English Abstract The incoming paradigm of the network (or systems) biology calls for a new high throughput tool for a wide scale study of protein-protein interactions. Mass spectrometry-based proteomics have experienced a great progress in recent years and have become an indispensable technology of elementary as well as clinical research. Glutamate carboxypeptidase II (GCPII; EC 3.5.17.21) is a transmembrane protein with two known enzymatic activities. Its expression is highly upregulated in some solid tumors and also in tumor-associated neovasculature in general. Nevertheless, none of the two enzymatic activities were shown to be physiologically relevant to these cells. Some facts point at a possible receptor function of GCPII, however, no specific binding partner has been found yet. In the search for potential binding partners and/or ligands of GCPII, a series of methods have been employed, including pull-down experiment, immunoprecipitation and mass spectrometry. Sample preparation and mass spectrometry data processing methodology was specifically developed in order to identify potential binding partners. As one of the outcome of that methodology, the interaction of β-subunit of F1 ATP synthase was selected for further detailed analysis as a putative ligand of GCPII.

The aim of the diploma thesis was to find optimal conditions of high pressure liquid chromatography for the detection and quantification of two common nucleotides, namely adenosine diphosphate and adenosine triphosphate, as well as to perform an analysis of these in real life samples of citrus fruits and plant extracts. Further aim of the project was to determine the limits of detection and quantification of adenosine diphosphate and adenosine triphosphate under the optimized conditions and using these to compare the sensitivity of given detectors. To achieve this HPLC-UV, capillary HPLC-DAD and HPLC-MS apparatus were used. With the help of HPLC with UV detection and capillary HPLC with diode array detector, the calibration curves of the mixture of analytes were measured and the limits of detection as well as quantification of adenosine diphosphate and adenosine triphosphate were determined. Separation of the analytes up to the base line using HPLC-UV and capillary HPLC-DAD was achieved under the conditions of ion pairing chromatography. Column C18 was chosen as an appropriate column. The mobile phase included phosphate buffer, acetonitrile and tetrabutylammonium bisulphate as an ion pairing reagent. The separation was performed with gradient elution. Conditions for analysis using LC-MS were...

In this work we report the formation of inclusion complexes of the pesticides 1-naphthylacetic acid, 1-naphthylacetamide, napropamide and carbaryl with cucurbit[n]uril (n=7 and 8) and -cyclodextrin hosts. We also report results of the photochemistry of these compounds when free in aqueous solution and included within these nano-containers. The formation of inclusion complexes was studied by electrospray ionization mass spectrometry (ESI-MS) which gave us information about stoichiometry of the complexes and about their reactivity in the gas phase. The photodecomposition and formation of products was followed by high performance liquid chromatography coupled to both mass spectrometry (HPLC-MS) and UV/VIS detector, and spectrophotometry. The studied CBs and their host-guest complexes were readily detected in the gas phase by ESI-MS, mainly as single and double charged ions containing either H+ , Na+ or K+ . The assignments were made based on the m/z values of the observed signals and further confirmed by fragmentation (tandem mass spectrometry - MSn ). All pesticides formed 1:1 (host:guest) complexes. Carbaryl, napropamide and 1-naphthylacetamide also formed 1:2 (host:guest) complexes. Under some solution chemistry conditions, namely for concentrations of cucurbiturils above 50 µM and at low acid and...

Bumblebees are important pollinators, commercially used in large-scale plant growing in greenhouses. Their males produce marking pheromones for mating, which attract young bumblebee queens. These pheromones are often a complicated mixture of chemicals, which is produced in the male labial gland, and the mixture itself is specific for each bumblebee species. The regulation of bumblebee sexual pheromone biosynthesis is largely unknown, and this Master's thesis is focused on the analysis of the mechanisms which lead to the regulation of the Bombus terrestris male pheromone's fat-acid and terpenes biosynthesis, specifically on stereospecific enzymatic reduction of double bond of farnesol. This thesis studies the influence of potential neurohormones on a specific enzymatic mixture, which is involved in the pheromone biosynthesis. Methods used in this project include biochemical, analytic and molecular-biology methods.

