Induktiv gekoppelte Plasma-Massenspektrometrie mit Laserablation (LA-ICP-MS) wird zunehmend für die Einzelzellanalyse eingesetzt, jedoch wird eine weitere verbreitete Verbreitung durch den geringen Durchsatz behindert. Daher wurde in dieser Arbeit der Durchsatz von Einzelzellen-LA-ICP-MS untersucht und verbessert. Zunächst werden die beiden möglichen Ablationsmodi, Bildgebung und Einzelpunktanalyse (SSA), hinsichtlich ihrer analytischen Gütezahlen (Signal-Rausch-Verhältnis, Präzision, Genauigkeit, Durchsatz) verglichen. Hierfür wurden adhärente 3T3-Fibroblastenzellen mit zwei Metallfarbstoffen angefärbt und mit beiden Methoden mehrere Dutzend Zellen vermessen. SSA zeigte überlegene Eigenschaften hinsichtlich Durchsatz und Nachweisgrenzen. Darüber hinaus wurde gezeigt, dass >400 Zellen analysiert werden müssen, um zufriedenstellende Statistiken für einen quantitativen Vergleich der Ergebnisse zu erhalten, was als zu mühsam befunden wurde. Daher wurde ein Einzelzellen-Arraying-Schritt integriert, um eine automatisierte LA-ICP-MS-Analyse zu ermöglichen. Hierfür wurden zwei Arrayingverfahren getestet: Zunächst wurde das mikrofluidische Arraying von Zellen getestet, jedoch verhinderte das Einklemmen von weichen PDMS-Chips eine erfolgreiche Anwendung, und eine Neugestaltung des Chips wäre erforderlich. Daraufhin wurde eine neuartige Technologie getestet, die auf dem Arraying von Tröpfchen in Verbindung mit der Bilderkennung von Zellen beruht, wobei ein Anordnungsdurchsatz von 550 Zellen pro Stunde und eine beispiellose Einzelzellengenauigkeit (> 99%) gefunden wurde. In einem Proof-of-Principle-Experiment wurde ein Zellarray von THP-1-Suspensionszellen mittels LA-ICP-TOF-MS analysiert und erstmals gleichzeitig endogene und exogene Isotope einzelner Zellen als Isotopen-Fingerabdrücke von Zellen mit Nachweisgrenzen von lediglich wenigen hundert attogramm. Schließlich wurden diese Ergebnisse mit der derzeit gebräuchlichsten Analysemethode Single-Cell (sc)-ICP-MS verglichen. / Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is increasingly used for single-cell analysis. However, a more widespread use of LA-ICP-MS in single cell analysis is hampered by its low throughput. Hence, in this work the throughput of single cell LA-ICP-MS was studied and improved. First, the two possible ablation modes, imaging and single spot analysis (SSA) of single cells using a large laser spot, are compared regarding their analytical figures of merit (signal to noise, precision, accuracy, throughput), as well as regarding ease of operation and data evaluation. For that, adherent 3T3 fibroblast cells were stained with two metal dyes and several dozen cells were measured using both modes. SSA showed superior characteristics regarding throughput and detection limits. Moreover, it was shown that >400 cells must be analyzed to reach satisfactory statistics for a quantitative comparison of results, which would have been too laborious. Thus, a single cell arraying step was integrated to enable automated LA-ICP-MS analysis. Two different arraying methods were evaluated: First, arraying via hydrodynamic front trapping of cells using a microfluidic device was tested, but clamping of soft PDMS-chips prevented successful arraying and it was concluded that a major redesign of the chip is necessary. Secondly, and a novel technology relying on a microdroplet arrayer in conjunction with image recognition of cells was tested and a moderate arraying throughput (550 cells per hour) and an unprecedented single-cell accuracy (>99%) was found. In a proof of principle experiment, a cell array of THP-1 suspension cells was analyzed using LA-ICP-TOF-MS and endogenic and exogenic isotopes of individual cells were detected for the first time simultaneously as isotopic fingerprints of cells with detection limits as low as hundred attogram. Finally, these results were compared to the currently more commonly used analysis method single-cell (sc)-ICP-MS.

