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Preparation and study of human NK cell receptor AICL
Nový, Jiří ; Vaněk, Ondřej (advisor) ; Novák, Petr (referee)
Natural killer cells, or NK cells are an integral component of innate immunity and fullfills the function of recognizing and killing tumor and virus-infected cells. Their function is regulated by signals produced by the interaction of inhibitory and stimulatory receptors on their surface with their specific ligands on the targer cell surface. NKp80 is an activating receptor of NK cells and forms specific complex with cell receptor AICL, both of which belong to the family of C-type lectin-like receptors. Overexpression of AICL receptor is preferably specific for tumor cells of myeloid character. This master's thesis describes the production of AICL mutated form by expression in Escherichia coli BL21 Gold (DE3) followed by isolation and in vitro renaturation of the target protein. In a previous study it was found that an odd number of cysteines in the extracelular lectin domain of AICL causes wrong folding of the protein. Substituting an odd cystein for serine at position 87 lead to stable soluble form of AICL with an even number of cysteines in conserved positions, typical for CTLD receptors. Correctness of the formation of disulfide bonds between cysteines was verified by mass spectrometry. Significant amount of the protein gained allowed for setting up a wide variety of crystallization conditions....
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Studies on interactions between natural killer cell lectin receptors and their protein ligands.
Hernychová, Lucie ; Novák, Petr (advisor) ; Drbal, Karel (referee)
NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.
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Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
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Hail detection with weather radar data
Skripniková, Kateřina ; Řezáčová, Daniela (advisor) ; Novák, Petr (referee) ; Müller, Miloslav (referee)
The thesis deals with testing of radar-based hail detection methods and their use in hail climatology of Czechia. Data from C-band Doppler radars were used. There is described the testing of the selected algorithms in the first part of the thesis. The algorithms were chosen according to the data availability. On the basis of 25 well documented hail events, suitable threshold values were found to form hail criteria. Because of the hail events considered, the work concentrates on severe hail with diameter from about 2 cm. In addition, available data from Germany (Baden-Württemberg) were included in testing the detection criteria. The best performing criteria were used to form a new combined criterion (COMBI). The second part of the work applies the hail criteria, in particular the COMBI criterion. The performance of the COMBI criterion is shown in four case studies. Then the COMBI criterion was determined from the radar data of the six years 2007-2012. All the days from the months May to August were evaluated. The point maximum of about one day with severe hail was found for Czechia. For the most of the Czech territory the value was not higher than a quarter of a day in a year. In the seasonal distribution the maximum of hail days belongs to June. And the diurnal distribution showed the maximum of severe hail...
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Modular design for in-atomizer preconcentration of hydride forming elements with AAS detection
Novák, Petr ; Kratzer, Jan (advisor) ; Korunová, Vlasta (referee)
Modular design of hydride trap-and-atomizer device for AAS is constructed and tested. Modular design enables to test different preconcentration surfaces easily. Efficient in- atomizer preconcentration allows to reach detection limits of hydride forming elements at ultratrace levels. Bismuth and arsenic were chosen as model analytes and their preconcentration efficiencies were quantified employing quartz and sapphire as preconcentration surfaces. The results reached in the modular design were compared to those found previously in the compact quartz trap-and-atomizer device. The performance of the modular design is fully comparable with that of compact trap-and-atomizer design. Modular design can thus be employed for testing of novel preconcentration surfaces.
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Soviet Economic and Military Aid to Egypt in 1960's and 1970's
Čáp, Jiří ; Litera, Bohuslav (advisor) ; Novák, Petr (referee)
This diploma thesis deals with relations between Soviet Union and Egypt in 1960's and 1970's, with focus on Soviet economic and military aid to this Arabian country. Soviet Union is perceived as a protector superpower, while Egypt is a client state, which receives generous financial and material aid. This work maps particular pe-riods of Soviet economic aid since early 1960's, when some great economic projects were gradually realized (Aswan Dam, Helwan metallurgic complex), the attention is also decicated to arms supplies and modernization of Egyptian army. The first break point influencing mutual relations is Egyptian defeat in Six-Day War in 1967, after which president Nasser was willing to deepen relations with Moscow and even to grant durable Soviet military presence on the Egyptian territory. The period after the inauguration of new president Anwar Sadat in 1970 can be characterized as gradual worsening of mutual relations. The aim of this thesis is to describe particular aspects of Soviet aid and evaluate it in scope of broader political and strategic interests of USSR.
