conventional fluorescence signal. In our screen we evaluated the in vivo drug response of 180 kinase inhibitors in zebrafish embryos...
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Diagnostic potential of extracellular vesicles
Klementová, Helena ; Holada, Karel (advisor) ; Živný, Jan (referee)
Extracellular vesicles (EV) are membrane particles produced by cells. Many published papers describe changes in the level of EV in body fluids under various pathological conditions. In our study, we analyzed the numbers of EV in the peripheral blood of patients with multiple sclerosis (MS) and in the umbilical cord blood of preterm newborns with inflammation using flow cytometry. We extensively optimized the detection method and verified the presence of EV in the samples using electron microscopy. We found a reduced number of T-cell and phosphatidylserine-positive EV in patients with newly diagnosed MS compared to healthy blood donors. In MS patients requiring a change in the treatment, the number of B-cell EV was lower compared to neurological controls. Similarly, the level of endothelial EV was lower in MS patients receiving Natalizumab treatment. In the umbilical cord blood of preterm newborns with inflammation, we observed significantly increased numbers of placental, endothelial and platelet EV compared to preterm newborns without inflammation. Compared to the full-term controls, the changes in EV numbers were not significant. The number of endothelial EV in umbilical cord blood correlated with the concentration of IL-6 in the venous blood of newborns. Likewise, the number of platelet and...
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Cloning, expression and characterization of recombinant growth factors
Svoboda, Ondřej ; Bartůněk, Petr (advisor) ; Živný, Jan (referee)
Cloning, expression and characterization of recombinant growth factors The role of growth factors and cytokines to regulate a variety of biological processes is ever increasing. Many studies have illustrated that this diverse group of proteins are capable of stimulating cellular growth, proliferation, survival, and cellular differentiation. This thesis mainly focuses on the cytokine interleukin-6 and its specific receptor (IL-6R). The IL-6R protein has been shown to exist as both a membrane-bound and soluble form. The soluble form of IL-6R (sIL-6R) signals via a mechanism called "transsignaling" that involves the receptor associating extracellularly with its ligand IL-6 to generate an IL-6/sIL- 6R agonistic complex. This complex subsequently binds directly to another IL-6 receptor, gp130 that in turn activates it to augment a specific response. It is known that the formation of the IL-6/sIL-6R complex can be greatly enhanced by converting it into a single molecule, called Hyper-IL-6. This fusion protein consists of a sIL- 6R located at the N-terminus and IL-6 at the C-terminus separated by a flexible linker sequence. It was more than ten years since human Hyper-IL-6 was first described and since then, this molecule has proven to be an ...
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Apoptosis of tumor cells : role of TRAIL and caspase 10
Truxová, Iva ; Živný, Jan (advisor) ; Anděra, Ladislav (referee)
One of the key features of cancer cells is the ability to escape programmed cell death (apoptosis). As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro-apoptotic genes have been reported in many cancers. Caspase 10 is an initiator caspase whose physiological function remains poorly understood. Also the ability of caspase 10 to substitute for caspase 8 in the death receptors apoptotic pathway is still controversial. However, the fact that some of the mutations found in CASP10 gene was associated with apoptosis defects (79, 81) suggest that caspase 10 could be also important in apoptosis initiation. In our lab, there was found a heterozygous mutation in CASP10 gene of Jurkat (human T-acute lymphoblastic leukemia) clone resistant to TRAIL (J-TR1). This mutation influence the amino acid composition close to the active site of the enzyme. The aim of this thesis was to confirm the mutation by ARMS-PCR and to determine if an overexpression of normal (unmutated) or mutated caspase 10 D in TRAIL sensitive and/or TRAIL resistant Jurkat cells (J-WT and/or J-TR1) will influence TRAIL induced apoptosis. Mutation was confirmed. We created J-WT and J-TR1 stable clones transfected by vector with unmutated or mutated CASP10 D (CASP10 D WT or CASP10 D MUT). CASP10 D MUT overexpression in J-WT...
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Molecular and functional characterization of the death receptor 6
Klíma, Martin ; Anděra, Ladislav (advisor) ; Živný, Jan (referee) ; Kovář, Marek (referee)
Death receptor-6 (DR6/TNFRsf21/CD358) is a receptor from the TNFR superfamily that likely participates in the regulation of proliferation and differentiation of T- and B-lymphocytes and neural cells. The 655-amino acid human DR6 is a type I transmembrane protein containing four cysteine-rich domains in its extracelular part and a death domain followed by the CARD-like region in its cytoplasmic part. Overexpression of DR6 in some cell lines leads to apoptosis, and/or to activation of nuclear factor NF-κB and stress kinases of the JNK family. In the first part of our work we focused on molecular characterization of DR6, including the analysis of its posttranslational modifications. We found that DR6 is an extensively posttranslationally modified protein including S-palmitoylation and both N- and O-glycosylation. Six N-glycosylation and one S-palmitoylation sites were precisely mapped to appropriate asparagines and cysteine respectively. The juxtaposed linker region (between cystein-rich domains and the transmembrane part), which also contains Ser/Thr/Pro-rich region with clustered putative O-glycosylation sites, is required for the plasma membrane localization of DR6. N-glycosylation, but interestingly not S-palmitoylation, may play a role in targeting of DR6 into detergent-resistant...
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