Electronic devices define our everyday lives. They are often large, rigid, and brittle. Modern medical science has come so far as to start using miniature electronic devices to monitor many types of diseases. Especially, neurological disorders pose obstacles hard to overcome when treating them but it also motivates finding methods that allow for continuous monitoring. Implementing a small electronic device inside the human body adds requirements on the device to be stretchable, biocompatible, and more. Not only is the device limited by these factors, but also, current fabrication methods are not efficient for creating nanoscale versions for these types of devices. patterninglaser ablation is a growing field for cutting out and pattern nano-materialistic devices with high precision and good repeatability. This project is focused on using a laser engraving tool from metaquip on different substrates. This project is focused on the development of methods for laser fabrication of soft neural electrodes. The requiered steps are the alignment of samples to assure good precision when engraving it with the laser engraving tool, that also will be called “laserpatterner”, finding good parameters for cutting out devices and pattern conductors for said devices. On top of that, a linear force stretching will be used to characterize samples that were cut, in the form of strips, using the laserpatterner. The stretching behavior of strips consisting of the elastomer polydimethylsiloxane, which in turn will be the insulator for silver nanowires, is examined in the stretching setup. Parameter optimization is relevant in all experiments done in this project and lay the foundations for cutting and patterning silver nanowires devices. All factors included will eventually lead to a good method for fabricating soft neural electrode devices and this project is stepping stone towards that goal. / <p>Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p>

The absence of a cheap and easily scalable synthesis technique for nitrogen-vacancy (NV) centers enriched nanodiamonds (NDs) is a critical factor for the development of devices based on this very peculiar nanoparticle. Indeed, the combination between the unique NV fluorescence properties and NDs characteristics allow to obtain a tool having quantum sensing capabilities, with nanometric spatial resolution, which is able to operate in a wide range of temperature, pressures and in harsh chemical conditions. NVenriched NDs applications in nanothermometry, nanomagnetometry and in bio-imaging have already been reported. However, most of the standard fluorescent NDs production techniques present common drawbacks: poor control in NDs size distribution and in nitrogen concentration, as well as the need of post-synthesis process to clean the NDs surface from impurities and to increase the NV density. In this thesis, an alternative method for fluorescent NDs synthesis based on pulsed laser ablation (PLA) of graphite is demonstrated. After the introductory chapters on NV-centers physics and NDs properties (Chapter 2 and 3), the demonstration that PLA is a viable route for synthesis of NDs is given in Chapter 4. In particular, PLA of graphite and of diamond-like carbon is performed in water. Here, a thermodynamic model taking into account the peculiar physical processes occurring during PLA is developed to explain NDs formation. Then, synthesis of NV-enriched NDs is demonstrated through PLA of graphite in a nitrogen atmosphere (Chapter 5) and in liquid nitrogen (Chapter 6). In both chapters, the thermodynamic model is adapted to explain diamond phase formation in a gaseous environment and in a cryogenic liquid. Furthermore, NV centers optical properties are fully characterized with optically detected magnetic resonance (ODMR) spectroscopy. Finally, in Chapter 7, fluorescent NDs are produced by laser ablation of N-doped graphite in water. This particular target is then used for a quantitative comparison between the other fluorescent NDs laser-synthesis, with the aim of establishing in which condition the highest NV-center formation efficiency is achieved.

