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The role of exocyst subunit AtEXO70E2 in autophagy and secretion
Moulík, Michal ; Sabol, Peter (advisor) ; Janda, Martin (referee)
Exocyst is a protein complex composed of eight subunits, evolutionarily conserved in yeasts, animals, and plants. The main function of exocyst is to mediate the tethering of secretory vesicles to the plasma membrane. However, the involvement of exocyst in some other processes, especially in autophagy, has been recently discovered. Plant exocyst is specific because most of its subunits have multiple paralogs. The most diversified subunit is EXO70, which is encoded by 23 paralogous genes in Arabidopsis thaliana. In this thesis, I dealt with subunit AtEXO70E2 (AT5G61010), which has been localized to double-membrane compartments considerably reminiscent of autophagosomes. These compartments were named EXPOs (for exocyst-positive organelles) and described as a component of unconventional protein secretion pathways. There are also hints that EXO70E2 could play a role in autophagic processes. However, details of this relationship remained unexplored. For my experiments, I used stably transformed lines of A. thaliana and transiently transformed leaves of Nicotiana benthamiana. I performed numerous colocalization experiments, applied various pharmacological treatments to the studied lines, and analyzed a mutant line in the EXO70E2 gene. According to my observations, protein EXO70E2 is expressed especially...
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Study of effects of tyrosine kinase inhibitors and their metabolites on tumour cell lines
Kolárik, Matúš ; Indra, Radek (advisor) ; Vinklářová, Lucie (referee)
Vandetanib, lenvatinib and cabozantinib are inhibitors of receptor tyrosine kinases approved to treat locally advanced or metastatic thyroid gland, kidney and liver cancers. These multi- kinase inhibitors, inhibit phosphorylation of tyrosine moieties of protein, thus modulate cell signalization in cancer cells. Metabolites of vandetanib, lenvatinib and cabozantinib were detected in vitro as well as in vivo in blood and urine. Cytochromes P450 and flavin monooxygenases were identified as primary enzymes participating in metabolism of these drugs. Literature lacks information regarding pharmacological efficacy of vandetanib, lenvatinib and cabozantinib metabolites. The aim of this diploma thesis was the investigation of pharmacological efficacy of N-oxides of vandetanib, lenvatinib and cabozantinib. The viability measurement under normoxic and hypoxic conditions was employed to determined their efficacy. The expression of enzymes of the first phase of xenobiotics metabolism (CYP 450 1A1, 1B1, 3A4 a CYP 450 oxidoreductase) and receptor tyrosine kinases RET and VEGFR2, as well as mechanism of changes in their expression were investigated using western blotting and flow cytometry. High performance liquid chromatography was utilised to investigate possible metabolism of tyrosine kinase inhibitors and...
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Autophagy in yeast
Nejedlý, Adam ; Palková, Zdena (advisor) ; Libus, Jiří (referee)
Autophagy is a process of degradation of the cell cytoplasm, proteins, and organelles. It appears mainly as a response to a lack of nutrients or damage of cell structures and components. Yeast Saccharomyces cerevisiae and also Komagataella phapphi are the main model organisms for the research of autophagy. Autophagy is classified by two main criteria. Firstly, we divide autophagy into macroautophagy and microautophagy. During macroautophagy, the phagophore vesicle, called the autophagosome, is created around sequestrated cargo and then fuses with the vacuole. Microautophagy is a process in which a vacuolar membrane directly surrounds and sequester degraded cargo. During non-selective (bulk) autophagy, a random non- specific portion of cytoplasm is degraded. On the other hand, selective autophagy serves as a pathway for the degradation of specific proteins and organelles. Autophagy research has nowadays a great medical significance thanks to the role of autophagy defects in a wide area of human diseases.
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Autophagy as a key mediator of beneficial effects of caloric restriction
Kačinská, Irena ; Novotný, Jiří (advisor) ; Funda, Jiří (referee)
Caloric restriction has long been known in the scientific world as a life-prolonging condition, and there is growing evidence that the effect of activation of non-selective autophagy is responsible for these effects. In this thesis, the molecular mechanisms of autophagy in the state of caloric restriction will be described, as well as its beneficial effects on the aging of the organism and various health problems associated with aging and the modern way of life will be summarized. Some risks that could result from unprofessional intervention in one's own body will also be mentioned. It is hypothesized that practicing caloric restriction under the supervision of an expert brings prevention of diseases of civilization diseases and diseases associated with aging and prolongs quality of life, which would, among other things, help reduce the cost of health and social care. Keywords: Autophagy, caloric restriciton, molecular mechanisms
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Characterization of promoter regions of HGSNAT and GBA genes, and a contribution to the study of pathogenesis of MPS IIIC and Gaucher disease
Richtrová, Eva ; Hřebíček, Martin (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...
