The chicken yolk sac and small intestine are connected through the yolk stalk and share many biological similarities. During the embryonic stage, the extra-embryonic yolk sac helps the embryo to absorb nutrients primarily in the last two weeks of incubation. The chicken yolk sac physically moves yolk contents from the yolk sac to the small intestine at the end of embryogenesis. This is the time when the small intestine replaces the yolk sac in assimilating nutrients for the embryo and later for the posthatch chicken. Additionally, both chicken small intestinal epithelia and the yolk sac secrete beta defensins for promoting intestinal health. Since there are heterogeneous cell types along the mammalian intestinal villus, which are derived from the intestinal stem cells in the crypts, we investigated if cells of the chicken yolk sac and small intestine have the same ontogeny as mammalian intestinal epithelial cells. In this dissertation, we mainly focused on the spatial expression of nutrient transporters (PepT1 and SGLT1), intestinal stem cell markers (Lgr5 and Olfm4), and avian beta defensins in the chicken yolk sac and small intestine during the embryonic and early posthatch stages. RNAscope in situ hybridization was used to identify the distribution of cells expressing PepT1 mRNA in both the chicken yolk sac and small intestine. PepT1 mRNA was found to be expressed by epithelial cells in both the yolk sac and small intestine. In the yolk sac, PepT1 mRNA was uniformly distributed in each endodermal epithelial cell along the villus-like structure. The pattern of PepT1 mRNA expression observed in the chicken yolk sac during the last 10 days of incubation revealed that PepT1 mRNA was increased from e11 to e13, and decreased from e15 to day of hatch. The peak of PepT1 mRNA expression was between e13 and e15, when the yolk sac reaches maximum absorptive area and the growth of the chicken embryo is at its fastest rate. However, the expression of PepT1 mRNA in the intestine was only detected in columnar enterocytes along the villus and not in goblet cells or cells in the crypts. The immunofluorescence assay confirmed that PepT1 protein was located at the brush border membrane of the enterocytes and that protein expression of PepT1 was restricted to the intestinal epithelial cells from approximately the middle to the tip of the villus. In order to identify intestinal stem cells, we used the known mammalian stem cell markers, Lgr5 and Olfm4. Both Lgr5 and Olfm4 are specifically expressed by cells in the chicken intestinal crypts, suggesting that they can be used as biomarkers for chicken intestinal stem cells. Dual labelling of PepT1 and Olfm4 mRNA on the same chicken intestinal sample revealed that there was a gap between PepT1-expressing enterocytes and Olfm4-expressing intestinal stem cells. The cells in this gap were presumably transit amplifying (TA) cells. Additionally, we also found that the TA cell zone along the intestinal villus was reduced during chicken growth. This TA cell population could be clearly detected at day of hatch and d1 posthatch but not later. The expression of SGLT1 mRNA was localized to yolk sac endodermal epithelial cells and showed a sharp increase at the end of incubation. This increase of SGLT1 mRNA coincided with the increase in glucose in the yolk, indicating that the chicken embryo needs glucose as energy for hatching. The mRNA expression profiles of various avian beta defensins have been examined by qPCR and in situ hybridization to investigate the immune function of the yolk sac and small intestine. We found that AvBD10 mRNA showed the highest expression level in the yolk sac and was expressed predominantly in the yolk sac endodermal epithelial cells. Additionally, the expression of AvBD10 mRNA showed a development-specific pattern, which increased from e9 to e11, and decreased from e13 towards day of hatch. The expression patterns of AvBD1, 2, and 7 mRNA were similar to each other. These three genes were found to be expressed by chicken heterophils distributed in the yolk sac blood islands and small intestinal blood vessels. Only a subset of heterophils, which might be activated, were able to express AvBD1, 2, and 7 mRNA. In the intestine, the expression of AvBD10 mRNA was localized to cells along the villus at e19 and day of hatch, but later to only a few cells located above the intestinal crypts. In summary, the endodermal epithelial cells are responsible for the absorptive and immune functions of the chicken yolk sac. The yolk sac mesoderm is critical for embryonic hematopoiesis and innate immunity. