|
|
Interaction of protein subunits SEC10 and SEC15 of the exocyst tethering complex
Bartáková, Anna ; Ryšlavá, Helena (advisor) ; Čermáková, Michaela (referee)
The exocyst is an evolutionarily conserved tethering complex involved in the regulation of the secretory pathway in eukaryotic cells. As an effector of Small GTPases, the exocyst contributes to efficient targeting of secretory vesicles to the sites of intense exocytosis at the plasmatic membrane through interactions with specific membrane phospholipids. The exocyst complex consists of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Each subunit binds at least two other subunits, among them the interaction of SEC10 and SEC15 is crucial for the exocyst function. The question remains how this particular interaction is evolutionarily conserved across different plant taxonomic groups. SEC10 and SEC15 genes from the three evolutionary groups of plants (green algae - Klebsormidium nitens, mosses - Marchantia polymorpha, angiosperms - Arabidopsis thaliana) were cloned into yeast two-hybrid vectors for studying protein interactions. Testing the interaction of SEC10 and SEC15 exocyst subunits from different plant species in the yeast-two hybrid system showed that despite partially different protein sequences of the tested subunits, their interaction across different evolutionary groups of plants remains very well conserved.
|
|
|
Detail enzymatic characterization of a model heme-containing oxygen sensor
Vojáčková, Lukrécie Sophie ; Martínková, Markéta (advisor) ; Čermáková, Michaela (referee)
This thesis focuses on heme-based gas sensors, particularly phosphodiesterase from the bacterium Escherichia coli, referred to as EcDOS. The enzyme catalyzes the degradation of c-di-GMP, an important signaling molecule in bacteria that affects cellular processes, such as bacterial motility or biofilm formation. The thesis deals with detailed enzyme kinetics of protein forms in different redox and ligand states of the heme iron ion [Fe(III), Fe(II) and Fe(II)-O2], as well as a mutant form of the enzyme (EcDOS H77A) which does not bind heme. Results confirmed that the EcDOS WT Fe(II)-O2 form has higher kcat values than the EcDOS form with the ferrous ion of heme in the reduced state. Other significant result was that the enzyme activity is affected not only by the state of the heme iron ion but also by the presence and concentrations of divalent metal cations. The presence of the metal cation is essential for enzyme function, and suitable metal ions that stimulate enzyme activity are Mg2+ , Mn2+ and Zn2+ or their mixtures, which act synergistically on enzyme activity under chosen conditions. Analysis by ICP-MS also showed that Zn2+ cations are natural components of the enzyme. Thus, for further kinetic studies, it would be appropriate to use Zn2+ or mixtures of metal ions that are physiological for...
|
|
|
The study of energetic metabolism in patients with mitochondrial translation defects
Hýbl, Martin ; Hansíková, Hana (advisor) ; Čermáková, Michaela (referee)
Mitochondria are semi-autonomous organelles that contain their own DNA. Human mitochondrial DNA (mtDNA) encodes a total of 37 genes: 13 subunits of oxidative phosphorylation complexes (OXPHOS), 22 transfer RNA (tRNA) molecules and 2 ribosomal RNA (rRNA) molecules. Pathogenic mutations in genes associated with mitochondrial translation are a common cause of mitochondrial disease. These mutations can be found in mtDNA or in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, mitochondrial tRNA-modifying enzymes and aminoacyl-tRNA synthetases. Mitochondrial aminoacyl-tRNA synthetases (mt-aaRS) are enzymes that catalyse the addition of single amino acids to specific tRNAs. The aim of the bachelor thesis was an introduction to the work in the tissue culture laboratory. To prepare samples for the following experiments, skin fibroblasts from five patients with mt-aaRS disorders (AARS2, DARS2, NARS2, SARS2) and control lines were cultured in glucose and galactose media. Subsequently, the procedure for determining the equilibrium amount of selected subunits of the OXPHOS complexes was optimized and applied to the analysis of fibroblasts from five patients with mt-aaRS disorder. When the cells were cultured in glucose medium, decreased levels of some subunits of complex...
|
|
|
Preparation of the high-affinity binding domain of protein B7-H6
Jeníček, Jakub ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
Natural killer cells are part of innate immunity and play a key role in defending the organism. Their role in the defense against tumors and anti-tumor therapy has been the subject of multiple research projects because tumors are among the most frequent causes of death worldwide. Tumor therapy is often complicated and invasive; therefore, finding new therapeutic approaches that target naturally occurring defense mechanisms is advantageous. One of the key mechanisms used by NK cells to recognize tumor cells and eliminate them is signaling via their receptor NKp30. The binding of an activation ligand to this receptor can induce the activation of a cytotoxic response, leading to the elimination of the tumor cell. One of the activating ligands that can bind to NKp30 is B7-H6, a cell surface protein found on certain types of tumors. However, the interaction between B7-H6 and NKp30 has not been wholly described yet. This thesis focuses on different methods, which can be used for obtaining the B7-H6 domain bearing a fluorescent label, that could be used to visualize NKp30 on the cell surface, thus allowing for further description of the interaction between these molecules. KEY WORDS NK cells, NKp30, B7-H6, sortase A, HEK293
|
|
|
Effect of pH and other factors on the metabolic conversion of tyrosine kinase inhibitors.
