Inhibition of hepatitis B virus replication using synthetic antiviral RNA interference activators

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg 2016Chronic liver infection by hepatitis B virus (HBV) may lead to devastating clinical conditions that include hepatocellular carcinoma and cirrhosis. Approved antiHBV drugs do not completely eradicate the infection, leading to continued viral persistence in infected individuals. Inhibition of HBV replication using synthetic activators of RNA interference (RNAi) may provide a feasible strategy of developing superior antiviral drugs. The aim of this study was to evaluate the therapeutic utility of novel 2’-O-guanidinopropyl (GP) modified synthetic small interfering RNAs (siRNAs) to counter HBV replication in cultured mammalian cells and mice. Initially, single GP moieties were placed at different nucleotide positions of the guide strand of a potent antiHBV siRNA. Some GP-modified siRNAs enhanced antiHBV activity in vitro following transient transfection of Human hepatoma 7 (Huh7) cells with siRNAs and pCH-9/3091, a replication competent HBV target plasmid. These siRNAs inhibited the secretion of Hepatitis B surface antigen (HBsAg) by up to 95% in Huh7 cells. The level of knockdown exhibited by some modified siRNAs was statistically significant relative to that displayed by unmodified siRNA3 which achieved HBsAg silencing of 73%. Additionally, modified siRNAs were also capable of reducing RNA containing the X sequence in vitro by 88-93%. Impressively, some of these knockdown levels were statistically significant when compared to unmodified siRNA3, which achieved HBx knockdown of 83%. Quantitation of interferon (IFN) response genes by reverse transcription quantitative polymerase chain reaction (RTqPCR) and evaluation of cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay revealed no evidence of innate immune stimulation or cytotoxicity in cultured cells, respectively. Modified siRNAs also displayed moderate stability in 80% foetal calf serum (FCS). Target specificity was markedly improved by GP-modified siRNAs, especially those with seed modifications (comprising nucleotide position 2 to 8 from the 5’ end of the guide strand). The siRNA-mediated mRNA cleavage product was detected from transfected cells using 5’ Rapid Amplification of cDNA ends (5’ RACE). In the hydrodynamic mouse model, co-injection of GP-modified siRNAs and HBV plasmid vector led to HBsAg suppression of approximately 80-92% at day 3 and 77-96% at day 5 post-administration. The HBV knockdown levels observed at day 3 were statistically significant when compared to those displayed by unmodified siRNA3 which achieved HBsAg silencing of 58% during the same time frame. Furthermore, both sets of siRNAs also suppressed the number of circulating viral particle equivalents (VPEs) by 88- 90% at day 3 post-injection. HBV silencing efficacy of 70-75% and 65% was achieved by modified and unmodified siRNAs, respectively at day 5 post-administration. Finally, antiHBV efficacy of GP-modified siRNAs was tested in HBV transgenic mice following delivery of these RNAi effectors using cationic polyglutamate (PG) adjuvant liposomes. Both groups of antiHBV siRNAs effected HBsAg knockdown that ranged from 70-86% at day 3 to 7 post-administration as siRNA lipoplexes in HBV transgenic mice. In contrast to the unmodified siRNA3, GP-containing siRNAs achieved durable HBsAg silencing of 70% at day 14 post-administration, while the unmodified siRNA3 displayed a shorter duration of activity. As with HBsAg data, the GP-modified siRNAs also displayed silencing efficacy that was similar to the unmodified siRNA, reducing the number of circulating VPEs by 95% from day 3 to 7 post-injection. However, the unmodified siRNA3 lost efficacy by day 14 post-administration, while the GP-modified siRNAs displayed prolonged suppression by reducing the number of circulating VPEs by 75% during the same time interval. Intrahepatic RNA levels were also assessed in transgenic mice, in which GP3 siRNA3 significantly suppressed surface and core RNA levels by 40 and 42%, respectively at day 18 post-injection. The unmodified siRNA3 suppressed surface RNA levels by 20% and core RNA levels by 25% at day 21 post-administration. Furthermore, GP4 siRNA3 silenced both surface and core RNA levels by 42% during the same time period. Additionally, intrahepatic RNA quantitation revealed no induction of IFN response genes by either unmodified or GP-modified siRNAs. In contrast to mice that had received GP-modified siRNAs, significant induction of proinflammatory cytokine release was observed in mice treated with unmodified siRNAs. The siRNA-mediated mRNA cleavage product was also detected from liver samples following 5’ RACE analysis. Neither GPmodified nor unmodified siRNAs significantly induced toxicity in injected mice. Collectively, our data provide evidence that utilisation of GP-modified siRNAs and an efficient hepatotropic non-viral delivery system may be used as a strategy to counter chronic HBV infection.MT201