The present research work mainly focuses on the fabrication and characterization of single and multilayer thin films based on Bismuth Vanadate (BVO) and Bismuth Titanate (BTO). The multi-target laser ablation technique was used to fabricate single layer thin films of BVO, BVN and BTO; and multilayers composed of BVO and BTO in different structures. The fabricated thin films exhibited dense microstructure and a sharp interface with the substrate. The lattice strain, surface roughness and grain size could be varied as functions of composition and individual layer thickness in different structure fabricated. The optical properties were studied by spectroscopic ellipsometry and optical transmission spectra. The various models that were used for ellipsometric data analysis gave an excellent fitting to the experimental data. The optical constants were determined through multilayer analyses of the films. The band gap of these films was studied by spectroscopic ellipsometry and optical transmission. The optical studies carried out on BVO-BTO bilayer indicated the presence of an interfacial layer in between the BVO and BTO layer, whose refractive index was different from that of the individual layers and is attributed to different nature of the interfacial layer. The ferroelectric nature of BVO films was confirmed by P-E hysteresis loop studies under different applied fields and at various probing frequencies. The same was corroborated via the C-V measurements of these BVO films which exhibited butterfly shaped C-V characteristics. Fatigue studies in these films indicated that the switchable polarization is essentially constant through 105 cycles, after which it starts increasing probably due to the ionic conduction in BVO thin films. The dielectric response of undoped and Nb doped BVO as well as BVBT ML thin films were studied over a wide range of temperatures. The BVO films exhibited remarkable dielectric dispersion at low frequencies especially in the high temperature regime. Further, the frequency and temperature dependence of the dielectric, impedance, modulus and conductivity spectra of these films were investigated in detail. The ac conductivity was found to obey well the double power law in case of ML, indicating the different contributions to the conductivity, the low frequency conductivity being due to the short range translational hopping and the high frequency conductivity is due to the localized or reorientational hopping motion. DC leakage conduction in BVO, BVN and BVBT ML thin films was studied over a wide range of temperatures and applied electric fields. The experimental data were analyzed in light of different models to investigate the dc conduction mechanism in these films which were broadly classified into electrode limited and bulk limited conduction processes. In the case of BVO thin films the dc leakage current exhibited an ohmic nature at low electric fields followed by an onset of the space charge limited conduction (>1). Further in case of BVN films, three distinct regions were observed in I-V characteristics signifying different types of conduction processes in these films. In case of BVBT ML thin films, bulk limited PF mechanism was found to determine the conduction behavior at moderate electric fields. At higher electric fields, a trap filled region was observed which was followed by SCL conduction at higher fields. Therefore the present observation indicates the presence of more than one bulk limited conduction process in BVBT ML thin films. BVO thin films exhibiting good structure and dense morphology were successfully prepared on p-type Si by chemical solution decomposition technique. The C-V characteristics were evaluated for Au/BVO/Si MFS structure which showed a typical high frequency feature of a conventional MFIS structure.

Les applications présentées dans cette thèse valorisent de diverses manières le principe d'ablation laser, c'est à dire l'arrachement de la matière d'une surface solide suite à l'irradiation avec un faisceau laser. Le plasma généré par irradiation laser impulsionnel a été utilisé pour le dépôt de couches minces ou de nanoparticules et pour l'analyse compositionnelle des couches d'épaisseur nanométrique. Nous avons synthétisé par dépôt laser combinatoire des librairies compositionnelles d'un oxyde mixte transparent d'In et Zn. En utilisant le plasma d'ablation pour le diagnostic compositionnel, nous avons déterminé les concentrations d'indium et de zinc dans les couches minces par spectroscopie laser. Des couches minces de bioverre ont été synthétisées par dépôt laser impulsionnel sur des substrats de titane. En contact avec des cellules ostéoblastes, les bioverres ont stimulé la prolifération et ont augmenté la viabilité. La prolifération des ostéoblastes cultivés sur les couches de bioverre a été 30% supérieure a l'échantillon de contrôle. On a déposé par PLD des couches minces ou nanoparticules adhérentes de ZnO sur des substrats textiles hydrophiles dans un flux d'oxygène ou sous vide pour obtenir des structures avec différentes mouillabilités. En augmentant le nombre d'impulsions laser de 10 à 100 nous avons observé la transition du recouvrement par des nanoparticules isolées vers des couches minces. En fonction de l'atmosphère environnant lors du dépôt, les couches minces et les nanoparticules ont changé leur mouillabilité, passant d'hydrophile en flux d'oxygène à un comportement superhydrophobe (angle de contact de 157°) en cas de dépôt sous vide. / The applications presented in this thesis exploit in different modes the principle of laser ablation, i.e. the material removal from a solid surface following irradiation with a pulsed laser beam. The plasma generated by laser ablation was used for thin films or nanoparticles deposition and for the compositional analysis of nanometric thin films. We synthesized by combinatorial pulsed laser deposition, thin film libraries of a complex oxide of In and Zn. Using the ablation plasma for compositional diagnostic, we determined the In and Zn concentrations in films by Laser Induced Breakdown Spectroscopy using a procedure based on the spectral luminance calculation of a plasma in local thermodynamic equilibrium. Thin films of bioactive glass were synthesized by pulsed laser deposition, magnetron sputtering and MAPLE on Ti substrates and tested the transfer accuracy by physico-chemical tests and their functionality in vitro. In contact with human osteoblast cells, the bioactive glasses stimulated their proliferation and enhanced their viability. The proliferation of osteoblasts cultivated on bioactive films was 30% superior to the control sample. ZnO thin films or nanoparticles were deposited on hydrophilic textile substrates in oxygen flux or in vacuum in order to obtain structures with different wetting behavior. Increasing the number of laser pulses from 10 to 100, we observed a coating transition from isolated nanoparticles to thin films fully coating the textile fibers. Function of the ambient atmosphere during experiments, the structures changed their wetting behavior, passing from hydrophilic in oxygen flux to superhydrophobic (157°) in case of deposition in vacuum.

