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Investigation of the role of the KEAP1-NRF2 antioxidant pathway in the therapy of secondary acute myeloid leukaemia
Myšáková, Michaela ; Pimková, Kristýna (advisor) ; Suttnar, Jiří (referee)
The development of therapy resistance is a long-standing problem in treating cancer, particularly in the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), where the hypomethylating agent 5-azacytidine (AZA) is the first choice of treatment. To enhance therapeutic efficacy, AZA is often combined with other agents such as pevonedistat (Pevo), a NEDDylation inhibitor targeting the ubiquitin-proteasome system. While initial results showed a synergistic effect of the AZA and Pevo combination in treating MDS and AML, dual resistance has been described, underlining the importance of understanding the mechanisms behind the resistance development. Our previous data demonstrated an essential role of redox homeostasis and antioxidant system represented by Nuclear factor erythroid 2-related factor 2 (NRF2) in AZA resistance. The Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway is the master regulator of antioxidative defence in cells crucial for maintaining redox balance. However, hyperactivation of NRF2 has been implicated in therapy resistance and cancer progression. We hypothesised that NRF2 is crucial in MDS/AML therapy resistance, particularly in resistance to combined AZA and Pevo therapy. We worked with cells sensitive and resistant to AZA and Pevo and monitored...
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Investigation of HSP70 oligomerization by structural mass spectrometry
Melikov, Aleksandr ; Novák, Petr (advisor) ; Jeřábek, Petr (referee)
Heat shock cognate protein 70 (HSC70) is a 71 kDa chaperone protein belonging to the ubiquitous family of heat shock proteins 70 (Hsp70). The representatives of this protein family are considered as molecular machines with ATP-hydrolase activity facilitating correct folding of spatial protein structure, both in normal and stressful conditions (hypoxia, heat shock, pH fluctuations etc.) In addition, HSC70 was identified as an uncoating enzyme for triskelion meshwork on the surface of clathrin-coated vesicles. Among other roles, HSC70 prevents protein aggregation and assists the polypeptide maturation, it facilitates the protein transport into organelles, such as endoplasmic reticulum and mitochondria. It is involved in targeting proteins for lysosomal degradation and in many other dramatically important cellular processes related to protein homeostasis. Therefore, the regulation of HSC70 and other HSP70 proteins is believed to be dramatically important, especially in a context of cellular stress. Based on the experimental observation, the mechanism of inactivation through oligomerization was hypothesized. The dimer and trimer species of Hsp70 proteins were identified both in case of prokaryotic and eukaryotic homologs. It was also speculated that Hsp40 cofactors promote oligomerization to even...
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Identification of new tissue-specific interaction partners of chromatin remodelling ATPase Smarca5
Arishaka, Yuliia ; Kokavec, Juraj (advisor) ; Děd, Lukáš (referee)
The regulation of chromatin structure is fundamental to a wide range of cellular processes, including transcriptional regulation, cell division, differentiation and DNA damage repair, and ATP-dependent chromatin remodeling complexes have been established as essential components of this regulatory network. Smarca5, as an ATPase/Helicase enzyme, has been shown to regulate chromatin structure by interacting with bromodomain and DDT-WHIM domain-containing partners to control the binding of chromatin-associated proteins and transcription factors to their specific DNA target sequences. In this work we identify a previously uncharacterized protein with a conserved N-terminal bromodomain and ISWI protein binding DDT-WHIM domain through co-immunoprecipitation and mass spectrometry in mammalian cell lines and establish it as a novel interaction partner of chromatin remodeling ATPase Smarca5. Furthermore, we have pinpointed the region required for Smarca5 interaction that corresponds to DDT-WHIM domain. We have furthermore attempted to identify additional interaction partners which may hint on the potential function of this novel chromatin complex and validated its expression in embryonic and postnatal tissues. This discovery represents a unique opportunity for further investigation into its potential...
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Proteomic identification of nuts in festive cookies
Kadeřábková, Lucie ; Kučková, Štěpánka (advisor) ; Kolář, Karel (referee)
This diploma thesis is devoted to the proteomic identification of selected nuts in festive cookies. Two types of mass spectrometry were used for the nut identification; their outcomes allow for comparison and completeness of the data. The theoretical part provides a comprehensive overview of the nutritional values of nuts and the issues of food fraud and authenticity. It concludes with a didactic view and potential applications in chemistry lessons and other school subjects. The experimental part analysed eleven different nuts, submitted to high temperatures, which simulated baking and seven different types of festive cookies (six mass-produced cookies and one type of cookie with priorly unknown content of nuts). Mass spectrometry MALDI-TOF was used with subsequent evaluation using relational database system PostgreSQL to obtain characteristic m/z values for each of the eleven heat-subjected nuts. These values were then compared to the m/z values of the festive cookies to identify declared nuts and nuts used in the "unknown" sample. This identification was proved with various success rates in four of six mass-produced samples. The success rate was derived from the number of unique m/z values found in each of the nuts. The "unknown" sample was correctly identified based on the presence of one third...
