A Thesis Submitted to the Faculty 0/ Engineering, University 0/ Lite Witwatersrand, Johannesburg in fulfilment 0/ the requirements /01' the Degree 0/ Doctor 0/ Philosophy. Johannesburg 1990. / This thesis studies fault tolerant strategies for real-time distributed computer control systems so as to propose an error recovery technique that renders individual processors on the network resistant to soft failures. The technique is effective for soft failures which have as certain maximum duration, and does not require the use of specialised hardware. Attention is focused on achieving resistance to soft failures in environments which have demanding time constraints such as those found in computer systems for process control, materials handling and automated manufacturing. [Abbreviated Abstract. Open document to view full version]

The South African National Antiretroviral Treatment Guidelines recommend the use of HIV-1 viral load assays for routine monitoring of HIV-1 positive patients receiving highly active antiretroviral therapy (HAART). This thesis describes the innovative approaches to developing more affordable HIV-1 diagnostics and monitoring assays for South Africa, which take into account the tiered laboratory infrastructure of this country. An in-house HIV-1 viral load assay – the LUX assay, was developed and evaluated with a view of implementing this more affordable option in high tier laboratories. The LUX assay represents quantitative real-time RT-PCR that utilizes the LightCycler® technology (Roche) in a novel combination with a LUXÔ primer. The assay showed good analytical sensitivity, specificity and reproducibility of its linear dynamic range of 4x102 to 4x106 RNA copies/ml. Preliminary clinical evaluation (n = 458) of the LUX assay showed good agreement with the COBAS Amplicor assay, and demonstrated its usefulness for long term monitoring of HAART patients. ELISA based viral load testing approaches were investigated as low cost and less technically complex alternatives for medium tier laboratories. The HiSens HIV-1 p24 Ag Ultra (Perkin Elmer) and the ExaVir™ Load Quantitative HIV-RT kits (CAVIDI) were compared with the Roche Amplicor assay. Both assays showed strong association with the Roche Amplicor assay, with R2 = 0.686 and R2 = 0.810, respectively (n = 117). These alternative assays seemed most useful in the serial monitoring of patients on HAART. Major drawbacks included the wide variability of both assays, insufficient sensitivity of the p24 antigen assay and low throughput of the RT assay. Development of a point-of-care HIV-1 RNA assay could address issues related to early and cost effective diagnosis of acute HIV infection. A novel isothermal amplification technique termed the Reverse Transcription Loop Dependant Amplification (RT-LDA) was developed as one component for a potential point-of-care HIV-1 RNA assay. The RT-LDA converted RNA into partially looped ssDNA amplicons, over a wide RNA concentration range (4x103 to 4x108 copies/ml) using a 1 hour incubation at 53ºC. The RT-LDA technology is fully compatible with a lateral flow detection system using dipsticks and highly suitable for point-of-care testing. Overall, this study demonstrates the feasibility of developing novel, more affordable HIV-1 testing options that would be appropriate for the tiered laboratory infrastructure present in South Africa. Evaluation of commercially available, less expensive alternative HIV viral load assays in local settings facilitates their implementation.

