Ce mémoire de thèse traite du contrôle et de la mise en forme des fronts de phase et d'énergie d'impulsions lasers brèves ultra-intenses.<br />La première partie est consacrée à la conception et la réalisation d'une boucle d'optique adaptative pour la correction des distorsions de surface d'onde sur la chaîne laser de puissance 100 Térawatts du Laboratoire pour l'Utilisation des Lasers Intenses. Cette boucle repose sur l'utilisation d'un dispositif à cristaux liquides comme modulateur de phase et d'un interféromètre à décalage comme senseur de front d'onde. L'association de ces deux dispositifs a permis la construction d'un système innovant de mesure et mise en forme de la surface d'onde d'impulsions lasers ultra-intenses. Il est démontré qu'il permet de corriger une surface d'onde présentant d'importantes distorsions et ainsi améliorer grandement la qualité de focalisation de faisceaux lasers. Cette boucle d'optique adaptative a été testée avec succès sur la chaîne 100 Térawatts et une correction de la surface d'onde de l'ordre de 60% a ainsi pu être démontrée.<br />La seconde partie du mémoire traite de la mise en forme d'une impulsion laser inhomogène brève permettant le pompage du milieu à gain (un plasma) d'un laser à rayons X. L'idée consiste à jouer sur le parallélisme du système de compression d'impulsions utilisé en fin de chaîne 100 Térawatts. Un modèle expliquant la génération d'une impulsion inhomogène laser brève à l'aide de ce compresseur à réseaux de diffraction est développé. Une campagne expérimentale Laser X a notamment permis de valider les prédictions théoriques annoncées par ce modèle et a montré la nécessité d'utiliser une telle impulsion inhomogène afin d'obtenir une émission laser X lorsque le pompage s'effectue par impulsion brève.

Ce travail de thèse se focalise sur l'usage de grands sondages pour extraire de l'information sur la cosmologie. Une photométrie de précision joue un rôle clé dans cette quête à travers la détermination de redshifts photométriques ainsi que la propagation des erreurs dans les résultats cosmologiques ; thème unissant les deux parties de cette thèse. Après une revue de la cosmologie et des mesures favorisant le modèle ΛCDM, ainsi qu'une description du Large Synoptic Survey Telescope (LSST), la première partie de ce travail se porte sur l'étude de la variation des principaux constituants de l'atmosphère sur la photométrie au sol, au niveau du site du LSST à Cerro Pachón, au Chili. Nous utilisons des données récentes sur l'ozone, la vapeur d'eau et les aérosols pour en construire une simulation de longue durée et estimer quantitativement l'influence des gradients spatiaux et temporels de ces constituants sur la procédure de la calibration du LSST. La deuxième partie de ce travail débute par une description théorique de l'effet de lentille gravitationnelle, avec un accent sur le lentillage faible. Après une comparaison des avantages et inconvénients inhérents aux mesures de cisaillement cosmique, nous explorons l'utilisation de l'amplification cosmique conjointement à la séparation tomographique en redshift permise par le LSST afin de contraindre les modèles. Nous trouvons que l'amplification cosmique, bien qu'affectée par le clustering intrinsèque, représente une sonde prometteuse du biais des galaxies et de l'énergie noire, complémentaire au cisaillement cosmique, et qui augmente la robustesse des contraintes cosmologiques provenant des mêmes relevés.

