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Utilization of micropreparative isoelectric focusing in the analysis of biological samples
Vlčková, Zuzana ; Flodrová, Dana (referee) ; Šalplachta, Jiří (advisor)
The aim of this Master Thesis is the optimization and verification of conditions of separation of proteins from biological materials. For the separation a developed technique called micropreparative isoelectric focusing is being used. The theoretical part describes analytical methods of biological materials with a focus on proteins which were used for the optimization of the developing system in general. This part of the Master Thesis also presents this method itself. Furthermore, basic characteristics of other techniques used to control the efficiency of the micropreparative isoelectric focusing are introduced. The experimental part depicts the individual steps of optimization of the micropreparative isoelectric focusing. The ideal separation procedure was firstly found out on the individual standard proteins, secondly on their mixture and lastly the optimised procedure was applied to a real biological sample. The efficiency of the micropreparative isoelectric focusing was verified by SDS-PAGE and MALDI-TOF mass spectrometry.
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Monitoring of vitamin E content in the samples of cereals
Cibulcová, Pavla ; Borkovcová,, Ivana (referee) ; Benešová, Karolína (advisor)
Thesis deals with the analysis of vitamin E in cereal samples, namely barley and wheat and malt produced from them. Selected samples were treated with low-temperature plasma, fungicide and biological treatment to elimination fungus and mycotoxins. The aim of the thesis was to find out the influence of the treatment, the combination of several types of treatment on vitamin E content in grain and malt. If the content of health-positive substances is not reduced and grain can be used for food, malt, or for feed purposes. The theoretical part focuses on the properties, occurrence, biochemistry and importance of vitamin E for living organisms. It also focuses on the properties of cereals that are a significant source of vitamin E. Malt production and sample treatment are also described. The experimental part describes the field experiment, individual types of treatment, extraction of vitamin E from the samples and determination of vitamin E content by high performance liquid chromatography with fluorescence detection. The results are compared with the current literature. It was found that the treatment did not have a significant negative effect on vitamin E content.
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Race: myth for the classification of human variability
Sládek, Vladimír
There is long history to use race concept and typological approach for classification of human diversity. Since the first use, the race concept is based on presumption that morphology and/or genetic background of living organism offer essential information about their genealogy. This essential information can be studied by race features; the race features are stabile both during the time period and reproductive cycles. Therefore, the race feature can be used to connect reproductive groups. However, evolutionary studies show that static race features are not present in biological organisms. Morphological as well as genetic features changed not only due to functional demand and selective factors but also by stochastic processes as for example by genetic drift. Therefore, a selection of race features to classify human variability is arbitrary process since there is no direct connection between consequent generations.
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Design and synthesis of BCA2 inhibitors.
Soukupová, Jitka ; Doležal, Martin (advisor) ; Musílek, Kamil (referee) ; Westwell, Andrew D. (referee)
BCA 2 (Breast Cancer Associated Protein2) is a novel monomeric RING (Really Interesting New Gene 2) - type ubiquitin E3 ligase. It was found to be overexpressed in 56 % of invasive breast cancers and its expression is correlated with a positive estrogen receptor status. The RING-type family of proteins possesses ubiquitin ligase activity and involves in protein degradation. Ubiquitylation is used to target proteins of different biological processes including proteosomal degradation or endocytosis. Polyubiquitination of target protein substrates is carried out by three classes of ubiquitin ligase enzymes, of which the diverse E3 ligase family catalyse the final step of ubiquitin transfer to specific lysyl residues of target proteins prior to proteosomal destruction. The RING-type proteins can be defined as unique linear series of conserved cysteine and histidin residues and binds two zinc atoms in a cross-brace arrangement. Studies of zinc-ejecting compounds have led to the identification of disulfiram, which has been used for alcohol aversion therapy for alcohol aversion therapy as an alcohol dehydrogenase inhibitor. In this thesis I describe the synthesis of three series of novel zinc-affinic compounds, in order to optimise selectivity of BCA2 inhibitors which could lead to the identification of...
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New Electrochemical Methods for Determination of Nitro and Oxo Derivatives of Fluorene
Vyskočil, Vlastimil ; Barek, Jiří (advisor) ; Ludvík, Jiří (referee) ; Heyrovský, Michal (referee)
4. Conclusion This Thesis represents a contribution to the search for new analytical methods applicable on environmental and biological samples. The current state- of-art concerning the formation, occurrence, and biological activity of fluorene and its nitro and oxo derivatives was summarized in Chapter 1. Further experimental work was focused on assessment of new approaches in the detection of the studied compounds. Attention was paid mainly to nitrofluorenes, fluorenone and nitrofluorenones, namely 2-nitrofluorene, 2,7-dinitrofluorene, 9-fluorenone, 2-nitro-9-fluorenone, and 2,7-dinitro-9- fluorenone and the possibilities of their determination using polarographic and voltammetric methods. Interactions of calf thymus dsDNA with 2-nitrofluorene and 2,7-dinitrofluorene at the DNA/SPCPE were investigated and subtle DNA damage under conditions of direct DNA-analyte interaction at room temperature and damage to DNA bases under condition of electrogeneration of short-lived radicals of nitrofluorenes at human body temperature were found. The obtained results can be summarized as follows: ● Modern polarographic and voltammetric methods at mercury electrodes developed for determination of trace amounts of 9-fluorenone and 2,7-dinitro-9-fluorenone (and other NPAHs generally) in concentration ranges from 2×10-8 to...
