Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22148号 / 医博第4539号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 坂井 義治, 教授 小西 靖彦, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This diploma thesis is focused on studies of changing of protein profile during barley malting. Substantial part of this work is devoted to the proteomics identification of barley proteins which change during malting and so become more stationary and they influence quality of beer (haze and foam in beer). For this experiment was used barley variety Jersey. In the theoretical part of this thesis there is information about beer, manufacturing of beer with description of important commodities for manufacturing of beer and information about barley malting and information about malting process. Next there is description of methods for separation of proteins (1D gel electrophoresis and 2D gel electrophoresis), MALDI TOF/TOF mass spectrometry and this use for the analysis and identification of proteins, the use of matrices and ways of the sample preparation. In the experimental part of this thesis there was carried out the optimisation of the dosage of sample for 1D gel electrophoresis and the optimisation of staining. The 15 % TRIS-HCl gel was the best, this gel was stained by Commassie Brilliant Blue G-250. For illustration of changes was made 2D gel electrophoresis. With help of method peptide mass fingerprinting and MS/MS protein of barley – protein Z, -amylase subtilisin inhibitor, -amylase a peroxidase were identificated. The analysis of barley extract intact proteins was carried out, this analysis was focused on changes of important barley protein LTP 1.

This work deals with the using and application of separation techniques for analysis of polymers degradation and polycyclic aromatic hydrocarbons. Thereby this work is separated to two special parts. In the first part, the degradation properties of synthetic biopolymers based on lactic acid, gylcolic acid and poly(ethyleneglycol) PLGA-PEG-PLGA and ITA-PLGA-PEG-PLGA-ITA (modified by itaconic acid) were studied. These copolymers (firstly their thermosensitive hydrogels) should be used for therapy of fractures in orthopedy (as adhesives). Therefore, the sol-gel and gel-sol phase diagrams were determinated for selected samples of copolymers. The samples forming gel at 37 C was used for other study. Polymer samples were depredated in phosphate buffer at 37°C. The degradation process of physical hydrogels was described by the decrease of molecular weight and the increase of concentration lactic acid and glycolic acid in phosphate buffer. The obtained results confirmed that the degradation of polymer modified by itaconic acid is faster process than no modified polymer and polymers with lower ratio PLGA/PEG degrade also faster than lower ration PLGA/PEG. The influence of pH it was also tested. The rate of degradation of polymers was follow pH 4,0

(EN) Vernix caseosa is a white creamy substance that covers the skin of a newborn. It is produced during the third trimester by the skin of the baby and remains there until the age of one or even two weeks. It is uniquely human. In utero, vernix protects the skin from maceration, during the birth it serves as a lubricant and after the delivery it protects the baby against infection and regulates the temperature. As vernix is produced in third trimester, prematurely born infants lack it and this may lead to, among other things, suffering from desiccation and therefore heat loss. It is important to study it thoroughly and to find a suitable substitute of vernix for the preterm infants. Vernix consists of lipids, proteins and 80 % water. This project is aimed at the lipids. Vernix is composed of 10 % of lipids. Basic analytical methods of pocessing vernix were searched. The methods of isolation, separation and transesterification have been optimized for the lipids. For separation, thin-layer chromatography has been chosen. The method of the lipid analysis of intact molecules by MALDI-TOF MS has been optimized for these lipids. The results were confirmed using fragmentation spectra and transesterification. Esterified lipids were measured by gas chromatography coupled with mass spectrometry detection....

Das Plattenepithelkarzinom stellt mit über 90% die häufigste maligne Neubildung innerhalb der Mundhöhle mit gravierenden funktionellen Einschränkungen für die betroffenen Patienten und hoher Mortalität dar. In den meisten Fällen bestehen bereits vor der malignen Transformation visuell erkennbare Veränderungen mit höherem Entartungspotential, sogenannte potenziell maligne Veränderungen. Die vorliegende Arbeit beschäftigt sich mit einem experimentellen Verfahren zur oralen Tumorfrüherkennung und Dignitätsabklärung von potenziell malignen Veränderungen der Mundschleimhaut. Grundlage bildet das Verfahren des „intact cell peptidome profiling“ (ICPP) mittels MALDI-TOF MS anhand von Präparaten oraler Bürstenbiopsien. Untersucht wurden 16 Proben oraler Plattenepithelkarzinome, 69 Fälle von oralem Lichen planus, 26 orale Leukoplakien, 21 andere orale Veränderungen sowie 56 Mundschleimhautläsionen an Fanconi-Anämie erkrankter Patienten. Die Methode eignet sich zur Unterscheidung zwischen gesunder Mundschleimhaut und maligne verändertem Gewebe, wie mit einer Sensitivität von 86 % und einer Spezifität von 100 % belegt wurde. Eine Differenzierung zwischen den einzelnen Mundschleimhautveränderungen und gesundem Gewebe konnte nicht erreicht werden. Die Untersuchungsreihe ist als vielversprechend einzuschätzen, um ein weiterführendes diagnostisches Verfahren zu entwickeln, dass den Untersucher durch eine objektive Messmethode unterstützen und Sensitivität und Spezifität der Bürstenbiopsie steigern könnte.