L’objectif de ce travail est de positionner l’imagerie terahertz comme un nouvel outil dédié à la métrologie et au contrôle non destructif. Ses propriétés étonnantes à la fois de forte pénétration dans les matériaux diélectriques et de longueur d’onde de dimension millimétrique voire submillimétrique en font un outil très enthousiasmant pour des applications comme le contrôle non destructif (CND).En premier lieu, nous avons présenté deux techniques d’imagerie : une consistant à déplacer l’objet, point par point, dans un faisceau terahertz focalisé pour reconstruire, pixel par pixel, une image en deux dimensions. L’autre technique repose sur l’utilisation d’un objectif et d’un capteur matriciel à l’état de l’art fonctionnant à température ambiante. Ainsi nous avons imagé des objets d’intérêts, sans déplacement mécanique. Bien que cette dernière technique soit beaucoup plus rapide que l’imagerie point par point, la qualité des images obtenues n’est pas comparable et ceci à cause de deux verrous. Ainsi, une partie du travail a consisté en l’étude des stratégies d’éclairage pour limiter les effets optiques interférentiels induits par la cohérence de la source. De plus, en déployant des simulations de toute la chaine optique avec le logiciel Zemax, de nombreuses images de qualité permettent d’envisager des applications en imagerie 2D (métrologie, CND) et en imagerie 3D. Ainsi, deux techniques complémentaires de reconstruction tomographique sont expérimentées sur des images obtenues en temps réel ; une technique inspirée de l’imagerie par rayon X et une technique utilisée en microscopie optique 3D qui exploite la profondeur de champ réduite de l’objectif. Enfin, plusieurs études spectroscopiques et métrologiques sont menées afin d’évaluer le comportement des matériaux et la dimension des objets à partir de leurs images THz en 2D ou avec leur reconstruction tomographique 3D. Les travaux effectués constituent les briques de base au déploiement de l’imagerie terahertz vers le domaine applicatif grâce à une qualité d’image incomparable et des acquisitions en temps réels. / The aim of this work is to position terahertz imaging as a new tool dedicated to metrology and non-destructive testing. Its astonishing properties of both high penetration in dielectric materials and wavelength millimeter or even submillimetric make it a very exciting tool for applications such as non-destructive testing (NDT).First, we presented two imaging techniques: one to move the object, point by point, in a focused terahertz beam to reconstruct, pixel by pixel, a two-dimensional image. The other technique is based on the use of a lens and a matrix sensor in the state of the art operating at room temperature. Thus we have imaged objects of interest, without mechanical displacement. Although this last technique is much faster than point-by-point imaging, the quality of the images obtained is not comparable and this because of two drawbacks. Thus, part of the work consists in the study of lighting strategies to limit the interferential optical effects induced by the coherence of the source. In addition, by deploying simulations of the entire optical chain with Zemax software, numerous quality images make it possible to consider applications in 2D (metrology, NDT) and 3D imaging. Thus, two complementary tomographic reconstruction techniques are tested on images obtained in real time; a technique inspired by X-ray imaging and a technique used in 3D optical microscopy that exploits the reduced depth of field of the lens. Finally, several spectroscopic and metrological studies are carried out in order to evaluate the behavior of the materials and the dimension of the objects starting from their THz images in 2D or with their 3D tomographic reconstruction. The work done is the foundation for deploying terahertz imagery to the application domain with unmatched image quality and real-time acquisition.

Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA contains a partially synthesised positive DNA stand and a gap region within the negative strand. After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An important objective of HBV therapy is the elimination of cccDNA, as its persistence within hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this replication intermediate is crucial. The objective of this study was to develop a method based on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells, only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA detection as it allows for the rapid simultaneous amplification and quantification of a specific DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy

The goal of the thesis was to characterize transcriptional profiles of in vivo and in vitro derived embryos during bovine minor and major embryonic genome activation and to identify mRNA transcripts newly synthesized during these stages. In our first work we have concentrated on the study of minor genome activation at the 4-cell stage of embryo. Using SSH, we have identified 31 amplicons homologous with already identified genes. We have selected 5 of these for detailed study of their expression during the whole period of preimplantation development: centromere protein, 350/400 kDa (CENPF, mitosin), splicing factor arginine/serine-rich 3 (SRFS3), high mobility group nucleosomal binding domain 2 (HMGN2) protein and eukaryotic translation initiation factors EIF4A2 a EIF4E. All these genes play an important role in the early embryo development. SRFS3 is the first described gene with an important function in preimplantation development, which is expressed already during bovine minor genome activation, and its transcription is α-amanitin sensitive during this period. We have selected CENPF gene for a more thorough study. By silencing its expression by the injection of CENPF dsRNA into the zygote, we have studied its function throughout the whole preimplantation development of bovine embryo....

