Peptide sequencing by surface-induced dissociation (SID) on a MALDI-Ion Mobility-orthogonal-TOF mass spectrometer is demonstrated. The early version of the instrument used for proof-of-concept experiments achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (ca. 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOFMS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (peptide mass mapping) and partial sequence analysis, (real time tandem mass spectrometry.) Optimization of the MALDI-IM-SID-o-TOF mass spectrometer for peptide sequencing is discussed. SID spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. Optimum collision energies differ for the various surfaces. The fragmentation patterns observed for a series of peptides and protein digests using the Nd:YAG laser (355 nm) for MALDI ion formation and an FSAM surface for ion activation is compared to the fragmentation patterns observed for CID and photodissociation. The fragmentation patterns observed in all cases are strikingly similar. Photodissociation produced a greater abundance of ions resulting from side-chain cleavages. As a general rule optimized SID spectra contain fewer immonium ions than either photodissociation or CID. Evaluation of an instrument incorporating a new hybrid drift cell is discussed. Spectra for a digest of hemoglobin is compared to that acquired with an ABI 4700 TOF-TOF. The performance of the instrument is also evaluated using a micro-crystal Nd:YAG laser (355 nm) for MALDI operated at 400 Hz. Experiments were performed to determine the sensitivity and overall performance of the instrument. The reproducibility of the MS/MS spectra for gramicidin S is shown to be 94% run-to-run. The best mobility resolution obtained for a neat deposition of the dye Crystal Violet was 60 t/∆t. Sensitivity was tested with the peptide fibrinopeptide A (m/z 1537, AA sequence ADSGEGDFLAEGGGVR). Data acquired for sixty seconds with approximately sixty femtomoles deposited. Abundant [M+H]+ ions where observed as well as [M+H]+-NH3 ions. The S/N for this short run was insufficient to identify any SID fragments

Buildings, maps and objects et cetera, can be modeled using a computer or reconstructed in 3D by data from different kinds of cameras or laser scanners. This thesis concerns the latter. The recent improvements of Time-of-Flight cameras have brought a number of new interesting research areas to the surface. Registration of several ToF camera point clouds is such an area. A literature study has been made to summarize the research done in the area over the last two decades. The most popular method for registering point clouds, namely the Iterative Closest Point (ICP), has been studied. In addition to this, an error relaxation algorithm was implemented to minimize the accumulated error of the sequential pairwise ICP. A few different real-world test scenarios and one scenario with synthetic data were constructed. These data sets were registered with varying outcome. The obtained camera poses from the sequential ICP were improved by loop closing and error relaxation. The results illustrate the importance of having good initial guesses on the relative transformations to obtain a correct model. Furthermore the strengths and weaknesses of the sequential ICP and the utilized error relaxation method are shown.

Recently, the limited-angle TOF-PET system has become an active topic mainly due to the considerable reduction of hardware cost and potential applicability for performing needle biopsy on patients while in the scanner. However, this kind of measurement configurations oftentimes suffers from the deteriorated reconstructed images, because insufficient data are observed. The established theory of Compressed Sensing (CS) provides a potential framework for attacking this problem. CS claims that the imaged object can be faithfully recovered from highly underdetermined observations, provided that it can be sparse in some transformed domain. In here a first attempt was made in applying the CS framework to TOF-PET imaging for two undersampling configurations. First, to deal with undersampling TOF-PET imaging, an efficient sparsity-promoted algorithm was developed for combined regularizations of p-TV and l1-norm, where it was found that (a) it is capable of providing better reconstruction than the traditional EM algorithm, and (b) the 0.5-TV regularization was significantly superior to the regularizations of 0-TV and 1-TV, which are widely investigated in the open literature. Second, a general framework was proposed for sparsity-promoted ART, where accelerated techniques of multi-step and order-set were simultaneously used. From the results, it was observed that the accelerated sparsity-promoted ART method was capable of providing better reconstruction than traditional ART. Finally, a relationship was established between the number of detectors (or the range of angle) and TOF time resolution, which provided an empirical guidance for designing novel low-cost TOF-PET systems while ensuring good reconstruction quality.