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which are functioning to absorb nutrients and secrete antimicrobial peptides. / Ph. D. / The chicken yolk sac and small intestine are connected to each other and share many biological similarities. Both chicken small intestinal and yolk sac epithelia play critical roles for nutrient absorption and immune defense. In this dissertation, the mRNA for nutrient transporters such as the peptide transporter, PepT1 and the sodium-glucose co-transporter, SGLT1 were found to be expressed by absorptive epithelial cells in both the yolk sac and small intestine. Additionally, both intestinal and yolk sac epithelial cells expressed avian beta defensins (AvBDs), which are important chicken host defense peptides. In the small intestine, there are a number of differentiated cell types that originate from stem cells in the crypt that express the known mammalian stem cell markers, Olfm4 and Lgr5 mRNA. However, in the chicken yolk sac, only the stem cell marker Lgr5 mRNA was expressed by endothelial cells. In summary, the yolk sac epithelial cells are responsible for the absorptive and immune functions for the embryonic stage. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which function to absorb nutrients and secrete antimicrobial peptides.

Corrosion of iron and copper pipes can produce leaks and loss of efficiency in the water distribution system, elevate levels of contaminants at the tap, and cost billions of dollars annually in pipe replacement or rehabilitation. In situ pipe rehabilitation using cement mortar, polyurethane, and epoxy is a commonly employed method of dealing with aging yet structurally sound pipes because it is less expensive and less invasive than replacing pipe infrastructure. Although epoxy has been shown to be an effective solution to pipe corrosion, little research has been conducted regarding its impact on a comprehensive list of water quality parameters. This research addressed that gap in the literature by conducting short-term immersion tests in which new epoxy linings were exposed to reference tap waters containing one of three disinfectant treatments: no disinfectant, free chlorine, or chloramines. As a comparison, an aged epoxy-lined field sample was also tested. Water exposed to the liners under stagnant conditions was analyzed for the following water quality parameters: pH, ammonia, alkalinity, hardness, metals, disinfectant consumption, total organic carbon (TOC), semi-volatile organic compounds (SVOCs), disinfectant byproduct (DBP) formation, and odor. Results of the study showed relatively low impacts on water quality, as all USEPA drinking water regulations were met. Impacts were highest during the first 24 hour exposure period during which time significant disinfectant consumption was shown (> 90% free chlorine consumed, 13% chloramines consumed), high TOC was leached (2.6-6.2 mg/L), trihalomethanes and haloacetic acids were formed (both < 15 ug/L), Bisphenol-A, an endocrine disrupter, was detected (< 35 ug/L), and odor was reported by panelists at a moderate intensity and described as sweet/chemical/burning/chlorinous. Impacts were much less after the initial 24 hours, although odor remained noticeable throughout the 30 day study. Overall, water quality impacts were greatest in chlorinated waters and both new and aged epoxy showed slight differences in results. / Master of Science

No description available.

Introduction : L'hybridation in situ en fluorescence (FISH) est souvent utilisée pour l'étude des populations microbiennes hétérogènes. Toutefois, cette méthode n'est pas adaptée pour détecter des bactéries sous forme de spores, vue leur grande résistance aux traitements de perméabilisation conventionnels. Cependant, les bactéries formant des spores ont un rôle écologique, économique et médical important. Le but de cette étude est de développer des protocoles de perméabilisation rapides afin de détecter des spores de Bacillus par FISH. Méthodes : Un protocole pour les spores de B. megaterium a été adapté et optimisé pour les modèles choisis (B. cereus, B. atrophaeus et B. megaterium). Des sondes universelles et spécifiques ont été utilisées lors de l'hybridation. L'effet du traitement de perméabilisation a été évalué à Laide de la microscopie électronique à transmission et à balayage. Par la suite, les protocoles ont été adaptés pour permettre l'entrée de grosses molécules (comme la streptavidine) afin de permettre l'utilisation de méthode d'amplification de signal. Résultats : Avec les protocoles développés, les spores de Bacillus ont été détectées avec des sondes par FISH. La microscopie électronique à balayage a permis d'observer les différences de surface entre les spores traitées et non traitées. Des spores de Bacillus ont été détectées avec les protocoles adaptés pour la streptavidine. Conclusion : Des protocoles efficaces ont été développés pour détecter rapidement des spores de Bacillus par FISH. En utilisant cette technique, il est possible de détecter des spores de Bacillus en moins d'une heure.