L’émergence des satellites très haut débit pour la couverture des zones rurales s’accompagne de nombreuses contraintes technologiques. Dans le cadre du plan France très haut débit, le projet THD-sat proposé par le CNES se base sur l’utilisation des bandes Q et V pour assurer les liaisons avec les stations au sol et libérer de la ressource sur la bande Ka communément utilisée par les satellites ancienne génération. Avec la montée en fréquence, les besoins en termes de filtrage deviennent très stricts nécessitant des considérations particulières. Le premier chapitre reprend le contexte de l’étude et expose les différents éléments permettant de justifier le choix de la technologie SIW qui profite à la fois des bons facteurs de qualité des modes volumiques se propageant dans le substrat et de l’aisance du procédé technologique et de l’intégration des structures planaires. Les performances des cavités SIW restent néanmoins intimement liées à l’épaisseur de substrat qui doit être augmentée pour atteindre de meilleurs facteurs de qualité. L’augmentation de l’épaisseur de substrat s’accompagne de deux principales limitations : le rallongement des fils de « bonding » utilisés pour le câblage du filtre à son environnement MIC d’épaisseur 254 μm et l’élargissement de la ligne d’accès 50 Ω induisant des problèmes de discontinuités et d’excitations de modes parasites. L’approche suivie consiste à considérer des formes 3D permettant l’adaptation de mode et d’épaisseur entre une ligne microruban sur substrat de 254 μm d’épaisseur et le SIW d’épaisseur plus importante. Une nouvelle transition 3D est dès lors imaginée. Le chapitre II reprend les différents procédés technologiques utilisés pour la mise en forme et la métallisation des substrats 3D. Les substrats considérés sont l’alumine et la silice fondue mis en forme par ablation laser et le thermoplastique COP mis en forme par moulage. La principale limitation de l’ablation laser concerne les épaisseurs de substrat accessibles. Nous nous limitons à 635 μm dans le cas de l’alumine et à 500 μm dans le cas de la silice fondue. Le moulage polymère permet de s’affranchir de cette limitation et de viser des substrats plus épais (2 mm pour la solution COP).Le chapitre III reprend les étapes de conception des différentes solutions de filtrage avec la nouvelle transition 3D. Des résultats de mesures de différents prototypes réalisés sont par ailleurs présentés. Ces résultats sont globalement encourageants mais nécessitent d’être davantage développés pour être mieux exploitables. / The emergence of satellite high-speed internet for the coverage of rural zones is accompanied by numerous technological constraints. The current trend is to use higher frequency bands to release the satellite capacity for users. The increasing frequency requires new considerations especially for filtering needs which become notably strict in terms of performance and integration in small integrated circuits. This work introduces filtering solutions based on high quality factor Substrate Integrated Waveguides (SIW) using a novel 3D transition for a better integration in widely planar Hybrid ICs.The first chapter introduces the study’s context and the different elements justifying the use of the SIW technology.In fact, these structures profit from both the good quality factors of TE-modes propagating in the substrate and the easy fabrication process and integration of planar circuits. However, to increase the SIW quality factor, the substrate’s height should be increased which induces interconnection limitations such as long bond wires with high parasitic effects and large microstrip access lines with discontinuity problems and the propagation of parasitic modes. The adopted approach consists in imagining 3D shapes providing both mode and thickness matching between a microstrip line etched on a thin substrate and a high substrate SIW.The second chapter introduces the different manufacturing processes used for the substrate’s shaping and metallization. Three substrates are considered: Alumina, fused Silica and Cyclo Olefin Polymer COC. Alumina is widely used in space applications and has a well-mastered process. For equivalent dielectric losses, fused silica has a lower permittivity for bigger structures with less manufacturing tolerance sensitivity. Both Alumina and fused silica substrates are shaped using a laser ablation. The reachable substrate’s height using this machining method is relatively low. The polymer solution (COP) is elaborated using a molding process allowing higher substrates heights.The last chapter outlines the design steps of the different solutions and the measurement results of the first prototypes. These results are on the whole encouraging but require further development.