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Cette thèse caractérise les propriétés optiques des matériaux plasmoniques microstructurés et procède à l’évaluation des paramètres analytiques afin de les employer comme plateforme de biodétection en spectroscopie de résonance des plasmons de surface (SPR). Aux dimensions micrométriques, les matériaux plasmoniques présentent des caractéristiques optiques propres aux nano- et macromatériaux. La cartographie physicooptiques en SPR de matériaux méso- et microscopiques s’est effectuée à l’aide de films structurés de motifs périodiques triangulaires et circulaires fabriqués par une technique modifiée de lithographie par nanosphères (nanosphere lithography, NSL). À partir de cette vue d’ensemble, quelques films structurés ont été sélectionné en fonction d’aspects analytiques tels que la sensibilité et la résolution face aux variations d’indice de réfraction (RI) pour déterminer le potentiel de ces matériaux comme plateforme de biodetection. Les propriétés optiques distinctes des films microstructurés proviennent d’interactions résonantes entre les modes de plasmons de surface (SP) localisé et délocalisé identifiés par la relation de dispersion en SPR ainsi que l’imagerie Raman. Les conditions de résonance des modes SP dépendant de paramètres expérimentaux (λ, θ, η) tel qu’observés numériquement par rigorous coupled wave analysis (RCWA) et empiriquement. Ces travaux démontrent la nature plasmonique distincte des micro-matériaux et leur potentiel d’intégration aux techniques analytiques SPR existantes. Les matériaux plasmoniques micrométriques furent également étudiés pour l’implémentation de la SPR à une pointe de microscopie à force atomique (atomic force microscopy, AFM) combinant ainsi la spectroscopie à l’imagerie topographique. Des travaux préliminaires se sont concentrés sur la signature spectroscopique de leviers en silicium (Si) et en nitrure de silicium (Si3N4), l’impact d’un revêtement d’or sur les pointes et l’influence de milieu environnant. Une image d’origine plasmonique a été obtenue avec des leviers en Si3N4 revêtus d’or en transmission dans un environnement aqueux, indiquant ainsi le potentiel de ces pointes comme micro-biocapteur SPR. Ces résultats préliminaires servent de fondement pour orienter les prochaines investigations dans ce projet. / This thesis characterizes the optical properties of microstructured plasmonic materials and evaluates analytical parameters to use them as biosensing platforms in surface plasmon resonance (SPR) spectroscopy. At microscopic dimensions, plasmonic materials present optical characteristics unique to nano- and macromaterials. A SPR physico-optic mapping of meso- and microscopic materials was performed using structured films with triangular and circular periodic patterns fabricate by modified nanosphere lithography (NSL) technique. From this overview, a few structured films were selected based on analytical aspects such as sensitivity and resolution with respect to the refractive index (RI) to determine the potential of these materials as biosensing platforms. The distinct plasmonic properties of microstructured films emerge from resonant interactions between localized and propagating surface plasmons (SP) modes identified by the SPR dispersion relation and by Raman imaging. The conditions of SP modes resonant interactions depend on experimental parameters (λ, θ, η) as observed numerically in rigorous coupled wave analysis (RCWA) and empirically. These works show the distinct plasmonic nature of micromaterials and their potential integration to existing SPR techniques. Plasmonic micromaterials were also studied for the implementation of SPR to an atomic force microscopy (AFM) cantilever, hence combining spectroscopy to topographic imaging. Preliminanry works were focused on the spectroscopic response of silicon (Si) and silicon nitride (Si3N4) cantilever, the impact of gold coating on the cantilever is tip, and the influence of the adjacent environment. An image of plasmonic nature was obtained in transmission spectroscopy with gold coated Si3N4 cantilever in water environment, thus indicating the potential of these cantilevers as micro-SPR sensing probes. These preliminary results provide a basis to guide future investigations in this project.

The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...

