In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

O diagnóstico rápido e eficiente da mastite subclínica é importante para reduzir a persistência da doença e os prejuízos decorrentes. O objetivo do estudo foi avaliar a técnica de espectrometria de massas (MS) por ionização e dessorção a laser assistida por matriz - tempo-de-vôo (MALDI-TOF) para identificação de bactérias causadoras de mastite subclínica bovina por dois métodos: 1) a partir de bactérias isoladas por cultivo icrobiológico; 2) a partir da recuperação de bactérias diretamente do leite, visando eliminar completamente a necessidade de cultivo microbiológico para identificação dos patógenos. O estudo foi composto por dois experimentos (1 e 2), no experimento 1 foram utilizadas 33 amostras de leite provenientes de animais das raças Gir e Holandesa de quatro fazendas leiteiras para a identificação icrobiológica e MALDI-TOF MS. As amostras com resultados conflitantes foram confirmadas por sequenciamento do gene 16S rRNA. Os resultados de cultura microbiológica foram Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) e Estafilococos coagulase negativo (ECN) (n = 10). Para todas as amostras de Streptococcus agalactiae, resultados similares foram observados para a identificação microbiológica e por MALDI-TOF MS. De 13 isolados de Staphylococcus aureus, 11 foram igualmente identificados por MALDITOF, 1 isolado foi identificado como Staphylococcus haemolyticus por sequenciamento do gene 16S rRNA, e o outro isolado isolado com resultado conflitante foi caracterizado como cultura mista de Staphylococcus aureus e Enterococcus faecalis. Em relação às amostras de ECN, todas as amostras do grupo foram identificadas por MALDI-TOF em nível de gênero (S. simulans, S. epidermidis, S. Haemolyticus, S. Chromogens e S. aureus coagulase negativa). No experimento 2 foi avaliado o método de recuperação de bactérias presentes em leite e sua identificação por MALDI-TOF MS utilizando o método de contaminação experimental de leite com Escherichia coli, Enterococcus faecalis e Staphylococcus aureus. A identificação de patógenos recuperados diretamente do leite foi possível quando a concentração de E. faecalis e S. aureus foi de 106 ufc/mL, e de 107 ufc/mL para E.coli. Concluímos que o uso de MALDI-TOF MS pode acelerar a identificação de patógenos causadores de mastite subclínica bovina podendo contribuir para a adoção de medidas de controle e tratamento mais adequado. / Rapid and efficient diagnosis of subclinical mastitis is important to reduce the persistence of the disease and its losses. This study aimed to evaluate the technique of mass spectrometry (MS) and desorption ionization by matrix assisted laser time-offlight (MALDI-TOF) for identification of bacteria causing bovine subclinical mastitis by two methods: 1) from bacteria isolated by microbiological culture, 2) from bacteria recovered directly from milk, to eliminate completely the need for microbiological culture for identification of pathogens. The study consisted of two experiments (1 and 2).In experiment 1, 33 milk samples from Gir and Holstein animals collected in four dairy farms were used for microbiological identification and MALDI-TOF MS. The samples with different results were confirmed by sequencing the 16S rRNA. These samples were identified by microbiological culture as Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) and Staphylococcus coagulase negative (SCN) (n = 10). For all strains of Streptococcus agalactiae, similar results were observed for microbiological identification and MALDI-TOF MS. From 13 isolates of Staphylococcus aureus, 11 were also identified by MALDI-TOF, one isolate was identified as Staphylococcus haemolyticus by 16S rRNA sequencing , and the other discrepant sample was charactezided a mixed culture of Staphylococcus aureus and Enterococcus faecalis. Regarding the SCN samples, all samples of this group were identified by MALDI-TOF at gender level (S. simulans, S. epidermidis, S. haemolyticus, S. Chromogens and S. aureus coagulase negative). In experiment 2, we evaluated the method of recovery of bacteria present in milk and their identification by MALDI-TOF MS using experimental method of contamination of milk with Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The identification of pathogens recovered directly from milk was possible when the concentration of E. faecalis and S. aureus was 106 ufc/mL and 107 ufc/mL for E.coli. We conclude that the use of MALDI-TOF MS can accelerate the identification of pathogens causing bovine subclinical mastitis may contribute to the adoption of control measures and appropriate treatment.

