Mon travail de thèse a consisté à poursuivre le développement de l’Imagerie par Spectrométrie de Masse (IMS), à la fois sur le plan méthodologique mais aussi à travers des applications biologiques.Dans une première partie est exposé le développement d’une méthode de préparation des échantillons de très petite taille pour l’imagerie chimique, et plus particulièrement pour l’imagerie TOF-SIMS. Cette méthode a pu être validée en étudiant des molécules de la famille des flavonoïdes dans différents types de graines d’Arabidopsis thaliana ne mesurant que 400 nm de diamètre. La seconde partie, dédiée aux applications biologiques, est divisée en deux sections. La première section regroupe deux sujets où il était question de détecter et de localiser, à l’aide de l’imagerie TOF-SIMS, la molécule active d’une crème anti-acné dans des coupes de peaux de cadavre humain, ainsi qu’un retardateur de flamme bromé, le décabromodiphényl éther, dans ses tissus cibles chez le rat. Dans la seconde section, nous avons étudié, en imagerie TOF-SIMS et MALDI-TOF, l’absorption des lipides durant la digestion et enfin, avec l’aide d’outils d’analyse statistiques, nous avons comparé les profils lipidiques d’échantillons sains et atteins de la mucoviscidose chez un animal modèle de la maladie.À travers ces différents projets, nous avons pu conclure que les imageries par spectrométrie de masse TOF-SIMS et MALDI-TOF sont deux techniques complémentaires et qui, combinées à l’analyse statistique, peuvent être de puissants outils. / My PhD’s work consisted in continuing the development of Mass Spectrometry Imaging (MSI) methods, in terms of methodology improvements but also through biological applications.The first part concerned the development of a novel sample preparation method dedicated to very small objects for chemical imaging, particularly for TOF-SIMS imaging. This method has been validated by studying different types of flavonoids in from seeds of Arabidopsis thaliana, with a size of 400 nm only. The second part, dedicated to biological applications, is divided into two sections. The first section includes two projects where the goals was to detect and locate, using TOF-SIMS imaging, the active molecule of an anti-acne cream in human skin sections, and a brominated flame retardant, the decabromodiphenyl ether, in target tissues in rats. In the second section, we have studied by MALDI-TOF and TOF-SIMS imaging the lipid absorption during the digestion, and finally, with the help of statistical analysis tools, we compared lipid profiles of healthy samples versus those from cystic fibrosis samples in a model animal of the disease.Through these projects, we have concluded that MALDI-TOF and TOF-SIMS imaging are two complementary techniques, and, when they are combined with statistical analysis, they can be powerful tools.

We have successfully implemented the time-resolved technique at frequency domain on a laser scanning microscope. The method is implemented via interfacing a lock-in amplifier with the laser scanning microscope and is used to investigate organic photonic devices (OPD). The available modalities include electroluminescence and optical beam induced current. In this way, temporal response of a device can be investigated at high spatial resolution. Despite of its now limited role in flat panel display, OPD presents as a versatile material with numerous potentials, with solar power panel as the most noted one recently. The investigation conducted enables us to better understand the fundamental dynamics of carriers unique to OPDs.

Parkinson's is a neurological condition in which parts of the brain responsible for movements becomes incapacitated over time due to the abnormal dopamine equilibrium. Freezing of Gait (FOG) is one of the main Parkinson's Disease (PD) symptoms that affects patients not only physically but also psychologically as it prevents them from fulfilling simple tasks such as standing up or walking. Different auditory and visual cues have been proven to be very effective in improving the mobility of People with Parkinson's (PwP). Nonetheless, many of the available methods require user intervention or devices to be worn, charged, etc. to activate the cues. This research suggests a system that can provide an unobtrusive facility to detect FOG and falling in PwP as well as monitoring and improving their mobility using laser-based visual cues casted by an automated laser system. It proposes a new indoor method for casting a set of two parallel laser lines as a dynamic visual cue in front of a subject's feet based on the subject's head direction and 3D location in a room. The proposed system controls the movement of a set of pan/tilt servo motors and laser pointers using a microcontroller based on the real-time skeletal information acquired from a Kinect v2 sensor. A Graphical User Interface (GUI) is created that enables users to control and adjust the settings based on the user preferences. The system was tested and trained by 12 healthy participants and reviewed by 15 PwP who suffer from frequent FOG episodes. The results showed the possibility of employing the system as an indoor and on-demand visual cue system for PwP that does not rely on the subject's input or introduce any additional complexities to operate. Despite limitations regarding its outdoor use, feedback was very positive in terms of domestic usability and convenience, where 12/15 PwP showed interest in installing and using the system at their homes.

Abstract Rapid and reliable identification of microorganisms is of paramount importance for advancing homeland security. Mass spectrometry is emerging as an effective bioanalytical tool having unique capabilities in handing complex mixtures. Matrix-assisted layser desorption / ionization time-of-flight mass spectrometry ¡]MALDI/TOF MS¡^is commonly used in the analysis of bio-molecules owing to its merits of high sensitivity, wide mass range, and ease-of-operation. The objective of the first part of the thesis is to develop an analysis method to characterize the genus, strains, and subspecies of the infections gastroenteritis bacteria by using MALDI/TOF MS. MALDI/TOF MS can be applied to classify different microoganisms based on examination of ion patterns from different microoganisms and unique distinguishing protein ions as biomarkers for different strains. The second section, bacteriophages MS2 and T4 were used as materials for identification of their specific protein markers. Protocols including sample preparation, purification, combined in gel digestion and post source decay to which the mass spectrometer brought for analytical specificity were employed. According to the experimental results, classification of different microorganisms based on examination of ion patterns is feasible. Furthermore, the peak at m/z 5255 in the MALDI mass spectra of MS2 analysis and the peak at m/z 8600 of T4 can provide the unique distinguishing signals. The experimental mass spectral peaks were submitted to the database search, and one of these peaks was matched to a tail fiber assembly helper protein. Thus, the method developed and the mass spectra presented in this thesis can be potentially applied to the practical virus analysis.