Um Amplificador Paramétrico Óptico (optical parametric amplifier - OPA) é uma fonte de luz coerente, de alta qualidade e sintonizável, baseada em processos ópticos não-lineares de segunda ordem. Alguns modelos possuem largura de banda estreita e um amplo intervalo de sintonia, podendo alcançar regiões que vão desde o ultravioleta até o infravermelho médio. A nossa motivação para construir este amplificador paramétrico óptico é sua utilização em experimentos de espectroscopia vibracional de superfícies através do processo óptico não-linear de segunda ordem, geração de soma de frequências (sum-frequency generation - SFG), que é uma técnica que exige fontes sintonizáveis no infravermelho médio e com altas intensidades de pico e largura de banda estreita. O objetivo desse trabalho foi projetar, montar e testar um amplificador paramétrico óptico capaz de produzir pulsos sintonizáveis de alta energia no infravermelho médio (&lambda; ~ 2,5 a 10 &mu;m) a partir de um laser de bombeio que fornece pulsos de 25 ps, com alta energia em &lambda; = 1064 nm. Para obter-se uma geração de infravermelho bastante eficiente, foi proposto um projeto inovador para amplificadores paramétricos de picossegundos, utilizando-se a geração de supercontínuo de luz branca como feixe sinal do estágio de amplificação paramétrica. O pulso de bombeio (&lambda; = 1064 nm) é dividido em duas partes: a primeira, de menor energia, é utilizada para gerar um pulso de alta largura espectral no infravermelho próximo (supercontínuo de luz branca de picossegundos). Uma fração espectral desse pulso é selecionada através de um monocromador e utilizada como semente do estágio de amplificação paramétrica. O cristal amplificador paramétrico (sulfeto de prata e gálio, AgGaS2) é então bombeado pelo restante do pulso de bombeio e simultaneamente amplifica a semente sintonizável no infravermelho próximo e gera um novo pulso de frequência complementar no infravermelho médio. Foram testados vários meios para geração de supercontínuo, mas os melhores resultados foram obtidos em uma cubeta de 10 cm de comprimento com uma mistura de água e água deuterada (3 % em volume de H2O em D2O) e em uma fibra fotônica não-linear com 2 m de comprimento. Usando o supercontínuo como feixe semente, observou-se amplificação paramétrica no caso do feixe gerado na fibra fotônica com um ganho de 260 vezes, mas não com o feixe gerado na mistura de água/água deuterada, presumivelmente pela maior instabilidade desse supercontínuo. / An Optical Parametric Amplifier (OPA) is a tunable light source of high quality, coherent radiation, based on second-order nonlinear optical processes. Some models have a narrow spectral bandwidth and a tuning range from the ultraviolet to the mid-infrared. The motivation for building this optical parametric amplifier is its use in vibrational spectroscopy of surfaces by a second-order nonlinear optical process, sum-frequency generation (SFG), which is a technique that requires tunable sources in the mid-infrared with narrow bandwidth and high peak intensities. The purpose of this work is to design, implement and test an OPA to generate tunable high energy pulses tuneable in the mid-infrared (&lambda; ~ 2.5 to 10 &mu;m) from a pumping laser that provides 25 ps pulses with high energy at &lambda; = 1064 nm. For an efficient mid-infrared generation, we propose an innovative design for picosecond parametric amplifiers, using the near infrared portion of a white-light supercontinuum pulse as the seed beam for the parametric amplifier. The pump pulse (&lambda; = 1064 nm) is divided into two parts: the first one, with lower energy, generates a high spectral width pulse in the near infrared (white-light supercontinuum picosecond pulse). A spectral fraction of this pulse is selected through a monochromator and is used as seed for the parametric amplification stage. The second part of the laser beam pumps the parametric amplifier crystal (silver gallium sulfide, AgGaS2) which simultaneously amplifies the tunable seed beam in the near infrared and generates a new pulse with complementary frequency in the mid-infrared. Several media were tested for supercontinuum generation, but the best results were obtained with a 10 cm long cuvette with a mixture of water and deuterated water (3 % volume of H2O in D2O) and with a 2 m long nonlinear photonic crystal fiber. Using the supercontinuum as a seed beam, we have obtained parametric amplification of the seed generated by the photonic fiber with a gain of 260 times, but not of the beam generated by the water mixture, presumably because of its significantly higher instability.

