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Il est intuitif qu'avec de nouveaux outils, il devient possible d'explorer de nouveaux domaines de la physique. Les champs électromagnétiques ultra-rapides sont l'un de ces outils, ils permettent de sonder la matière à de nouvelles échelles de temps, à la fois pour développer de nouvelles applications et pour la recherche fondamentale. Néanmoins, ces champs constituent en eux-mêmes un phénomène méritant d'être analysé et étudié. Le travail présenté ici est divisé en deux parties, dont la première s'occupe de la génération et amplification de lasers ultracourtes. L'amplification paramétrique optique est discutée dans les différentes configurations, notamment dans le cas où le signal a une dérivé angulaire spectrale. On discute aussi deux oscillateurs à blocage de mode en phase. Ont présente aussi une nouvelle manière d'étudier son comportement en fonction des dimensions de la sous-cavité. La mesure de ces phénomènes représente également un défi en raison de l'échelle temporelle extrêmement réduite à laquelle ces phénomènes se produisent, échelle bien trop petite pour des méthodes de mesure traditionnelles. Dans ce manuscrit, nous avons abordé deux techniques de mesure bien connues: l'autocorrélation interférométrique (IAC) du second ordre et la corrélation croisée du 3ème ordre (TOCC). Avec l'IAC et une mesure de la puissance spectrale du champ, il est possible de reconstruire intégralement le champ électrique tandis que le TOCC associé à l'autocorrélation en intensité détermine le profil en intensité de manière unique, et ont présente des algorithmes que font la reconstruction avec un haut contraste. Nous avons par ailleurs étudié la réalisation d'une nouvelle configuration de corrélateur croisé monocoup.

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 city of Wellington has a high population concentration and lies within a geologically active landscape at the southern end of the North Island, New Zealand. Wellington has a high seismic risk due to its close proximity to several major fault systems, with the active Wellington Fault located in the north-western central city. Varying soil depth and properties in combination with the close proximity of active faults mean that in a large earthquake rupture event, ground shaking amplification is expected to occur in Thorndon, Te Aro and around the waterfront. This thesis focuses on the area bounded by Thorndon Overbridge in the north, Wellington Hospital in the south, Kelburn in the west, and Oriental Bay in the east. It includes many of the major buildings and infrastructural elements located within the central Wellington commercial area. The main objectives were to create an electronic database which allows for convenient access to all available data within the study area, to create a 3D geological model based upon this data, and to define areas of different seismic subsoil class and depth to rock within the study area at a scale that is useful for preliminary geotechnical analysis (1:5,000. Borelogs from 1025 holes with accompanying geological and geotechnical data obtained from GNS Science and Tonkin & Taylor were compiled into a database, together with the results from SPAC microtremor testing at 12 sites undertaken specifically for this study. This thesis discusses relevant background work and defines the local Wellington geology. A 3D geological model of the central Wellington commercial area, along with ten ArcGIS maps including surficial, depth to bedrock, site period, Vs30, ground shaking amplification hazard and site class (NZS 1170.5:2004) maps were created. These outputs show that a significant ground shaking amplification risk is posed on the city, with the waterfront, Te Aro and Thorndon areas most at risk.