Le virus ostreid herpesvirus type 1 (OsHV-1), peut être considéré comme un des agents infectieux majeur affectant les élevages d’huîtres creuses, Crassostrea gigas, en France. Des différences de sensibilité à l’infection ont également été observées au sein de cette espèce. Des travaux précédents suggèrent un lien entre la base génétique et la survie des animaux face à l’infection. Dans ce contexte, l’objectif principal du travail de thèse était de mieux comprendre les interactions entre l’huître creuse et OsHV-1, et plus particulièrement, les bases moléculaires du cycle viral. Nos résultats montre que le virus est capable de se répliquer chez l’hôte quel que soit son stade de développement, et sa sensibilité. Cependant, la cinétique de multiplication est plus rapide chez des individus sensibles comparés aux moins sensibles. Il apparaît également que chez les individus survivants, le virus ne soit plus détectable après une phase de réplication active. Cette observation laisse suspecter (i) une rémission avec une élimination du virus ou (ii) une persistance du virus sans symptômes détectables. Ces résultats mettent en lumière la possibilité du virus de circuler au sein des individus survivants. Ces individus peuvent excréter des particules virales et intervenir ainsi dans le processus d’infection en milieu naturel. L’ensemble de ces résultats représentent une premier contribution à la compréhension du cycle d’ OsHV-1 chez l’huître creuse, plus particulièrement au niveau moléculaire. / In France, Ostreid herpesvirus type 1 (OsHV-1), can be considered one of the major infectious agents in Pacific oysters, Crassostrea gigas. Susceptibility differences to infection were observed in this species. Previous work suggested that the genetic basis and the survival animals to infection were related. In this context, the main objective of this thesis was to understand the interactions between oysters and OsHV-1, in particular, the molecular basis of the viral cycle. Our results shows that the virus is able to replicate in the host regardless of its stage of development or its susceptible. However, multiplication kinetic is faster in susceptible individuals compared to less susceptible individuals. After a active replication, it would appear that the virus is no detectable in survival individuals. This observation suggests (i) a remission with elimination of the virus or (ii) a virus persistence without detectable symptoms. These results highlight the ability of the virus circulating in the host without causing mortality. These individuals can excrete viral particles and interfere with the infection process in field. All these results represent a first contribution to the understanding of OsHV-1 cycle in Pacific oysters, particularly at the molecular level.

A área de pesquisa e prototipagem de soluções para Sistemas de Potência compartilha, com outras áreas da engenharia, vários problemas relacionados a software, principalmente, os seus custos e o seu tempo de desenvolvimento. Mais especificamente, nas aplicações com sistemas embarcados e dispositivos computacionais em tempo real, a presença de fatores como a complexidade dos algoritmos, os requisitos críticos de desempenho e as restrições impostas pelo hardware da aplicação, fazem com que o desenvolvimento do software evolua de forma muito lenta. Finalmente, quando um projeto está terminado, seu software já é obsoleto ou completamente incompatível para uso em novos projetos, com um hardware novo. Esse trabalho apresenta uma possível solução a esses problemas, por meio de um arcabouço de software para aplicações em tempo real. Através de uma metalinguagem de descrição de fluxos de dados baseada em blocos, e de ferramentas de compilação e interpretação, esse arcabouço permite abstrair o projeto, a implementação e os testes da aplicação, do desenvolvimento das demais partes do dispositivo, favorecendo um projeto sistemático em módulos, o futuro reuso de códigos, a fácil manutenção de algoritmos e a execução de testes cruzados, entre plataformas, com previsibilidade de resultados. O arcabouço foi elaborado e testado em um cenário de Sistemas de Potência, com a criação de um relé digital de proteção contra sobrecorrentes, com aquisição de dados via barramento de processo da norma IEC 61850. Entretanto, toda a metodologia desse trabalho pode ser aplicada a qualquer outra área correlata, mediante a extensão de sua metalinguagem. / The research and prototype area for Power System solutions share, among other engineering disciplines, several problems concerning software, mainly their developing time and costs. More specifically, in embedded system and real time computing devices applications, the presence of factors, such as the algorithm complexity, the critical performance requirements and other restrictions imposed by the application hardware, makes the software development to slowly evolve. Finally, when a project is over, its software is already obsolete or completely incompatible for use in other projects, with a new hardware. This work presents a possible solution to these problems, through a software framework for real time applications in Power Systems. Through a block based data stream description metalanguage, and compiling and interpreting tools, this framework allows the application design, implementation and testing procedures to be abstracted from the development of other device parts, permitting a systematic and modular project, the code reuse in the future, easy algorithm maintenance and the execution of cross-platforms tests with predictable results. The framework was created and tested in Power System scenarios, especially in the construction of a digital protection overcurrent relay, with data acquisition through the IEC 61850 process bus. However, the entire methodology of this study could be applied to any other related area, by extending its metalanguage with the appropriate building blocks.