Environmental field analysis provides advantages that allow real-time decisions, interactive sampling and cost effective solutions to the problems faced at the time of investigation. Gas chromatography (GC), a widespread technique for the determination of organic pollutants in the environment, has also shown to be useful in environmental field analysis. Thermal desorption of solid environmental sample provides a technique for liberation of volatile analytes from the samples without the need for solvent extraction. Combining the thermal desorption technique with a field gas chromatograph (GC) thus provides the possibility of on-site determination of organic contaminants in soils. However, to better characterize trace level contaminants in complex sample matrices, laboratory analysis using analytical instrument with great separation and resolution power is required. Comprehensive two-dimensional gas chromatography (GC X GC) is such a powerful analytical tool that provides enhanced separation and resolution capacity for the task. The project presented here involves the development and validation of a field method for the analysis of total polychlorinated biphenyls (PCBs) in soils, and determination of individual PCB congeners in the same samples by further laboratory analysis. The field analytical system developed was a field portable GC interfaced with a thermal desorber. The identification of PCB congeners was realized by a GC X GC system with a time-of-flight mass spectrometer (TOF-MS) as a detector. The field method was developed by optimizing and characterizing the method using PCB standards, followed by the application of the developed method to environmental soil samples. Finally, analyses of PCB congeners in environmental soil samples were performed using the GC X GC system.

Environmental field analysis provides advantages that allow real-time decisions, interactive sampling and cost effective solutions to the problems faced at the time of investigation. Gas chromatography (GC), a widespread technique for the determination of organic pollutants in the environment, has also shown to be useful in environmental field analysis. Thermal desorption of solid environmental sample provides a technique for liberation of volatile analytes from the samples without the need for solvent extraction. Combining the thermal desorption technique with a field gas chromatograph (GC) thus provides the possibility of on-site determination of organic contaminants in soils. However, to better characterize trace level contaminants in complex sample matrices, laboratory analysis using analytical instrument with great separation and resolution power is required. Comprehensive two-dimensional gas chromatography (GC X GC) is such a powerful analytical tool that provides enhanced separation and resolution capacity for the task. The project presented here involves the development and validation of a field method for the analysis of total polychlorinated biphenyls (PCBs) in soils, and determination of individual PCB congeners in the same samples by further laboratory analysis. The field analytical system developed was a field portable GC interfaced with a thermal desorber. The identification of PCB congeners was realized by a GC X GC system with a time-of-flight mass spectrometer (TOF-MS) as a detector. The field method was developed by optimizing and characterizing the method using PCB standards, followed by the application of the developed method to environmental soil samples. Finally, analyses of PCB congeners in environmental soil samples were performed using the GC X GC system.

Hemoglobin plays an important role in transporting oxygen in human beings and other mammals. Hemoglobin is a tetrameric protein composed of two alpha and two beta subunits. The £\ and £] subunits are both necessary and the stoichiometric ratio of the two dislike subunits is critical for hemoglobin to perform its oxygen-carrying function properly. To better understand the coupling between the £\ and £] subunits and the subunit disassembly pathway, p-hydroxymercuri-benzoate (PMB) has been used to react with the cysteine residues in hemoglobin. The hemoglobin tetramer becomes unstable and disassembles into £\ and £] subunits when the cysteine sites are perturbed upon reacting with PMB. There are three kinds of cysteine residues, £]93, £\104 and £]112, in human hemoglobin. The reactivity of different cysteine residues with PMB and their reaction sequence have been studied via the Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The resonance Raman spectroscopy has been used to investigate the structural changes of hemoglobin accompanying the PMB-modification under the oxygenated and deoxygenated conditions. At last, a hemoglobin subunit disassembly mechanism is proposed and the site-specific roles of cysteine residues in human hemoglobin are discussed in detail.