Deficiência na enzima lisossomal glucocerebrosidase (GCR) resulta na doença de Gaucher. O tratamento atual consiste na administração da enzima exógena, produzida em células CHO. Porém, o medicamento disponível no mercado é extremamente custoso. Neste trabalho, propusemos a clonagem e a expressão da GCR humana em células CHO, visando a obtenção de um clone celular produtor para viabilizar a produção futura da enzima, a um custo menor, no Instituto Butantan. A expressão estável da GCR recombinante foi obtida a partir da transfecção de células CHO-dhfr- com o plasmídeo pED de expressão em células de mamíferos contendo o cDNA da GCR, seguido de amplificação gênica por MTX. A GCR foi detectada no extrato celular (~ 64 kDa) e secretada para o sobrenadante (63-69 kDa) em ensaios de western blotting, usando o anticorpo policlonal anti-GCR gerado neste trabalho. A enzima secretada hidrolisou o substrato 4-MUG e a sua produtividade foi estimada em 5,14 pg/célula/dia para o melhor subclone produtor, selecionado para a produção futura da GCR em larga escala. / Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher\'s disease. Current treatment consists on enzyme replacement therapy by the administration of recombinant GCR produced in CHO cells. However, the medicine available in the market is extremely expensive. In this work, we proposed the cloning and expression of human GCR in CHO cells, in order to obtain a productive cellular clone for future production of GCR enzyme at a lower cost at the Butantan Institute. The stable expression of recombinant GCR was obtained after transfection of CHO-dhfr- cells with pED mammalian expression vector containing the GCR cDNA, followed by gene amplification with MTX. The GCR was detected by western blotting analysis, either as cell-associated (~ 64 kDa) or as secreted forms (63-69 kDa), using the anti-GCR polyclonal antibody produced in this work. The secreted enzyme was active on 4-MUG and was produced at a level of about 5,14 pg/cell/day for the best producer subclone, selected for subsequent steps of GCR production on large scale in next future.

Introdução O diagnóstico de derrame pleural maligno (DPM) se baseia no achado de células tumorais no líquido ou no tecido pleural. Resultados falsos positivos ou falsos negativos influenciam na escolha da melhor conduta terapêutica a ser tomada, além de alterar substancialmente o prognóstico desses pacientes. A sensibilidade do exame citológico é geralmente inferior a 70%, motivo pelo qual, métodos complementares são frequentemente associados. Fatores como tipo histológico, sítio primário e grau de invasibilidade do tumor são os principais responsáveis por esta variação. Dentre os exames complementares propostos, destacam-se a dosagem de marcadores tumorais no líquido pleural (LP), as técnicas citoquímicas, imunocitoquímicas e de marcadores de proliferação celular em células do LP, a análise da ploidia de DNA por citometria de fluxo (CF) ou estática (CE) e, mais recentemente, as técnicas de citogenética e de biologia molecular, como a técnica de hibridação in situ por fluorescência (FISH) e a técnica de amplificação multiplex por sondas ligação - dependentes (MLPA) estas, capazes de detectar alterações em regiões gênicas consideradas \"alvo\" para o desfecho neoplásico. Objetivos 1) Padronizar as técnicas de DNA ploidia, FISH e MLPA em amostras frescas de líquido pleural; 2) Testar a eficiência diagnóstica dos métodos da DNA ploidia e da FISH no diagnóstico de derrame pleural maligno e 3) Avaliar alterações no número de cópias no gene EGFR em metástases pleurais utilizando a técnica de MLPA. Métodos Foram incluídos 200 pacientes adultos portadores de derrame pleural (DP) com indicação de toracocentese. O diagnóstico histológico foi o padrão ouro para malignidade. Características clínicas, radiológicas, histológicas e de seguimento clínico foram considerados para a exclusão de malignidade, de maneira que 130 casos foram classificados como malignos e 70 como benignos. As 200 amostras de LP foram submetidas ao exame citológico e à FISH utilizando sondas centroméricas para os cromossomos 11 e 17. A análise da ploidia de DNA por CF foi realizada em 45 casos de DP e a MLPA com o kit do gene do receptor do fator de crescimento epidérmico (EGFR) em 50 casos. Resultados A análise da ploidia de DNA por CF apresentou sensibilidade inferior ao exame citológico, com especificidade próxima (57,0%vs 96,2%; 70,0% vs 66,7%, respectivamente). A FISH isoladamente apresentou sensibilidade de 98,5% e especificidade de 98,6% e de 98,0% e 99,% quando associada ao exame citológico, com apenas um caso falso positivo e dois casos falsos negativos. A técnica de MLPA, padronizada para LP, demonstrou alterações na