Legal Regulation of Animal Protection Ex Situ in USA and the Czech Republic This thesis aims to compare the legislation of animal protection ex situ in the Czech Republic and USA. The content is systematically divided in two sections. First section focuses on different law systems, which is international, national and local legislation. Second section explains the law used in different institutions that provide ex situ protection, such as zoos, aquariums, wildlife sanctuaries, etc. The thesis describes different legal approaches and uses several examples for a practical comparison. In its conclusion the thesis sums up the negatives and positives of each legislation and their overall quality and efficiency. The thesis also points out some of the desirable solutions to some current problems and a possible development in the area.

15 samples of gem opals were analyzed by laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) and inductively coupled plasma mass spectrometry (ICP-MS). For this study were selected the most representative samples from Slovakia, Australia, Peru, Ethiopia, Mexico, USA and the Czech Republic. These samples were obtained from private collections. The study focuses on the geochemical relationships between various forms of opal originating from igneous and sedimentary environments based on the results from LA ICP-MS and ICP-MS. The major and trace element compositions are unique for each studied opal locality and important for understanding numerous aspects of opal formation. Major element analysis shows that opals are essentially pure SiO2 (77 - 92 wt. %). Al, Fe, Ca, K, Na and Mg are the main elemental impurities. The concentrations of the trace elements vary strongly depending on the opal locality. Differences were found, e.g., in the concentrations of REE and some refractory elements, which might be indicative for determining the provenance of opals. Many studies have been published explaining the origin of luminescence, microstructures and the thermal properties of opals. However, there are very few comprehensive studies comparing gem opals from the most important historical and...

La cuisson est un procédé universel de transformation de la matière première carnée en aliment. Le chauffage de la matrice musculaire conditionne diverses qualités organoleptiques, technologiques, sanitaires et nutritionnelles des viandes cuites. La cuisson étant appliquée de plus en plus fréquemment en conditions industrielles standardisées, il est pertinent de modéliser certains mécanismes clés dans le déterminisme de ces qualités afin d’optimiser le procédé. Pour cela, une démarche expérimentale originale a été mise en place fondée sur une analyse quantitative, locale, dynamique et in situ de la viande pendant la cuisson. Cette démarche ne fait pas d’hypothèse réductionniste en étudiant un échantillon intact à l’échelle de l’aliment consommé, ni d’hypothèse simplificatrice en prenant en compte les variations spatiales de la température dans l’échantillon. Elle s’appuie sur des développements originaux en imagerie par résonance magnétique nucléaire à haut champ et en traitement d’images pour cartographier la déformation et la quantité d’eau. Des modèles robustes liant température, déformation et quantité d’eau ont été obtenus pour des muscles de teneur variable en tissu conjonctif. Les résultats montrent principalement une augmentation de la déformation avec la température en plusieurs phases dont les caractéristiques dépendent de la composition du muscle, et une diminution de la quantité d’eau avec la température. Tous ces résultats sont discutés et interprétés au regard du comportement à la température des différents composants du muscle. Ce travail montre d’abord que l’imagerie dynamique, quantitative et multiparamétrique permet de décrypter des mécanismes intervenant lors de la cuisson des viandes sans établir des hypothèses réductrices lors de l’interprétation de ces phénomènes. Elle a conduit de plus, à