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The development of new radical cross-linkers for structural characterization of proteins
Karpíšek, Michael ; Kukačka, Zdeněk (advisor) ; Chmelík, Josef (referee)
Proteins are important biomolecules because they carry out many essential functions. Since the structure of these biomolecules is usually closely linked to their function, there are numerous techniques to determine their three-dimensional structure. Exploiting the advantages of mass spectrometry, chemical cross-linking coupled to mass spectrometry is one of the methods that is used for structural characterization of studied molecule. Currently there is no reagent that targets and links all of the aromatic proteinogenic residues found in the sequence of proteins. In this work, we verified the reactivity of newly developed cross-linker towards aromatic residues and sulfhydryl group of cystein. This new reagent is based on the so-called Togni reagents that serve as trifluoralkylation agents. First, we monitored the impact of several different conditions on the composition of the reaction products using apomyoglobin as a model protein and electrosprey ionization as the method of choice. Subsequently we employed bottom up approach to find out which residues were modified. For this purpose we chose apomyoglobin and two other proteins that contain cysteine in its reduced form - RhoA and HSC70. Based on obtained results, we could confirm the ability of the new cross-linker to modify both aromatic residues...
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Studying protein structure and interactions by structural mass spectrometry
Portašiková, Jasmína Mária ; Man, Petr (advisor) ; Vrbacký, Marek (referee)
Transmembrane channels and transporters of the ClC protein family are present across all living organisms. They are found on the cytoplasmic and lysosomal membranes of the cells, where they participate in maintaining ion homeostasis. When dysfuncional, they lead to serious health complications. To develop treatment for these diseases, it is essential to describe transport mechanism of ClC proteins. The antiporter ClC-ec1 from E.coli is used as a model protein for the entire ClC protein family. This homodimeric protein, which transports one proton against two chloride ions, has a separate transport path in each monomer. Based on the crystal structure, it is believed that during transport the protein alternates between outward and inward-facing conformations. Conversion to the outward-facing conformation of the protein is accompanied by the protonation of three glutamates located in the transport path. To study these conditions, a QQQ mutant was designed that has these glutamates replaced by glutamines. Until now, the study of the transport mechanism of ClC-ec1 has mainly relied on studies based on X-ray crystallography. Crystallography provided static images, which did not contain sufficient information about protein dynamics. Therefore, to study transport mechanism of ClC-ec1, we chose a dynamic...
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Proteomic resolution of cereals
Nyškovská, Kristýna ; Kučková, Štěpánka (advisor) ; Kolář, Karel (referee)
This bachelor thesis deals with the proteomic differentiation of cereals (amaranth, sown barley, sown maize, qinoa, grain sorghum, sown oats, sown wheat, sown buckwheat, sown millet, sown rice, spelt, sown rye). Cereals are abundant in the human diet and are one of the main sources of protein and carbohydrates. The aim was to determine whether it is possible to distinguish between different types of cereals and to compare the difference between cereals and pseudocereals based on the protein composition of their grains using the mass spectrometric method MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization - Time of Flight. The theoretical part deals with the introduction of cereals, pseudocereals and their nutritional values, application and analytical method MALDI-TOF MS. The experimental part deals with the collection of different cereal species and their subsequent analysis. The samples were mechanically processed and digested using the enzyme trypsin. The cleaved peptides were measured by MALDI-TOF MS and the obtained spectra were subsequently processed using mMass and PostgreSQL (version 14.2). In this way, specific m/z values were determined that are characteristic of each cereal analyzed. From these values, a table was compiled that summarizes these characteristic values. Which allows the...
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Possibilities of pathogen identification using MALDI-TOF mass spectrometry
Fusková, Lucie ; Strouhalová,, Dana (referee) ; Šalplachta, Jiří (advisor)
This bachelor thesis focuses on the utilization of MALDI-TOF MS technique for the identification of plant pathogens, specifically targeting molds of the genus Fusarium affecting cereals. Molds pose a serious threat to agriculture and the food industry. Current identification methods are time-consuming and often fail to provide precise results. Mass spectrometry offers a rapid and reliable alternative. Identification via MALDI is characterized by its high accuracy, sensitivity, and speed compared to commonly used methods. The thesis addresses the optimization of the method using cultured fungal strains and the identification of molds in infected cereals. The results of the study demonstrate the potential of utilizing MALDI technique for the identification of plant pathogens.
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