Abstract: Neurodegenerative diseases are associated with progressive loss of structure or function of neurons which results in cell death. Recent evidence indicate that all neurodegenerative disorders, sporadic or transmissible, may have a common pathological mechanism at the molecular level. This common feature consists of protein aggregation and accumulation of harmful aggregates in neuronal cells resulting in cellular apoptosis and neurotoxicity. Neurodegenerative diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory and other abilities. This diverse group of diseases includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases or transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis. In my project I worked on the molecular mechanism of protein aggregation, propagation and neurotoxicity in Parkinson's disease and prion disease. Prion disease and PD are associated with misfolding and aggregation of PrPc and a-Synuclein (a-Syn), respectively. Despite being two important neurodegenerative disorders, molecular mechanisms of a-Syn or PrPC aggregation and amyloidogenesis are still unclear in PD and prion disease. Furthermore, the toxic protein species in PD have not been characterized yet. In this study we characterize the mechanism of a-Syn and PrPc misfolding in a physiological-like cell free condition in the absence of a-Syn aggregates, PrPc ggregated isoform (Pre's), denaturants or acidic environment. A number of studies indicate that dimerization of PrPc or a-Syn may be a key step in the aggregation process. To test this hypothesis we verified if enforced dimerization of PrPc or a-Syn may induce a conformational change reminiscent of the conversion of PrPc or a-Syn to PrPR' or a-Syn aggregates, respectively. We used a well-described inducible dimerization strategy where a dimerizing domain called FK506-binding protein (Fv) was fused to PrPc or a-Syn in order to produce chimeric proteins Fv-PrP and a-SynF'''. A divalent ligand AP20187 was used to induce protein dimerization. Addition of AP20187 to recombinant Fv-PrP in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet (13-Sheet) structure and simultaneous aggregation of the proteins. However, non-dimerized PrP formed 13-Sheet conformation in very slower rates. In the presence of AP20187, we also report a rapid random coil into 13-sheet conformational transformation of a-SynF" within 24 h, whereas wild type a-Syn showed 24 h delay to achieve P-sheet structure after 48 h. Electron microscopy experiments demonstrated that dimerization induced amyloid fibril formation after 48 h for both Fv-PrP and a-Syr?", whereas in the absence of dimerizing ligand AP20187, PrP or a-Syn converted into amyloid fibrils after 3 days or even later. Dimerization-induced Fv-PrP aggregates were partially resistant to PK digestion which is a characteristics of the naturally occurring PrPR'. The rates of amyloidogenesis in the presence of dimerization was also characterized by Thioflavin T (ThT) fluorescence probing. Whereas the stable structure of Fv-PrP showed no ThT binding for over 60 h of incubation at 37°C, the addition of AP20187 to Fv-PrP resulted in a time-dependent increase in ThT binding. As for a-SynR, dimerization accelerated the rate of ThT binding and amyloid formation comparing to the slower amyloidogenesis rate of wild type a-Syn in the absence of dimerizer AP20187. The impact of dimerization on a-Syn aggregation was further determined by Fluorescence ANS probing, indicating a higher affinity of dimerization-induced a-SynF" aggregates for binding to ANS comparing to wild type a-Syn aggregates. These results indicate that dimerization increases the aggregation and amyloidogenesis processes for Fv-PrP and a-SynF". Both Fv-PrP and a-SynF" amyloids were successfully propagated in vitro by protein misfolding amplification (PMCA) cycle. These results ar in agreement with the theory that all protein aggregates in neurodegenerative diseases propagate with the same molecular mechanism. Neurotoxicity of recombinant Fv-PrP and a-SynF" aggregates was determined in cellulo and in vivo, respectively. Aggregates of Fv-PrP were toxic to cultured cells whilst soluble Fv-PrP and amyloid fibres were harmless to the cells. When injected to the mice brain, both a-Syni" and a-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity in the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by PMCA method, providing the first evidence for the in vitro propagation of synthetic a-Syn aggregates. These results suggest an important role for protein dimerization in aggregation and amyloidogenesis, and therefore, in the pathology of PD and prion disease. The similarities between aggregation, amyloidogenesis and toxicity of PrPC and ct-Syn provide further evidence on the existance of a prion-like mechanism in all neurodegenerative disorders. // Résumé: Les maladies neurodégénératives sont associées à la perte progressive des propriétés structurales ou fonctionnelles des neurones, ce qui engendre la mort des cellules. De récentes études indiquent que tous les désordres neurodégénératifs, sporadiques ou transmissibles, peuvent avoir un mécanisme pathologique commun au niveau moléculaire. Ce dispositif commun se compose de l'agrégation de protéines, de la propagation des agrégats, et de l'accumulation d’agrégats toxiques dans les cellules neuronales, menant à l'apoptose et à la neurotoxicité cellulaire. Les maladies neurodégénératives peuvent affecter la pensée abstraite, les mouvements habiles, les sentiments émotifs, la connaissance, la Mémoire et d'autres capacités cognitives. Ce groupe divers de maladies inclut la maladie d'Alzheimer (AD), de Parkinson (PD), de Huntington (HD), les maladies à prions ou encéphalopathies spongiformes transmissibles (TSEs) et la sclérose latérale amyotrophique (ALS). [symboles non conformes]