Die Laserablation gekoppelt mit der Massenspektrometrie mit induktiv gekoppeltem Plasma ist eine vielversprechende direkte Feststofftechnik, die sich jedoch bei der Analyse von Kunststoffen wegen des Mangels an matrixangepassten zertifizierten Referenzmaterialien nicht durchsetzen konnte. Vorherige Arbeiten belegen polymerabhängige Abtragsraten. Das oft als interner Standard verwendete Intensitätssignal des Kohlenstoffisotopes 13C zur Korrektur dieser Unterschiede wird in der Literatur kritisch diskutiert. In dieser Arbeit als ein Teil des BMBF-geförderten MaxLaP-Projektes wurden matrixangepasste Standards auf Polyethylen- und Acrylnitril-Butadien-Styrolbasis entwickelt. In diese Standards wurden Br, Cd, Cu, Cr, Fe, Sb in organischer und anorganischer Form ihrer Verbindungen mittels Extrusion eingearbeitet. Die quantitative Zusammensetzung der Materialien wurde mittels ETV-ICP-OES, DC-arc-OES, RFA und ICP-MS nach Mikrowellendruckaufschluss überprüft. Die Verfahren wurden für die Kunststoffanalyse optimiert. Die mikroskopische Homogenität der Einarbeitung wurde mittels µ-SYRFA und LA-ICP-MS untersucht. Zur Untersuchung der Matrixeffekte während der LA-ICP-MS und der matrixunabhängigen Kalibrierung für Kunststoffe wurden der Einfluss der chemischen Verbindung der Additive, die Größe, der bei der Laserablation gebildeten Partikel und die Art des Kunststoffes auf die Laserablation analysiert. Die Korrektur des verschiedenen Materialabtrages über die Verwendung des 13C-Signals konnte für 21 verschiedene Kunststoffe erfolgreich durchgeführt werden. Allerdings zeigen die zugesetzten Additive ein nicht identisches Verhalten hinsichtlich Transport und Ionisierung. Weitere Ergebnisse belegen eine Anreicherung der Additive in verschiedenen Partikelgrößen sowie eine deutlich unterschiedliche Partikelbildung bei Ablationen von verschiedenen polymeren Matrices, was zu einer verstärkten Elementfraktionierung bei einer nicht matrixangepassten Kalibrierung führt. / Laser ablation coupled to a mass spectrometer with inductively coupled plasma (LA-ICP-MS) is a promising direct solid sampling technique. Due to the lack of matrix matched standard materials laser ablation is not well established in polymer analysis. In a recent study a polymer dependent interaction with the laser beam was reported, which resulted in a polymer depending ablation rate. The usage of the carbon-13-signal intensity as internal standard to correct these differences as commonly applied has been critically discussed in literature. In this work as part of a BMBF-supported MaxLaP-project (matrix effects during laser ablation of polymers) matrix matched standards based on polyethylene and acrylonitrile butadiene styrene were developed. In these materials Br, Cd, Cu, Cr, Fe and Sb were incorporated as organic and inorganic compounds through extrusion. Quantitative composition of the materials was determined by ETV-ICP-OES, DC-arc-OES, XRF and ICP-MS after high pressure microwave digestion. Analytical methods were optimized for trace analysis in plastic matrices. Microscopic homogeneity was examined by µ-SyXRF as well as LA-ICP-MS. In order to investigate the matrix effects and to determine the possibility of a matrix independent calibration for plastic materials, the influence of the chemical form of the additives, size of the formed particles and the type of the plastic on the LA-ICP-MS measurements were analyzed. The correction of the material uptake by the carbon-13-signal was successfully applied for 21 different types of plastic. However, different incorporated additives show a different transport and ionization behavior. Furthermore, our results confirm a different enrichment of the additives depending on particle size and a significantly different particle formation for different types of plastic, which leads to a more pronounced elemental fractionation by not using a matrix matched calibration.

