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The content of components of ATP synthasome in different rat tissues and in patients with defects in ATP synthase
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Kalous, Martin (referee)
The complexes of oxidative phosphorylation (OXPHOS) are situated in the inner mitochondrial membrane in higher structural and functional complexes, so-called supercomplexes, which facilitates substrate channeling. ATP synthase is also able to organize in higher structures. In mammalian mitochondria, ATP synthase is usually present in a dimeric form. There is evidence of its trimerization and even tetramerization. Furthermore, it seems that ATP synthase catalyzing the phosphorylation of ADP to ATP, adenine nucleotide translocator (ANT) ensuring the exchange of ADP for newly synthesized ATP across the inner mitochondrial membrane and phosphate carrier (PiC) allowing the import of inorganic phosphate (Pi) into the matrix of mitochondria are assembled in a supercomplex called ATP synthasome. This association among the components of phosphorylative apparatus seems to increase the efficiency of processes leading to the ATP synthesis. First, we studied amounts of the components of phosphorylative apparatus in connection with various ATP synthase contents among mitochondria isolated from nine rat tissues. Mitochondrial proteins were separated by denaturing electrophoresis (SDS-PAGE) and their content was analyzed using specific antibodies. In agreement with our expectations, the highest content of...
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Analysis and identification of proteins in organ dysfunction using proteomic methods
Tůma, Zdeněk ; Matějovič, Martin (advisor) ; Lopot, František (referee) ; Hernychová, Lenka (referee)
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...
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Analysis of autenthicity of food products with fruit component
Prachárová, Adriana ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of this thesis was to determine the authenticity of fruit food for infants using molecular and instrumental methods. In the experimental part, plant DNA isolations from fruit leaves (peaches, apricots, plums and apples) and bananas were performed. Further, DNA was isolated also from five commercial products, and from model mixtures that were prepared in terms of content identical to the commercial mixtures. The isolated DNA was characterized and verified by qPCR with plant DNA-specific ITS2 primers. Three triple primer pairs were selected, and their specificity was evaluated when performing multiplex PCR. This method makes it possible to detect more types of fruit in one reaction, reducing the economic and time requirements for detection. As none of the selected primer pairs were sufficiently specific for the apricot, the evidence from the plum and peach was further realized using duplex PCR. High resolution melting curve analysis was used for better DNA type recognition. Subsequently, agarose gel electrophoresis was performed to analyse the fragment lengths. Furthermore, experiments have been made to identify some specific phenolic substances in commercial and model fruit mixtures by HPLC. Since phenolic substances are degradable under unsuitable storage conditions, the presence of individual compounds was not detected by this method.
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Structural study of the ASK1:thioredoxin complex.
Pšenáková, Katarína
5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...
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The cancer cell proteome and its changes after anti-cancer drug treatment
Tylečková, Jiřina
Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...
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Changes of gluten proteins during beer processing
Porubiaková, Otília ; Mikulíková, Renata (referee) ; Márová, Ivana (advisor)
The aim of thesis was monitoring of changes in the content of gluten proteins in the biotechnological process of beer production. During the production process of wheat and barley beer, the samples were collected and analysed using the electrophoresis and immunoassay method. The results of the analyses were compared with commercial Czech beers. The theoretical part contains description and composition of gluten proteins, malt and beer technology, the changes that occur in this process, and methods of gluten proteins analysis. The experimental part contains procedures for laboratory production of barley and wheat beer and analyses of gluten proteins. To identify the individual gluteal protein fraction acid and SDS electrophoresis methods were used. For quantification, enzyme immunoassay was used and evaluated spectrophotometrically. The identification of the gluten‘s fractions by electrophoretic methods has been shown to be less specific for samples with lower content of gluten proteins and for barley specimens. A decrease in the concentration of gliadins and glutenins in the beer production process was demonstrated. A significant change was found during wort production with 98% decrease of gluten content compared to the feedstock and during the fermentation, when the gluten concentration dropped below 10 mg/kg. This value is acceptable from the legislation for products labelled „gluten-free“.
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Structural study of the ASK1:thioredoxin complex.
Pšenáková, Katarína
5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...
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Analysis and identification of proteins in organ dysfunction using proteomic methods
Tůma, Zdeněk ; Matějovič, Martin (advisor) ; Lopot, František (referee) ; Hernychová, Lenka (referee)
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...
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Pulse proteolysis in evaluation of conformational stability of cytochromes b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...
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Structural study of the ASK1:thioredoxin complex.
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Štěpánek, Miroslav (referee)
5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...
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