sequência do gene do EGFR em 28,2% dos casos malignos. Nenhuma amostra de líquido pleural dos casos benignos (controle) apresentou alteração no número de cópias e/ou rearranjos estruturais. Conclusão A análise citogenética de amostras frescas de líquido pleural por FISH é um valioso complemento ao exame citológico no diagnóstico de derrame pleural maligno, particularmente nos casos em que o resultado da citologia oncótica é inconclusiva / Introduction The diagnosis of malignant pleural effusion (MPE) is based on the finding of tumor cells in the pleural fluid or tissue. False positive or false negative results influence the choice of the best therapeutic approach to be used with these patients and substantially change their prognosis. The sensitivity of the cytology is generally lesser than 70%, for which complementary methods are often associated. Factors such as tumor histological type, staging, primary site and potential of invasiveness are responsible for this variation. Among the proposed ancillary tests, we highlight the dosage of tumor markers in pleural fluid (PF), the cytochemical and immunocytochemical techniques, including markers of cell proliferation, DNA ploidy analysis by flow cytometry (FC) or static cytometry (EC) and more recently, the cytogenetics and molecular techniques, as the fluorescence in situ hybridization (FISH) and the multiplex ligation - dependent probe amplification (MLPA), capable of detecting changes in gene regions considered \"target\" for the neoplastic outcome. Objectives 1) To standardize the techniques of DNA ploidy, FISH and MLPA in fresh samples of pleural fluid; 2) To test the diagnosis efficiency of DNA ploidy and FISH in the diagnosis of malignant pleural effusion and 3) To evaluate changes in the copy number of the EGFR gene by using the MLPA technique in cases of pleural metastases. Methods We included 200 adult patients with pleural effusion and clinical indication for thoracentesis. The histological diagnosis was considered the gold standard for malignancy. Clinical follow-up, radiological and histological characteristics were considered for exclusion of malignancy, which ranked de cases as 130 malignant effusions and 70 as benign ones. All cases were submitted to cytology and FISH using centromeric probes for the chromosomes 11 and 17. Analysis of DNA ploidy by FC was performed in 45 cases and the MLPA for epidermal growth factor receptor (EGFR) gene in 50 cases. Results DNA ploidy analysis showed less sensitivity than PF cytology, with similar specificity (57.0% vs 96.2% and 70.0% vs 66.7%, respectively). FISH alone had a sensitivity of 98.5% and specificity of 98.6%, and of 98.0% and 99% when associated with cytology. Only one false positive and two false negative cases were observed. The MLPA technique, standardized for PF, showed changes in the EGFR gene in 28.2% of the malignant cases. No samples of pleural fluid from benign cases (control) showed changes in copy number and/or structural rearrangements. Conclusion The cytogenetic analysis of fresh pleural fluid samples by FISH seems to be a valuable method to be associated to cytology in the diagnosis of malignant pleural effusion, particularly in cases of inconclusive cytological results

Cette thèse explore les caractéristiques uniques offertes par le mélangeur Josephson dans le domaine émergeant de l’optique quantique micro-onde. Nous avons démontré que le mixeur Josephson pouvait jouer trois rôles majeurs pour le traitement de l’information quantique. Nous avons conçu et fabriqué un amplificateur à la limite quantique avec la meilleure efficacité quantique démontrée à cette date. Cet outil crucial peut être utilisé pour la mesure microonde de systèmes mésoscopiques dont les circuits supraconducteurs. En particulier, cela nous a permis de réaliser avec succès la stabilisation de trajectoires d’un bit quantique supraconducteur par rétroaction basée sur la mesure. Ensuite, nous avons montré comment ce circuit peut générer et distribuer des radiations micro-ondes intriquées par conversion paramétrique spontanée sur des lignes de transmissions séparées dans l’espace et à des fréquences différentes. En utilisant deux mixeurs Josephson, nous avons fourni la première démonstration d’intrication non- locale entre deux champs propageants dans le domaine micro-onde, les états dits EPR. Finalement, nous avons utilisé le mixeur Josephson dans le mode de conversion de fréquence. Il se comporte alors comme un interrupteur, permettant d’ouvrir ou fermer dynamiquement l’accès à une cavité de haut facteur de qualité. L’ensemble constitue une mémoire quantique. En combinant cela avec la génération d’intrication, nous avons mesuré la distribution, le stockage et la