Gadoliumbasierte Kontrastmittel werden in der Magnetresonanztomographie verwendet und gelangen nach der Exkretion durch behandelte Patienten in Oberflächengewässer, da sie von Klärwerken nur unzureichend entfernt werden. Das Verhalten der Kontrastmittel in der Umwelt und deren Wechselwirkung mit Organismen wurde in dieser Arbeit aufgrund der Toxizität des freien Gd-Ions und der damit verbundenen möglichen Gefahren, wie der Anreicherung in der menschlichen Nahrungskette, näher untersucht. Die bildgebenden elementanalytischen Methoden Laserablation mit induktiv gekoppelter Plasmamassenspektrometrie (LA-ICP-MS) und Röntgenfluoreszenzanalyse mit Synchrotronstrahlung (SRXRF) wurden genutzt, um verschiedene biologische Systeme hinsichtlich ihrer Aufnahme, Verteilung und Abgabe von Gd-basierten Kontrastmitteln zu untersuchen. Zuvor wurden beide Methoden für diese Anwendung analytisch charakterisiert und verglichen. Die Nachweisgrenzen von Gadolinium wurden mittels LA-ICP-MS (0.78 pg) und SRXRF (89 pg) unter jeweils optimierten Bedingungen durch die Kalibrierung über Rückstände eingetrockneter Elementstandardtropfen bestimmt. Anhand von Fadenalgen konnte die Aufnahme und Abgabe der Gd-basierten Kontrastmittel gezeigt werden. Die Abhängigkeit von der Konzentration des Kontrastmittels in der Expositionslösung und die Unabhängigkeit der zeitlichen Aufnahme innerhalb eines Zeitraums von 1-7 Tagen wurden an Wasserlinsen studiert. Gadolinium konnte in einem Kresseblatt quantifiziert werden. Die Verifizierung der LA-ICP-MS-Ergebnisse erfolgte mit der SRXRF und der ICP-MS nach Aufschluss. Auch die Aufnahme und Abgabe von Gd in Wasserflöhen wurde beobachtet. Die Lokalisierung des Gds wurde mittels 3D µ-Computertomographie durch den Vergleich mit einem unbelasteten Wasserfloh aufgeklärt. In allen Untersuchungen wurde Gadolinium in den exponierten Modellorganismen nachgewiesen, woraus geschlussfolgert werden kann, dass die Kontrastmittel aus der Umgebung aufgenommen werden. / Gadolinium based contrast agents are used for magnetic resonance imaging. After their excretion by medicated patients they reach surface water passing waste water treat-ment plants where they are not removed sufficiently. The behavior of the contrast agents in the environment and the interaction with organisms was investigated in this work due to the toxicity of the free Gd3+ ion and the associated risks, such as accumulation in the human food chain. In this work, the two elemental analytical imaging methods laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and synchrotron radiation X-ray fluorescence analysis (SRXRF) have been used to investigate the uptake, distribution, and excretion of Gd-based contrast agents by various biological systems. Both methods were analytically characterized and compared for this application. The detection limits of gadolinium were determined under optimized conditions by LA-ICP-MS and SRXRF. With calibration by remains of dried elemental standard droplets detection limits of 0.78 pg absolute amount of gadolinium (LA-ICP-MS), respectively 89 pg (SRXRF) were reached. Based on filamentous algae as water plants the uptake and the excretion of Gd-based contrast agents were revealed. The dependence on concentration of the contrast agent in the exposition solution and the independence of temporal uptake within one to seven days were studied for duckweed. By LA-ICP-MS gadolinium was quantified in a leaf of cress plant. The verification of the results was performed by SRXRF and ICP-MS after digestion. Furthermore, the uptake and distribution of Gd-based contrast agents in higher organisms (water flea) were observed. The exact location of gadolinium was resolved by three-dimensional μ-computed tomography by the comparison of an exposed with a Gd-free water flea. In all studies, gadolinium was detected in the investigated exposed model organisms. It can be concluded that the contrast agents were taken from the environment.

