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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Signaling systems of TLR receptors in microglia
Prokop, Jonáš ; Novotný, Jiří (advisor) ; Filipp, Dominik (referee)
Emerging importance of TLR signalling in microglia has been suggested by various studies of neuroinflammation. Both MyD88 dependent and TRIF dependent pathways used by TLR induce secretion of proinflammatory cytokines in microglia by the activation of transcriptional factors such as NF-κB. This in turn polarizes the whole microglial population to a proinflammatory phenotype. Under physiological conditions microglia express various anti-inflammatory and growth factors and serve in the maintenance of the CNS. Once activated, microglia are connected to neuroinflammation and in the case of overactivation to neurodegeneration. Research of TLR signalling in microglial populations is important for our understanding of complex pathologies of the central nervous system. Moreover, exact knowledge of associated mechanisms might prove very useful in finding new therapeutic approaches. Key words: Microglia, signalling systems of toll-like receptors, Myd88, TRIF, neuroinflammation, neurodegeneration
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Canonical and non-canonical signalling triggered by activated TRAIL receptors in human cells
Nahácka, Zuzana
TRAIL ligand can trigger apoptosis of permissive human cells via engagement of its two pro- apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Its ability to induce apoptosis independently on p53 status and to selectively kill cancer cells in vitro and in vivo made this ligand an attractive target in cancer research. However, acquired resistance of primary cancer cells, unsatisfactory outcome of clinical trials and recent studies arguing that TRAIL might under specific conditions promote cancer progression, opened new plethora of questions, which need to be addressed. Though both receptors DR4 and DR5 are ubiquitously expressed, different types of tumours show preference for either of the receptors. The relative participation of DR4 and DR5 in TRAIL- induced signalling is still largely unknown. To analyse TRAIL receptor-specific signalling, I prepared Strep-tagged, trimerised variants of recombinant human TRAIL ligands with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, I examined a contribution of individual pro-apoptotic receptors to TRAIL-induced signalling pathways. I found that in TRAIL resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeded comparably in both DR4- and...
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Canonical and non-canonical signalling triggered by activated TRAIL receptors in human cells
Nahácka, Zuzana ; Anděra, Ladislav (advisor) ; Rudolf, Emil (referee) ; Vondráček, Jan (referee)
TRAIL ligand can trigger apoptosis of permissive human cells via engagement of its two pro- apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Its ability to induce apoptosis independently on p53 status and to selectively kill cancer cells in vitro and in vivo made this ligand an attractive target in cancer research. However, acquired resistance of primary cancer cells, unsatisfactory outcome of clinical trials and recent studies arguing that TRAIL might under specific conditions promote cancer progression, opened new plethora of questions, which need to be addressed. Though both receptors DR4 and DR5 are ubiquitously expressed, different types of tumours show preference for either of the receptors. The relative participation of DR4 and DR5 in TRAIL- induced signalling is still largely unknown. To analyse TRAIL receptor-specific signalling, I prepared Strep-tagged, trimerised variants of recombinant human TRAIL ligands with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, I examined a contribution of individual pro-apoptotic receptors to TRAIL-induced signalling pathways. I found that in TRAIL resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeded comparably in both DR4- and...
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Potassium deficiency in plants - signs and responses
Štočková, Hana ; Tylová, Edita (advisor) ; Konrádová, Hana (referee)
Plants receive K+ mainly from the soil through the root system. In soil, K+ occurs primarily in minerals such as mica and potassium feldspar. The availability of K+ for plant uptake depends on the form in which K+ occurs in soil. There are forms directly available, slowly available, and unavailable for plant and the transition of K+ between these pools may occur. In plant, K+ is very mobile and it occurs in high amount in cells. It is the most prominent cytoplasmic cation. It affects high number of metabolic processes, including photosynthesis, osmoregulation, and activation of enzymes. K+ shortage can cause changes in plant morphology, anatomy and metabolism. K+ deficiency can be manifested by leaf deformation, decreased leaf area, necrosis, short internodes, reduced rate of photosynthesis, etc. K+ deficient plant is also more vulnerable to pathogens and its resistance to abiotic stress factors such as drought, low temperatures, or salinity is decreased. Deficiency of K+ reduces the biomass and overall yield in agricultural crops, so K+ fertilizers, both soil and foliar, are needed. The thesis focuses on the manifestations of K+ deficiency in plants and summarizes the recent findings on mechanisms of K+ deficiency perception and signal pathways leading to the response to this deficiency. Key...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Analysis of cell signaling mediated by the adapter protein Daxx
Švadlenka, Jan ; Anděra, Ladislav (advisor) ; Forstová, Jitka (referee) ; Stopka, Tomáš (referee)
2 Abstract Multifunctional adapter protein and histone chaperone Daxx has been described in nu- merous cellular processes, including the regulation of apoptotic and stress signalling, antiviral response and processes connected to chromatin (e. g. transcription). Its influ- ence on chromatin-related processes is mainly carried out by several associated en- zymes, such as DNA-methyltransferase-1, histone deacetylases and chromatin- remodelling ATPase ATRX. In the complex with ATRX Daxx functions as a chaperone of histone-3.3, maintaining the constitutive heterochromatin e. g. at centromeric and telomeric regions. The main aim of this Thesis was a better understanding of the Daxx cellular functions through identification and functional characterization of its novel interacting proteins. Using the yeast two-hybrid screen, several such new Daxx-interacting proteins were identified. These proteins were mainly nuclear, connected to the regulation of chroma- tin-related processes. More detailed analysis focused on the interaction of Daxx with chromatin-remodelling ATPase Brg1. This interaction was confirmed both in vitro and in the cells, where Daxx and Brg1 associated mainly in high molecular weight pro- tein complexes. These likely chromatin-remodelling complexes contain, in addition to Brg1, several...
