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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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Preparation of proteases for protein structure studies.
Jirečková, Barbora ; Man, Petr (advisor) ; Vaňková, Pavla (referee)
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a method allowing the study of protein structure and dynamics. Its spatial resolution is given by the proteolysis step that is included in the HDX-MS workflow. Most widely used pepsin has however some limitations and use of a single protease often does not provide optimal spatial resolution. Several publications have emphasized the importance of the alternative proteases nepenthesin-2 (Nep-2) and aspergillopepsin (XIII) cleaving, in contrast to pepsin, after basic amino acids. In studies targeting proline-rich proteins, another enzyme, prolyl endoprotease from Aspergillus niger (AnPEP), is gaining importance. This work focuses on the characterization of immobilized AnPEP in combination with pepsin, aspergillopepsin or Nep-2 for their application in HDX-MS. First, columns with only one protease were tested on a set of model proteins. It was found that immobilized AnPEP did not have optimal cleavage characteristics compared to the other proteases. In order to combine the advantages of the proteases mentioned above, the model proteins were digested using columns with AnPEP coimmobilized with pepsin, Nep-2 or XIII and also using two protease columns in series (always AnPEP column with pepsin, Nep-2 or XIII column in both...
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Expression of recombinant soluble form of NKR-P1A receptor.
Vaňková, Pavla ; Man, Petr (advisor) ; Mrázek, Hynek (referee)
NK cells are large granular lymphocytes and they are part of the innate immune system. In defense against tumor or virus-infected cells they activate their cytotoxic mechanisms and by production of cytokines NK cells can mobilize assistance in form of adaptive immune response. They create a large area of interest because of finding of ligands to their receptors and understanding these interactions can lead to significant discoveries. In particular, in medicine, where it can be applied i.a. for transplantation, in the fight against tumors or HIV. This thesis focuses on NKR-P1A receptor, which belongs to NKR-P1 family with other six receptors. This protein is activation receptor and recently its novel isoform was discovered. New isoform differs in transmembrane domain, whose considerable part is lacked. Aim of the thesis was prepare production plasmid encoding mNKR-P1A ISO2 followed by test of expression.
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New Challenges for Directors of secondary Schools in the Czech Republic and France - Project management in the sphere of education
Vaňková, Pavla ; Trunda, Jiří (advisor) ; Voda, Jan (referee)
This diploma thesis explores the issue of project activity which has become a new challenge for school directors in EU member states for the last few years. The projects not only contribute to the fulfillment of the tasks of the educational process, but also to the development of the school environment. The aim of this thesis is to compare the use of project possibilities within the research sample of directors of secondary schools in the Czech Republic and France. Theoretical background specifies the project and its cycle together with the strategy, tools and subsidy opportunities within the educational and regional policy of the European Union. The empirical part investigates the differences between the activity and success in the use of grant funds within the sample of directors of Czech and French schools and views on the form of international cooperation in the Comenius sub-program of lifelong learning.
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The study of structure and dynamics of protein molecules and protein complexes.
Vaňková, Pavla ; Man, Petr (advisor) ; Obšilová, Veronika (referee) ; Hernychová, Lenka (referee)
5 Abstract Inside the cellular milieu, proteins are reaching their native conformation with the assistance of molecular chaperones. These high mass dynamic complexes perform other functions that are contributing to the stability of the cellular proteome. Transport of proteins into the target compartment or defence against oxidative stress rank among these as well. Dynamic structural changes of chaperones Hsp70 and NQO1 and co-chaperone Tomm34 that are participating in above mentioned processes and their interactions with ligands are characterized here by the combination of structural mass spectrometry, biophysical and molecular biology techniques. In this thesis the ATP-dependent dimerization of human inducible isoform Hsp70 (HSPA1A) and the characterization of its evolutionarily conserved dimerization interface were described. Further, highly conserved amino acid residues crucial for allosteric movements were identified. These residues create a network of contacts stabilizing both extreme conformations of HSPA1A (low and high-affinity) during the ATPase cycle. One of the co-chaperones that can affect Hsp70 ATPase cycle is TPR co-chaperone Tomm34. This co-chaperone is participating on the transport of the nuclear encoded mitochondrial precursors into the mitochondria. Here we described regulation of this...