Le sélénium est un micronutriment essentiel pour des nombreux organismes vivants, y compris l’homme. Son rôle est lié à sa présence dans des sélénoprotéines sous forme d’un acide aminé, génétiquement encodé – la sélénocystéine. Il y a 25 sélénoprotéines encodées dans le génome humain. Leurs fonctions, la cinétique et la hiérarchie d'expression se trouvent au cœur des problématiques de recherche concernant le sélénium et la santé humaine. Il existe également un autre type de protéines où le sélénium est inséré par un remplacement partiel du soufre dans la méthionine mais aussi, potentiellement, dans la cystéine. Ces protéines suscitent l’intérêt dans les sciences de nutrition comme source de sélénium biodisponible dans l’alimentation naturelle et supplémentée. L'objectif de cette thèse a été la mise au point de méthodologies analytiques visant la spéciation du sélénium incorporé dans les protéines à l’échelle du protéome entier. Une procédure inédite a été développée pour la détection globale de protéines séléniées dans des gels d’électrophorèse bidimensionnelle par l’imagerie d’ablation laser ICP MS (spectrométrie de masse plasma à couplage inductif) permettant de s’affranchir de l’utilisation de l’isotope radioactif 75Se. Les autres avancées comprennent la mise en place d’un couplage robuste de HPLC capillaire avec l’ICP MS pour la détection des sélénopeptides dans des microvolumes de digestats trypsiques des protéines extraites du gel ainsi que la mise en place des protocoles d’identification des protéines séléniées par la spectrométrie de masse électrospray en tandem utilisant la trappe orbitale (Orbitrap). Les méthodes développées ont permis (i) la caractérisation de la part du protéome sélénié contenant la sélénocystéine chez la levure séléniée, (ii) l’identification des protéines majeures qui accumulent le sélénium dans le blé, et (iii) le dosage semi quantitatif et la caractérisation globale des sélénoprotéomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) dans les lignées cellulaires. / Selenium is an essential micronutrient for many living organisms including man. Its role is related to selenoproteins which contain genetically encoded selenocysteine. There are 25 selenoproteins encoded in the human genome. Their function, expression kinetics and hierarchy have been a topic of intense research in life sciences. There is another type of proteins which contain selenium inserted non-specifically by partly replacing sulphur in methionine and, potentially, cysteine. They are of interest in nutrition science as source of bio-available selenium in natural and supplemented foods. The goal of this Ph.D. was the development of methodologies for the analysis of selenium-containing proteins on the entire proteome scale. A novel procedure was developed for their global detection in 2D electrophoretic gels par laser ablation inductively coupled plasma mass spectrometry (ICP MS) imaging permitting to avoid the use of the radioactive 75Se. The other developments included (i) a robust capillary HPLC – ICP MS coupling allowing the detection of Se-containing peptides in microliter volumes of the digests of proteins extracted from the gel and (ii) protocols allowing the targeted identification of the Se-containing proteins by a parallel capillary HPLC - electrospray Orbitrap MS/MS. The methods developed allowed (i) the characterisation of the selenocystein-containing part of the selenoproteome of Se-enriched yeast, (ii) identification of the major Se-accumulating proteins in wheat, and (iii) semiquatitive analysis and global identification of the selenoproteomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) expressed in different human cell lines.

