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Im Rahmen der Untersuchung wurde bei 27 Patienten, die klinische Zeichen der Karies, aber keine pulpitischen Symptome zeigten, kariöses Dentin mit Hilfe eines sterilen Exkavators entnommen. Das durchschnittliche Alter der Patienten beträgt 41 Jahre und der Durchschnittswert des DMF-T-Index 12,5. Die Untersuchungsgruppe bestand zu 55,5 % aus männlichen Probanden sowie zu 44 % aus Rauchern. Nach Isolierung von 107 Reinkulturen aus den Patientenproben erfolgte die Identifizierung der oralen Streptokokken mittels eines mikrobiologischen Standardtests (RapidID-32Strep der Firma BioMérieux) und MALDI-TOF-MS-Analyse. Parallel wurden speziesspezifische PCRs der Dentinproben für S. sanguinis, S. constellatus, S. intermedius, S. anginosus, S. mutans, S. salivarius, S. oralis, S. mitis, S. gordonii und S. parasanguinis durchgeführt. Mittels MALDI-TOF-MS-Analyse konnten insgesamt sechs verschiedene Spezies oraler Streptokokken in den Dentinproben nachgewiesen werden. Am häufigsten kamen Vertreter der Mitis-Gruppe vor (in 89 % der Dentinproben), gefolgt von S. gordonii und S. sanguinis (zu 52 % und 26 % vertreten). Die MALDI-TOF-MS-Methode erwies sich als geeignetere der mit Kultivierung verbundenen Nachweismethoden. Ihre Ergebnisse wurden durch selektive PCRs einzelner Subkulturen und DNA-Sequenzierung bestätigt. Mittels der speziesspezifischen PCRs der Dentinspäne wurden zehn verschiedene Spezies oraler Streptokokken identifiziert. Vertreter der Mutans-Gruppe wurden so zu durchschnittlich 44 %, S. salivarius zu 37 % nachgewiesen. Es zeigte sich ein signifikantes Vorkommen von S. anginosus in Proben, die ebenfalls S. mutans enthielten (p= 0,00213). Alle drei Verfahren sind zur Untersuchung klinischer Proben geeignet, wobei die MALDI-TOF-MS-Analyse die genaueste Differenzierung auf Speziesebene ermöglicht. Die Non-Mutans-Streptokokken S. oralis, S. gordonii und S. anginosus scheinen die Mikroflora von kariösem Dentin zu dominieren. Sie übertrafen in der vorliegenden Arbeit in ihrem Vorkommen S. mutans in mehr als der Hälfte der untersuchten Proben. Diese Beobachtung stützt die erweiterte ökologische Plaquehypothese.

Les enzymes et leur diversité d’actions sont appréciées dans des domaines d’applications variés allant del’agroalimentaire à la thérapeutique. Ainsi, une attention toute particulière est portée à leur étude afin d’améliorer uneaction (contre le vieillissement de la peau, antivirale, anticancéreuse…) ou un procédé de synthèse. Ce projet derecherche s’inscrit dans une démarche de développement d’outils analytiques basés sur la spectrométrie de masse,permettant le suivi rapide et sensible d’interactions enzyme-ligand.Dans une première étude, l’approche TLC couplée à une détection par UV a été évaluée pour la déterminationde constantes enzymatiques de l’enzyme invertase. Cette approche couplée à un MALDI/TOF MS a permis d’identifierdes substrats spécifiques de l’invertase au sein d’extraits de plantes. Pour preuve de concept, l’interactioncellobiohydrolase II–ligand est présentée dans le cadre de l’identification d’inhibiteur par TLC-MALDI/TOF et TLCENALDIMS.En seconde étude, nos travaux ont porté sur la caractérisation directe de différentes enzymes kinases, puis auxsuivis des réactions de phosphorylation de nucléosides /tides endogènes. Ces études, basées sur des approches « offline» (Flow Injection Analysis, FIA) et « on-line » (Frontal Affinity Chromatography, FAC) couplées à unspectromètre de masse haute résolution, ont été réalisées au moyen de ces kinases libres et immobilisées. Dans le cadrede la recherche de nouveaux candidats médicamenteux antiviraux, le suivi d’une phosphorylation spécifique desmolécules de synthèse, au regard de souches humaine ou virale de kinase, a également été évalué par ces deuxméthodologies. / Enzymes are very appreciated and useful in various application fields from agri-business to therapeutic due to theirdiversity of actions. Therefore, their action mechanisms are widely studied in order to enhance an action (anti-aging ofskin, antiviral, antitumorous) or a synthesis process. This research project is part of the approach to propose analyticaltools based on mass spectrometry, allowing rapid and sensitive follow-up of enzyme-ligand interactions.In a first study, the Thin-Layer Chromatography (TLC) approach coupled with UV detection was evaluated forthe determination of invertase kinetic constants. This approach coupled with a MALDI / TOF-MS led to theidentification of invertase substrates in plant extracts. As a proof of concept, the cellobiohydrolase II - ligand interactionwas presented in the framework of the identification of inhibitor by TLC-MALDI / TOF and TLC-ENALDI MS.In the second study, our work aimed at developing a direct method for the determination of kinetic parametersof kinases and following-up the phosphorylation reactions of endogenous nucleosides / tides. These studies, based on“off-line” (Flow Injection Analysis, FIA) and “on-line” (Frontal Affinity Chromatography, FAC) approaches coupledwith a high-resolution mass spectrometer, were carried out using free and immobilized kinases. In the context of thesearch for new antiviral drug candidates, a specific phosphorylation of synthetic molecules regards to human or viralkinase was also evaluated by these both approaches.