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Specifics of children from homoparental families
Sedláčková, Anna ; Šulová, Lenka (advisor) ; Štětovská, Iva (referee)
The diploma thesis deals with topic of lesbian and gay families and specifics of children from these families. The theoretical part of the work is supported by studies from the USA and the Great Britain in particular. This part is focused on definition of homoparental family and its types, specifics of upbringing and relations within. It also deals with the topic of coming-out. It gives account of the family roles - biological mother, biological father, social mother / stepmother. The second half of the theoretical part describes psychological, gender and social development of the children from lesbian families. There are also mentioned some children's feelings and views of their nontraditional family and homosexuality of their parents. The research presented in the empirical part is based on individual interviews and projective techniques. The study group comprises 10 children between the age 4 and 15. In the study group there are children from previous heterosexual relationship as well as children from planned lesbian families. Participants to the sample were recruited via advertising and snow ball technique. The research is qualitative and its outcomes are elaborated in the form of case study report. The list of crucial themes elaborated through clustering method is other outcome. Results of research...
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Computational Investigations of Biomolecular Systems and Comparison with Experiments in Various Environmental Conditions
KHABIRI, Morteza
Computational methods were used to study two different types of biological systems. The first study is related to the effect of three different organic solvents (formamide, acetone and isopropanol) on the structure and behavior of three globular proteins. These enzymes belong to the haloalkane dehalogenase family: DhaA, LinB, and DbjA. Moreover, the effect of mutation in the presence of DMSO was also investigated in two variants of DhaA; DhaA57 (L95V+A172V) and DhaA80 (Thr148Leu+Gly171Gln+Ala172Val+Cys176Phe). The simulation results showed that except for DhaA80, organic solvents entered the active site and influence its hydration. Not only the active site but also the enzyme?s hydration shell is influenced by organic molecules. The results showed that the water molecules are stripped out from the enzyme surfaces. It seems that the dual nature of organic molecules makes them favorable to solvate the enzymes. Radial Distribution Function (RDF) of the different parts of each organic molecule reveals that the behavior of each organic solvent in the vicinity of hydrophobic surfaces is similar to their behavior at the air-water interface. Structural analysis of root-mean-square-deviations (RMSD) and B-factors reveals that the flexibility of the enzymes decreased in the presence of most organic solvents, mainly in the CAP domain. Changes of other structural properties like radius of gyration and total solvent accessible surface areas are minimal. DbjA exists as a dimer and is more influenced by organic molecules. They penetrate to the amino acid network between monomers and influence their motion. The second study is the interaction of voltage-gated potassium channel Kv1.3 wild type and its mutants (Kv1.3_V388C, Kv1.3_V388C_H399T) with scorpion toxin (ChTX). Since there is no structure for Kv1.3, 94% sequence identity with Kv1.2 structure was used to make a homology model based on the Kv1.2 structure. MD structural analyses reveal that mutation of V388C changes the stability of selectivity filter by interrupting the amino acid network interactions behind the selectivity filter. The interaction of ChTX is also affected by the single mutant. ChTX is able to block wild type and double mutant channels but cannot occlude the pore entirely. Introducing the second point mutation H399T in the pore region reverts the structural changes back to the wild type. These results are entirely consistent with experimental results. Additionally, the binding energy of ChTX with the wild type mKv1.3 was investigated by the potential of mean force method, in the presence and absence of KCl solution. The results both in experiment and simulation show that, even though the unbinding process and dissociation rate is changing in the present of K+ ions, the binding energy is independent of K+ concentration. All together, the combination of computer simulations together with experiments provides new knowledge about channel-toxin interactions which could be helpful for drug design.
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The fluorescence assay of aminopeptidase activity as an alterantive indicator of water quality?
Vrba, Jaroslav ; Svoboda, J. ; Fuksa, J. K.
Recent water quality/pollution control uses standard chemical, biological or microbiological indicators that provide information on water status with a delay of several days. Though they characterise direct impact of released compounds on water quality in a recipient, they do not well reflect consequent reaction of the ecosystem on increased pollution. Following the Water Framework Directive 2000/60/EC we focused on such alternative microbial indicators that would allow for instant evaluation of ecological status of running waters and ongoing in-stream processes. Recently, activities of different extracellular enzymes have been suggested as indicators of both trophic status of lakes and organic or faecal pollution of streams. This contribution evaluates an indication potential of the extracellular aminopeptidase assay.
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Analysis og gene expression in prokaryotic and eukaryotic model organisms by proteomic gel-based separation tools
Petráčková, Denisa ; Weiser, Jaroslav (advisor) ; Nešvera, Jan (referee) ; Stulík, Jiří (referee)
This PhD thesis showed the applicability of a gel-based proteomic separation tool, 2-D electrophoresis in three independent projects. Supplemented with results obtained using different techniques the proteomic studies enabled a global imaging of proteoms in the studied biological systems. Comparing total proteoms of E. coli 61 protein changes were identified and connected with the development of the bacterial population in the presence of an antibiotic compound, erythromycin. This classic proteomic approach included sample extraction, optimization of its 2D separation followed by 2D gel analysis and protein identification by MS methods. A disadvantage of this work was an enourmously large amount of data to be analyzed by computer analysis. For the study of membrane proteom of B. subtilis during a pH induced stress, on the other hand, a modification of isolation techniques for membrane and membrane associated proteins was required first to improve the subsequent protein separation by 2-D electrophoresis. The optimalization of protein extraction included changes in detergents used for protein solubilization and a prolongation of time periods in the protein solubilization protocol. 5 relevant protein changes were then described that play a role in the bacterial response to pH stress. The proteins were...
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