Recentes pesquisas têm investigado modelos de garantia de desempenho baseados em restrições temporais, parametrizadas pela especificação de limites superiores de tempo médio de resposta. Este trabalho estende o desenvolvimento da política de escalonamento de temporeal EBS, aplicável a esse problema, apresentando um mecanismo de controle de admissão de requisições em aplicações com tais requisitos. A abordagem baseia-se em um método adaptativo capaz de administrar o nível de degradação do sistema, de forma a isolar o efeito do comportamento de um usuário sobre a qualidade de serviço oferecida aos demais usuários. Também é proposta uma modificação na implementação do algoritmo originalmente definido para a EBS, de forma a diminuir sua complexidade temporal. Resultados de simulação demonstram a efetividade dos mecanismos propostos / In recent research works performance guarantee models based on temporal constraints with specified response-time upper bounds have been investigated. This work extends the development of the EBS real-time scheduling policy, applicable to this problem, by proposing an admission control mechanism. The introduced approach is based on an adaptive model which, based on the system degradation level, tries to isolate the impact of the behavior of a given user upon the quality of service offered to the other users. Its also proposed a new algorithm to reduce the complexity order of the original EBS implementation. Simulation results illustrate the effectiveness of proposed methods

Este trabalho descreve o projeto e a implementação de instrumentação eletrônica dedicada para neurobiofísica, com foco para pesquisas em codificação neural com invertebrados. Foram desenvolvidos sistemas de tempo real, controlados por computador, para a aquisição de tempos de disparo de spikes neurais oriundos de neurônios do cérebro de moscas, e para a geração de sinais de controle de estímulos visuais gerados a uma taxa de varredura vertical de 500Hz, usado para estimulações visuais em invertebrados. Estes sistemas possuem hardware dedicado, incluindo front end analógico, hardware digital baseado em FPGAs, hospedado em microcomputador IBM/PC compatível, que prove a interface do sistema com o usuário. Dois protótipos foram construídos, sendo o primeiro com ênfase em hardware dedicado, e o segundo com ênfase em sistema operacional de tempo real para controle do hospedeiro. Ambos sistemas estão sendo utilizados no laboratório Dipteralab do IFSC, para o estudo da transmissão da informação no duto óptico da mosca / This work describes the project and implementation of custom build electronic instrumentation to be used in neurobiophysical research, more precisely in neural coding experiments with invertebrates. A hard real time computer controlled integrated system was developed capable to acquire the timestamps of evoked neural spikes of a fly submitted to visual stimuli. The visual stimuli generator is a custom build system connected to the hard real time computer capable to generate images at a frame rate of 500Hz. Dedicated hardware was developed, including an analog front end and FPGA based digital circuits. The IBM/PC compatible computer hosted hardware provides the system interface with the end user. Two prototypes were build, one based on dedicated hardware and the other based on real time routines executed on the host computer. Both systems are in use at the Dipteralab of our institute to study the information transmission of the optic flow signal in fly.