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Role of cFLIP/CFLAR protein in the activation and regulation of cell death
Ksandrová, Marie ; Anděra, Ladislav (advisor) ; Macůrková, Marie (referee)
Programmed cell death as a natural mechanism plays essential role in development and homeostasis maintenance through removal of damaged, unwanted or dangerous cells. The cell death is an irreversible proces must be strictly regulated and thus defects in the regulation of cell death could lead to serious/fatal diseases. There are also one of the reasons why cell death regulating mechanisms are subjects of intense research nowadays. c-FLIP is one of several important cell death regulators. Three distinct isoforms of c-FLIP were detected on the protein level in human organism (long c-FLIPL and two shorter variants c-FLIPS and c-FLIPR) that interact their main cellular partner procaspase 8. The functional consequences of their interaction (enhancement or suppression of procaspase 8 selfprocessing) depend on the cellular level of c-FLIP, expressed splice variants and extracellular signaling. As c-FLIP is an important component of cell death signaling pathways (apoptosis and regulated necrosis), its expression and functional posttranslational modifications are strictly regulated by several mechanisms. Upregulation of c-FLIP levels has been found in various types of tumor cells that are often resistant to anticancer treatment.
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A study of signaling and cytoprotective potential of cannabinoid GPR55 receptors in PC12 cells
Pavluch, Vojtěch ; Novotný, Jiří (advisor) ; Ostašov, Pavel (referee)
At the end of the 20th century it was known that cannabinoid drugs interact with two receptors, CB1 and CB2. Subsequent pharmacological studies have confirmed that there are other receptors interacting with cannabinoids. GPR55 is a transmembrane G protein coupled receptor, which together with the receptor GPR18 and GPR119 belong to a group of new cannabinoid receptors and is involved in the function of the endocannabinoid system. In addition to some of cannabinoid substances, it is stimulated primarily phospholipid lysofosfatidylinositolem. LPI-dependent signaling GPR55 plays an important role in the regulation of many physiological and pathological processes, such as pain, inflammation, cell proliferation, or endothelial function. It was found that LPI confers tolerance to ischemic brain damage and has a cytoprotective effect on the pyramidal cells. The aim of the study was to determine whether the application of five ligands induce phosphorylation of protein kinase ERK 1/2, Akt and activate the GTPase RhoA and whether activation of the receptor GPR55 has cytoprotective effect in model cell line PC12, in which hypoxic conditions were simulated by adding CoCl2. For working methods were used SDS-PAGE, Western bloting and colorimetric measurement. Pharmacological studies in recent years have shown...
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The role of membrane cholesterol in delta-opioid receptor signaling Correlation with plasma membrane structure
Brejchová, Jana ; Svoboda, Petr (advisor) ; Novotný, Jiří (referee) ; Teisinger, Jan (referee)
Study of HEK293 cells stably expressing fusion protein between delta opioid receptor (δ-OR) and pertussis toxin-insensitive mutant of Gi1α protein, δ-OR-Gi1α (Cys351 -Ile351 ), provided the following results. Decrease of plasma membrane cholesterol content (cholesterol depletion) induced by cyclic oligosaccharide β-cyclodextrin did not affect binding of specific δ-OR agonist, [3 H]DADLE. Neither the maximum number of binding sites nor the affinity of [3 H]DADLE binding was changed by cholesterol depletion. However, the ability of δ-OR to activate cognate trimeric G proteins was impaired. EC50 value of agonist-stimulated [35 S]GTPγS binding was an order of magnitude higher. This effect was observed in case of both control and pertussis toxin-treated cells. It means that cholesterol depletion markedly reduced the efficiency of functional coupling of δ-OR to endogenously expressed pertussis toxin-sensitive G proteins of Gi/Go family as well as covalently bound Gi1α (Cys351 -Ile351 ) protein. Unchanged plasma membrane cholesterol content is therefore important requirement for proper δ-OR function. Detection of the effect of cholesterol depletion on the functional activity of δ-OR was supported by the analysis of changes in biophysical state of plasma membrane using fluorescent membrane probes,...
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