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Cellular protein interactions studied by advanced fluorescence imaging methods
Belejová, Sára ; Heřman, Petr (advisor) ; Krůšek, Jan (referee)
This thesis studies an important tumor suppressor, p53, and its interac�on partner, nucleophosmin (NPM), in living cells. Proteins are studied using fluorescence confocal microscopy techniques such as fluorescence life�me imaging and fluorescence anisotropy measurements. The primary focus of the research is on a specific variant of the p53 protein called p53-L344P, which is generated by a point muta�on from its original form (p53wt). We inves�gate the oligomeriza�on state of p53-L344P in vivo, which appears to be monomeric, confirming the results of in vitro experiments from other studies. Further, we show that p53wt and p53-L344P can form complexes with each other. We compare the interac�on of the NPMmutA protein with p53wt and p53-L344P proteins. Our findings reveal that the L344P mutant is not transferred from the nucleus to the cytoplasm in the presence of NPMmut, as is p53wt. Furthermore, we inves�gate the oligomeriza�on state of p53wt when it is in the cytoplasm and propose avenues for further research into this interac�on.
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Impact of NMDA antagonists on neuroplasticity as a biological phenomenon
Kalivodová, Michaela ; Valeš, Karel (advisor) ; Krůšek, Jan (referee)
Neuroplasticity is a fundamental biological phenomenon that accompanies us throughout our lives, both in health and illness. By studying neuroplasticity, we can understand serious neurodegenerative diseases, help remodel the nervous system after damage, or make our actions more efficient in today's world. The first chapter of this thesis will discuss neuroplasticity in the physiological state, describing the development of nervous system, learning and memory, different types of neuroplasticity, and other ways to modify neurogenesis. The following chapter deals with the role of neuroplasticity in disease, its impairment in neuropsychiatric diseases. The most common ones will be described, such as depression, schizophrenia, Alzheimer's disease and Parkinson's disease. The next part of the thesis will discuss the molecular biology of NMDA receptor including structure, activation and excitotoxicity of the NMDA receptor, which is distinct from other receptors. Subsequently, selected NMDA antagonists competitive, non-competitive, uncompetitive, as well as glycine agonists and other selected natural modulators of the NMDA receptor will be described. Finally, this thesis will present the molecular and cellular mechanisms of the BDNF and mTOR pathways, knowledge of which provides a basis for further...
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The role of nociceptive synaptic transmission modulation
Heleš, Mário ; Paleček, Jiří (advisor) ; Rokyta, Richard (referee) ; Krůšek, Jan (referee)
Pain represents a major symptom in a multitude of medical conditions and can often become the main negative factor in a patient's low quality of life. The complex issue of pain management is further underscored by the reduced efficacy of conventional analgesics in conditions such as neuropathic pain. Neuropathic pain, unlike acute nociceptive pain, originates from damage to the peripheral or central nervous system and often develops into chronic pain syndrome. Most analgesics available today provide only limited and unsatisfactory analgesia in chronic neuropathic pain and are often associated with severe adverse effects. Modulation of nociceptive transmission in spinal cord dorsal horn (SCDH) stands out in recent research as a pivotal mechanism, especially in chronic pain development and maintenance. The major aim of this doctoral thesis was to investigate how pain-associated processes interfere with opioid-induced analgesia, with the main focus on the interaction between chemokine (C-C motif) ligand 2 (CCL2), transient receptor potential vanilloid type 1 (TRPV1), and μ-opioid receptor (MOR). To achieve a better insight into opioid signaling in SCDH we studied the following issues: (I.) How does CCL2 modulate MOR-mediated effects on nociceptive synaptic transmission in SCDH neurons and in vivo...
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Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee) ; Osička, Radim (referee)
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
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Properties and interactions of nucleoproteins studied by advanced lifetime-based imaging methods
Vacková, Anežka ; Heřman, Petr (advisor) ; Krůšek, Jan (referee)
The main aim of this thesis was implementation and testing of an additional imaging method for investigation of protein-protein interactions in our laboratory and to extend already established heteroFRET (Förster resonance energy transfer) method used with the fluorescence lifetime imaging (FLIM). We performed homoFRET experiments with both the steady-state and time-resolved anisotropy-imaging methods. The presence of homoFRET between fluorophores was done by partial fluorophore bleaching and by the "red edge" analysis. We compared the methods, especially their sensitivity, experimental requirements, complexity, and informational value. We effectively applied steady-state and time-resolved anisotropy imaging to six proteins of interest; both proved comparably effective and reliable as heteroFRET-FLIM for detection of the protein interactions. The red edge method appeared problematic due to background contribution, which could not be suppressed with the current setup and instrumentation. We validated steady-state and time-resolved anisotropy imaging by studying well- described oligomerizing nucleophosmin variants (NPMwt and NPMmut) and noninter- acting mutant NPMcut, expressed in living HEK-293T cells. Then, we also probed nu- cleolin (NCL) and free fluorescent tags NowGFP and mVenus, aiming to find...
