Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.

Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.

This diploma thesis is focused on the issue of nonylphenol, degradation product of surfactants. Nonylphenol however most often arises in wastewater treatment plants predominantly during the microbial degradation of nonylphenol ethoxylates used in industry. It is a substance highly bioaccumulative, toxic predominantly to aquatic organisms. Furthermore, it belongs among hormone disruptors. The detailed research was prepared, on the basis which experimental part of diploma thesis was solved. Nonylphenol was determined in samples of the wastewater. The solid phase extraction (SPE) was used for the isolation of the analyte and purification of the extract. The final determination was performed on the gas chromatography with tandem mass spectrometry with time of flight analyzer (TOF). All steps of the preanalytical and the analytical procedure were optimized.

Synthetic musk compounds are artificial organic substances commonly used as fragrant constituents of personal care products as parfums, cosmetics, detergents, in-house cleaning and washing agents. Given their large area of applications and their ability to be persistent, they leaked into all parts of ecosystem, especially aquatic one. There have been a big focus on studiyng these compounds, their properties and fate in the different parts of ecosystems in the last years. The aim of master's thesis was carry out the determination of five representatives (habanolide, exaltolide, ambrettolide, musk MC4 and ethylene brassylate) of macrocyclic musk compounds in waste water samples from influent and effluent of three water treatment plants (WWTP Brno-Modřice, WWTP Lednice and WWTP Mikulov). One of the main tasks was to perform a method optimisation for the determination of selected musk compounds in waste water and evaluation and interpretation of the results. Solid phase microextraction (SPME) technique was used for the extraction of analytes, consequently gas chromatography-mass spectrometry was used for analysis of selected analytes. Following evaluation the removal efficiency of musk compounds in the waste water treatment plant was assessed.

This work was focused on the determination of musk compounds in various components of the environment. Synthetic musk compounds are organic substances commonly used as fragrant constituents of detergents, perfumes, cosmetics and other personal care products (PCPs). These compounds contaminate the aquatic environment through municipal wastewater from sewage treatment plants. The result is accumulation of these analytes in the environment and their occurrence in food chain. Synthetic fragrances are commonly detected also in human body; these substances generally belong among relatively new persistent organic pollutants (POPs) because of their persistence, bioaccumulation, and potential toxicity. The aim of this work was to optimize the methods for the determination of selected synthetic fragrances in wastewater (municipal, industrial) and in fish muscle. These analytes were determined in wastewater [the wastewater treatment plant (WWTP) Brno – Modřice, cosmetic manufacturing plant] and in fish Squalius cephalus that had been caught in the river Svratka. The selected analytes (14 substances) originate from the group of nitromusk, polycyclic musk and linear musk compounds. Solid phase microextraction (SPME) [for wastewater samples] and pressurized solvent extraction (PSE) [for fish samples] were used for isolation of analytes from real samples. The identification and quantification of analytes were carried out by high resolution gas chromatography coupled with mass spectrometry detection (GC-MS). The results show that galaxolide (g.L-1) and tonalide (tenths of g.L-1) were polycyclic musk compounds which were found at the highest concentration level in influent and also in effluent samples from the WWTP Brno – Modřice. The most extended nitromusk compound was musk ketone (hundredths of g.L-1). Linear musk compounds were detected in all influent samples (g.L-1). The overall removal efficiency of synthetic musk compounds from wastewater in the WWTP varies within the following ranges: nitromusk compounds 34.4 % – 85.4 %, polycyclic musk compounds 36.7 % – 53.3 % and linear musk compounds 90.5 % – 99.9 %. On the basis of the results it can be stated that wastewater (effluent from the WWTP) is the primary source of musk compounds, especially of the polycyclic ones in the environment. It has been also shown that ultrafiltration used in a cosmetic plant is a highly efficient technology for removing nitromusk and polycyclic musk compounds from industry wastewater. The results from evaluation of the fish contamination by musk compounds show that galaxolide was the substance which reached the highest concentration levels (tens of g.kg-1 l.w.). The results corroborate ubiquitous occurrence of musk compounds in the environment and reduction of using of nitromusk compounds due to their potential toxic properties. It has been shown that the insufficiency of the purifying procedures used in the WWTP Brno – Modřice has a significant influence on the concentration level of polycyclic musk compounds (tonalide, galaxolide) in fish caught from the river Svratka.