This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...

A comprehensive understanding of physiological role of proteins requires knowledge of their three-dimensional structure, dynamics and protein-protein interactions. Chemical cross-linking in combination with mass spectrometry represents an alternative approach to standard methods for protein structure elucidation (X-ray crystalography, NMR spectroscopy) and enables characterization of interaction interface within protein complexes in their native states. The presented thesis is mainly focused on novel cross-linking methodology based on the in vivo incorporation of methionine analog with photo-reactive functional group (photo-Met) into the sequence of studied protein (so called protein photo-nanoprobe). Interaction between two molecules of 14-3-3zeta protein was used as a model to test and optimize the protein photo-nanoprobe production. The findings confirmed usefulness of this approach for mapping the protein-protein interactions. The photo-initiated cross-linking was used to detect the heterooligomeric membrane structures of cytochromes P450 2B4 and b5 and the molar ratio of cytochromes within individual complexes was assessed. The chemical cross-linking in combination with mass spectrometry was employed to characterize the interaction of their catalytic domains and two mutual orientations of...

In dieser Arbeit werden spektroskopische und spektrometrische Methoden diskutiert, um Verfahren auf Grundlage von umfassenden, molekularen Informationen für die Charakterisierung, Klassifizierung und Identifizierung von Pollen zu entwickeln. Es ist bereits bekannt, dass Raman- und Infrarot-Spektroskopie spezifische Fingerabdrücke der chemischen Zusammensetzung von Pollen liefern können. Im Rahmen dieser Arbeit werden nun die oberflächenverstärkte Raman-Streuung (surface-enhanced Raman scattering, SERS), die matrix-assisted laser desorption/ionisation time-of-flight-Massenspektrometrie (MALDI-TOF MS) und die Fourier-Transform-Infrarot-Spektroskopie (FTIR-Spektroskopie) einzelner Pollenkörner für die taxonomische Klassifizierung eingesetzt. Neben der Anwendung zur taxonomischen Klassifizierung können spektroskopische und spektrometrische Methoden auch zur Erkennung von Unterschieden, die durch Umwelteinflüsse verursacht werden, eingesetzt werden. Als ein erstes Beispiel dieser Anwendung werden cherry leaf roll virus (CLRV) infizierte Birkenpollen untersucht und mit Pollen von Kontrollpflanzen verglichen. Die Ergebnisse dieser Arbeit zeigen, dass spektroskopische und spektrometrische Methoden vielversprechend für die Pollenanalytik in vielen verschiedenen Forschungsgebieten sind. / The investigation of pollen grain samples for characterization, classification and identification is still an analytical task that is both challenging and time-consuming, since it is mainly based on the morphological characteristics of the pollen grains. In order to develop approaches that are based on extensive molecular information and that lead to an automated classification, in this thesis, a combination of spectroscopic and spectrometric tools is discussed. It is already known that a fingerprint analysis of the chemical composition of pollen samples can be accomplished by Raman and infrared spectroscopies. Here, surface-enhanced Raman scattering (SERS), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and Fourier transform infrared (FTIR) spectroscopy of single pollen grains are successfully applied for the taxonomic classification of pollen samples. In addition to the application for taxonomic classification, it is conceivable that spectroscopic and spectrometric data could also be used to investigate chemical changes caused by environmental influences. As a first example for such an application, cherry leaf roll virus (CLRV) infected birch pollen are investigated and compared to samples from control plants. The results in this work indicate that spectroscopic and spectrometric methods are powerful analytical tools that may be useful for improved pollen investigation in different areas of research.