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Blütenstaub ist eine der Hauptursachen für allergische Erkrankungen des Menschen. Insbesondere die Matrix-unterstützte Laser-Desorption/Ionisation-Flugzeit Massenspektrometrie stellt eine geeignete Technik dar, um solch komplexe, biologische Proben zu charakterisieren. Bei MALDI-TOF MS-Analysen werden die Bestandteile detektiert, die zuvor durch die Matrixauftragung und/oder während der Laser-Desorption von der Oberfläche des Pollenkorns abgelöst werden. Hier zeigte sich, dass die Verwendung eines leitfähigen Klebebandes zur Probenpräparation sowohl die Fixierung der Pollenkörner als auch die Extraktion der Analyten aus diesen verbessert. Die gezeigten Ergebnisse belegen, dass sich Pollen verschiedener Pflanzenarten über den Vergleich von massenspektrometrischen Peakmustern differenzieren lassen. Zur Beurteilung der spektralen Muster wurden in dieser Arbeit multivariate, statistische Verfahren, speziell die Hauptkomponentenanalyse verwendet. Diese hebt Unterschiede in den Datensätzen hervor und ermöglicht eine varianzgewichtete Darstellung und verbesserte Klassifizierung. Zur effizienten multivarianten Analyse wurde dieses Verfahren in eine graphische Benutzeroberfläche eingebunden. Neben der individuellen Charakterisierung einzelner Pollenproben liegt eine weitere Herausforderung in der massenspektrometrischen Analyse von Pollenkornmischungen, da es durch Vermischungs- und Suppressionseffekte zur Diskriminierung einzelner, möglicherweise charakteristischer MS-Peaks kommen kann. Deshalb wurde in dieser Arbeit die bildgebende MALDI Massenspektrometrie angewendet. So konnten in einem Modellsystem einzelne Pollenkörner unterschiedlicher Arten nachgewiesen und ihren ursprünglichen Positionen in der Probe zugeordnet werden. Der im Rahmen der Arbeit vorgestellte Ansatz stellt eine neue, zuverlässige Methode zur Pollenbestimmung dar, die nicht auf der individuellen, visuellen Beurteilung, sondern auf einer spektrometrisch-analytischen Basis beruht. / Pollen represent one of the major causes for allergies in humans. Matrix-assisted laser desorption/ ionization time-of-flight is a suitable technique to characterize such complex biological samples. In pollen analyses, MALDI-TOF MS detects components that are extracted from the grain surface by the matrix solution and during laser desorption. Like any biological structure, pollen grains contain specific proteins, peptides, lipids and carbohydrates that produce characteristic signal patterns in the mass spectrum. The sample preparation applied in this work showed that the use of a conductive adhesive tape improves both the fixation of the pollen grains and the extraction of the analytes from them. The shown results prove that pollen of different plant species can be differentiated by comparing their respective mass spectral patterns. For the evaluation of such spectral patterns multivariate statistical methods, and in this work especially principal component analysis, are needed. PCA highlights differences in data sets and enables variance-weighting and improved classification. For efficient multivariate analysis, this method was integrated into a graphical user interface. In addition to the individual characterization of pollen samples, the mass spectrometric analysis of pollen grain mixtures posed a further challenge, since mixing and suppression effects can lead to discrimination of individual, possibly characteristic MS-peaks. For this purpose, the MALDI MS Imaging technique was applied. For the evaluation of such imaging data sets, further multivariate methods were applied, investigated, and evaluated. Thereby, in a model system individual pollen grains of different species could be identified and assigned their original position in the sample. The presented approach represents a new, reliable method for pollen determination, based on spectrometric-analytical basis rather than an individual visual assessment.