Devido à sua múltipla utilidade, a cana-de-açúcar (Saccharum spp.) é amplamente cultivada em mais de 127 países tropicais e subtropicais. No Brasil, a cultura apresentou expansão em área plantada nos últimos anos em virtude da crescente demanda por biocombustíveis. Porém, a produção de mudas sadias tende a ser um fator limitante a este aumento de demanda, já que diversas doenças são transmitidas por toletes contaminados. Dentre estas, destaca-se o raquitismo da soqueira (RSD), causado pela bactéria fastidiosa Leifsonia xyli subsp. xyli (Lxx) (KAO e DAMANN, 1980). De acordo com a literatura, a transmissão de Lxx se dá através de toletes contaminados e/ou instrumentos de corte, não havendo relatos sobre sua transmissão por sementes. Desta forma, seu controle se baseia no princípio da exclusão, através do uso de toletes tratados termicamente como fonte de material propagativo. Porém, não se conhece o efeito do tratamento no título de Lxx, uma vez que os relatos de eficiência da termoterapia baseiam-se em avaliações qualitativas. Em face destas informações, este estudo objetivou avaliar a transmissão de Leifsonia xyli subsp. xyli por sementes e também quantificar o efeito da termoterapia no título de Lxx em duas variedades de cana-de-açúcar através da PCR quantitativa em tempo real. No ensaio de transmissão via sementes, a bactéria foi detectada em altas incidências (>87%) em plantas de 90 dias oriundas de sementes coletadas de plantas infectadas e em quantidades estimadas superiores a 51 células por 100ng de DNA total de sementes. Além disso, aos 270 dias após o plantio, a bactéria foi isolada de 6 plantas. A identidade das colônias foi comprovada através de PCR convencional utilizando primers específicos para Lxx. Dois ensaios foram conduzidos para quantificar o efeito da termoterapia na população bacteriana. O título de Lxx foi determinado em folhas de plantas de duas variedades de cana-deaçúcar oriundas de toletes tratados termicamente ou não aos 90 dias após o plantio. O título inicial presente nos colmos utilizados como fonte de toletes interferiu na eficiência do tratamento, que reduziu a população bacteriana somente no ensaio onde os colmos apresentaram elevado título bacteriano inicial. Além disso, 70 e 55% das plantas oriundas de toletes tratados termicamente no primeiro e no segundo ensaio, respectivamente, apresentavam células de Lxx, porém em menores quantidades quando comparadas a plantas oriundas de toletes não tratados. Com isso, conclui-se que Lxx é transmitida em elevada frequência via sementes e a termoterapia, embora não tenha efeito erradicante pronunciado, é capaz de reduzir o título bacteriano no tecido vegetal. / Due to its multiple uses, sugarcane (Saccharum spp.) is widely cultivated in more than 127 tropical and subtropical countries. In Brazil, sugarcane cropping has increased in recent years because of the growing demand for biofuels. However, the production of healthy seedlings tends to be a limiting factor to this increase, since contaminated setts transmit many diseases. Among these, the Ratoon Stunting Disease (RSD) is a major disease caused by the fastidious bacterium Leifsonia xyli subsp. xyli (Lxx) (KAO and DAMANN, 1980). According to the literature, the transmission of Lxx occurs through contaminated setts and/or cutting tools, but there are no reports of its transmission by seeds. Therefore, its control is based on the principle of exclusion using heat-treated setts as source of propagative material. However, the quantitative effect of the treatment on the bacterial titer is not known, since reports of thermotherapy efficiency are based on qualitative assessments. This study aimed to evaluate the transmission of Leifsonia xyli subsp. xyli by seeds and to quantify the effect of thermotherapy on Lxx in two sugarcane varieties using real-time quantitative PCR. In the test of disease transmission by seeds, the bacterium was detected in high incidences (>87%) in 90-day old plants generated from seeds collected from infected plants in quantities exceeding 51 cells per 100ng of total DNA of seeds. In addition, at 270 days after planting, the bacterium was isolated from six plants. The colony identity was confirmed by conventional PCR using Lxx-specific primers. Two trials were conducted to quantify thermotherapy effect on bacterial population. Lxx titers were determined 90 days after planting in plant leaves of two sugarcane varieties originated from setts subjected or not to heat-treatment. The initial bacterial titer in the stalks affected the treatment efficiency, which significantly reduced Lxx titers only in the trial where the stalks presented high initial bacterial levels. Moreover, 70 and 55% of the plants originated from heat-treated setts in the first and second tests, respectively, were infected with Lxx, cells; however, in smaller amounts compared to plants from untreated setts. Thus, it is concluded that Lxx is transmitted in high frequency by seeds and that the thermo treatment, although not efficient as an eradicating treatment, reduces the bacterial population in the plant tissue.