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Characterization of recombinant fragment of an antibody against CD3 marker.
Písačková, Jana ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...
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Preparation and crystallization of a metabolic repressor LutR from Bacillus subtilis
Soldánová, Anna ; Maloy Řezáčová, Pavlína (advisor) ; Kutá-Smatanová, Ivana (referee)
Metabolic transcriptional repressors are proteins controlling transcription of specific genes involved in bacterial metabolism. These proteins typically consist of two domains: N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain. When an effector (usually a metabolite molecule) binds to the protein, the conformation of the protein is changed. This causes a change in affinity to its DNA operator and that subsequently modulates the transcription of genes of the specific metabolic pathway. LutR belongs to the GntR family of bacterial transcriptional regulators. In undomesticated strain RO-NN-1 of Bacillus subtilis, LutR regulates transcription of genes required for L-lactate utilization. Interestingly, LutR from laboratory strains PY79 and 168 has a different function. Due to a mutation, it lacks the first 21 amino acids and this alters its DNA recognition specificity. This LutR variant acts as a global regulator and regulates many genes associated with transition from exponential growth to stationary phase of bacterial population. Knowledge of 3D structure of LutR with DNA could elucidate the impact of this short deletion in LutR DBD on the mechanism of DNA recognition. In this work, the DBD of LutR from undomesticated strains of B. subtilis was prepared by heterologous...
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Zero liquid discharge systems
Štylárková, Petra ; Máša, Vítězslav (referee) ; Touš, Michal (advisor)
The bachelor thesis contains a description of zero liquid discharge systems. It describes the process, which has three main parts, it is pretreatment, preconcentration and evaporation. In each parts there is a basic description of the principle of the metod, their use etc.
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Expression and purification of recombinant proteins of human Aichi virus and other kobuviruses
Ludvík, Tomáš ; Bouřa, Evžen (advisor) ; Petrvalská, Olívia (referee)
Viral 2Apro plays many important functions in the life cycle of virus. It is protease that is involved in the cleavage of the viral polyprotein in the VP1/2A region. The structure of protein contains active site, thanks to this the protein is able to bind various substrates in the host cell, which is essential for viral infection. With the help of 2Apro , the virus can bypass the host immune response and stop the production of key host proteins. Therefore, 2Apro is suitable target for antiviral intervention. In this work were prepared two recombinant proteins from Aichi virus and Coxsackievirus B3. These are viruses of the family Picornaviridae, which belong among the human pathogens that in some cases cause serious diseases. Aichi virus is the cause of gastroenteritis and Coxsackievirus B3 in the worst cases causes dilated cardiomyopathy. Cardiomyopathy leads to poor heart function due to it is enlargement and can lead to heart failure. (In Czech) Key words: recombinant proteins, 2Apro , Aichi virus, Coxsackievirus B3
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Problematics of active compounds recrystalization in pharmaceutical preparations
Máčalík, Tomáš ; Holas, Ondřej (advisor) ; Vraníková, Barbora (referee)
1 Abstract Charles University in Prague, Faculty of Pharmacy in Hradci Králové Department of: Pharmaceutical technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Tomáš Máčalík Title of Thesis: Problematics of active compounds recrystalisation in pharmaceutical preparations The topic of the thesis is the recrystallization of active substances in pharmaceutical preparations. The aim of this work is to demonstrate this phenomenon on a real particular pharmaceutical product, in which it has occurred. It includes a detailed description of the manufacturing process including all mandatory conditions, possible critical points, investigations and measures taken. The thesis consists of two parts. The first describes in details the current state of knowledge. It is based on the findings of the available literature on the subject. The basic concepts of crystallization and polymorphism are explained in detail, which form the basis for the subsequent case study. The case study includes a detailed description of a specific medicinal product. Subsequently, it's production, good manufacturing practice and gradual investigation of the discovered technological incompatibility are explained step by step. The result of the work points out that the issue of recrystallization and polymorphism is very closely related to...
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Crystallization of coating systems based on epoxy resins.
Seidlová, Michaela ; Dohnálek,, Pavel (referee) ; Petránek, Vít (referee) ; Drochytka, Rostislav (referee) ; Bydžovský, Jiří (advisor)
Crystallisation of epoxy resins based on bisphenol A and bisphenol F is the natural property of these oligomers. However, manufacturers of paints and other systems based on these epoxy resins, strive to slow down the crystallisation process as much as possible, thereby prolonging the shelf life and improving the competitiveness of their products. The main aim of the thesis is generalisation of the factors influencing the crystallisation process of epoxy resins based on bisphenol A and bisphenol F, verifying new approaches to the possibility of influencing the crystallisation process and determining the possibility of using a certain degree of epoxy resins crystallinity based on bisphenol A and bisphenol F in the preparation and production of epoxy resinbased coating systems.
