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Study of hyaluronan-albumin systems using light scattering methods and calorimetry
Sereda, Alena ; Hermannová, Martina (referee) ; Chytil, Martin (advisor)
This thesis, which is a continuation of the previous Bachelor thesis, is dedicated to the study of polyelectrolyte complexes between hyaluronan (HA) and bovine serum albumin (BSA). Interactions between HA and BSA and a formation of complexes were studied by SEC-MALLS method, where a molar mass, a root mean square (rms) radius, a hydronamic radius and an intrinsic viscosity of particles of the system were defined. Furthermore the interactions were studied by calorimetric measurement ITC, where thermodynamic character of complex formation was determined. Additionally, dynamic and electrophoretic light scattering methods (DLS and ELS) were applied, where hydrodynamic radius and a value of the zeta potential were defined. Also the effect of higher temperature on the character and complex formation was examined by SEC-MALLS and ITC measurements. The interactions were confirmed at any of the used media, but with different efficiency. It was also proved that complexes become smaller in their radii and viscosity with growing BSA concentration. Furthermore it was proved, that the high ionic strength hinders surface charges of HA and BSA molecules and minimizes their mutual interactions. At the higher ionic strength the radii of the complexes, their intrinsic viscosity and zeta potential are increased. The higher temperature has only minimal effect on the formation of the complexes.
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Preparation of mutated forms of protein B7-H6
Malá, Viktorie ; Vaněk, Ondřej (advisor) ; Bělonožníková, Kateřina (referee)
NK cells are an essential part of the immune system. They are the so-called natural killers and carry several receptors on their surface. Two types of receptors are crucial for immune surveillance, according to their function, called inhibitory and activating. Inhibitory receptors provide auto-tolerance. The second type is activating receptors, whose activation signals toward initiating a cytotoxic response or involving other immune system components (e.g., macrophages). The activating receptor NKp30 (also known as NCR3 or CD337) is a protein of the Natural Cytotoxicity Receptors (NCR) family. It is one of the primary human NK cell activating receptors with potential use in targeted anticancer therapy. NKp30 has an activating ligand protein B7-H6, which is not found on the surface of healthy cells. It is exclusively found on highly stressed cells, e.g., infected or malignantly transformed cells. If a malignantly transformed cell carries B7-H6 on its surface, in most cases, it is recognized and eliminated almost at its formation. Not all tumour cells are B7-H6 positive, so it is possible to visualize them to the immune system through surface decoration with B7- H6. Moreover, such a ligand can be modified to amplify the immune response. This work is based on Pekar L. et al. (2020) publication, which...
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Advanced methods of assaying lectin-carbohydrate interaction
Červený, Jakub ; Bosáková, Zuzana (advisor) ; Pavlíček, Jiří (referee)
A reliable and reproducible quantification and detailed characterization of the interactions between a biomedically relevant protein and its synthetic carbohydrate ligand can provide new information for the design of better diagnostic and therapeutic tools. A high sensitivity and specificity are essential requirements for the feasibility of an analytical method. These parameters can be difficult to achieve for highly complex systems such as lectin-saccharide complexes. On the other hand, carbohydrate-based diagnostics and therapeutics offer the great advantage of detecting biologically active lectin receptors, which cannot be achieved with antibodies. In this work we demonstrate the use of the BLI technique to monitor lectin- carbohydrate interactions for a multivalent system. Using functionalization of a novel construct of galectin-1 with an in vivo biotinylated AVI tag, suitable immobilization on a biosensor was achieved without the loss of its lectin activity. Due to the high sensitivity of this technique, kinetic parameters of interaction with prepared multivalent neoglycoproteins based on LacNAc epitopes were obtained in the micro- to nanolar KD range. The complementarity of this method was demonstrated by parallel ITC and competitive ELISA measurements. The neoglycoprotein with a lower LacNAc...
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Study of intermolecular interactions by isothermal titration calorimetry
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
Calorimetric methods are used to studying the mechanism of regulation and control of biological processes at the molecular level. Isothermal titration calorimetry (ITC) is used for monitoring the heat exchange of molecular interactions in an environment with constant temperature. This method is also preferred because of its ability of direct measurement of thermodynamic parameters associated with complex formation. ITC measures mostly the energetics of biochemical reactions or molecular interactions protein-protein, protein-ligand, or enzyme-substrate. Main goals of this bachelor thesis were: (I) to acquire working skills with nano isothermal titration calorimeter; (II) to prepare the DNA-binding domain of human transcription factor FOXO4 (FOXO4-DBD); and (III) to study thermodynamic aspects of the interaction between FOXO4-DBD and the double-stranded DNA containing specific binding motif. FOXO4 protein is one of the four members of a "O" subgroup of forkhead transcription factors FOX, which play an important role in many cellular processes including stress resistance, age control and oncogenesis. Forkhead DNA- binding domain recognizes sequences containing the core motif 5'-(A/C)AA(C/T)A-3' with the binding stoichiometry 1:1. Results of this bachelor thesis show that FOXO4-DBD binds the...