Das Monitoring von Landwirtschaftsflächen ist eines der Kernthemen der zukünftigen EnMAP Mission, einem deutschen, hyperspektralen Fernerkundungssensor, dessen Start für 2015 geplant ist. In Vorbereitung dieser Mission gehören die Erweiterung und Entwicklung objektiver, robuster sowie zuverlässiger Methoden zur Ableitung biophysikalischer Parameter zu den Hauptaufgaben. Die für das Monitoring von Vegetation notwendige hohe zeitliche Auflösung wird durch ein stufenloses Schwenken von bis zu +/-30° quer zur Flugrichtung erreicht. Daraus resultiert, dass die Daten durch variierende Ein- und Ausstrahlungsgeometrien stark beeinflusst werden. Daher ist eine detaillierte Kenntnis der bidirektionalen Reflexionsfunktion (engl. bidirectional reflectance distribution function, BRDF) der beobachteten Oberflächen notwendig, um diese Einflüsse auf das Reflexionssignal zu identifizieren und anschließend zu korrigieren. Zu diesem Zweck wurde in dieser Arbeit eine Methodik entwickelt, die es ermöglicht, auf der Basis von simulierten Spektren realistische BRDF Szenarien zu modellieren und oberflächenspezifische Korrekturfunktionen abzuleiten. Die Methodik besteht aus drei aufeinander aufbauenden Komponenten. Im ersten Schritt erfolgt die Modellierung der BRDF von landwirtschaftlichen Vegetationsbeständen. Im zweiten Schritt wird der Einfluss der Bestandsarchitektur auf die BRDF analysiert. Darüber hinaus wird untersucht, inwiefern sich Variationen in der BRDF auf die quantitative Ausprägung von Vegetationsindizes auswirken. Solche Indizes sind eine häufig genutzte Möglichkeit zur Quantifizierung biophysikalische Parameter im Rahmen empirischer Verfahren. Aufbauend auf den gewonnenen Erkenntnissen wurden im dritten Schritt Korrekturfunktionen für ausgewählte Vegetationsindizes entwickelt, um Schrägblickbeobachtungen in Nadirbeobachtungen zu transformieren. Abschließend wurde die entwickelte Methodik auf simulierte, sensorspezifische Spektren übertragen. / Monitoring of arable crops is one of the core applications of the upcoming spaceborne EnMAP mission, a German hyperspectral imaging spectrometer scheduled for launch in 2015. During the present preparatory phase one of the primary tasks is the development of accurate, robust and reliable retrieval methods for biophysical canopy parameters. Monitoring of crop canopies requires a frequent temporal coverage. In case of EnMAP, this will be realised by an off-nadir pointing of the sensor up to +/-30° across to the flight direction. The off-nadir pointing leads to data strongly influenced by varying acquisition geometry. Therefore, detailed knowledge of bidirectional reflectance distribution functions (BRDF) of the observed surfaces is necessary to identify and to correct BRDF influenced reflectance signals. For this purpose, a methodology was developed that allows modelling of realistic BRDF scenarios and of surface-specific correction functions. This methodology consists of three consecutive parts. In the first part, modelling of the BRDF of crop canopies was performed. In the second part, the influence of canopy architecture on the BRDF was analysed. Additionally, the BRDF related dependencies of vegetation indices were investigated. Such indices are widely used to quantify biophysical canopy parameters based on empirical methods. In the third part, correction functions were developed for selected vegetation indices to enable a transformation from off-nadir into nadir observations. These correction functions incorporate results obtained in the second part of the methodology. Finally, the developed methodology was applied on sensor-specific simulated spectra.

This thesis is focused on the development of an analytical method for the determination of volatile fatty acids in aqueous samples using GC-MS/MS. The theoretical section provides general information on a selected group of acids, their significance in the biogas plants, the means of their derivatization and the analytical techniques used. The practical part mainly deals with optimizition of various parameters in the derivatization reaction to achieve optimal conditions. Finally, the analytical method was successfully validated and applied to the analysis of a real sample.

This diploma thesis deals with the ongoing research under the auspices of the Department of Applied Physics and Technics Faculty of Education, University of South Bohemia in České Budějovice, in which the author of the work was actively participated. The thesis is divided into theoretical and experimental part. The theoretical part contains information from natural polymers, especially cellulose, plasma technology and infrared spectrometry. The subject of the experimental part of the thesis is research that deals with the functionalization of cellulose using a microwave plasma discharge on the apparatus CX-22. In the present research was also conducted to process optimization of functionalization with the liquid precursor hexamethyldisiloxane (HMDSO).