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Study of the limestone decarbonation process
Koporcová, Diana Mária ; Všianský,, Dalibor (referee) ; Dvořák, Karel (advisor)
The bachelor thesis deals with a research and a description of decarbonation process of limestone during the lime burning. The goal of the bachelor thesis is to try to find a connection between characteristics of input material and course of the decarbonation process, which is monitored by high temperature XRD analysis. There is an evaluation of the microstructure of emerging calcium oxide, especially by the size of the crystallites, in the experimental part.
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Salts of 2-aminoethanol and ferrocene-based acids
Zábranský, Martin ; Štěpnička, Petr (advisor) ; Němec, Ivan (referee)
Combination of the compact but sterically flexible ferrocene scaffold with intermolecular binding potential and conformational variability of (2-hydroxyethyl)ammonium structural motif was utilised in the construction of solid crystalline materials. Crystallisation in systems containing ferrocenecarboxylic, 2-ferrocenylacetic, 3-ferrocenylpropionic, 3-ferrocenyl- acrylic, 3-ferrocenylacrylic, 3-ferrocenylpropiolic, ferrocene-1,1'-dicarboxylic or ferrocene- sulfonic acid with 2-aminoethanol afforded crystals of the corresponding salts. The resulting crystalline products were characterised with the usual methods (proton nuclear magnetic resonance, infrared spectroscopy, elemental analysis) and their crystal structures were determined by means of single-crystal X-ray diffraction analysis. The crystal structures of the salts mentioned above usually contain rather complicated two dimensional networks of charge-assisted hydrogen bonds. With the aim of studying potentially more simple hydrogen-bonded structures, additional crystallisation experiments were conducted in systems of ferrocenecarboxylic acid and 2-(methylamino)ethanol or 2-(dimethylamino)ethanol. These experiments yielded simple salt of the former amine and adducts of salts of both bases with ferrocenecarboxylic acid in the ratio of 1:1. The...
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Structure Analysis of Plant Bifunctional Nuclease TBN1
Kovaľ, Tomáš ; Dohnálek, Jan (advisor) ; Brynda, Jiří (referee) ; Schneider, Bohdan (referee)
This work is dedicated to thorough structural analysis of plant bifunctional nuclease TBN1, the representative of plant nuclease I group. TBN1 along with homologous nucleases from this family plays an important role in plant cell life cycle and also shows considerable anticancerogenic effects. Two variants of TBN1 (wild type and N211D mutant) were studied. Properties of both variants in different solutions were analyzed. Both were successfully crystallized. Structures of both types of TBN1 were solved using X-ray diffraction. The phase problem was solved by Multi-wavelength anomalous dispersion using Zn2+ ions natively present in TBN1. Structural properties of TBN1 such as fold, active site composition, effect of glycosylation and surface electrostatic potential distribution were analyzed. Reaction mechanism of TBN1 was proposed on the basis of structural properties and comparisons with similar structures.
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Quantitative description of textures in volcanic rocks: Case study of trachybasalt from Dubičná
Ditterová, Hana ; Dolejš, David (advisor) ; Rapprich, Vladislav (referee)
- iii - ENGLISH ABSTRACT Quantitative description of textures, in particular, the distribution of grain sizes, is becoming a powerful tool for interpreting kinetic processes that govern crystallization of igneous rocks. Variation in grain size are frequently represented by logarithm of population density vs. linear crystal size, where the majority of igneous rocks forms a linear trend. Its slope provides an estimate of characteristic grain size, or growth rate and crystallization time, whereas the intercept provides the nucleation density. The log-linear trend is predicted to result from kinetic nucleation and growth processes only, whereas its modifications such as kinks or curvature are due to mechanical separation or accumulation of crystals, magma mixing or Ostwald ripening (coarsening) in the presence of melt. In order to investigate the true, three-dimensional crystal size distribution, the raw two-dimensional data acquired from thin sections or rock slabs must be processed by various stereological methods. We illustrate application of quantitative textural measurements to distribution and origin of clinopyroxene crystals in a trachybasaltic lava flow from Dubičná near Úštěk. This effusion belongs to Cenozoic volcanic products in the Bohemian Massif, specifically to its main Eocene-Miocene stage in...
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