(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.

In infected patients, the rapid identification of pathogens is critical. After a long period of slow technological improvement, the microbiology laboratory is now undergoing significant evolution. This work evaluated the contribution of recent MALDI-TOF MS technology in terms of the diagnosis and clinical management of patients and its implementation in the laboratory of tomorrow. The studies were conducted over a 3.5-year period, mostly in the iris public hospital network of Brussels. <p>First, we confirmed the accurate performance of MALDI-TOF MS in the identification of routine isolates, regardless of whether the Biotyper (92.7% correct species identification) or VITEK MS (93.2%) (n=986) commercial system was used, and demonstrated the supremacy of this technology over conventional identification techniques for fastidious bacteria, including Campylobacter and related organisms (98.3%, 72.2% and 79.9% correct species identification by Biotyper, Vitek NH Card and API Campy, respectively; n=234). <p>Second, we showed that the direct MALDI-TOF MS identification of bacteria from positive blood cultures was not only feasible but also led to an 24-h reduction in the time-to-identification. In an adult population, more than 13% of the direct identifications from positive blood cultures resulted in the faster adaptation of the antimicrobial treatment. <p>Third, we demonstrated that MALDI-TOF MS could easily be implemented in a network, which was associated with significant cost savings and reduction in the time-to-identification. Finally, our promising Blastocystis subtyping results suggest that the number of MALDI-TOF MS applications may be increased.<p>In the future, automation of the technique will make its use in clinical laboratories even easier, eliminating the use of conventional identification techniques. Improvement of the preanalytical procedures is also important to make MALDI-TOF MS a suitable instrument for resistance and toxicity mechanism detection and subtyping. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

The catfish industry suffers losses primarily due to enteric septicemia of catfish and columnaris disease caused by Edwardsiella ictaluri and Flavobacterium columnare, respectively. Understanding the host-pathogen interactions is vital for prevention and eradication of these diseases. Hence, the overall objective of this study was to analyze whole cell proteomes of these two bacteria, and to determine the changes in E. ictaluri protein expression against in vitro iron-restriction and host serum treatment. High-throughput proteomic analysis of these bacteria was conducted using two-dimensional liquid chromatography followed by electrospray ionization tandem mass spectrometry (2-D LC ESI MS/MS) and two-dimentional gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-oflight mass spectrometry (2-DE MALDI TOF/TOF). Identified proteins were clustered into functional groups using clusters of orthologous groups, and subcellular locations as well as possible functional relationships were determined. A total of 788 unique E. ictaluri and 621 unique F. columnare proteins were identified, which represented 12 and 28 pathways, respectively. Vertebrate hosts tend to chelate free iron of their body and make the environment hostile for bacteria. Hence, reduced availability of iron may cause significant stress for pathogens and is considered a signal that leads to alteration in virulent gene expression. Similarly, E. ictaluri might use the catfish blood stream effectively for quick systemic invasion. Hence, exposure to catfish serum components might reveal the ability of E. ictaluri to protect against host defense mechanisms. Using two-dimensional difference gel electrophoresis, responses of E. ictaluri due to in vitro iron-restriction and host serum treatment were determined. A total of 50 and 19 proteins were identified to be differentially expressed due to in vitro iron-restriction and catfish serum treatment, respectively. Among the differentially expressed proteins, several putative virulent determinants, immunogenic proteins, chaperones, and housekeeping genes were noted. To initiate functional studies, four differentially expressed E. ictaluri genes (lamB, glyS, malE, and sdhA) were mutated by inrame deletion. Results from this study provided experimental evidence for many predicted proteins. In addition, identification of differentially expressed proteins provided targets for further functional analysis, which could help elucidate pathogenic mechanisms of E. ictaluri.

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The mechanochemical polycondensation between a diamine and a dialdehyde constitutes a sustainable alternative to classical solvent-based polymerization reactions. This process not only allows for a higher conversion and a shorter reaction time as compared to standard solvent-based syntheses of this conjugated polymer, but the reaction can also be adjusted by the energy introduced via the ball mill.

