The presented thesis deals with optimization of parameters speciation analysis based on a selective hydride generation in a direct transfer mode of hydrides and on-line atomization in an atomic absorption spectrometer. In the first part of the thesis processes during generation were investigated. Important indicator of a reactions taking place in a generator is volume of hydrogen (created by decomposition of NaBH4) which is subsequently introduced to the atomizer. In this part of the thesis a new approach to determine a flow rate of hydrogen delivered to the atomizator was developed. An influence of relevant paremeters of the atomizers and atomic absorption spectrometers on basic analytical characteristics of arsenic determination based on hydride generation was performed in the second part. Three atomic absorption spectrometers were used and three sources of radiation were compared. The most important analytic characteristic was the limit of detection which is influenced by standard deviation of measurement values of blank samples and by sensitivity. The best values of the limit of detection were achieved with electrodeless discharge lamps. Key words: hydride generation, arsenic, speciation analysis, atomic absorption spectrometry

Speciation analysis of arsenic based on selective hydride generation and detection by atomic fluorescence spectrometry have been studied in this work. It was found that under optimum conditions of atomization in the flame in gas shield atomizer, sensitivity was approximately twice higher and detection limit was about four times lower compared to miniature diffusion flame, which is a standard atomizer for atomic fluorescence spectrometry. The conditions to generate hydrides from both inorganic forms of the arsenic, i.e. from arsenite and arsenate, with the same efficiency have been found in the batch arrangement, by using 1 mol dm-3 hydrochlorid acid and 1% solution of tetrahydridoborate. To determine only trivalent form, TRIS buffer at pH 6.00 was used together with 1% sodium tetrahydridoborate. The detection limits found for inorganic arsenic, i.e. for arsenite and arsenate, respectively, were 15 ng dm-3 and 9 ng dm-3 . It was found that in the batch arrangement under these conditions it is possible to generate corresponding hydrides methylarsonate and dimethylarsonate with the same efficiency as from the inorganic form. Finally, it was found when slightly changing the gas-liquid separator design in order to introduce the mixture of tetrahydridoborate with hydrochloric acid to the bottom of the...

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

Normotensive hydrocephalus (NPH) is a neurodegenerative disease that occurs mainly in patients of high age. The disorder causes accumulation of cerebrospinal fluid (CSF), which leads to enlargement of ventricles and pressure exerted on cerebral structures. Clinical manifestations (gait disorders, development of dementia, incontinence) can be easily confused with symptoms of other neurodegenerative diseases; unlike other such disorders, however, NPH can be treated by surgery, if diagnosed in time. Patients are indicated for the procedure by a lumbar drainage test. There are currently no reliable laboratory biomarkers known that could be the basis of NPH diagnostics. In the past, steroids proved to be linked to neuronal activity in neurodegenerative diseases with the help of specific diagnostic markers. An instrumental method has been developed for the purposes of this thesis and it was used to gauge the level of certain steroids in CSF in a sample group of NPH patients and a control sample group of healthy individuals. A significant difference has been found in levels of aldosterone and cortisone. Aldosterone was higher in NPH sufferers, while cortisone levels were higher in the control group. It is crucial to differentiate patients with NPH from patients with similar clinical manifestations during...

The aim of this work was development of high-performance liquid chromatography method with DAD detection for determination of nicotine in tobacco. Standard operating procedure used by World Health Organization was chosen as comparison of the developed method. Optimized high performance chromatography method is suitable for determining nicotine in tobacco. Limit of detection for this method was 0,0003 mg/ml and limit of quantification was 0,0010 mg/ml. Optimization of preparation of samples was significant part of this thesis. Sample preparation procedure was made substantially easier in comparison to other commonly used methods. Nicotine content was determined from real tobacco leaves samples, cigarette tobacco filler, nicotine cartridge for electronic cigarettes and pipe tobacco. Satisfactory relative standard deviation was achieved for all types of samples. Next part of this thesis focused on study of determining polyphenols using high-performance liquid chromatography with diode array detector. Chosen analytes were chlorogenic acid, caffeic acid, rutine, scopoletine and quercitrine. Among the five tested analytes, the highest sensitivity was achieved for chlorogenic acid and caffeic acid. All of the analytes achieved low limits of detection and quantification. Key words Liquid chromatography,...