Au regard des nombreux avantages en terme de rapidité, de coût, de sensibilité et de fiabilité de la spectrométrie de masse MALDI-TOF nous avons cru pouvoir l’appliquer à l’étude des cellules eucaryotes intactes, en particulier à l’étude des cellules immunitaires. Nous avons ainsi montré que cette approche était applicable à l'analyse globale des cellules eucaryotes y compris des cellules immunitaire circulantes. En outre, elle a permis de caractériser les multiples facettes d'activation des macrophages humain en analysant les données avec le logiciel R, la librairie « MALDIquant » et des algorithmes spécifiques. Les empreintes peptidiques/protéiques induites par les agonistes M1, IFN-γ, TNF, LPS et LPS + IFN-γ ou les agonistes M2, IL-4, TGF-β1 et IL-10, sont distinctes des macrophages non stimulés et spécifiques de chaque agoniste. La spectrométrie de masse MALDI -TOF peut ainsi être utilisée pour caractériser les sous-types de macrophages M1 et M2. En outre, les empreintes induites par des bactéries extracellulaires (streptocoque du groupe B, Staphylococcus aureus) sont spécifiques et similaires à celles induites par l'IL-4. Les réponses des macrophages à des bactéries intracellulaires (BCG, Orientia tsutsugamushi, Coxiella burnetii) sont également uniques. La spectrométrie de masse MALDI-TOF sur cellules entières a ainsi révélé donc les multiples facettes d'activation des macrophages humains. Enfin, des résultats préliminaires montrent que notre approche pourrait être utilisée en clinique en analysant les cellules circulantes de la réponse immune. / In view of the many advantages in terms of speed, cost , sensitivity and reliability of the MALDI -TOF mass, we thought we could apply it to the study of intact eukaryotic cells, in particular the study of cells immune . We have shown that this approach is applicable to the global analysis of eukaryotic cells including circulating immune cells. In addition, it allowed us to characterize the many faceted of human macrophage activation by analyzing the data with the R software library " MALDIquant " and specific algorithms. The protein/peptide fingerprint induced by the M1 agonists : IFN - γ , TNF , LPS and LPS + IFN - γ or M2 agonists : IL- 4 , TGF - β1 and IL- 10 are distinct to unstimulated macrophages and specific for each agonist. MALDI -TOF Mass spectrometry can then be used to characterize the subtypes M1 and M2 macrophages . In addition, fingerprints induced by extracellular bacteria ( group B streptococcus , Staphylococcus aureus ) are specific and closed to those induced by IL -4 . The responses of macrophages to intracellular bacteria (BCG, Orientia tsutsugamushi , Coxiella burnetii ) are also unique. Mass spectrometry MALDI -TOF of whole cell revealed therefore the multifaceted activation in human macrophages . Finally, preliminary results show that our approach could be used clinically for the analysis of circulating cells in the case of host-pathogen interaction.

Einleitung Rodentibacter (R.) pneumotropicus und R. heylii sind wichtige Pneumonie-, Otitis- und Mastitiserreger in Labormäusen. Auf Grund der hohen Prävalenz und Relevanz für tierexperimentelle Studien sind gut etablierte diagnostische Methoden entscheidend. Die bisherigen Möglichkeiten der Diagnostik sind jedoch nicht zufriedenstellend. Auch die Differenzierung zu Muribacter (M.) muris, einer apathogenen Pasteurellaceae, ist nicht mit allen Methoden möglich. Die Pathogenese von Infektionen mit Rodentibacter spp. ist bis heute nicht endgültig beschrieben und auch Virulenzfaktoren sind nur in Form rekombinanter Proteine in vitro untersucht. Ziele der Untersuchung Diese Doktorarbeit verfolgte die Ziele, den direkten Erregernachweis wichtiger muriner Pasteurellaceae über MALDI-TOF MS zu etablieren und zu evaluieren und indirekte spezifische und sensitive Nachweisverfahren (ELISA) von Labormausinfektionen mit R. pneumotropicus und R. heylii im Rahmen eines Verbundprojektes zu entwickeln. Des Weiteren sollte ein neu identifiziertes Immunogen eines hochvirulenten R.