<p>The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.</p><p>The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.</p><p>In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.</p>

The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein. The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization. In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.

Pseudoexfoliation (PEX) syndrome is an age-related condition characterized by the production and accumulation of extracellular fibrillary material in the anterior segment of the eye. PEX predisposes for several pathological conditions, such as glaucoma and complications during and after cataract surgery. The pathogenesis of PEX is not yet fully understood. It is multifactorial with genetics and ageing as contributing factors. We aimed to study the proteome in aqueous humor (AH) in PEX in order to increase the knowledge about its pathophysiology. Therefore, we developed sampling techniques and evaluated separation methods necessary for analyzing small sample volumes. Other objectives were to study the lens capsule in eyes with PEX regarding small molecules, and to investigate the association between PEX and cataract surgery in a population-based 30-year follow-up study. Samples of AH from eyes with PEX and control eyes were collected during cataract surgery. In pooled, and individual samples, various liquid based separation techniques and high resolution mass spectrometry were utilized. For quantitation, various methods for labeling, and label free techniques were applied. Lens capsules were collected from some of the patients, and analysed by imaging mass spectrometry. A cohort of 1,471 elderly individuals underwent a comprehensive ophthalmological examination at baseline. Medical information was obtained by questionnaires, and from medical records. Incident cases of cataract surgery were identified by review of medical records. In the initial study, several techniques were explored for protein detection, and a number of proteins were identified as differentially expressed. In the individually labelled samples, changes in the proteome were observed. Eyes with PEX contained higher levels of proteins involved in inflammation, oxidative stress, and coagulation, suggesting that these mechanisms are involved in the pathogenesis in PEX. The levels of β/γ-crystallins were significantly increased in PEX, which is a novel finding. In the lens capsules from individuals with PEX, changes in the lipid composition was observed with time-of-flight secondary ion mass spectrometry. These changes remain to be elucidated. By multivariate analysis, lens opacities were the first, and PEX the second most important predictor for cataract surgery, the later accounting for a 2.38-fold increased risk for cataract surgery.