Čillíková, Olívia ; Indra, Radek (advisor) ; Čermáková, Michaela (referee)
Targeted anti-tumor therapy is a modern and effective approach to cancer treatment. This type of therapy includes several groups of anticancer drugs. The second most commonly used group of targeted chemotherapeutic substances are tyrosinkinase inhibitors. These are small- molecular agents that target specific signalling pathways of the tumor cell, thereby inhibiting it's growth, proliferation and angiogenesis. Representatives of this drug group include vandetanib and sunitinib. Vandetanib is primarily used for the treatment of medullary thyroid cancer and sunitinib has been approved for the treatment of gastrointestinal stromal tumor and advanced renal cell carcinoma. The diploma thesis focuses on the effect of pH and temperature on the in vitro metabolism of vandetanib and sunitinib using first-phase biotrasnformation enzymes. Differently premedicated and control rat liver microsomes were used in the experiments. Human and rat recombinant cytochromes P450 were used for comparison. The primary purpose of the experiments was to find the experimental pH and temperature optimum of the in vitro metabolism of vandetanib and sunitinib. These experiments also investigated the effect of pH and temperature on the drugs themselves and their stability. The effect of pH and temperature on the metabolism of the...
|
|
|
Analysis of the education system in the organization
Čermáková, Michaela ; Svobodová, Zuzana (advisor) ; Kursch, Martin (referee)
The subject of this work will be to describe and evaluate the current education system in the selected organization and analyze the educational needs of employees, their motivation and barriers for further education. The work will be divided into two parts. It is the theoretical part and the empirical part. The theoretical part describes the basic concepts associated with personnel management, tasks and strategies of human resources management and the importance of education for organization. It will also focus on the motivation of employees for further education and barriers that prevent employees from education. The issue of objectives and philosophy of education, education system, education process and methods of education will be addressed. The text will draw on domestic and foreign literature. The next chapter will analyze the system of education of employees in a selected organization that will rely on the case study and subsequently the interpretation of the obtained data. The research will be carried out in the first phase in the form of a questionnaire survey, followed by the second phase, namely direct interviews with employees of the given branch of the organization and their manager responsible for education. In the end, all findings resulting from the research will be summarized, the...
|
|
|
Funding of a Non-profit Organization in the Cultural Area
ČERMÁKOVÁ, Michaela
The theoretical part of this thesis describes non-profit sector, non-profit organization as well as the types of non-profit organizations. The thesis places strong emphasis on the description of specific methods of funding non-profit organizations, particularly in the cultural area. The practical part characterizes the selected non-profit organization and performs a financial analysis in this organization. It describes the financial resources of the organization and compares them over time. On the bases of the obtained results, the effectiveness of the use of financial resources is examined, the dominant resource is found and also overall financial management is assessed. Finally, some suggestions for obtaining others financial resources are recommended.
|
|
| |
|
|
Recombinant expression of the mouse NK cell receptor NKR-P1C
Rudenko, Anastassiya ; Vaněk, Ondřej (advisor) ; Čermáková, Michaela (referee)
NK cells are at the forefront of the body's defense against various threats, such as viruses or cancer cells. NK cells can recognize and destroy harmful cells in several ways that are very different from the defense mechanisms of, for example, T- or B-lymphocytes. NK cells carry a wide range of activating and inhibitory receptors on their surface that distinguish healthy cells from harmful or infected cells. NKR-P1 (natural killer receptor-protein 1) is the first discovered family of NK cell receptors similar to C-type lectins, which includes both activating and inhibitory receptors. NKR-P1C is an activating receptor capable of forming homodimers on the surface of NK cells. The major ligands of the NKR-P1 receptor family are the immunoenvasins of murine cytomegalovirus and other molecules structurally distinct from MHC class I glycoproteins. This work focuses on the recombinant expression of the NKR-P1C receptor protein. Using the pUT7 plasmid, we prepared a stably transfected HEK293T cell line carrying the gene for expression of the extracellular domain of NKR-P1C fused to the Fc fragment of IgG using the PiggiyBac system, and between them, they carry the TEV protease cleavage site. The protein was purified by affinity and gel permeation chromatography, and its purity was subsequently verified by...
|
|
|
Drama as the Prevention of Bulying
Čermáková, Michaela ; Svobodová, Radmila (advisor) ; Holemá, Irena (referee)
The diploma thesis deals with the method of structured drama and its help in the prevention of bullying. Specifically, the aim is to understand the role of a silent witness as a person who can prevent bullying by stepping out of this role. The theoretical part defines the term bullying, describes the forms, causes and characteristics of bullying among pupils of the first grade of the primary school. This part of the thesis also gives a definition of the chosen method of structured drama, its procedures and techniques. The practical part deals with the created structured drama and its reflection. The questionnaire survey then examines the attitude shift of selected pupils in their decision to step out of the role of the silent witness. The period immediately preceding the realization of structured drama and the period after a few months are the initial and terminal phases of the examined attitude shift. KEYWORDS: Structured drama, drama education, methods and techniques, bullying, silent majority, prevention, the first grade of the primary school, decision-making
|
guest ::
RSS feed.