-heylii-Stammes geno- und phänotypisch analysiert und charakterisiert werden. Material und Methoden In der ersten Publikation wurden Feldisolate und Referenzstämme der Spezies R. pneumotropicus, R. heylii, R. ratti und M. muris mittels 16S rRNA identifiziert und daraus Referenzspektren zur Erweiterung der MALDI-TOF MS-Datenbank erstellt. Die Evaluierung fand mittels Abgleiches mit einer etablierten Multiplex-PCR und bereits publizierter Stämme statt. Für die zweite Publikation wurden BALB/c- und C57BL/6-Mäuse mit R. heylii SF27GVG oder M. muris infiziert. Als Readout Parameter wurde ein klinischer und pathologischer Score eingesetzt. Weiterhin erfolgte eine quantitative oder semiquantitative bakteriologische Untersuchung unterschiedlicher Gewebe und Körperflüssigkeiten sowie eine serologische Untersuchung auf Serokonversion mit unterschiedlichen ELISAs. Das durch Immunoproteomics identifizierte R. heylii Immunogen A (RhiA) wurde nach in silico Analysen in trunkierter Form rekombinant exprimiert und zur Etablierung eines ELISA genutzt. Die phänotypische Charakterisierung fand mittels Western Blot, Durchflusszytometrie und Immunhistochemie statt. Hierfür wurde ein RhiA-spezifischer IgY-Antikörper eingesetzt. In der dritten Publikation wurde das Protein CARLO-1 kloniert und rekombinant exprimiert. Der neu etablierte ELISA wurde mit Hilfe von Rekonvaleszenzseren verschiedener Tierversuche evaluiert und außerdem die Konservierung des entsprechenden Gens in R. heylii und R. pneumotropicus-Stämmen analysiert. In der vierten Studie wurde das für BALB/c-Mäuse bereits etablierte R. pneumotropicus Infektionsmodell eingesetzt, um die Schutzwirkung einer Inaktivatvakzine vor einer Infektion der Lunge mit diesem Pathogen zu ermitteln. Ergebnisse Die erweiterte MALDI-TOF MS-Datenbank ermöglicht eine sichere Differenzierung zwischen R. heylii, R. pneumotropicus, R. ratti und M. muris, wie die Übereinstimmungen zu Ergebnissen der Multiplex-PCR zeigen. Im Tierversuch zeigte sich, dass R. heylii SF27GVG hoch virulent ist, obwohl dieser Stamm keines der bekannten RTX-Proteine trägt. Das identifizierte Immunogen RhiA dieses Stammes liegt oberflächenassoziiert vor, wird in vitro und in vivo exprimiert und hat strukturelle Ähnlichkeiten zu PnxIII. Es kann in infizierten Lungen assoziiert mit Bakterienrasen nachgewiesen werden und ist als RTX-Adhäsin möglicher Weise an der Biofilmproduktion beteiligt. RhiA ist hoch spezifisch für R. heylii (ELISA-Spezifität 100%). Es wird aber nur von wenigen der untersuchten Stämme gebildet. Der CARLO-1-ELISA zeigt eine Sensitivität von 93,3% und eine Spezifität von 100%. Das kodierende Gen ist in allen untersuchten R. heylii und R. pneumotropicus-Stämmen konserviert. Mit dem in einer vorausgegangenen Dissertation etablierten R. pneumotropicus Infektionsmodell konnte die protektive Wirkung einer neuen Inaktivatvakzine erfolgreich bestätigt werden. Schlussfolgerungen Nach Erweiterung der Datenbank können R. heylii, R. pneumotropicus und M. muris mittels MALDI-TOF MS differenziert werden. RhiA ist ein hochspezifisches, repetitives, Oberflächen-assoziiertes und in Lungenläsionen exprimiertes RTX-Immunogen, das von einem hochvirulenten R.-heylii-Pathotyp gebildet wird. Durch die Unterstützung von Kooperationspartnern konnte der CARLO-1-ELISA als spezifisches und sensitives indirektes Nachweisverfahren für Infektionen mit R. pneumotropicus oder R. heylii etabliert und evaluiert werden. Weiterhin wurde erstmalig gezeigt, dass sich mit dem R. pneumotropicus Infektionsmodell die protektive Wirkung einer Inaktivatvakzine aufzeigen lässt.:I. Inhaltsverzeichnis I II. Abkürzungsverzeichnis III 1 Einleitung 1 2 Literaturübersicht 2 2.1 Taxonomie 2 2.1.1 Pasteurellaceae 2 2.1.2 Rodentibacter spp. 5 2.1.3 Muribacter muris 5 2.2 Klinik und Virulenzfaktoren 7 2.2.1 Pasteurellaceae 7 2.2.2 Rodentibacter heylii und Rodentibacter pneumotropicus 11 2.3 Diagnostik 13 2.3.1 Pasteurellaceae 13 2.3.1.1 Monitoring 13 2.3.1.2 Direkte Diagnostik 14 2.3.1.3 Indirekte Diagnostik 20 2.3.2 Rodentibacter heylii und Rodentibacter pneumotropicus 22 2.3.3 Muribacter muris 23 3 Publikationen 25 3.1 Differentiation of Rodentibacter pneumotropicus, Rodentibacter heylii and Muribacter muris by MALDI-TOF MS 25 3.2 Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain 31 3.3 Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice 43 3.4 Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicus—A New Method for the Generation of Bacterial Vaccines with Increased Efficacy 57 4 Diskussion 69 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 8 Anhang 93 8.1 Anhang zu „Identification of a large repetitive RTX immunogen in a highly virulent Rodentibacter heylii strain” 93 8.2 Anhang zu “Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice“ 102 8.3 Anhang zu “Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicus—A New Method for the Generation of Bacterial Vaccines with Increased Efficacy” 123 Danksagung 125