The fungus Cunninghamella elegans has in many studies shown to have abiotransformation similar to the metabolism of mammals. If the biotransformation isgeneral, it enables the production of metabolites by the fungus and the use asreference material. The purpose of the project were to examine whether themetabolic process of C. elegans is general, with respect to the formation ofglucosides, and can be applied to different classes of drugs. During the project, theanalyses were performed on a UPLC Q-TOF, run in both MSE and MSMS mode. Themobile phase used consisted of MeOH and 0.1 % formic acid in MQ water. Toincrease the concentration of possible glucosides, the samples were subjected to anacidic or alkaline SPE. Glucosides were detected in the fungal incubates of diclofenac,buprenorphine, norbuprenorphine and oxazepam. For diclofenac, besides twodifferent glucosides (diclofenac glucoside and hydroxylated diclofenac glucoside), ahydroxylated metabolite and a hydroxylated metabolite conjugated with sulfate werediscovered. In the samples containing buprenorphine, the phase I metabolitenorbuprenorphine was also encountered. Further, in the fungal incubates ofdexamethasone a defluorinated metabolite was identified, which is a metabolicpathway never before described for C. elegans.ISSN: 1650

Peptide sequencing by surface-induced dissociation (SID) on a MALDI-Ion Mobility-orthogonal-TOF mass spectrometer is demonstrated. The early version of the instrument used for proof-of-concept experiments achieves a mobility resolution of approximately 20 and TOF mass resolution better than 200. Peptide sequences of four peptides from a tryptic digest of cytochrome c (ca. 1 pmol deposited) were obtained. The advantage of IM-SID-o-TOFMS is that a single experiment can be used to simultaneously measure the molecular weights of the tryptic peptide fragments (peptide mass mapping) and partial sequence analysis, (real time tandem mass spectrometry.) Optimization of the MALDI-IM-SID-o-TOF mass spectrometer for peptide sequencing is discussed. SID spectra obtained by using stainless steel, Au grids, and fluorinated self-assembled monolayers (F-SAM) on Au are compared. Optimum collision energies differ for the various surfaces. The fragmentation patterns observed for a series of peptides and protein digests using the Nd:YAG laser (355 nm) for MALDI ion formation and an FSAM surface for ion activation is compared to the fragmentation patterns observed for CID and photodissociation. The fragmentation patterns observed in all cases are strikingly similar. Photodissociation produced a greater abundance of ions resulting from side-chain cleavages. As a general rule optimized SID spectra contain fewer immonium ions than either photodissociation or CID. Evaluation of an instrument incorporating a new hybrid drift cell is discussed. Spectra for a digest of hemoglobin is compared to that acquired with an ABI 4700 TOF-TOF. The performance of the instrument is also evaluated using a micro-crystal Nd:YAG laser (355 nm) for MALDI operated at 400 Hz. Experiments were performed to determine the sensitivity and overall performance of the instrument. The reproducibility of the MS/MS spectra for gramicidin S is shown to be 94% run-to-run. The best mobility resolution obtained for a neat deposition of the dye Crystal Violet was 60 t/∆t. Sensitivity was tested with the peptide fibrinopeptide A (m/z 1537, AA sequence ADSGEGDFLAEGGGVR). Data acquired for sixty seconds with approximately sixty femtomoles deposited. Abundant [M+H]+ ions where observed as well as [M+H]+-NH3 ions. The S/N for this short run was insufficient to identify any SID fragments