Mainz, Universiẗat, Diss., 2002.

Frankfurt (Main), Universiẗat, Diss., 2001.

Kiel, Universiẗat, Diss., 2001.

Urinary tract infection (UTI) is a very common infection in humans and a majority is caused by Escherichia coli. UTI are commonly treated empirically. However, empiric treatment has become more problematic due to increased antibiotic resistance to commonly used antibiotic agents. It is therefore desirable with short turnover times for antimicrobial susceptibility testing and species identification to improve antibiotic treatment at an early stage. Matrix Assisted Laser Desorption/Ionisation Time of Flight (MALDI-TOF) can provide species identification faster than former routine methods. This study compared direct and standard susceptibility testing using disk diffusion on Enterobacteriaceae (EB) from urine samples. The possibility to standardise the inoculum for direct susceptibility testing via a pellet obtained by a series of centrifugations was also evaluated, as well as direct species identification with MALDI-TOF from the pellet. Results from direct susceptibility testing from urine samples with EB, performed either directly from the urine or with a standardised inoculum, correlated well to those obtained with standardised susceptibility testing using EUCAST disk diffusion methodology with few errors, of which most were associated with Proteus mirabilis. The concept of standardising the inoculum for direct susceptibility testing to 0.5 McFarland was labour intensive and did not improve the results further. However, direct species identification from the urine pellet using MALDI-TOF showed good correlation to routine identification. Of 238 samples, an EB was correctly identified in 148 samples using MALDI-TOF.

Die klassische Identifizierung von obligat anaeroben Bakterien ist mit einem hohen Labor- und Zeitaufwand verbunden. Um festzustellen, ob die Identifizierung mittels Matrix-unterstützter Laser-Desorption/Ionisation und Massenspektrometrie mit Flugzeitanalysator (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MALDI-TOF MS) ein Verfahren ist, um obligate Anaerobier eindeutig zu identifizieren, wurde mit der vorliegenden Arbeit die Identifizierung von unterschiedlichen Spezies der B. fragilis Gruppe mittels MALDI-TOF MS untersucht. Hierfür wurden 105 obligate Anaerobier der B. fragilis Gruppe aus der Stammsammlung des Institutes für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig untersucht. Es fanden sich für die untersuchten Erreger Spektren mit sehr guter Auflösung. Eine Identifizierung und Differenzierung war eindeutig möglich. Unter Verwendung dieser Daten wurde eine Referenzdatenbank erstellt. Die erhaltenen Ergebnisse wurden mittels einer verblindeten Studie überprüft, wobei 52 von 53 (98,1%) der untersuchten Stämme eindeutig identifiziert werden konnten. Dies schließt ebenfalls die Identifizierung und Differenzierung von 15 Metronidazol-sensiblen/ Enterotoxin-negativen, 8 Metronidazol-resistenten/ Enterotoxin-negativen und 8 Metronidazol-sensiblen/ Enterotoxin-positiven B. fragilis Stämmen ein. Die Identifizierung mittels MALDI-TOF MS ist somit eine zuverlässige Methode zur Identifizierung von obligaten Anaerobiern der B. fragilis Gruppe. Weiterhin finden sich Hinweise, dass ein Nachweis von Resistenz-, Virulenz- und Pathogenitätsfaktoren mittels MALDI-TOF MS bei diesen Erregern möglich ist.

Die klassische Identifizierung von obligat anaeroben Bakterien ist mit einem hohen Labor- und Zeitaufwand verbunden. Um festzustellen, ob die Identifizierung mittels Matrix-unterstützter Laser-Desorption/Ionisation und Massenspektrometrie mit Flugzeitanalysator (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MALDI-TOF MS) ein Verfahren ist, um obligate Anaerobier eindeutig zu identifizieren, wurde mit der vorliegenden Arbeit die Identifizierung von unterschiedlichen Spezies der B. fragilis Gruppe mittels MALDI-TOF MS untersucht. Hierfür wurden 105 obligate Anaerobier der B. fragilis Gruppe aus der Stammsammlung des Institutes für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig untersucht. Es fanden sich für die untersuchten Erreger Spektren mit sehr guter Auflösung. Eine Identifizierung und Differenzierung war eindeutig möglich. Unter Verwendung dieser Daten wurde eine Referenzdatenbank erstellt. Die erhaltenen Ergebnisse wurden mittels einer verblindeten Studie überprüft, wobei 52 von 53 (98,1%) der untersuchten Stämme eindeutig identifiziert werden konnten. Dies schließt ebenfalls die Identifizierung und Differenzierung von 15 Metronidazol-sensiblen/ Enterotoxin-negativen, 8 Metronidazol-resistenten/ Enterotoxin-negativen und 8 Metronidazol-sensiblen/ Enterotoxin-positiven B. fragilis Stämmen ein. Die Identifizierung mittels MALDI-TOF MS ist somit eine zuverlässige Methode zur Identifizierung von obligaten Anaerobiern der B. fragilis Gruppe. Weiterhin finden sich Hinweise, dass ein Nachweis von Resistenz-, Virulenz- und Pathogenitätsfaktoren mittels MALDI-TOF MS bei diesen Erregern möglich ist.