Opportunities in Software Engineering Research for Web API Consumption

Nowadays, invoking third party code increasingly involves calling web services via their web APIs, as opposed to the more traditional scenario of downloading a library and invoking the library's API. However, there are also new challenges for developers calling these web APIs. In this paper, we highlight a broad set of these challenges and argue for resulting opportunities for software engineering research to support developers in consuming web APIs. We outline two specific research threads in this context: (1) web API specification curation, which enables us to know the signatures of web APIs, and (2) static analysis that is capable of extracting URLs, HTTP methods etc. of web API calls. Furthermore, we present new work on how we combine (1) and (2) to provide IDE support for application developers consuming web APIs. As web APIs are used broadly, research in supporting the consumption of web APIs offers exciting opportunities.Comment: Erik Wittern and Annie Ying are both first author

How does Internationalisation affect the productivity of R&D activities in large innovative firms? A conditional nonparametric investigation

This work explores the relationship between multinational R&D and innovation productivity among top corporate knowledge and R&D producers by adopting a twofold concept of internationalisation: (1) the firm’s degree of R&D internationalisation, and (2) the firm’s geographic diversification. We model the patent production process with an appropriate and robust conditional Data Envelopment Analysis (DEA) estimator, using a unique database of firms that matches financial indicators and patent information. Our results reinforce the fundamental role of internationalisation in the knowledge production process when the internationalisation process is properly and strategically managed. We interpret our empirical evidence through the theoretical lens of the learning theory of internationalisation, and we postulate that a high R&D intensity is a key driver to overcoming the challenges of internationalisation

Fluctuaciones minerales en pastos tropicales : I. Braquiaria en los Llanos Orientales

Mineral variation in Tropical grasses. I. Braquiaria in Llanos Orientales. This experiment was conducted at La Libertad experimental Station (Meta), using Brachiaria decumbens from August 78 to October 79. It was determined the nitrogen and mineral contents of weekly samples from areas with cattle under continuous grazing, during one year, (2,4 head per hectare). The content of minerals during the year in Brachiaria decumbens was high enough to supple the animal requeriments. Only the nitrogen, phosphorus, magnesium, copper and zinc were in siib-normal or deficient conditions. Season variations of Ca and Mg were found with high values in dry seasson but they were not significant. The Fe and Mn levels were high in both seasons, but significantly higher in dry season. This findings suggest that in areas planted with Brachiaria Decunib ens it might be necessary to supply only a few mineral elements (Ca. P. Cu and Zn) to guarantee the mineral requeriments for grazing cattle.Se determinó el contenido de proteina y elementos minerales en pasto Braquiaria en muestras tomadas semanaimente durante un afio de 4 praderas bajo pastoreo continuo, con una carga de 2,4 animales por hectárea, en la Estación Experimental La Libertad, Meta. Se encontró que el pasto Braquiaria puede suministrar hierro, magnesio y rnanganeso en cantidades suficientes, para llenar las necesidades de los animales en pastoreo y que solo el nitrógeno, fósforo, cobre, magnesio y zinc están en niveles sub-normales o deficitarios. Las variaciones estacionales muestran una tendencia a incrementar los valores en Ia época de sequia aunque no en forma significativa; solo el fósforo y magnesio se mantienen constantes durante el año. Los niveles de hierro y manganeso se presentan demasiado altos como para constituir problemas por deficiencias, aunque lo podrían ser por exceso. Estos elementos fueron significativamente más altos en verano. El bajo contenido de minerales encontrado en invierno concuerda con lo registrado en Ia literatura, de que el aumento de follaje verde de los forrajes produce Ia disminución en ci contenido de minerales. Este hecho se complica cuando los animales incrementan el consumo de forraje y las necesidades de minerales. Lo hallado sugiere garantizar Ia suplementación de sal mineral, principalmente en época de lluvia.Pastos y forraje

Valor nutritivo de pastos tropicales, 5: Pasto angleton (Dichanthiumaristatum, (Poir) C. E. Hubbard) anual y estacional.