libération sur demande d’un état intriqué. Ceci est un pré-requis pour jouer le rôle de nœud au sein d’un réseau quantique. / This thesis work explores unique features offered by the Josephson mixer in the upcoming field of microwave quantum optics. We have demonstrated three major roles the Josephson mixer could play in emerging quantum information architectures. First, we have designed and fabricated a state-of-the-art practical quantum limited amplifier with the best quantum efficiency achieved to date. This tool is crucial for probing mesoscopic systems with microwaves, and in particular superconducting circuits. Hence, it has enabled us to realize successfully the stabilization of quantum trajectories of a superconducting qubit by measurement-based feedback. Second, we have shown how this circuit can generate and distribute entangled microwave radiations on separated transmission lines at different frequencies. Using two Josephson mixers, we have provided the first demonstration of entanglement between spatially separated propagating fields in the microwave domain, the so-called Einstein-Podolsky-Rosen states. Finally, we have used the Josephson mixer as a frequency converter. Acting as a switch, it is able to dynamically turn on and off the coupling to a low loss cavity. This feature allowed us to realize a quantum memory for microwaves. In combination with the ability to generate entanglement, we have measured the time-controlled generation, storage and on-demand release of an entangled state, which is a prerequisite for nodes of a quantum network.

Nile tilapia, Oreochromis niloticus, is one of the most important farmed fish globally. One of the most serious bacterial diseases constraining global tilapia production is Francisellosis caused by Francisella noatunensis subsp. orientalis (Fno). Although outbreaks of Fno are increasing worldwide, there are no licenced commercial vaccines to prevent the disease for use on tilapia farms. Thus, the current treatment of choice is the use of antibiotics combined with increasing water temperature up to 30°C. Studies investigating the diversity of circulating Fno isolates and the immune response of tilapia elicited by vaccination against piscine francisellosis are lacking. In addition, the current conventional and molecular tools used for detection of Fno have many drawbacks, making detection of Fno a challenging process. In this study, five clinical isolates of Fno from diverse geographical locations (UK, Costa Rica, Mexico, Japan and Austria), previously characterised by morphology, genotype, antimicrobial susceptibility and virulence, were used in a proteomic study. The whole proteomic cell profile of the five isolates were homogenous by one-dimension sodium dodecyl polyacrylamide gel electrophoresis (1D-SDS-PAGE), while minor differences in the intensity of 15 proteins between the strains were observed by two-dimension SDS-PAGE (2DE), including some important virulence related proteins. The UK isolate was the most significantly different isolate when compared to the other Fno isolates in the current study. The Fno UK isolate had significantly higher abundance of 10/15 of the significantly expressed proteins including four of the essential pathogenicity and virulence related proteins (IglC, GroEL, DnaK, ClpB) compared to the other used Fno isolates. The antigenic profiles of the five Fno isolates were studied by 1D western blotting using tilapia hyper immune sera which recognised an immunodominant band of a molecular weight of ~ 17-28 kDa in all tested Fno isolates. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI/MS/MS) identified 47 proteins in this antigenic band. Some of the identified proteins are associated with Fno pathogenicity. 2D western blot analysis of the vaccine isolate (Fno UK) revealed differential antigen recognition between sera from vaccinated and non-vaccinated fish following experimental challenge (26 antigenic spots recognised by sera from vaccinated fish; 31 antigenic spots recognised by sera from vaccinated and challenged fish and 30 antigenic spots recognised by non-vaccinated and challenged fish). The identity of these proteins was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of them are known Francisella virulence related proteins. Bioinformatics analyses revealed diverse categories of proteins with high biological functions, however the vast majority of these proteins are involved in energy production and metabolic pathways of the bacteria. This detailed analysis will facilitate the development of cross-strain protective subunit Fno vaccines and antigen-targeted Fno diagnostics. The outer membrane proteins (OMPs) of the same five Fno isolates were extracted using the ionic