Eine traumatische Läsion am artikulären Knorpel stellt eine noch ungelöste Herausforderung für den behandelnden Arzt dar. Bei den betroffenen Patienten handelt es sich häufig um junge, sportlich aktive Menschen im Arbeitsprozess {Hjelle et al. 2002}, bei denen eine längerfristige Belastungs- und Bewegungseinschränkung oder sogar eine Verminderung der Erwerbsfähigkeit zwingend vermieden werden muss. Jedoch existiert für den traumatischen Gelenkknorpelschaden derzeit noch keine Therapie mit sehr gutem, funktionellem Langzeitergebnis {Richter 2005}. Die konservativen Therapieformen haben immer eine narbige Ausheilung zur Folge. Aber auch mit der chirurgischen Basisversorgung, bestehend aus Debridement und Lavage des betroffenen Gelenks, kann keine Ausheilung erreicht werden. Regenerierende Verfahren, die auf der Grundlage der Penetration des subchondralen Knochens basieren, sollen durch das Einspülen von körpereigenen Stammzellen in den Defekt eine autologe Regeneration induzieren. Langzeitstudien zeigen, dass trotz der oft erreichten, guten funktionellen Ergebnisse histomorphologisch keine Wiederherstellung von intaktem, hyalinem Gelenkknorpel erreicht werden kann {Gaissmaier et al. 2003, Bernholt/Höher 2003}. Vielversprechende neue Therapiekonzepte liefert derzeit das „Tissue Engineering“ am Gelenkknorpel. Vor allem die Autologe Chondrozytentransplantation (ACT) eröffnet vollkommen neue Behandlungsstrategien. Bei der ACT werden arthroskopisch patienteneigene Chondrozyten gewonnen, die in dreidimensionale Gele eingesät und in Zellkultur gebracht werden. Durch verschiedene Stimulationstechniken werden die Zellen zur Proliferation und Produktion von Extrazellulärer Matrix (ECM), insbesondere Kollagen und Proteoglykan, angeregt. Nach drei bis sechs Wochen Kultivierung erfolgt die Implantation in den aufbereiteten Knorpeldefekt des Patienten. Aktuelle Studien konnten zeigen, dass das Transplantat mittelfristig ohne Narbenbildung in den bestehenden Gelenkknorpel einwächst und so die Inkongruenz des Gelenks, welche präarthrotisch wirkt, aufhebt {Brittberg et al. 1996, Peterson et al. 2000, Horas et al. 2000}. Um dieses neuartige Verfahren schnell im klinischen Alltag zu etablieren, bedarf es ausgereifter analytischer Methoden, die eine Qualitätsprüfung des biotechnologisch hergestellten Knorpels ermöglichen. MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) ist eine schnelle und sehr sensitive Methode, um die molaren Massen von Stoffen genau zu bestimmen und komplex zusammengesetzte Proben zu analysieren. Ziel dieser Arbeit war es, MALDI-TOF Massenspektrometrie als Analyseverfahren zu nutzen, um die Zusammensetzung des natürlichen Knorpels zu bestimmen und die hier gewonnenen Erkenntnisse auf biotechnologisch hergestellten Knorpel anzuwenden. Somit war die Überprüfung der Anwendbarkeit dieser massenspektrometrischen Untersuchungsmethode auf das komplexe biologische System Knorpel die erste Fragestellung in dieser Arbeit. Es erfolgte daher zunächst die Anpassung und Optimierung der Präparations- und Messmethoden mit dem Ziel, standardisierte Protokolle festzulegen, welche zu reproduzierbaren Spektren führen. Es schloss sich die Analyse der kommerziell verfügbaren Hauptbestandteile der ECM - Proteoglykane und Kollagene – mittels MADI-TOF MS an. Hierzu wurden Chondroitinsulfat und Kollagen verschiedener Typen enzymatisch verdaut und analysiert. So konnten Referenzspektren erstellt werden, welche die Grundlage für die Analyse des wesentlich komplexeren Systems des natürlichen Knorpels bildeten. Durch den Einsatz von Trypsin zur Hydrolyse nach thermischer Denaturierung des Kollagens wurde die Differenzierung zwischen den verschiedenen Kollagentypen ermöglicht. Es schlossen sich Studien zur Quantifizierung der enzymatischen Verdauungsprodukte der ECM an, welche das Ziel verfolgten, neben der stofflichen Zusammensetzung Aussagen zu den