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Molecular mechanisms of apoptosis induction in cancer cells.
Fenclová, Tereza ; Novák, Petr (advisor) ; Milichovský, Jan (referee)
Cancer diseases are now the third leading cause of death (20% of all deaths). It is therefore important to find new ways of getting tumor cells effectively and specifically disposed of and a promising path is targeted therapy. One of the most frequently deregulated protooncogenes in cancer is C-MYC, which makes it suitable as an effective target for treatment. However, the development of such targeted active ingredients is very expensive, so in this thesis we investigate natural substances that have been used for the treatment of cancer in ancient China. We examined the substances shikonin, cnicin and artemisinin. The results show that shikonin induces apoptosis of tumor cells by reducing the expression of C-MYC and activating tumor suppressor kinase MST1. Cnicin reduces the expression of C-MYC as well, but activates MST1 only weakly. Artemisinin, on the other hand, increases expression of C-MYC and doesn't activate MST1, thus operates on inducing apoptosis of tumor cells by a completely different mechanism. (In Czech)
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Estimation of the survival function in the reliability analysis
Vojtěch, Jonáš ; Novák, Petr (advisor) ; Hurt, Jan (referee)
Present Bachelor thesis deals with the basic concepts and methods used in the survival analysis. Both nonparametric and parametric approaches to the estimation of the survival function are described. Nonparametric Kaplan Meier method is presented in order to estimate the survival function and consequently derive its basic properties. From the point of the probability distributions used in the analysis of reliability, exponential, Weibull's and logarithmic-normal distri- butions are applied. Parameters in the parametric approach to the estimation of the survival function are determined by the modification of maximum likelihood method for censored data. From the tests that are proper for the comparison of distribution of the duration of survival of more groups, nonparametric logrank test and parametric likelihood ratio test are mentioned. In the last section of the Bachelor thesis the theoretical findings are illustrated on simulated as well as actual data using Mathematica 9. Keywords: survival function, Kaplan-Meier estimator, logrank test, maximum likelihood method, likelihood-ratio test 1 Literatura 2 Seznam obrázků 3 Seznam tabulek 4
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Poisson process
Kráľová, Veronika ; Beneš, Viktor (advisor) ; Novák, Petr (referee)
Poisson process is a random set, which analyses an occurrence of the independent random points in a certain set. An object of study are probabilistic properties of this random set. Basic definitions and properties are formulated for the Euclidean spaces of arbitrary dimension and used to simulate different types of Poisson processes. We provide the algoritm procedures and source codes for each simulation. Statistical test of complete spatial randomness (CSR) verifies in conclusion, if the simulated processes are really homogeneous Poisson processes with specific properties.
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Mass Spectrometry-Based Identification of a Potential Binding Partner of Glutamate Carboxypetidase II
Tužil, Jan ; Konvalinka, Jan (advisor) ; Novák, Petr (referee)
English Abstract The incoming paradigm of the network (or systems) biology calls for a new high throughput tool for a wide scale study of protein-protein interactions. Mass spectrometry-based proteomics have experienced a great progress in recent years and have become an indispensable technology of elementary as well as clinical research. Glutamate carboxypeptidase II (GCPII; EC 3.5.17.21) is a transmembrane protein with two known enzymatic activities. Its expression is highly upregulated in some solid tumors and also in tumor-associated neovasculature in general. Nevertheless, none of the two enzymatic activities were shown to be physiologically relevant to these cells. Some facts point at a possible receptor function of GCPII, however, no specific binding partner has been found yet. In the search for potential binding partners and/or ligands of GCPII, a series of methods have been employed, including pull-down experiment, immunoprecipitation and mass spectrometry. Sample preparation and mass spectrometry data processing methodology was specifically developed in order to identify potential binding partners. As one of the outcome of that methodology, the interaction of β-subunit of F1 ATP synthase was selected for further detailed analysis as a putative ligand of GCPII.
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