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Structural characterization of intracellular form of mice protein Nkr-p1a
Vaňková, Pavla ; Novák, Petr (advisor) ; Kolenko, Petr (referee)
NK cells are a component of innate immunity system, which is derived from lymphoid progenitor. By a sophisticated receptor repertoire, which is expressed on their surface, they provide a surveillance against pathogenic, virus infected or tumour cells. Simultaneously they produce cytokines, thereby are involved in adaptive immune response. This work is focused on the study of structure of mice soluble mNkr-p1a isoform. Recently this short isoform was identified at the transcriptional level by a member of our laboratory and it is designated as isoform 2. The aim was to produce mNkr-p1a iso2 protein in the prokaryotic expression system and to perform its renaturation and purification in vitro. In the next phase of work, the obtained product was analyzed by the mass spectrometry methods. Recieved results made us think about that our protein is in unfolded state. This assumption was refuted by following biophysical methods, nuclear magnetic resonance, circular dichroism and dynamic light scattering measurement. Keywords: NK cells Receptor mNkr - p1a Short isoform mNkr - p1a iso2 Alternative splicing Protein biosynthesis Recombinant protein production Protein purification Mass spectrometry Disulfide bond Chemical cross-linking NMR, CD, DLS 5
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New Challenges for Directors of secondary Schools in the Czech Republic and France - Project management in the sphere of education
Vaňková, Pavla ; Trunda, Jiří (advisor) ; Voda, Jan (referee)
This diploma thesis explores the issue of project activity which has become a new challenge for school directors in EU member states for the last few years. The projects not only contribute to the fulfillment of the tasks of the educational process, but also to the development of the school environment. The aim of this thesis is to compare the use of project possibilities within the research sample of directors of secondary schools in the Czech Republic and France. Theoretical background specifies the project and its cycle together with the strategy, tools and subsidy opportunities within the educational and regional policy of the European Union. The empirical part investigates the differences between the activity and success in the use of grant funds within the sample of directors of Czech and French schools and views on the form of international cooperation in the Comenius sub-program of lifelong learning.
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Expression of recombinant soluble form of NKR-P1A receptor.
Vaňková, Pavla ; Man, Petr (advisor) ; Mrázek, Hynek (referee)
NK cells are large granular lymphocytes and they are part of the innate immune system. In defense against tumor or virus-infected cells they activate their cytotoxic mechanisms and by production of cytokines NK cells can mobilize assistance in form of adaptive immune response. They create a large area of interest because of finding of ligands to their receptors and understanding these interactions can lead to significant discoveries. In particular, in medicine, where it can be applied i.a. for transplantation, in the fight against tumors or HIV. This thesis focuses on NKR-P1A receptor, which belongs to NKR-P1 family with other six receptors. This protein is activation receptor and recently its novel isoform was discovered. New isoform differs in transmembrane domain, whose considerable part is lacked. Aim of the thesis was prepare production plasmid encoding mNKR-P1A ISO2 followed by test of expression.
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Selected biblical symbols, their artistic and theological meaning
Vaňková, Pavla ; Liguš, Ján (advisor) ; Bravená, Noemi (referee)
This Diploma Thesis deals with selected biblical symbols and their theological and graphic meaning. The subject has been reduced to biblical symbols concerning the representation of Christ , particularly in early christian period. It comes out of my own experience as a creative artist with the work with symbolism. The choice of the subject has been also inspired by a deeper personal interest in theological significance of symbols. I have attempted in the Diploma Thesis to point out the importance and linkage of symbols in arts. In present days, when the arts cease to be communicatory in thein statements, I wanted to return into the first centuries of early christian creations and start to draw from this living source. I have chosen the symbolic representation of Christ in the first centuries of christian history. The symbols have been arranged into chapters according to creative development of representation of Christ from the first centuries up to the time before the division of Christianity. I have used the analytic-explicit methodology. The theological significance of symbols is based on a czech translation of Bible from the year 1987.
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