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

Penicillium nordicum è un importante contaminante di salumi, rappresentanando il 10 % e il 26 % della popolazione di Penicillium spp . isolati , rispettivamente dall'aria e dai prodotti carnei stagionati in un'indagine gestita in Italia ( Battilani et al. , 2007). Diverse colonie di P. nordicum isolate dai salumi hanno dimostrato di essere importanti produttori di ocratossina A , OTA ( Sansom e Frisvad , 2004 . Pietri et al, 2006 ; . Battilani et al , 2010). Attualmente, l'impostazione appropriata delle condizioni ambientali (temperatura, umidità relativa e circolazione dell'aria ), è l'unico strumento accettato per impedire la crescita incontrollata di P. nordicum all'interno degli impianti di stagionatura attraverso una accurata analisi dei punti critici di controllo e l’ideazione di un relativo piano HACCP (Hazard Analysis and Critical Control Points) ben struttutato ( Asefa et al , 2011; Virgili et al , 2012). Anche se il sistema HACCP è stato applicato con successo nel settore alimentare ci sono rischi per la sicurezza alimentare non attentamente considerati. Questo è particolarmente vero per quanto riguarda i rischi micotossigeni associati ai prodotti alimentari di origine animale. Il termine "rischi micotossigeni" è utilizzato da Asefa et al. ( 2011) per descrivere lieviti patogeni e metaboliti secondari tossici prodotti da specie fungine tossigene che contaminano i prodotti alimentari e incidono sulla sicurezza alimentare. La maggior parte dei piani HACCP nelle attività di trasformazione alimentare, come ad esempio la produzione di formaggi e di prodotti carnei stagionati, tiene in considerazione principalmente il rischio derivante da agenti batterici (Arvanitoyannis e Mavropoulos, 2000; Barbuti e Parolari, 2002) anche se tali prodotti alimentari vengono spesso contaminati da funghi micotossigeni e dai loro metaboliti (Spotti et al 1989; Spotti et al , 2001a; Battilani et al 2007). Pertanto, dovrebbe essere cruciale definire un piano HACCP specificamente incentrato sui rischi micotossigeni. L'identificazione, il controllo e la standardizzazione della micoflora superficie dei salumi è fondamentale per preservare la sicurezza delle produzioni e la salute dei consumatori . Questo è il contesto in cui deve essere valutata l’efficacia e l’affidabilità per l’identificazione delle popolazioni di Penicillium spp di interessante per la produzione alimentare. In questo contesto , il progetto di ricerca di questa tesi di dottorato ha cercato di approfondire le conoscenze su tali tematiche con l'intento di limitare il rischio micotossigeno nella catena di produzione dei prodotti carnei stagionati. Sono stati affrontati i seguenti argomenti: 1 . studio della composizione e dinamica della microflora fungina presente sulla superficie dei salumi (prodotto testato, salame) e l'aria di ambienti di stagionatura tenendo conto dell'influenza di alcuni parametri di processo (inoculo starter, temperatura, fase produttiva). 2 . sviluppo di un metodo MALDI TOF MS per l'identificazione di Penicilium a livello di specie per le prospettive future di screening diretti della microflora presente sui salumi. 3 . confronto e integrazione di diverse tecniche, come l'analisi morfologica, l’analisi molecolare e l’analisi tramite spettrometria di massa, per l'identificazione delle specie di Penicillium presenti nei salumi. 4 . valutazione dei lieviti selezionati, isolati dalla superficie di prosciutto crudo, per competere con P. nordicum ed inibire l'accumulo di OTA nella prospettiva del loro uso come starter superficiali con funzione di agenti di biocontrollo. / Penicillium nordicum is an important contaminant of cured meat products, representing 10% and 26% of the Penicillium spp. isolated, respectively, from the air or the products in a survey managed in Italy (Battilani et al., 2007). Several P. nordicum cured meat isolates proved to be important producers of ochratoxin A, OTA (Sansom and Frisvad, 2004; Pietri et al., 2006; Battilani et al., 2010). Currently, the appropriate setting of environmental conditions (temperature, relative humidity and air circulation), is the only accepted tool to prevent the uncontrolled growth of P. nordicum inside dry-curing plants through a carefully structured Hazard Analysis Critical Control Point (HACCP) plan (Asefa et al., 2011; Virgili et al., 2012). Even if the HACCP system has been successfully applied in the food industry, there are food safety hazards not carefully considered. This is especially true with regard to mycotoxigenic hazards associated with animal food products. The term “mycotoxigenic hazards” is used by Asefa et al. (2011) to describe pathogenic yeasts and toxic secondary metabolites of toxigenic moulds that contaminate food products and affect food safety. Most HACCP plans in food processing activities, such as the production of cheese and dry-cured meat products, considered mainly bacterial agents (Arvanitoyannis and Mavropoulos, 2000; Barbuti and Parolari, 2002), even if such food products get often contaminated with mycotoxigenic fungi and their metabolites (Spotti et al 1989; Spotti et al., 2001a; Battilani et al 2007). Therefore, it should be crucial to define a HACCP plan specifically focused on the mycotoxigenic hazards. The identification, control and standardization of the surface mycoflora of cured meat products is mandatory to preserve the productions safety and the consumers health. This is the context of the effectiveness and reliability evaluation for the Penicillium spp. identification methods of interesting species for food production. In this context, the research project of this PHD thesis tried to fill some gaps of knowledge with the attempt to limit the mycotoxigenic risk in the cured meat products chain. The following topics were faced: 1. study of the composition and dynamic of fungal microflora present on the surface of cured meat products (salami) and the air of seasoning environments taking into account the influence of some process parameters (starter inoculum, curing temperature, stage of seasoning). 2. development of a MALDI TOF MS method for the identification of Penicilium at species level for future direct screening perspectives of the microflora present on cured meat products. 3. comparison and integration of different techniques, as morphological, molecular and mass spectral analysis, for the identification of Penicillium species in cured meat products. 4. evaluation of selected yeasts, isolated from dry-cured ham surface, to compete with P. nordicum and to inhibit OTA accumulation in the perspective of their use as surface starter biocontrol agents.

Antalet monoklonala antikroppar som används i läkemedel ökar kraftigt. Dessa läkemedel är dyra och risken för förfalskning är stor. Behovet att utveckla en metod för snabb och precis identifiering av monoklonala antikroppar är därför brådskande. För identifiering utfördes analyser med Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) på nio monoklonala antikroppar. Fokuset var att undersöka huruvida signifikanta fysiokemiska egenskaper och unika aminosyrasekvenser var närvarande och kunde urskiljas. Olika analyser med MALDI-ToF-MS användes till att både separera de monoklonala antikropparna baserat på dess fysiokemiska egenskaper, och annotera aminosyrasekvenser innehållande nyckelfragment. Med metoderna baserade på kapillärelektrofores uppnåddes också separation. CZE föredras framför CGE då mängden data som erhålls från CZE är större och provberedningen är enklare. Sammanfattningsvis utformades ett protokoll för identifieringsprocessen, vilket inleds med MALDI-ToF-MS-analyser av monoklonala antikroppar på reducerad form mot kända referenser. Därefter är en hypotes formulerad utifrån vilka antikroppar som ser mest lika ut. Slutligen analyseras dessa med CZE för fastställning av den monoklonala antikroppens identitet. / The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.