des développements méthodologiques applicables à d’autres champs et ouvre la voie à d’autres investigations dans le domaine de l’optimisation qualitative des produits carnés transformés. / Cooking is a general process which transforms the meat raw material into food. The heating of muscle matrix influences different organoleptic, industrial, health and nutritional qualities of cooked meat. Cooking being applied more and more frequently in standardized and industrial conditions, it makes sense to model some key mechanisms which determine the latter qualities in order to optimize the process. For this purpose, an original experimental approach has been developed based on a quantitative, local, dynamic and in situ analysis of the meat during cooking. This approach is not based on any reductionist hypothesis by studying an intact sample at the scale of the consumed food, nor by the simplifying assumption of taking into account the spatial variations of temperature in the sample. It is based on original developments in nuclear magnetic resonance imaging at high-field and on image processing in order to map deformation and the water content. Robust models linking temperature, deformation and water content were obtained for muscles differing from their content in connective tissue. The results mainly show a deformation increase with the temperature in several phases whose characteristics depend on the muscle composition, and a decrease in the water content with temperature. All these results are discussed and interpreted thanks to the temperature behavior of the various muscle components. This work first shows that quantitative, multi-parametric and dynamic imaging can decipher the mechanisms involved during meat cooking, without formulating simplifying assumptions in the interpretation of these phenomena. Furthermore, it has led to methodological developments applicable to other fields and paves the way for further investigations in the field of quality optimization of processed meat products.

Les nanostructures ont des propriétés mécaniques qui diffèrent de celles des matériaux massifs. La compréhension des propriétés mécaniques aux échelles nanométriques requièrent la mise en place d’essais mécaniques combinés à des observations structurales. Au cours de cette thèse nous avons développé un microscope à force atomique (AFM) dédié permettant de solliciter mécaniquement un nano-objet unique sur une ligne de lumière synchrotron. Les possibilités offertes par cette nouvelle approche expérimentale sont démontrées sur deux exemples de sollicitation mécanique in situ: (i) la nanoindentation in situ de cristaux d’or combinée à la diffraction cohérente des rayons X; (ii) la flexion trois points de nanofils d’or associée à la micro-diffraction de Laue. Ces expériences permettent d'accéder au comportement élastique ainsi qu’au comportement plastique du nanomatériau et permettent de déterminer la limite d'élasticité et le type de défauts induits par le chargement mécanique. / Nanostructures were found to exhibit different mechanical properties compared to their bulk counterpart. For obtaining further insight into the mechanical behaviour on the nanoscale, mechanical tests are combined with observation techniques allowing for monitoring the structural evolution. Within this thesis a special atomic force microscope has been developed which is compatible with different X-ray diffraction techniques at synchrotron sources for in situ mechanical testing on single nano-objects. The great potential of the new experimental approach is demonstrated on two kinds of in situ mechanical tests: (i) in situ nano-indentation on Au crystals with coherent X-ray diffraction. (ii) In situ three point bending tests on Au nanowires with μLaue diffraction. These experiments give access to the elastic as well as the plastic behavior of the nanomaterial and allows for determining the elastic limit and the type of defects induced by the mechanical loading.