This disertation deals with geographical authentification of different types of spices and spice preparations. Investigated spice species were garlic (Allium sativum), ground pepper (Capsicum anuum), and dried carrot (Daucus carota). Theoretical part of the disertation describes main qualitative parameters of the examined species and production technology, means of food adulteration and statistic methods used in chemometrics. The aim of this dissertation was to verify the hypothesis whether it is possible to use targeted analytical techniques commonly used in quality control of spices and condiments, and statistical processing of measured data to distinguish samples of spices and condiments of different geographical origin. The use of non-targeted analysis was also investigated. Samples of garlic and ground pepper were used for targeted analysis. The examined parameters were dry matter and moisture of the sample, ash content, total phenolic content according to Folin-Ciocaulteu, carbohydrate content, alliin concentration, total nitrogen content, total color pigment (ASTA), pH of aqueous extract, total fat content, and concentration of selected elements (Ca, Cu, Fe, K, Mn, Mg, Na, P, Zn). The instrumental techniques used were molecular absorption spectrometry, inductively coupled plasma optical emission spectrometry and high performance liquid chromatography. The obtained data were statistically processed by analysis of variance (ANOVA) and principal component analysis (PCA). Using statistical analysis significant differences between samples that came from more distant areas were found. However, samples from closer areas could not be distinguished. The researched hypothesis could not be unequivocally confirmed or refuted. Metabolic fingerprint of carrot samples was determined using non-targeted analysis. Metabolic analysis was performed using the tandem LC-TOF-MS technique. The data were processed by recursive peak extraction (BRE) and subsequently uvaluated with PCA. The samples were divided into clusters according to their origin. Targeted and non-targeted techniques have great potential in verifying the geographical authenticity of different types of spices. However, the main condition is consistent and sufficient sampling, guaranteed information on the origin of the sample and obtaining a sufficient amount of input data for statistical analysis.

In total, 84 yeast strains (originated from water, plants, fruits and soil) were selected for MALDI-TOF biotyping. All strains were cultivated on malt agar and YPD medium. Samples for biotyping were processed according to methods of Bruker Daltonik GmbH company, Institute of Chemistry of SAS and combination of these methods. Single strains were identified based on the analysis of intracellular ribozomal proteins using MALDI-TOF mass spectrometry. In case of ambiguous results, the DNA was isolated and the D1/D2 26S rRNA domain sequencing was performed. The strain identification was carried out by comparing its mass spectra with spectra of sequenced strains using MALDI Biotyper 3.0 software. The mutual similarity of strains was considered by score value, which was the result of the analysis. In total, 18 strains from 84 were previously sequenced and used as model strains for comparison with unknown isolates. Altogether 51 strains were definitely taxonomically categorized into 18 phylogenetic groups at the species level. The MALDI-TOF biotyping was repeated for overall 6 strains because of ambiguity of results. The taxonomic classification of 15 strains was not clearly determined and, therefore, these strains were suggested for D1/D2 26S rRNA domain sequencing. It was not possible to identify one strain, based on the results of sequencing, therefore, the DNA isolation was repeated. In the case of 8 strains, the results were identical with originally designed taxonomic classification. Conversely, the remaining 6 strains were identified as species. For 20 selected strains the basic characteristics were determined using microbiological methods. The shape of colonies growing on solid medium and appearance of cultures in liquid medium was assessed. Furthermore, the radial growth constant and the presence of urease were determined. Finally the microscopic observation of cells and the fermentation test for carbohydrate substrates were performed.