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Study of intermolecular interactions by isothermal titration calorimetry
Pšenáková, Katarína ; Obšil, Tomáš (advisor) ; Alblová, Miroslava (referee)
Calorimetric methods are used to studying the mechanism of regulation and control of biological processes at the molecular level. Isothermal titration calorimetry (ITC) is used for monitoring the heat exchange of molecular interactions in an environment with constant temperature. This method is also preferred because of its ability of direct measurement of thermodynamic parameters associated with complex formation. ITC measures mostly the energetics of biochemical reactions or molecular interactions protein-protein, protein-ligand, or enzyme-substrate. Main goals of this bachelor thesis were: (I) to acquire working skills with nano isothermal titration calorimeter; (II) to prepare the DNA-binding domain of human transcription factor FOXO4 (FOXO4-DBD); and (III) to study thermodynamic aspects of the interaction between FOXO4-DBD and the double-stranded DNA containing specific binding motif. FOXO4 protein is one of the four members of a "O" subgroup of forkhead transcription factors FOX, which play an important role in many cellular processes including stress resistance, age control and oncogenesis. Forkhead DNA- binding domain recognizes sequences containing the core motif 5'-(A/C)AA(C/T)A-3' with the binding stoichiometry 1:1. Results of this bachelor thesis show that FOXO4-DBD binds the...
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Study of hyaluronan-albumin systems using light scattering methods and calorimetry
Sereda, Alena ; Hermannová, Martina (referee) ; Chytil, Martin (advisor)
This thesis, which is a continuation of the previous Bachelor thesis, is dedicated to the study of polyelectrolyte complexes between hyaluronan (HA) and bovine serum albumin (BSA). Interactions between HA and BSA and a formation of complexes were studied by SEC-MALLS method, where a molar mass, a root mean square (rms) radius, a hydronamic radius and an intrinsic viscosity of particles of the system were defined. Furthermore the interactions were studied by calorimetric measurement ITC, where thermodynamic character of complex formation was determined. Additionally, dynamic and electrophoretic light scattering methods (DLS and ELS) were applied, where hydrodynamic radius and a value of the zeta potential were defined. Also the effect of higher temperature on the character and complex formation was examined by SEC-MALLS and ITC measurements. The interactions were confirmed at any of the used media, but with different efficiency. It was also proved that complexes become smaller in their radii and viscosity with growing BSA concentration. Furthermore it was proved, that the high ionic strength hinders surface charges of HA and BSA molecules and minimizes their mutual interactions. At the higher ionic strength the radii of the complexes, their intrinsic viscosity and zeta potential are increased. The higher temperature has only minimal effect on the formation of the complexes.
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PROBLEMS OF ELECTRONIC COMMUNICATION WITH FOCUS ON YOUTH
MAJEROVÁ, Aneta
The topic of this bachelor thesis is ?Problems of electronic communication with focus on youth. ? Electronic communication is today presented by information technology communication (ITC), whose rapid development in recent years led to great changes in ways of human communication. ICT include PC, e-mail, cell phone, internet, electronic data exchange or online shopping etc. Apart from many indisputable advantages we also need to perceive many risky phenomenon brought by ICT. Special focus is needed to be given to the part of population most endangered by these risks ? children and teenagers. Bachelor thesis is divided into theoretical and practical part. The theoretical part presents summary of information about the present state of this topic. The content of the present state is electronic communication in general, its progress and it?s modern forms, in conclusion there are specificity and risks of using electronic ways of communication by youth defined. The practical part is determined by partial aims of research and hypothesis set in advance. The partial aims are chosen in sequence to the main aim of the thesis. The main aim of the thesis is to define problems and possible hazards of electronic communication within its recent trends with focus on youth. The research was done quantitatively by method of asking by anonymous questionnaire. Part of questions of my questionnaire was used before in Uwe Buermann?s questionnaire, therefore following interpretation included not only comparison of theoretical knowledge with outcomes of my own research, but mostly comparison of my outcomes with selected outcomes of Uwe Buermann. Interpretation is set in chapter Discussion. In conclusion of thesis I generally review the most important outcomes of research and suggest proceeding which would lessen riskiness of some phenomenoms on the internet. I give there also possible contributions of my thesis.
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