detergent sarkosyl. The OMP fraction of the different isolates were separated via 1D-SDS PAGE and the digested peptides of the UK isolate were analysed by LC/ESI/MS/MS. High degree of similarity was observed in the OMP profile of the five Fno isolates with an abundant protein band at 17-28 kDa, which was found to be antigenic by 1D western blot using convalescent tilapia sera. LC/ESI/MS/MS analysis of the OMPs of the Fno UK isolate identified 239 proteins, including 44 proteins in the antigenic band (17-28 kDa). Comparison between the proteins identified in the immunogenic band of whole cell lysate and OMP fraction of the Fno UK isolate showed 30 common proteins between the two preparations, 17 proteins were identified only in the whole cell extract and 14 were identified only in OMP fraction. Outer membrane proteins (e.g. Omp-A), virulence related proteins such (e.g. IglC) and other stress related proteins (e.g. AhpC/TSA family peroxiredoxin) were more abundant in the OMP fraction than the whole cell lysate. In silico analysis enabled prediction of the function and location of the OMPs identified by Mass-spectrometry. The findings of this study provide preliminary data on bacterial surface proteins that exist in direct contact with the host immune defence during infection and offering an insight into their potential role as novel targets for Fno diagnostics and vaccine development. The efficacy of an injectable whole cell oil-adjuvanted vaccine was evaluated against challenge with heterologous Fno isolates in Nile tilapia, Oreochromis niloticus. Three duplicate groups of 130 healthy Nile tilapia (~15 g) were intraperitoneally (i.p.) injected with the vaccine, adjuvant-alone or PBS followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection to all immunised tilapia groups with a significantly higher relative percent survival (RPS) of 82.3% against homologous challenge, compared to 69.8% and 65.9% after heterologous challenge. Protection correlated with significantly elevated specific antibody responses and western blot analysis demonstrated cross-isolate antigenicity with sera from fish post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by quantitative real-time polymerase chain reaction (qPCR) in conjunction with significantly greater expression of IgM, IL-1β, TNF-a and MHCII 72 hours post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to those of adjuvant-alone and control fish. The latter results suggested stimulation of protective immune responses following vaccination. In addition, a whole cell formalin killed autogenous immersion vaccine against Fno was developed using the same isolate used for the injectable vaccine. Duplicate tanks of 35 tilapia fry were immersed in the vaccine or in sterile Modified Muller Hinton broth (MMHB) diluted in tank water (1:10 dilution) for 30 s and at 30 days post-vaccination (dpv), all fish groups were immersion challenged with the homologous Fno isolate and monitored for 21 days. A moderate RPS of 43.7% was provided by the vaccine. Serum IgM levels were below the threshold in 30 % of the vaccinated fry 30 dpv. Also, the IgM levels of the vaccinated fry were not significantly different from control fry 21 days-post challenge. A recombinase polymerase amplification (RPA) assay was developed and validated for rapid detection of Fno. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in fish farms, but also by screening 78 Nile tilapia and 5 water samples collected from UK and Thailand. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other species of bacteria tested. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The Fno-RPA was rapid, giving results in approximately 6 min in contrast to the qPCR that required approximately 90 min to reach the same detection limits. Moreover, the RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to control the infection through prompt on-site detection of Fno. The overall results of this study indicated that Fno isolates from different origins share a high degree of homology in their proteomic and antigenic profile. Proteomic characterisation data of Fno isolates has contributed to understanding the diversity of Fno isolates and assisted in identifying suitable candidates for developing an effective Fno vaccine. / Moreover, this study has proven the efficacy of a cross protective Fno injection vaccine in tilapia fingerlings, with further optimisation needed for immersion vaccination of fry, and given insights into the immune response of tilapia to vaccination against francisellosis. In addition, it provided a rapid, sensitive, specific and robust molecular tool for detection of Fno that can assist surveillance and control of piscine francisellosis on tilapia farms.