This work is focused on the study of penetration of pesticides and pharmaceuticals in the aquatic environment. The aim was to develop and verify the optimal method for long-term monitoring of the occurrence of selected pesticides and pharmaceuticals in surface waters. Two frequently used analgesics ibuprofen and diclofenac, and carbamazepine that is used to treat epilepsy, were chosen from the group of drugs. A group of 41 pesticides were selected for monitoring too. The attention was focused on a group of urea pesticides and phenoxyacetic acids, as well as on selected pesticides falling to the group of priority substances or identified as environmental quality standards. Some of pesticides were selected because their consumption in the Czech Republic exceeds 30 tons a year. Liquid chromatography with tandem mass spectrometry (LC / MS / MS) was used for the determination of selected analytes in surface waters. Two optimized analytical method were developed. Ten analytes were determined in negative ion mode and 34 analytes in positive ion mode. Solid phase extraction (SPE) was used for purification and concentration of the samples. Developed methodology was validated and used for monitoring of selected compounds in samples of surface waters from the Morava River basin. Samples were collected on the rivers Morava and Svratka monthly for two years. Samples from the river Blata and stream Nivnička were collected for one year. Surface water samples were also collected from the river Litava. Four samples were taken throughout the length of the flow during one day. Smaller streams were chosen for monitoring because of the probability of occurrence of pesticides. They flow abundantly agriculturally cultivated areas. Drugs ibuprofen and diclofenac were detected in all taken samples, carbamazepine mostly. Among the most commonly occurring pesticides were bentazon, diuron, isoproturon, carbendazim, MCPA, MCPP, propiconazole, tebuconazole and 2,4-D.

This work is focused on the determination of polar pesticides in surface and waste waters. In this study Mecoprop-P, MCPA, Dichlorprop-P, 2,4-D and MCPBA in differently loaded surface waters and communal waters from sewage treatment plants were analyzed. These herbicides are included in the group identified as environmental quality standards. Some of herbicides (2,4-D and MCPA) were selected because their consumption in the Czech Republic exceeds 30 tons a year. Samples were collected prom three rivers near town of Jaroměř (Elbe, Úpa and Metuje Rivers), from one river near the town of Brno (Svratka River) and from inflow and outflow of two sewage treatment plants (WWTP Jaroměř, WWTP Brno – Modřice). Particular rivers were chosen for monitoring due to the probability of occurrence of herbicides, because they flow through agriculturally cultivated areas. Solid phase extraction (SPE) was used for the isolation of target compounds and their concentration. Gas chromatography with tandem mass spectrometry (GC/MS/MS) was used for the determination of target analytes in surface and waste waters. Selected herbicides were detected in all collected samples.

La plupart des bactéries vivent en communautés organisées, appelées biofilms, augmentant leur résistance aux traitements antibiotiques. Ainsi, la formation de biofilms sur les organes et matériels médicaux est considérée comme la cause de la majorité des infections bactériennes. Il est alors important de trouver des traitements pour empêcher ou perturber la formation de ces biofilms. Il a été proposé que les porines de P. Stuartii, Omp-Pst1 et Omp-Pst2, peuvent s’auto-assembler via un steric-zipper, étape responsable du développement initial du biofilm. Ainsi, notre objectif est de synthétiser des inhibiteurs d’interactions entre porines pour limiter ce contact intercellulaire. Nous avons développé des molécules peptidomimétiques basées sur la séquence LGNYR, active dans les deux porines. Pour cela, des réactions de chimie click sur phase solide ont été mises en oeuvre afin de synthétiser des analogues de cette séquence, comme la CuAAC pour introduire un motif triazole, dans une position variable au sein du peptide. Nous avons ainsi développé une méthode rapide et efficace afin de réaliser cette réaction par l’utilisation d’un catalyseur cuivre(I)-N-hétérocyclique carbène stable à l’air. Similairement, de nouvelles conditions ont aussi été mises au point en phase solide afin d’obtenir régiosélectivement des peptides comportant un motif isoxazole 3,4- ou 3,5-disubstitué, par la réaction entre un alcyne et un oxyde de nitrile. Ces cycloadditions 1,3-dipolaires nous ont ainsi permis d’obtenir une première librairie de peptidotriazoles et de peptidoisoxazoles. Il sera enfin possible d’étudier les biofilms grâce à la synthèse de sondes fluorescentes basées sur les inhibiteurs montrant de fortes affinités avec la cible, couplées à un fluorophore dérivé de coumarine. / In the environment, most of bacteria live as organized communities, known as biofilms, enhancing their resistance to antibiotic treatments. Thus the formation of biofilms on organ and indwelling medical devices is considered to cause the majority of bacterial infections in the human body. Thus, to enhance antibiotics efficacy, there is a high need to find treatments to prevent or disrupt biofilm formation. It has been proposed that P. stuartii porins, Omp-Pst1 and Omp-Pst2, can self-associate through a steric zipper, being responsible for the initial development of biofilms. Thus, our objective is to synthesize porin’s self-matching interactions (PSMI) inhibitors to counterfeit this intercellular contact. We developed peptidomimetic molecules based on the LGNYR sequence, that have shown to be active in both porins. Then we used click chemistry to synthesize on solid phase analogues of this sequence, as the solid phase Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to introduce a triazole moiety into the peptide chain at different positions. We thus developed a fast and efficient method to perform this reaction using a stable copper(I)-N-heterocyclic carbene catalyst. Similarly, new conditions were developed on solid phase to synthesize regioselectively peptides containing a 3,4- or 3,5-disubstituted isoxazole moiety, through the reaction between an alkyne and a nitrile oxide. These 1,3-dipolar cycloadditions allowed us to developed a first library of peptidotriazoles and peptidoisoxazoles. We also obtained fluorescent probes based on the inhibitors showing higher affinity for the target as tools to study biofilm formation.