L'objectif central de cette thèse est d'exploiter les propriétés quantiques du champ électromagnétique pour développer de nouveaux dispositifs de communication. L'étude porte sur les composantes de quadrature (variables quantiques continues) d'un mode impulsionnel du champ lumineux. Une démonstration expérimentale de cryptographie quantique avec des états cohérents a été réalisée. Le dispositif se base sur des impulsions modulées en amplitude et en phase et comportant en moyenne une centaine de photons. Pour chaque impulsion lumineuse, une détection homodyne résolue en temps permet de mesurer une composante de quadrature particulière avec une forte efficacité. Une clé secrète a ainsi été transmise à un débit de 1.7 Mbits/s en l'absence de pertes et 75 kbits/s pour une transmission présentant des pertes de 3.1 dB, ce qui ouvre la voie pour des applications de cryptographie quantique à hauts débits. Afin d'étudier l'utilisation de spécificités quantiques, nous avons développé une source impulsionnelle d'états comprimés et d'états intriqués. Cette source utilise des conversions non-linéaires d'impulsions ultrabrèves intervenant dans un cristal mince de niobate de potassium. Suivant la configuration, la réduction du bruit en quadrature est de 2.7 dB sous le niveau de bruit quantique standard, ou les corrélations entre les quadratures des faisceaux intriqués sont de 2.5 dB. Grâce à ce dispositif, nous avons mis en oeuvre la première expérience de "dégaussification", pour transformer des impulsions de vide comprimé en des états non-gaussiens. Ce protocole est directement lié à la distillation de l'intrication de variables continues, qui permet d'améliorer la portée des dispositifs de cryptographie. Enfin, des schémas sont étudiés pour réaliser des tests complets des inégalités de Bell avec des variables continues mesurées par des détections homodynes.

Du fait de leurs fortes propriétés non-linéaires, les structures photoniques semi-conductrices sont très intéressantes pour le traitement tout-optique du signal. La structuration à petite échelle permet de localiser la lumière et donc d'exalter les phénomènes non-linéaires, mais avec un pouvoir d'exaltation différent suivant l'ordre de la non-linéarité, introduisant ainsi une nouvelle hiérarchie des régimes d'interaction. Après une description théorique présentant le renforcement des processus non- linéaires par la localisation dans les semi-conducteurs, nous démontrons expérimentalement et quantitativement, pour la première fois, l'influence de la localisation sur l'exaltation des non- linéarités telles que l'effet Kerr, l'absorption à deux photons et les effets de porteurs libres, par l'étude d'un guide à cristal photonique de GaAs. Nous résolvons le problème de l'amplification Raman d'impulsions dans un nanoguide de silicium, en développant un modèle analytique permettant de montrer l'influence des effets de modulation de la phase non-linéaire sur la chute du gain Raman effectif dans le silicium. Ce modèle est validé expérimentalement. Finalement, nous abordons l'application de la localisation à la commutation non- linéaire tout-optique. Nous introduisons la notion de longueurs non-linéaire de commutation et d'absorption permettant d'étudier différents matériaux. Nous montrons que la commutation par effet Kerr pur est impossible pour le Si et le GaAs (contrairement à l'AlGaAs et au GaN), mais qu'une commutation par effet de porteurs libres y est envisageable pour certaines géométries de microstructures.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

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