Les bactéries, organismes les plus abondants de notre planète, ont un rôle fondamental dans le fonctionnement des écosystèmes. En dépit de leur importance, la caractérisation des communautés bactériennes (microbiotes) demeure encore aujourd’hui très incomplète. Ceci a pour origine l’impossibilité de complètement décrypter taxonomiquement et fonctionnellement les microbiotes de ces écosystèmes et donc à appréhender la diversité bactérienne dans son ensemble que ce soit par des approches culturales (avec seulement 1% de bactéries cultivables) ou par des approches metagénomiques limitées par les biais d’extraction, de séquençage et d’analyse. Les travaux entrepris dans le cadre de cette thèse visent à développer une nouvelle voie exploratoire passant par le fractionnement des microbiotes afin d’en étudier séparément les génomes des différentes populations ou groupes de populations, leur somme devant permettre de reconstituer un metagénome complet. Cet objectif requiert le développement d’un outil pour la sélection spécifique de bactéries (sur des critères taxonomiques ou fonctionnels) et leur isolement du reste des microorganismes non ciblés. Les travaux de thèse ont porté sur le développement d’une approche de marquage magnétique des bactéries basée sur l’hybridation moléculaire (hybridation in situ) complétée par celui d’un outil de tri microfluidique. Deux méthodes ont été développées, MISH et HCR, ciblant le gène de l’ARNr 23S, chacune reposant sur la formation, lors du processus d’hybridation, d’une structure secondaire en arborescence (MISH) ou ordonnée (HCR) permettant le greffage de nanoparticules magnétiques. Les résultats obtenus illustrent le potentiel des deux approches d’abord pour le marquage spécifique de bactéries cibles (E.coli et Pseudomonas putida) en conditions de culture au laboratoire puis dans un second temps dans des échantillons de sol. Le tri microfluidique a également été optimisé par le développement d’un nouveau dispositif de tri magnétique permettant la séparation des cellules marquées sous flux continu faisant appel à l’injection d’un polymère composite magnétique pour intégrer au fond du microcanal une série de bandes parallèles magnétiques. La fonctionnalité du dispositif a été démontrée, sa simplicité de fabrication en faisant un outil de choix pour une application en routine dans les laboratoires d’écologie microbienne. En dépit de résultats prometteurs toute cette nouvelle approche d’étude de la diversité bactérienne environnementale nécessite encore de nombreuses étapes d’optimisation. / Bacteria, the most abundant organisms on our planet, have a fundamental role in ecosystem functioning. Despite their importance, the characterization of bacterial communities is today still incomplete. This is due to the impossibility of completely decomposing taxonomically and functionally the microbial communities of these ecosystems and as a consequence to apprehend the whole bacterial diversity, either by cultural approaches (with only 1% of culturable bacteria) or by metagenomic, a limited approaches cause by biases in extraction, sequencing and analysis. The work undertaken in this thesis aims to develop a new exploratory pathway through the fractionation of microbiota in order to study separately genomes of different populations or groups of populations. Their sum should enable reconstitution of complete metagenome. This objective requires the development of a tool for the specific selection of bacteria (on taxonomic or functional criteria) and their isolation from the rest of the non-target microorganisms. The thesis work focused on the development of a magnetic labeling approach for bacteria based on molecular hybridization (in situ hybridization) complete by development of a microfluidic cell-sorting tool. Two methods have been developed, MISH and HCR, targeting the 23S rRNA gene, each based on the formation, during the hybridization process, of a secondary random structure (MISH) or organized structure (HCR) enabling binding to magnetic nanoparticles. Results obtained illustrate the potential of the two approaches initially for the specific labeling of target bacteria (E.coli and Pseudomonas putida) under laboratory conditions and then in soil samples. The microfluidic sorting was also optimized by the development of a novel magnetic cell-sorting device allowing the separation of the labeled cells under continuous flow using the injection of a magnetic composite polymer to integrate a series of magnetic parallel strips at the bottom of the microchannel. The proper functioning of the sorting device has been demonstrated, its simple production making it a tool of choice for a routine application in laboratories of microbial ecology. Despite promising results all this new approach for studying environmental bacterial diversity is still requiring many optimization steps.