Le saumon Atlantique fait partie du patrimoine écologique et économique du bassin de l’Adour. Dans le cadre de la gestion actuelle du saumon dans ce bassin, l’origine natale des géniteurs, le taux de retour des individus d’origine piscicole, le taux de homing sur chaque sous-bassin ou encore le soutient par des géniteurs extérieurs au bassin de l’Adour sont des thématiques qui restent sans réponses. Ce projet propose donc de tester le potentiel de la géochimie des otolithes sur le saumon Atlantique du bassin de l’Adour. Nos travaux démontrent que la variation géographique de la composition chimique de l’eau dans 12 rivières colonisées par le saumon, associée à un enregistrement dans l’otolithe proportionnel à la signature géochimique du milieu de vie, permettent de discriminer l’origine géographique des individus. La combinaison des signatures élémentaires (Sr:Ca et Ba:Ca) et surtout l’isotopie du Sr (temporellement plus stable et sans fractionnement biologique) dans les otolithes améliore la précision du classement à l’échelle de la rivière de développement. En se basant sur la transmission de signatures géochimiques (élémentaires et isotopiques) transgénérationelles entre la femelle reproductrice et les otolithes des embryons produits par cette dernière, nous avons discriminé avec succès les individus nés en rivière de ceux nés en pisciculture. Le classement des géniteurs (180 individus) selon leur rivière natale a confirmé que le sous bassin du gave d’Oloron, et plus particulièrement le gave d’Ossau, reste le lieu qui produit le plus de saumon de retour. De façon non négligeable, le gave de Pau contribue lui aussi au renouvellement de la population (10 d’origine piscicole et 6 d’origine naturelle). 18 saumons sur 180 sont issus de l’alevinage (soit 10%); la majorité s’étant développée dans le sous-bassin du gave de Pau. Par ailleurs, nous avons mis à jour l’existence de périodes au cours de la vie des juvéniles (changements de milieu: sac vitellin—milieu extérieur et pisciculture—rivière) durant lesquelles l’enregistrement du Ba dans l’otolithe n’est pas en relation avec la chimie de l’eau. L’originalité de notre approche est d’avoir étudié l’influence des facteurs endogènes et environnementaux chez des poissons ayant vécu dans le milieu naturel ou ayant séjourné en milieu naturel contrôlé. Nos travaux mettent l’accent sur la complexité de l’intégration du rapport Ba:Ca dans l’otolithe et démontrent l’utilité des éléments traces et des isotopes du Sr comme « tag naturel » pour distinguer l’origine natale du saumon Atlantique. / The Adour basin holds one of the largest populations of Atlantic salmon in southern Europe exploited by commercial and sport fisheries. Determining the relative contributions of individual rivers and hatcheries to the Adour basin populations becomes crucial to understand key sources that contribute the most to its persistence. We successfully used Sr:Ca, Ba:Ca and 87Sr:86Sr ratios as natural tags for determining the natal origins of adults from 12 tributaries. Success in discriminating between fish from different sites was greatest using Sr isotopes since the latter remained relatively constant across years at a given location. Geochemical signatures from core regions of the otolith were also used to identify fish from hatchery or naturally spawned sources. The predominance of adults spawned in the Ossau River among returning adults corresponded with long-term juvenile production trends in the Ossau River. Despite the limited upstream accessibility of the Pau River, our study demonstrated that Atlantic salmon recruits can successfully leave this river to join the adult population in the Adour basin. We observed relatively clear separation between hatchery and wild juveniles using both Sr:Cacore (wild > 2.5 and hatchery < 0.80) and 87Sr:86Srcore (wild < 0.710 and hatchery > 0.710). The return of hatchery reared fish as adult spawners represented 10% of the total sampled fish we analyzed. Almost all adults, previously identified as belonging to the Ouzom River, were hatchery produced. Adults originated from the Pau River were either wild or hatchery reared fish. We also conducted field controlled experiments that characterized the elemental uptake process in juvenile Atlantic salmon otoliths during freshwater residency. Physiological effects influenced Ba deposition. Ba:Ca otolith profiles from hatchery-reared and field collected fish were characterised by a peak at yolk absorption mark. Hatchery-reared fish stocked in a river also displayed a peak of Ba:Ca following transfer which was not related to the water chemistry. Our experiment revealed a 20-day lag time between initial Ba:Cawater changes and Ba:Caotolith saturation. Results suggested that such effects should be considered during any attempts to determine rivers of origin of Atlantic salmon based on otolith elemental composition or reconstruct the movement of individual fish among and within streams.