L'objectif de cette thèse est d'appliquer la méthode d'identification bactérienne par spectrométrie de masse MALDI-TOF pour une utilisation en routine dans un laboratoire de microbiologie clinique. Dans un 1er temps et de manière prospective, nous avons évalué la performance et le coût-efficacité de l'identification bactérienne de routine par MALDI-TOF par rapport aux techniques conventionnelles d'identification phénotypique. Durant la période des 16 semaines d'étude, nous avons comparé la performance de la technique par MALDI-TOF aux techniques conventionnelles d'identification phénotypique comprenant la coloration de Gram, la galerie API ANA et le Vitek 2. En cas de résultats discordants entre ces deux techniques, l'identification était réalisée par biologie moléculaire. Nous avons montré que le MALDI-TOF est un moyen efficace et rentable pour l'identification des bactéries de routine. Le MALDI-TOF peut être utilisée en 1ère intention dans l'identification bactérienne avant l'ensemble de techniques phénotypiques. Dans un 2ème temps, nous avons évalué rétrospectivement la performance et le coût-efficacité de l'utilisation exclusive de MALDI-TOF en diagnostic bactériologique de routine en comparaison avec les techniques conventionnelles. En analysant les données des 11 dernières années, nous avons montré que le MALDI-TOF est efficace et tout à fait adaptée pour l'identification d'espèce bactérienne en routine. Nous avons également prouvé que MALDI-TOF est un outil puissant pour identifier les espèces bactériennes rarement impliquées dans les infections humaines. Cette technique pourrait être une alternative aux méthodes moléculaires dans le laboratoire clinique. / The objective of this thesis is to apply the method of bacterial identification by MALDI-TOF MS in daily practice in a routine clinical microbiological laboratory. Firstly, we prospectively evaluated the performance and the cost-effective of bacterial identification by MALDI-TOF in comparison with conventional phenotypic identification methods. During a 16-week study, we compared the performance of MALDI-TOF with conventional techniques of identification including Gram staining, API ANA identification strip and automated identification using the Vitek 2. The unmatched identifications between MALDI-TOF and conventional methods were resolved by molecular identification. In this study, we showed that MALDI-TOF was an effective tool and less expensive for the rapid identification of bacterial species in clinical microbiology laboratory. MALDI-TOF can be used in first intention for identification before Gram staining or other phenotypic identification techniques based on physicochemical properties of bacteria. Secondly, we retrospectively evaluated the performance and the cost-effectiveness of the exclusive use of MALDI-TOF in bacteriological diagnosis in comparison with conventional phenotypic identification. 11-year retrospective analysis of data showed that MALDI-TOF was efficient and completely adapted for the routine identification of bacterial species. We also showed that MALDI-TOF had capacity to identify bacterial species that were rarely involved in human diseases. This technique could be an alternative to molecular methods in the clinical laboratory.

Das magenbesiedelnde Bakterium Helicobacter pylori gehört zu den am weitesten verbreiteten Infektionserregern. Obwohl die Infektion meist lebenslang symptomlos verläuft, kann H. pylori bei einigen Menschen schwere Erkrankungen bis hin zum Magenkarzinom verursachen. Ziele dieser Arbeit waren Magenkarzinom-assoziierte Antigene für einen diagnostischen Test zu finden und Methoden zur Untersuchung von Spotkompositionen mittels MALDI-TOF/TOF Massenspektrometrie zu entwickeln. Im ersten Teil der Promotion wurden die Antigenerkennungsmuster von 30 Magenadenokarzinom- mit 30 Ulkus duodeni-Patienten mithilfe hochauflösender zweidimensionaler Immunoblots von H. pylori Lysat verglichen. Diese fokussierte Gegenüberstellung eignet sich gut für diese Fragestellung, da beide Erkrankungen von diesem Bakterium verursacht werden, aber nur sehr selten gemeinsam auftreten. Durch univariate statistische Analysen wurden 14 Magenkarzinom korrelierte Spots gefunden (p / The stomach-colonizing bacterium Helicobacter pylori is one of the most widespread infectious agents. Although infection mostly persists unnoticed, it may cause serious diseases like gastric carcinoma. Aims of this project were to find gastric carcinoma-associated antigens for a diagnostic test and to develop methods to analyze spot compositions using MALDI-TOF/TOF mass spectrometry. In the first part of this project antigen recognition patterns of 30 gastric carcinoma- and 30 duodenal ulcer- patients were compared using high-resolution two-dimensional immunoblots of H. pylori lysate. This focused comparison lent itself to this question because both diseases are caused by the bacterium but rarely occur conjointly. Utilizing univariate statistical tests 14 gastric carcinoma-associated spots were found (p