relativen Anteilen der einzelnen Bestandteile zu erlauben. Nachdem die grundsätzliche Eignung der Methode gezeigt werden konnte, diente der zweite Teil der hier dargelegten Forschungsarbeit der Beantwortung der Frage, ob die Erkenntnisse, welche bei der Analyse der Einzelbestandteile gewonnen wurden, auf natürliches Knorpelgewebe anwendbar sind. Der artikuläre Schweineknorpel ähnelt im Aufbau und den biomechanischen Eigenschaften dem menschlichen Knorpel. Da er zudem noch günstig und in adäquaten Mengen zur Verfügung gestellt werden kann, wurde der Gelenkknorpel des Schweins als Modellgewebe für die Anwendbarkeit der Methode auf natürlichen Knorpel genutzt. Ziel war es, die Hauptbestandteile der ECM einwandfrei zu identifizieren, Untersuchungen zur Quantifizierung anzustellen und Unterschiede zwischen den Knorpeltypen verschiedener Spezies nachzuweisen. Im letzten Abschnitt dieser Arbeit erfolgte die Analyse von biotechnologisch hergestelltem Knorpel und somit die Anwendung der bisher erworbenen, vor allem einem theoretisch-wissenschaftlichem Interesse folgenden Erkenntnisse auf eine praktisch-klinische Fragestellung nach der tatsächlichen Beschaffenheit des zu transplantierenden Konstrukts. Dazu wurden Konstrukte, welche zum einem aus dreidimensionalen Agarosegele bestückt mit Chondrozyten, zum anderen aus Kollagengel mit eingesäten Stammzellen oder Chondrozyten bestanden, untersucht. Dieser Schritt erfolgte in Zusammenarbeit mit dem Biotechnologisch-Biomedizinischen Zentrum Leipzig. Ziel war es, die bis dahin etablierten Probenpräparations- und Messbedingungen auf den künstlichen Knorpel anzuwenden und mit Referenzspektren von Bestandteilen der ECM sowie des natürlichen Schweineknorpels zu vergleichen. Durch die Analyse des Materials zu verschiedenen Kultivierungszeitpunkten sollte eine Aussage über die Qualität und die Menge der de novo synthetisierten ECM erreicht werden. Mit dieser Arbeit wurde die Grundlage geschaffen, die MALDI-TOF MS als geeignetes analytisches Verfahren zur Bestimmung der Konstruktqualität einzuführen. Perspektivisch sollte es so möglich sein, die Qualität der Konstrukte, welche für die Autologe Chondrozytentransplantation bestimmt sind, zu bewerten und zu überwachen.

High level of agricultural products processing and especially better storage of products currently represent one of the hottest topics. At the same time, there is a lot of effort to grow crops without chemical substances. Root vegetables, which are consumed a lot, are prone to many harmful and damaging influences. There are several risk factors, including a long vegetation period. This creates an opportunity to use natural substances, in particular essential oils. Their effects are used in various different fields because they are safe for the environment as well as for the human health and the area of food commodities treatment. This study focuses on antibacterial activity testing of several essential oils: satureja, cinnamon, clove, thyme and oregano. Carrot, garden parsley and celery were chosen as representatives of root vegetables. The antibacterial activity was measured by the broth microdilution method. Even though the vegetables were inoculated with pathogenic bacteria, putrefaction did not develop in the specific places. Therefore an isolate from the parsley and the celery was then used to identify several other microorganisms by the MALDI TOF mass spectrometry. Consequently, the minimum inhibitory concentration of essential oils was again tested against these bacteria in in vitro conditions. There was a demonstrable positive result: the most frequent minimum inhibitory concentration of the cinnamon essential oil was 0.128 mg/ml. At this level the essential oil inhibited eight out of the seventeen tested microorganisms. The other tested essential oils showed some inhibition activity at least against one bacterium in in vitro conditions.