This work is focused on the interactome study of 14-3-3ζ protein, a regulatory protein found in all eucaryotic cells. An important 14-3-3 protein feature is the ability to bind a number of structurally and functionally distinct protein ligands. This link is usually implemented through phosphorylated serine and threonine motifs. The first aim of this work is the preparation of sufficient amount of recombinant 14-3-3ζ protein with incorporated photoactivatable analogue of methionine (foto-Met, L-2-amino-5,5- azihexan acid). The four different conditions of recombinant expression in auxotrophic E. coli B834 (DE3) strain were tested to obtain a protein with a maximal rate of photoactivatable methionine analogue incorporation into the sequence 14-3-3 protein. The second aim is to study the methionine 121, 160 and 218 participation in the 14-3-3ζ protein binding groove and finding of potential covalent bond with the phosphorylated peptide 251-266 of Raf-1 kinase (phosphorylation on Ser259). The photo-initiated cross-linking method was used (photolysis), to form a reactive biradical of methionine analogue capable to attack any amino acid residues in close vicinity (till 5Å). Finally, the products of photo-initiated cross-linking were analyzed by cross-linking reactions using MALDI-TOF MS, LC-MS and...

The diploma thesis focuses on analysis of fatty alcohols with mass spectrometry. Theoretical part describes distribution, main properties, usage and analytical methods for analysis of fatty alcohols. Experimental work focuses on the search for a proper derivatisation procedure for fatty alcohols, which would enable their detection with soft ionisation techniques of mass spectrometry, as well as their structural analysis. The main aim of the structural analysis is to find the methyl branching in the fragmentation spectra of derivatives of the fatty alcohols.

Raman spectroscopy is an irreplaceable method for chemical and structural characterization of many substances. This is especially true for carbonaceous matter. It is non-destructive and capable of determining the crystallinity of the studied carbonaceous phase, and subsequently, with the aid of empirical relations, also the temperature of formation of the rock under study. Until very recently, the most of the Raman spectroscopy measurements was conducted only single point-wise on grains chosen on purpose, e.g., in an optical microscope. Alongside with the evolution and affordability of the Raman spectroscopic technique it is now very appealing to utilize the possibility of Raman mapping coupled to the automated analysis of large quantities of acquired spectra. The aim of this study was to critically evaluate the pros and cons of such an approach on selected rocks with varying carbon content and crystallinity of the carbonaceous phase. The samples range from graphite, Karelian shungite, elemental carbon-rich carbonates, alpine schist to eclogite. In general, we can rate the large area mapping as suitable in many cases, however, there are certain issues associated with the method, especially concerning the sample preparation and automated analysis. Several actions are proposed to limit the pitfalls...

(EN) The presented dissertation is devoted to hydride generation from arsenic species and its application for speciation analysis based on atomic fluorescence detection. Hydride generation from toxicologically relevant arsenic species was optimized in order to achieve a 100% efficiency. The resulted experimental setup was subsequently used for speciation analysis of arsenic in human urine by high performance liquid chromatography with detection by atomic fluorescence spectrometry. The accuracy of the developed method was verified by comparative analyses of human urine samples collected from five individuals with an independent reference method. The cleavage of As-C bond during the reaction of methylated arsenic species with tetrahydridoborate(1-) (THB) in acidic media was studied in detail. Pronounced demethylation of methylated arsenic species was found during the reaction of THB with HCl, H2SO4, and HClO4 while hydride generation from CH3COOH or TRIS buffer after prereduction with L- cysteine resulted in the exclusive formation of the corresponding hydrides. Firstly, this phenomenon can endanger the accuracy of arsenic speciation which is based on hydride generation of substituted arsanes. Secondly, the more complex arsenic species can be converted to the hydride. That was demonstrated on hydride...

Dolichol is a membrane lipid, which carries monnosaccharides and glycans for N-linked protein glycosylation and glycosylphosphatidylinositol-anchor biosynthesis occuring in endoplasmic reticulum. Its structure is composed of isoprenoid units. Dolichol is present in all tissues and in most of the membrane organelles of eukaryotic cells. Recently some types of congenital disorders of glycosylation have been described as a consequence of dolichol biosynthesis and metabolism defects, which are not detectable by standard methods. The aim of this diploma thesis was to analyze dolichol content in urine and in different tissues from patients with deficiency in dolichol biosynthesis by mass spectrometry and to study the impact of these defects on energetic metabolism. Biological material for this study consisted of urine samples from 76 controls with age ranging from 1 months to 81 years, 6 patients with congenital disorders of glycosylation and 43 patients with suspicion of congenital disorder of glycosylation; samples of frontal cortex, liver, muscle and heart tissues from 2 patients with mutation in NUS1 gene and controls. Urine samples were stored at -20 řC and tissue homogenates were stored in -80 řC until analysis. Lipid fraction after extraction was separated by liquid chromatography. Dolichols were...