The topic of this diploma thesis was the speciation analysis of the toxicological important forms of the arsenic in urban particulate matter samples. The detection technique used in this work was atomic absorption spectrometry with quartz atomizer. Acetic acid, ammonium acetate, hydroxylamine, potassium hydroxide and sodium carbonate are recommended leaching agents for extraction of metals from urban particulate matter samples. Results of the presenting work are following: Acidic extraction reagents can be not recommended for the speciation analysis of the arsenic, because these reagents lower sensitivity of the determination of As3+ and As5+ too and destroy the quartz atomizer. Alkaline extraction reagents don't lower sensitivity of the determination of As3+ and these reagents don't destroy the atomizer, but lower sensitivity of the determination of As5+ was obtained. Recommended reagents aren't useful for extraction of the metals from urban particulate matter samples for the speciation analysis.

The growing resistance of bacteria to traditional antibiotics promotes the interest in finding new substances for their production. Antimicrobial peptides have comparable effect to conventional antibiotics, but a different mechanism of action and they do not provoke bacterial resistance. These peptides were characterized in all forms of multicellular organisms. Hymenoptera venom contains many biologically active substances including antimicrobial peptides. For this reason, this thesis focuses on the acquisition of antimicrobial peptide sequences from selected species of bumblebees (Bombus terrestris, B. hortorum, B. hypnorum, B. pratorum, B. lucorum, B. lapidarius, B. humilis and B. bohemicus). The isolation from the venom glands was performed by high performance liquid chromatography with reversed phases. Subsequent analysis was performed using the methods of mass spectrometry, matrix-assisted laser desorption/ionization with time of flight analyzer and electrospray ionization connected with hybrid linear ion trap analyzer with orbitrap. The sequences for the found peptides were determined by tandem mass spectrometry methods "de novo" and Edman degradation. In this work we characterized 17 sequences of peptides extracted from bumblebee venom glands for which antimicrobial activity was determined...

The aim of the thesis was to analyze the historical pharmaceutical preparations, including the determination of the active substance and identify theirs possible degradation products. A historical pharmaceutical preparation of naloxone was analyzed by mass spectrometry. Historical pharmaceutical preparations of adrenaline and ephedrine were analyzed by UHPLC-MS and were quantified using a calibration curve. In the historical injection solution of naloxone, "NARCAN", dated around 1980, there were no significant degradation products and the measured mass and UV spectrum was consistent with the spectrum of naloxone. The analyzed sample of naloxone was stable even after 35 years of storage. In the analyzed historical injection solution of adrenaline, "Adrenalin Hydrochlor., Dr. Heisler" (dated between 1917 and 1938) was determined 5.26 ± 0.11 % of the declared amount of adrenaline. In the measured spectras were noticeable degradation products, which have not been described in the literature yet and their identification was beyond the scope of this paper. The analyzed sample of adrenaline was almost completely degraded during about ninety years. The stability test carried out with four standard solutions of adrenaline proved influence of oxygen, light, temperature and time on the degradation of adrenaline. In...

Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.

Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...

Vernix caseosa is a natural biofilm which covers the skin of a human fetus from the third trimester of pregnancy. It has hydrating, regenerating and anti-microbial effects. The components responsible for these properties of vernix caseosa could be used in the pharmaceutical and cosmetic industries. For this reason, the total composition is analyzed. The lipid components of vernix caseosa consist of squalene, wax esters, sterol esters, 1,2-diol diesters, triacylglycerides, free fatty acids, fatty alcohols, cholesterol, diacylglycerides, monoacylglyceridesů and phospholipids . This study is focused on structure analysis of the 1,2-diol diesters of vernix caseosa. Conditions were optimized for the HPLC separation. Nova -Pak C18 column was used and a gradient of acetonitrile:ethyl acetate was chosen as a mobile phase. Before entering the APCI source ammonium formate was added; ammonium adducts [M +18]+ were formed. Eight scan events was set in MS method. One for the full scan spectrum, second for MS2 spectrum of the precursor ions and six MS3 data-dependent spectra. The position of the double bonds of unsaturated 1,2-diol diesters was determined by fragmentation of their [M+C3H5N]+ adducts. There were identified over 2200 of 1,2-diol diesters differing in lengths of alcohol and fatty acid chains, and...