The overall thrust of this dissertation is to gain a comprehensive understanding over the factors that govern the performance and behavior of ultra-thin, cross-linked polymer films. Poly(NIPAAm) was used as a model polymer to study volume phase transition in surface tethered networks. Poly(NIPAAm) undergoes a reversible phase transition at approximately 32°C between a swollen hydrophilic random coil to a collapsed hydrophobic globule state, thought to be caused by increased hydrophobic attractions between the isopropyl groups at elevated temperatures. We present a simple photochemical technique for fabricating structured polymer networks, enabling the construction of responsive surfaces with unique properties. The approach is based on the photo-cross-linking of copolymers synthesized from N-isopropylacrylamide and methacroyloxybenzophenone (MaBP). In order correlate layer swelling to the MaBP content, we have studied the swelling behavior of such layers in contact with aqueous solutions with neutron reflection. The cross-linked networks provide a three-dimensional scaffold to host a variety of functionalities. These networks serve as a platform which can be used to amplify small local perturbations induced by various stimuli like temperature, pH, solvent, ionic strength and peptide modified hydrogels to bring about a macroscopic change. Neutron reflection experiments have shown that the volume-phase transition of a surface-tethered, cross-linked poly(NIPAAm) network coincided with the two-phase region of uncross-linked poly(NIPAAm) in solution. Parallel measurements with ATR-FTIR investigating the effect of temperature, pH and salts suggest that the discontinuous transition is the result of cooperative dehydration of the isopropyl groups, with water remaining confined between amide groups in the collapsed state as weakly hydrogen bonded bridges. Hybrid polymers with specific peptide sequences have shown specific response to external cues such as pH and metal ions exhibiting unique phase behavior.

Le but de cette thèse a concerné le développement de deux types de structures oligomériques contraintes, basées soit sur des dérivés non naturels d’acides aminés cycliques, soit sur des dimères cycliques de Peptide Nucleic Acids (PNA), en vue d’analyser leur tendance à adopter des conformations pré-organisées, imitant les conformations actives des protéines ou des acides nucléiques. La première partie de cette thèse a trait à l’élaboration de synthons clefs dérivant d’acides α-aminés cycliques non naturels (acides 4-oxopipécoliques 6-substitués), à leur utilisation potentielle dans la synthèse de peptidomimétiques contraints, homogènes et hétérogènes, ainsi qu’à leurs limites dans cette utilisation. Dans le cadre de ce travail, une nouvelle voie de synthèse permettant d’accéder à des résidus N-protégés d'acides 4-oxo-pipécoliques 6substitués a été mise au point. La deuxième partie de cette thèse concerne l’élaboration de dimères d’α-PNA (di-α-PNA) cycliques, dans lesquels les chaînes latérales de deux monomères d’α-PNA consécutifs sont «agrafées» via un pont lactame. Une stratégie de synthèse a tout d'abord été développée en phase liquide, puis appliquée à la synthèse en phase solide de di-α-PNA «agrafés» modèles, incorporant des bases nucléiques thymine. / The purpose of this thesis concerned the development of two kinds of constrained oligomeric structures, based either on unnatural cyclic α-amino acids derivatives or on cyclic Peptide Nucleic Acid (PNA) dimers, in view of analyzing their propensity to adopt pre-organized conformations mimicking the active conformations of proteins or nucleic acids. The first part of this thesis reports on the elaboration of new cyclic αamino-acids (6-substituted 4-oxopipecolic acids) building blocks, their potential use in the synthesis of constrained homogenous and heterogeneous peptidomimetics as well as their limitations in this use. As part of this work, a new methodology has been developed for the synthesis of N-protected 6-substituted 4-oxo-pipecolic acids residues. The second part of this thesis reports on the elaboration of constrained α-PNA dimers (di-α-PNA), in which the side-chains of two consecutive α-PNA monomers are “stapled” via a lactam bridge. A synthetic orthogonal strategy has been first developed in liquid-phase then applied to the solid-phase synthesis of models “stapled” di-α PNA incorporating thymine nucleobases.