Neste trabalho é descrito o desenvolvimento de sistemas eletroquímicos (EQ) acoplados com a RMN de baixo e alto campo para análises in situ. Para a análise EQ-RMN de baixo campo, foi construído um sistema eletroquímico para ser utilizado tanto em um espectrômetro RMN-DT (B0= 0,23 T) quanto em um sensor unilateral (B0= 0,4 T). A platina foi utilizada como eletrodo de trabalho e contraeletrodo e um fio de prata como eletrodo de referência. A reação de eletrodeposição do cobre foi monitorada por meio do valor do tempo de relaxação transversal (T2), adquirido através da sequência de CPMG, em intervalos de 10 minutos durante a eletrólise, a potencial constante, no tempo de 3 horas. Ambas as análises, EQ-RMNDT e EQ-RMN Unilateral, apresentaram bom desempenho analítico. No entanto, foi observado que a análise de EQ-RMN Unilateral permitiu o monitoramento da reação eletroquímica com um volume detectável de amostra muito menor, em torno de 300 uL, do aquele para a RMN-DT (2,8 mL). O efeito do campo magnético (B0= 0,4 T) sobre o processo eletrolítico foi avaliado pela técnica de microscopia eletrônica de varredura (SEM). A análise SEM mostrou que o eletrodepósito de cobre obtido sob o efeito do campo apresenta uma morfologia rugosa. Para a análise in situ de EQRMN de alto campo, o sistema eletroquímico foi desenvolvido em um tubo de RMN de 10 mm de diâmetro, para ser utilizado em um espectrômetro de 9,4 T. A fibra de carbono foi o material mais adequado para ser utilizado como eletrodo de trabalho. A sequência de Precessão Livre no Estado Estacionário (SSFP, Steady State Free Precession), que não tem dependência do tempo de relaxação longitudinal T1, e permite a aquisição de centenas de espectros por segundo, foi utilizada para monitorar a eletrólise do 9-cloroantraceno por RMN de 13C. A célula EQ-RMN de alto campo construída possibilitou a aquisição de espectros de RMN de 1H e SSFP de 13C durante a eletrólise do 9-cloroantraceno. O Método da Diagonalizacão Filtrada (FDM, Filter Diagonalization Method) foi utilizado para processar sinais no domínio do tempo de SSFP 13C, para resolver os problemas de anomalia de fase e melhorar a resolução espectral. Os sistemas eletroquímicos construídos apresentaram um bom desempenho analítico para o interfaceamento com a RMN, e as metodologias empregadas propiciaram o monitoramento em tempo real de uma reação eletroquímica. / In this work it is described the development of electrochemical (EC) systems coupled to the Low and High-field NMR for in situ analysis. By low-field NMR-EC analysis, it was constructed an electrochemical system to be utilized both TD-NMR spectrometer (B0= 0.23 T) and unilateral sensor (B0= 0.4 T). Platinum was used as working electrode and counter electrode and a silver wire as reference electrode. The copper electrodeposition reaction was monitored by Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. The measurements of the transverse relaxation time (T2) were collected in the interval of 10 minutes during electrolysis, constant potential, in 3 hours time. Both in situ analyses, TD-NMR-EC and Unilateral NMR-EC, demonstrated good analytical performance. However, the Unilateral NMR-EC allowed the monitoring copper concentration during electrolysis using detectable volume much smaller, approximately 300 uL, than TD-NMR (2.8 mL). The magnetic field effect (B0= 0.4 T) on electrolytic process was available by scanning electron microscopy (SEM) technique. The SEM analysis showed copper electrodeposits obtained on the field effect observes a rough morphology. For the in situ high-field NMR-EC analysis, the electrochemical system was developed in a tube NMR of 10 mm diameter to be utilized 9.4 T spectrometer. Carbon fiber was the most appropriated material to be used as working electrode. The Steady-state free precession (SSFP) sequence that has no dependence on the longitudinal relaxation time T1, allows it to acquire up to hundreds of spectra per T1 value, it was utilized to monitor the electrolysis of 9-chlroanthracene for 13C NMR. The high-field NMR-EC cell constructed allowed the acquisition of the 1H NMR and 13C SSFP spectra during the electrolysis of 9-chlroanthracene. The Filter Diagonalization Method (FDM) was used to process signals in the time domain of 13C SSFP, to solve problems of phase anomalies and improve the spectra resolution. The electrochemical systems constructed showed good analytical performance for coupling with NMR and the methodologies employed provided of the monitoring in real time of the electrochemical reaction.