Atomization of arsine in a novel hydride atomizer for atomic absorption spectrometry (HG-AAS) was thoroughly optimized. This plasma atomizer is based on a dielectric barrier discharge (DBD). Sensitivity and detection limit reached 0.48 s ng-1 As and 0.16 ng ml-1 As, respectively, under optimum atomization conditions (Ar discharge using a flow rate of 60 ml min-1 Ar, DBD power 17 W). Analytical figures of merit reached in DBD are comparable to those found in an externally heated quartz tube multiatomizer (MMQTA) that was chosen as a model of conventional approach to hydride atomization in HG-AAS. An extent of interferences (Se, Sb, Bi) during As determination was investigated comparing both MMQTA and DBD atomizers. The later one was found to be more resistant towards interferences. A simple preconcentration of As in a DBD atomizer was reached after oxygen introduction into the Ar plasma in the DBD resulting in analyte retention in the atomizer followed by its volatilization once the oxygen flow is switched off. Preconcentration efficiency of 100 % was reached and detection limit improvement by a factor of ten was achieved (0.01 ng ml-1 As, preconcentration period 300 s).

Radioactivity is an important part of the environment. In the years 2014 - 2015 a survey on medicinal radioactive springs was carried out in the Tanvald granite body. During extensive radiohydrogeochemical exploration in the surrounding area of Schindler spring, the outcrop of U - mineralization has been found at a new construction site in the Kokonín fault. It is possible to find "hot" pieces of uranium ore fragments in the quaternary cover in this place. The occurrence of the hydrothermal (vein) type of uranium in the Krkonoše - Jizera is new as it hasn't been mentioned in literatures before. After an agreement with the owners, detailed radiometric survey proceeded in the surrounding area of two affected houses by using gamma spectrometry and emanometry at the dense net. There is a wider area between two houses with activities of 222 Rn over 1 MBq/m3 and on the ore outcrop was measured 3.3 MBq/m3 . A quaternary solifluction flow with an increased radioactivity creeps down under the House 1. The highest uranium contents reached up to 291 ppm eU (= 3 595 Bq/kg 226 Ra) on the uranium ore lens outcrop. The average of gamma dose rate is 85,6 nGy/h with maximem 261 nGy/h on the outcrop. A further radiometric survey through the entire Kokonín fault line was carried out in an area of roughly 0.25 x 1 km2...

This thesis deals with optimization of conditions of photochemical generation of volatile species of nickel for atomic absorption spectrometry. The volatile species of nickel were generated in the flow arrangement, when sample was injected to a stream of a reaction medium. Either formic acid was used as the reaction medium or formic acid with the addition of formate anions. Two types of a generator were tested, a generator with a PTFE tube wrapped around a mercury UV lamp and a high efficiency generator with an inside channel. At the same time, two types of a gas-liquid separator were tested. Diffusion flame was used as an atomizer due to its high robustness. Quartz atomizer didn't provide higher sensitivity and measurements had worse repeatability. All the parameters affecting atomization in the diffusion flame were optimized (flow rates and composition of gases, observation height). In the next section, conditions of generation (irradiation time, HCOOH concentration, addition of formate anions) were optimized. Interferences of inorganic acids in photochemical generation were investigated as well. It was found out that the generator with the inside channel provided slightly higher sensitivity and thus generation efficiency than the generator with the PTFE tube wrapped around. Transmission of...

Capillary electrophoresis (CE) is highly efficient separation method based on the different migration velocity of ions in liquid media in electric field. It is commonly used in analytical laboratories and due to the different separation principle it is applied as a complementary method to the chromatographic methods (HPLC and UHPLC). Beside the applicability of CE for quantitative/qualitative analysis, the method can be used also for physico-chemical characterization of compounds (e.g. determination of acid dissociation constants of weak electrolytes or stability constants of complexes). This work is focused on the applicability of CE methods for determination of physico- chemical characteristic of compounds (acid dissociation constants of triazole fungicides and stability constants of dibenzo-18-crown-6, benzo-18-crown-6 and 18-crown-6 ether complexes with metal ions in hydro-organic solvent mixtures) and on the possibility to use affinity CE (ACE) with electrospray ionization-mass spectrometric detection (ACE- ESI/MS) for the study of non-covalent interactions of compounds. For the online hyphenation of CE and ESI/MS, two highly sensitive CE-ESI/MS interfaces were tested: i) porous tip and ii) nano-sheath liquid flow. The ability of the CE-ESI/MS interfaces to effectively decouple spray and...