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Autophagy in adipose tissue
Somova, Veronika ; Krulová, Magdaléna (advisor) ; Zouhar, Petr (referee)
Autophagy is an essential mechanism of recycling, which represents a path leading to utilization and reorganization of intracellular components (nutrients, organelles, etc). Recent studies on adipose tissue have shown, that autophagy plays crucial role in differentiation of the adipocytes precursors and in phenotypic tissue transformations. The aim of this thesis is to summarize the characteristics of autophagy in terms of various adipose tissues as well as to present the regulation of autophagy as a potentially useful in therapy focused on obesity treatment. However, with the regulation of autophagy, there are risks that will also be considered in the present thesis. Keywords: autophagy, adipose tissue, therapy
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The role of proteostatic mechanisms in neurodegenerative diseases
Zezulová, Kristýna ; Vodička, Petr (advisor) ; Marková, Vendula (referee)
Protein homeostasis (proteostasis) plays an important role in maintaining normal cell function and viability. Neurons are particularly vulnerable to proteostasis dysregulation, resulting in damage, dysfunction, and neuronal death, as manifested in many neurodegenerative diseases. One of them is Huntington disease, hereditary neurodegeneration with autosomal dominant inheritance. Expansion of the CAG repeats in the huntingtin gene is translated into an abnormally long glutamine chain in huntingtin protein, leading to disruption of neuronal proteostasis. The primary affected area of the brain is the striatum of the basal ganglia. Disease is progressive, the onset of symptoms usually occurs in adulthood, and after many years leads to the death of the patient. Despite intensive research, disease pathology is still not fully understood, treatment is still only symptomatic and new studies, together with a deeper understanding, also raise many new questions. Through the complexity of the issue, the study of proteostasis in neurodegeneration can bring not only possible implications for therapy, but also could go deeper into the understanding of stress, memory or aging processes.
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Study of Cellular Toxicity of Representative Nanoparticles in Tissue Cultures.
Filipová, Marcela ; Holada, Karel (advisor) ; Benson, Veronika (referee) ; Hubálek Kalbáčová, Marie (referee)
Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...
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Mitophagy in Huntington's Disease
Šonský, Ivan ; Hansíková, Hana (advisor) ; Macůrková, Marie (referee)
Mitochondrial dysfunctions contribute to the progression of many neurodegenerative diseases, including Huntington's disease (HD). In HD, mutation in the huntingtin gene (HTT) results in the expansion of CAG repeats, causing the growth of the polyglutamine tract. This growth is responsible for the gain of toxicity function of the protein. The turnover of dysfunctional and damaged mitochondria is mediated via mitophagy - a selective form of autophagy. Additionally, mitophagy impairments have recently been described to play a key role not only in neurodegenerative diseases. The protrusion of mitophagy results in the clustering of defective mitochondria, organelles which are responsible for fulfilling the energetic demands of neural cells. The most distinctive impact of the impairment is on the striatal medium spiny neurons and results in the development of motor and cognitive dysfunctions. This thesis describes how HD affects mitophagy and reveals the biggest obstacle of mitophagy - disruption of mitochondria targeting into emerging autophagosomes caused by the abnormal interaction of mHTT and p62. Induction of mitophagy at this stage could be crucial for the future therapeutic research of HD. Generally, initiation of mitophagy could become a relevant therapeutic target for many other...
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The role of autophagy in neurodegenerative processes
Marková, Veronika ; Novotný, Jiří (advisor) ; Čermák, Vladimír (referee)
The characteristics of many neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's disease is the accumulation of proteins and damaged organelles in the cytoplasm. Unfortunately, they are not sufficiently eliminated by autophagy. The basal autophagy, that maintains the cellular homeostasis, is disturbed in neurodegeneration. The process of autophagy becomes saturated and unable to remove all the toxic substances. Therefore, other degradation mechanisms are activated, aiming to restore the homeostasis. However, the neuronal cells are damaged under certain conditions leading to their death. The reduction in the number of neurons in specific brain areas may cause severe ataxias and dementias. Better understanding of autophagocytosis and its role v pathogenesis of neurodegenerative diseases may contribute to more effective treatment of these serious diseases in the future. Key words: autophagy, neurodegeneration, Alzheimer's disease, Parkinson's disease, Huntington's disease
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