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Identification and characterization of flagellar tip proteins in Trypanosoma brucei
Pavlisková, Hana ; Varga, Vladimír (advisor) ; Čajánek, Lukáš (referee) ; Hashimi, Hassan (referee)
The tip of the eukaryotic flagellum is one of the most important regions of the flagellum in many eukaryotic cells. Several important functions have been associated with the flagellar tip. However, its protein composition remains largely unknown. The main aim of this thesis was to identify flagellar tip proteins and characterize them. Using the parasitic model organism Trypanosoma brucei and the TrypTag.org project, a unique resource localizing every protein encoded in the trypanosome's genome, we were able to identify, for the first time, the complete catalog of flagellar tip proteins in a eukaryotic organism. In T. brucei the full complement comprises 78 proteins localizing exclusively to the flagellar tip or being highly enriched there. To characterize these proteins, we established new reagents and approaches. First, we developed antibody markers labeling the tip of an assembling trypanosome flagellum via recognizing the flagella connector. This enabled us to study the tip-specific processes. Second, we developed a rapid and cloning-free approach for tagging and inducible overexpression of trypanosome proteins. We demonstrated that the approach is well suited for overexpression of large proteins, such as some of the flagellum tip proteins. This enables the study of overexpression phenotypes and...
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The development of new isotope-coded cross-linkers for label free quantification of proteins
Procházková, Valérie ; Kukačka, Zdeněk (advisor) ; Vaňková, Pavla (referee)
Elucidating the structure of proteins in living organisms is an important step in describing their role. One method of structural biology is chemical cross-linking in combination with mass spectrometry, which is a commonly used tool for characterisation of the tertiary structure of proteins or protein-protein interactions. This method is based on the reaction of a cross-linking agent consisting of two reactive groups linked by an arm of a defined length to form a covalent bond. The main aim of the project was to verify whether chemical cross-linking using istopically labeled cross-linking agents can be used for quantification of two different structural states. The reagents used in this work were DSPU and an isotopically labeled form of DSPUx. Both the DSPU and DSPUx reagents contain a labile urea molecule in their structure, which is cleaved into characteristic fragments during collision-induced dissociation, allowing the identification of cross-linked peptides. Using top-down and bottom-up approaches, it was found on selected proteins (insulin, human carbonic anhydrase, and bovine serum albumin) that DSPU and DSPUx reagents interact with selected peptides and proteins and are suitable for quantification of structural changes. Subsequently, structural differences in the presence (holoform) 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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