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The Role of Bmh Proteins in the Regulation of Yeast Enzyme Neutral Trehalase Nth1.
Macáková, Eva ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Žáčková, Markéta (referee)
118 10. Summary Neutral trehalase 1 is a yeast enzyme from the family of hydrolases, which catalyzes hydrolysis of trehalose to two glucose molecules. Trehalose is a non-reducing disacharide, which serves as a carbon source in a yeast cells as well as a stress metabolite. When a cell is under stress conditions it accumulate trehalose and through the recovery process the trehalose is hydrolyses by trehalases. The main subject of our study was Nth1 from S. cerevisiae. It was published earlier (Panni, S., et al., 2008), that Nth1 must be phosphorylated by PKA and in the presence of 14-3-3 protein to be active. The activity of Nth1 also slightly increases in the presence of Ca2+ ions (Franco, A., et al., 2003). 14-3-3 proteins are family of acid regulatory proteins, which participates in variety of processes in the cells, like regulation of the cell-cycle, cell metabolism, transcription, apoptosis etc. They have more than 400 known binding partners, which include transcription factors, signalling molecules, enzymes and others. They control the regulation of their binding partners through phosphorylated motives in sequence by changing conformation of the binding partner, revealing or masking specific sequence or by mediation of protein-protein interactions. There are many isoforms of 14-3- 3 proteins through all...
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Pathological pain states, the role of synaptic modulation at spinal cord level
Nerandžič, Vladimír ; Paleček, Jiří (advisor) ; Krůšek, Jan (referee)
(English) Modulation of synaptic transmission in dorsal horn of spinal cord plays a key role in nociceptive signalling. Recent studies have indicated a great importance of presynaptic TRPV1 receptors (transient receptor potential vanilloid) in spinal cord. These receptors act as molecular integrator of nociceptive stimulation on periphery. The way of their activation and the effect on modulation of the synaptic transmission are not clarified yet. Previous studies demonstrated the influence of many inflammatory mediators and cytokins on TRPV1 receptors. The aim of our research was to show changes in activation of presynaptic TRPV1 receptors in the spinal cord following the application of endogenous agonist N-oleoyl dopamine (OLDA) in a model of peripheral neuropathy, after incubation with cytokine TNFα and to show the effect of precursor of anandamide N-acylphosphatidylethanolamine (NAPE). In our experiments, we have recorded miniature excitatory postsynaptic currents (mEPSC) from neurons of acute spinal cord slices by the patch-clamp method. The first series of experiments tested sensitivity to application of the endogenous agonist OLDA 5 days after evoking peripheral neuropathy. The frequency of mEPSC increased significantly - to 250 % of base level after applying a low concentration of OLDA (0,2...
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Changes of intracellular pH in yeast cells under stress conditions
Divín, Radek ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...
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Inhibition of nicotinic acetylcholine receptors by tacrine and its derivatives
Skřenková, Kristýna ; Krůšek, Jan (advisor) ; Doležal, Vladimír (referee)
Nicotinic acetylcholine receptors are ligand-gated ion channels which are located on neuromuscular junction and in central and perifric nervous system. Activity of nicotinic receptor might be modulated by variety of pharmacological agents. In this work, we have focused on the study of the inhibition effect of tacrine and its derivatives on the nicotinic acetycholine receptors of muscle and neuronal type. These derivatives function as acetylcholinesterase inhibitors and also interact with nicotinic acetylcholine receptors. The majority of current forms of treatment of Alzheimer's disease is based on cholinesterase inhibitors. We have studied the mechanism of tacrine and its derivatives by using patch clamp method in the configuration of whole-cell recording. Powered by TCPDF (www.tcpdf.org)
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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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