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Light and its effect on plant proteome
Luklová, Markéta
Light is one of the main environmental factors largely determining plant life. Light provides energy resources for photosynthesis and it is also the most important medium of perception as it represents the main source of information about plant surroundings. My diploma thesis entitled “Light and its effect on plant proteome” briefly summarizes present knowledge about the light perception in plants, its interpretation of light quality, quantity, and direction via photoreceptors. These components trigger a whole range of changes, both on molecular and developmental levels, but the complex molecular mechanisms regulating system of light signalling in plants are far from being resolved. Here, I followed effects of different light parameters on plant proteome, namely, diurnal variations, low- and high-light intensity, spectral composition, and a change in the light period duration. To study these effects two model organisms were selected (i) Arabidopsis thaliana as a representative plant model with the most annotated genome/proteome and (ii) Solanum lycopersicum as a representative plant model significant for agriculture and biotechnology. Proteome changes in response to different light factors were analyzed by LC-MS. In total, the analyses revealed over 100 and 300 light-responsive proteins in Arabidopsis and S. lycopersicum, respectively. Proteomics provided highly sensitive snapshots of protein abundance in response to light and selected experiments have been complemented with a GC-MS metabolite profiling, conductivity measurements, and fluorescence microscopy.
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Fast methods for analysis of migrants from food-contact materials
Vavrouš, Adam ; Čabala, Radomír (advisor) ; Jelínek, Ivan (referee) ; Bajerová, Petra (referee)
(AJ) Food contact materials pose a risk to human health due to a variety of contaminants which they can release into food. These compounds, migrants, include aromatic hydrocarbons, dialkyl phthalates, bisphenols, printing ink photoinitiators, and perfluoroalkyl compounds. The determination of all these substances can be expensive and time-consuming since universally applicable analytical approaches are not available nowadays. This work attempted to develop methods for the simultaneous determination of migrants from all of the mentioned groups in paper-based food contact materials and fatty food. A total of 68 contaminants were studied in paper products using liquid chromatography and gas chromatography coupled with tandem mass spectrometry detection. All analytes were isolated simultaneously using the modified "QuEChERS" method. This method demonstrated acceptable recovery and repeatability for most analytes in the validation study; LOQs ranged from 1.3 to 220 µg/kg. Analysis of 132 real paper products confirmed the occurrence of almost all studied analytes, which were often present in complex mixtures and at concentrations up to 628 mg/kg. A total of 41 potential contaminants were monitored in fatty foods by liquid chromatography with tandem mass detection. Contamination of the mobile phase by...
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Development of components for processing and visualization of proteomic data in KNIME environment
Schneiderová, Anna ; Tauš, Petr (referee) ; Mgr. David Potěšil, Ph.D (advisor)
Proteomics investigates the structure and function of proteins. The proteomic data processing involves several sequential steps, from data transformation and normalization to statistical data evaluation. An integral part of processing proteomic data is also their visualization, which is key for example in the control of input data quality and assessment of statistical evaluation outputs. In the Zbyněk Zdráhal Research Group, a software container is used with KNIME application for reproducible processing of proteomic data. As a part of the bachelor’s thesis, a request survey for new components for KNIME application was carried out. An interactive Volcano plot was chosen for implementation from the proposed components. Python programming language using Dash library was used for implementation. The application can be run independently or as a component of the KNIME environment for working with data from KNIME workflows. The interactive Volcano plot application makes it easy to identify and filter proteomic data.
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Using of techniques of mass spectrometry in the analysis of natural compounds
Vopelková, Alžběta ; Cahlíková, Lucie (advisor) ; Šafratová, Marcela (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Bc. Alžběta Vopelková Supervisor: doc. Ing. Lucie Cahlíková, Ph.D. Title of diploma thesis: Using of techniques of mass spectrometry in the analysis of natural compounds This diploma thesis deals with the topic " Using of techniques of mass spectrometry in the analysis of natural compounds ". The aim of the work was to create a research work and describe the profile of alkaloids in individual genera of the family Amaryllidaceae. The introduction to the theoretical part summarizes the analyzes gas chromatography (GC). Alkaloids have been classified according to their heterocyclic parts and the family Amaryllidaceae has been described. The work was based on professional texts by Czech and foreign authors. Used sources focused on the analysis of alkaloids in bulbs, or in the aboveground parts of plants in this family, using gas chromatography with mass detection (GC-MS). Key words: mass spectometry, analysis, natural compounds
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Clinically relevant protein-protein interactions participating in process of bacterial pathogenesis B. pertusis
Málek, Albert ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Whooping cought in human population was strongly suppressed during the 20th century. But in the past few years, the incidence of whooping cough began to rise. The origin of this disease is a pathogenic gram-negative bacteria Bordetella pertussis, which is becoming resistang to currecently used antibiotics or vaccination. B. pertussis attacks human respiratory system. One of it's virulent factors is adenylate cyclase toxin (ACT). It's secreted extracellularly from bacteria and binds to cytoplasmic membrane of host cells and translocates adenylatecyclase domain (dAC) to cytosol. This enzymatic domain is activated by non- covalent interaction with eukaryotic Calmodulin (CaM). After it's activation, dAC in high concentrations synthesizes cAMP, triggering host cell's apoptosis. We studied protein interaction of dAC with CaM by the PIXL method (Photo Induced Cross Linking) and mass spectrometry (MS). Mutant of dAC, with photo-methionine (pM), incorporated in position of Leucine 240 (dACL240pM) was expressed in transformed E.coli cells B834 in mineral medium containing pM. Expressed protein was isolated by affinite chromatography and characterized by MS (determined incorporation of pM was approximately 50 % in the final protein preparation). We performed a photochemical cross-linking with isolated...
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Noncanonical human eIF4Es in and out of the RNA granules
Frydrýšková, Klára ; Pospíšek, Martin (advisor) ; Půta, František (referee) ; Valášek, Leoš (referee)
Eukaryotic translation initiation factor eIF4E1 (eIF4E1) plays a pivotal role in the control of cap-dependent translation initiation, occurs in P- bodies and is important for the formation of stress granules (SG). Human cells encompass two other non-canonical translation initiation factors capable of cap binding although with a lower affinity for the cap: eIF4E2 and eIF4E3. Here, I investigated the ability of individual eIF4E family members and their variants to localize to SGs and P-bodies in stress-free, arsenite and heat shock conditions. Under all tested conditions, both eIF4E1 and eIF4E2 proteins and all their variants localized to P-bodies unlike eIF4E3 protein variants. Under both arsenite and heat stress conditions all tested variants of eIF4E1 and the variant eIF4E3-A localized to SGs albeit with different abilities. Protein eIF4E2 and all its investigated variants localized specifically to a major part of heat stress-induced stress granules. Further analysis showed that approximately 75% of heat stress-induced stress granules contain all three eIF4Es, while in 25% of them eIF4E2 is missing. Large ribosomal subunit protein L22 was found specifically enriched in arsenite induced SGs. Heat stress-induced re- localization of several proteins typical for P-bodies such as eIF4E2, DCP-1, AGO-2...
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Photodissociation studies of xanthene dyes, iron(III) azido complexes and hemithioindigo molecular switches in the gas phase
Navrátil, Rafael ; Roithová, Jana (advisor) ; Žabka, Ján (referee) ; Volný, Michael (referee)
Electronic excitation triggered by the absorption of light enables numerous chemical, physical and biological processes and transformations. Accordingly, full control over the processes involving excited molecules requires an in-depth knowledge of electronic UV/vis spectra and potential energy surfaces. Unsurprisingly, most electronic spectra are acquired in the condensed phase in which molecules are dissolved and most transformations occur. However, our knowledge of excitation, transformations and processes at the level of isolated molecules is still limited, partly because such studies require unconventional experimental approaches and equipment. This Thesis describes experimental methods for recording electronic spectra of isolated molecules in the gas phase by ion spectroscopy, which combines mass spectrometry with optical spectroscopy. Using these methods, experimental factors which affect the electronic excitation and therefore the electronic spectra of ions were determined and evaluated for various fluorescent xanthene dyes, iron-containing complexes and molecular pho- toswitches. Furthermore, factors which govern photochemical processes, such as photo- oxidation, photoreduction and photoisomerization, were also analyzed in detail, with surprisingly different outcomes from previous studies...
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Metabolipidomic profiling of white adipose tissue by UPLC-MS/MS
Rombaldová, Martina
Obesity is a serious problem in society today [1,2]. It might seems to have been caused simply by excess consumption of food compared to energy expenditure but obesity is actually a complex metabolic disorder centred on adipose lipid metabolism and cellular signalling systems linked to it [3]. Understanding the biology of adipose tissue (AT) is very important for the identification of novel and potential therapeutic targets in order to prevent and treat obesity-related disorders [4]. We utilized analytical approach liquid chromatography coupled to mass spectrometry (LC-MS) to study adipose tissue metabolism. Also, we were especially interested in the effect of omega-3 polyunsaturated fatty acids (PUFA) on that metabolism. Rodent and cell line experiments were performed and analyses were done of white adipose tissue (WAT), serum/plasma samples or cells as well as milk samples from mothers. At first, we established several ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for analysis of acylcarnitines (AC), amino acids (AmA) and other metabolites. Importantly, these methods were able to distinguish isobaric species of AC which is not usually possible. Using these approaches we uncovered several acylcarnitines, i.e. long chain AC, carnitine, acylcarnitine C4 and...
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Nuclear receptors in regulation of gene expression, development and metabolism in Celegans elegans.
Yilma, Petr ; Kostrouch, Zdeněk (advisor) ; Pompach, Petr (referee) ; Vaněk, Ondřej (referee)
5 Abstract Genetic mechanisms of regulation of gene expression form the basis for proper development, function of organisms and their responses to variable life conditions. However, they are relatively slow. Life processes that require a fast response to the changing environmental and metabolic conditions are mostly executed on the level of proteins especially their posttranslational modifications and protein- protein interactions.The goal of the experimental work that led to the presented thesis consisted in exploitation of the model organism Caenorhabditis elegans for analysis of regulation of gene expression by transcription factors from the protein family of nuclear receptors. The model system C. elegans enables very efficient experimental procedures in the field of genetics, genomics and functional analysis of phenotypes. In the experimental work connected with this thesis, I studied the regulation of gene expression under specific experimental conditions from the perspective of advanced functional proteomics and I focused on the employment of separation methods and methods of advanced proteomics, especially by mass spectrometry.In the first part of the work, I characterized the nuclear receptor NHR-60 on the protein level. This nuclear receptor is expressed as two protein forms with a mass of 50 kDa...
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