An intercomparison exercise of the world-harmonized light-duty vehicle test procedure (WLTP) aiming at measuring ethanol, formaldehyde and acetaldehyde emissions from a flex-fuel light-duty vehicle using E85 was conducted in the Vehicle Emission Laboratory (VELA) at the European Commission Joint Research Centre (EC-JRC), Ispra, Italy. The instruments used during the intercomparison allowed online measurements of these compounds directly from the diluted exhaust. Measurements were done either in real time or immediately after the test. The measurement and analysis of exhaust emissions over the world-harmonized light-duty vehicle test cycle was done by means of Fourier transform infrared spectroscopy (FTIR), proton transfer reaction-mass spectrometry (PTR-Qi-ToF-MS), photoacoustic spectroscopy (PAS) and gas chromatography (GC). Results showed that online systems can perform measurements from the vehicle diluted exhaust assuring a good repeatability (within instrument variance) and reproducibility (between instrument variance) of the results. Measurements from all the instruments were in good agreement (|Z-score| < 2). Results showed that online systems can perform measurements from the vehicle diluted exhaust assuring the reproducibility and repeatability of the results. Results obtained measuring at the tailpipe using a FTIR were in good agreement with those acquired measuring at the constant volume sampler (CVS). Considering the low sensitivity of the current technique used to measure hydrocarbons emissions towards oxygenated compounds (flame ionization detector; FID), non-methane organic gases (NMOG) were calculated applying their FID response factors to the measured emissions of ethanol, acetaldehyde and formaldehyde. NMOG resulted to be up to 74% higher than measured non-methane hydrocarbons (NMHC).

Since the late 18th century, chemicals have been industrially produced, and used by consumers. Today, the number of registered chemicals are over 150 000 in North America and Europe alone, and the number is predicted to increase. Industrial or anthropogenic chemicals can, directly or indirectly, be released into the ecosystem during their lifetime, where they can cause harm to human health and the environment. Depending on their properties, chemicals can travel far away from its source, causing global contamination. Through this, the Arctic region becomes a sink for many different types of contaminants. Because of the danger certain chemicals pose, techniques to detect and identify them in environmental samples have evolved during recent years. In these cases, non-targeted screening methods are commonly used to characterise contaminants in samples.In this study, surface sediment samples were collected on three locations in the Hudson Bay (Canada). The samples were analysed using two different instruments: a comprehensive two-dimensional gas chromatograph coupled to a high resolution time-of-flight mass spectrometer (GC×GC-HR-ToF-MS) and a gas chromatograph coupled with a Orbitrap mass spectrometer (GC-Orbitrap-MS). After data acquisition and processing, certain components were identified in both datasets, and their semi-quantitative concentrations were calculated.Overall, 32 compounds were detected and identified in the Orbitrap dataset, and 17 of these were also detected in the GC×GC dataset. The concentration was determined semi-quantitively for the identified compounds and ranged from 0.005–333 ng/g dry weight (d.w.) for the Orbitrap dataset, and 0.013–278 ng/g d.w. for the GC×GC dataset, which was below, or in the lower half, of concentration ranges from previous studies. Overall, the data processing for Orbitrap data seems to be more advanced and evolved than for GC×GC data, causing differences between the results from the two instruments.

Objectives: Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass-Spectrometry holds promise as a non invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may improve the early diagnosis of oral cancer tremendously. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. Material and Methods: In the first step, a data base consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as a basis for further classification of the blind study composed of additional 26 samples including HNSCC, oral lesions and healthy mucosa. Results: By analyzing spectral patterns of the blind study, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. Conclusion: ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to a more impartial early diagnosis of HNSCC.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12847号 / 論農博第2799号 / 新制||農||1027(附属図書館) / 学位論文||H26||N4864(農学部図書室) / 31430 / (主査)教授 宮川 恒, 教授 西田 律夫, 教授 植田 和光 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM