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Narušení metabolismu proteinů a jeho efekt na signalizaci cytokininů
Dufek, Martin
Cytokinins are N6 substituted adenine derivatives that affect many aspects of plant growth and development. A multistep phosphorelay systém, including hybrid sensor kinases, histidinecontaining phosphotransfer proteins and two sets of response regulators, is the key part of cytokinin signaling. However, a recent evidence indicates a crucial role for the proteasomeubiquitin systém (UPS) in the cytokinin response. Here, in this thesis entitled 'Protein metabolism disruption and its effect on cytokinin signaling' the major protein degradation mechanisms are outlined and the present-day model of cytokinin metabolism and signaling is discussed. In the experimental part, the UPS-cytokinin interaction is probed in a growth response experiment, an LC-MS proteome analysis and by the datamining of previously published proteomics data. The results indicate an interesting dosage-dependent balance between cytokinin- and proteasome-mediated signaling, and a huge impact of proteasome inhibition on cytokinin response proteins. Key words: proteasome, ubiquitin, growth response, protein degradation, LC-MS, proteome
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Diurnální variace v proteomu rostlin
Dobešová, Markéta
The proteome is dynamic and constantly changing. However, some of these changes will repeat over the period of time due to biorhythms. This periodicity in a protein abundance is maintained by the inner clock and regulation of expression, translation and degradation. This thesis entitled "Diurnal variations in plant proteome" summarizes present-day understanding of the plant inner clock and reviews the mechanisms of proteasome-mediated targeted degradation. In the experimental part, the diurnal variations in proteome of Arabidopsis thaliana were analyzed via an LC-MS profiling of wild-type (Col-0) and transgenic Arabidopsis seedlings with an inhibited proteasome degradation pathway. In total, profiles of 199 most abundant proteins were compared. Out of these 25 % were not significantly affected by diurnal rhythms and 72% had a significantly different response in seedlings with the inhibited proteasome patway.
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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Stanovení acetylcholinu pomocí LC-MS ve vzorcích mozkových mikrodialyzátů LC-MS/MS
Vrobel, Ivo ; Solich, Petr (advisor) ; Kujovská Krčmová, Lenka (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Ivo Vrobel Supervisors: Prof. RNDr. Petr Solich, CSc; Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague Prof. Seppo Auriola, MSc.(Chem.) Marko Lehtonen; Department of Pharmaceutical Chemistry, School of Pharmacy, University of Eastern Finland in Kuopio Title of master's thesis: LC-MS/MS analysis of acetylcholine in brain microdialysis samples Novel fast and simple LC-MS/MS method of ACh quantification in brain microdialysis samples utilizing stable-isotope-labeled IS was developed. The chromatographic step is based on revered-phase mode of pentafluorophenylpropyl (PFPP) column. The satisfactory retention of ACh is achieved with highly aqueous mobile phase containing 0.05% of the ion-pairing agent TFA and 4% of ACN in 4 min analytical run. Ionization of ACh and IS with low background noise and tolerant towards use of TFA was performed with atmospheric pressure thermospray ionization (APTSI). The selectivity of ACh and IS detection was obtained by SRM modes of MS/MS in the linear ion trap mass analyzer. The performance of developed method was cross validated to the validated method used in the laboratory for ACh measurements. The set of microdialysis...
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Structural study of the DCL-1 receptor using mass spectrometry
Růžičková, Barbora ; Kavan, Daniel (advisor) ; Mrázek, Hynek (referee)
DCL-1 (CD302) is a type I transmembrane C-type lectin receptor, which is expressed on monocytes, macrophages, granulocytes and dendritic cells. However, its extracellular domain lacks the amino acids motives essential for carbohydrate binding in the presence of calcium ions, suggesting that it does not have the classic binding capacity found in other C-type lectin receptors such as the mannose receptor. No exogenous or endogenous ligands have been identified yet, though. Due to internal colocalization with F-actin we can assume, that this unconventional lectin receptor plays a role not only in endocytosis and phagocytosis but also in the cell adhesion and migration. The receptor DCL-1 was first identified as a genetic fusion partner of human DEC-205 multilectin receptor in Hodgkin's lymphoma cell lines. The experimental part of this thesis deals with the characterization of disulfide bonds and data acquisition for validation of DCL-1 crystal structure. First the production and refolding conditions were optimized to obtain the highest amount of DCL-1 protein, precisely its extracellular domain. These optimal conditions were used to prepare the protein for in-gel digestion using specific endopeptidases in the presence of cystamine followed by LC-MS analysis. DCL-1 disulfide bonds were determined by comparing...
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Structure and reactivity of anions of aromatic hydrocarbons
Obluková, Michaela ; Čabala, Radomír (advisor) ; Jašík, Juraj (referee)
This bachelor thesis deals with research about negative ions reactivity of aromatic hydrocarbons which occur in Titan's ionosphere - Saturn's moon. As an experimental method there was used tandem mass spectrometry with electrospray ionization technique, quadrupole mass analyzer and collision cell. The aim of this thesis was to contribute to the general understanding of processes in the Titan's ionosphere. Particularly there were performed experimental studies to created isomeric benzyne radical anions (o-, m-, p- C6H4˙- ) which were generated from phthalic, isophthalic and terephthalic acid and subsequently their reactions were explored with selected neutral gases in collision cell (C2H2, HC3N, CH3CN, SO2, O2).
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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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Preparation of hyaluronan oligosaccharides with N-acetylglucosamine at both ends of the chain using enzymatic catalysis
Gadová, Martina ; Hermannová,, Martina (referee) ; Šílová, Tereza (advisor)
This bachelor thesis deals with preparation of odd-numbered hyaluronan oligosaccharides with N-acetylglucosamine at both ends of the chain using enzymatic catalysis by -Glucuronidase. The theoretical part focuses on present knowledge of hyaluronic acid and it fragments. It focuses on properties, enzymatic hydrolysis, application in different branches of science, but also analysis of oligosaccharides. One chapter is devoted to properties of enzyme -Glucuronidase. The experimental part deals with preparation of oligosaccharides type HANN. The reaction conditions for preparation of HA oligosacharides were optimized (pH, temperature) and the progress of enzymatic hydrolysis and the mixture composition after digestion were monitored by method UHPLC.. After optimization the mixture of oligosaccharides HANN was prepared and consenquently it was analysed by methods UHPLC and LC-MS. All the data were acquired in company Contipro Pharma, a.s., Dolní Dobrouč, Czech republic.
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