El experimento se llevó a cabo en una hacienda de clima cálido del departamento del ToIima con períodos definidos de lluvia y sequía y donde los pastos sufren cambios estacionales en su valor nutritivo. Las muestras se tomaron de potreros de pasto ángleton sometidos a pastoreo rotacional con ganado cebù cruzado y correspondieron a las porciones que los animales consumían. Los contenidos medios de proteína (7,5%) se encuentran en el límite subnormal del requerimiento de los animales pero gran parte del verano y el último mes de lluvia mostraron niveles deficitarios de estas sustancias. Los porcentajes de digestibilidad fueron moderadamente altos aunque no presentaron diferencias significativas entre estaciones (70,6% para lluvia y 66,5%para sequía). Las fracciones de la pared celular como la fibra en detergente neutro (69,9017o), la fibra en detergente ácido (47,27% ), la celulosa (33,14%) y la lignina (9,917o) constituyen valores que afectan marcadamente el consumo y la utilización del forraje. Las ecuaciones lineales calculadas fueron significativas al nivel del 1% para la digestibilidad y la energía digestible. Las regresiones múltiples mostraron que la proteína cruda, la libra en detergente neutro, la hemicelulosa y la celulosa afectaron en 67% la digestibilidad y en 74% la energía digestible. Las regresiones múltiples con los anteriores componentes permiten predecir de manera confiable la digestibilidad y la energía digestible de este pasto tropical.This experiment was carried out in a tropical area of the Tolima department with dry and wet periods where the grasses change drastically the nutritive value. The samples were taken manually from the paddocks with animals in rotational grazing conditions with two animals per hectare. The crude protein content during the year was 7,5%. This quantity is close to the limit of nutritional requeriment for growing steers. However, during the dry season and the last month of the wet season the amount of crude protein was highly deficient. The dry matter digestibility was relatively high in both seasons but without differences between seasons. The cell wall constituents as NDF (69,90%), ADF (47,27% ), cellulose (33,14% ) and lignin (9,9%) were high enough to affect the dry matter intake and grass utilization by the animal. The simple regression coeficients were significant (P < 0,01) between chemical components and the dry matter digestibility and digestible energy. The multiple regression equations showed that the crude protein, neutral detergent fiber, hemicellulose and cellulose affected the digestibility with 67% and the digestible energy with 74%. The multiple regression with the chemical components make it possible to predict the dry matter digestibility and the digestible energyPastos y forraje

Valor nutritivo de pastos tropicales, 4: Pasto Carimagua-1(Andropogon gayanus Kunth) anual y estacional

El pasto Carimagua-1 (Andropogon gayanus, Kunth) presenta buenas características agronòmicas y gran capacidad para adaptarse a suelos ácidos de baja fertilidad y para tolerar sequías prolongadas. En Colombia se adapta muy bien a suelos de sabanas planas de los Llanos Orientales. El material de estudio se obtuvo en praderas bajo pastoreo continuo con carga animal media de 2,0 reses por hectárea. Las muestras se tomaron manualmente cada 15 días. Los componentes químicos variaron durante todo el año con grandes diferencias estacionales. Los valores de proteína cruda, FDN, FDA y lignina son factores determinantes de la calidad nutritiva del pasto Carimagua-l. La digestibilidad in vitro y la energía digestible presentan valores bajos durante las estaciones de lluvia y sequía aunque con poca diferencia lo que significa que la cantidad de forraje disponible y la oportunidad de seleccionar fracciones ricas en nutrientes podrían permitir aumentos de peso en los animales durante la época de lluvias y persistencia del peso durante la época de sequía. Las regresiones simples entre componentes químicos y digestibilidad de la materia seca fueron significativas. Cuando se determinaron las regresiones múltiples con las mismas variables se encontraron coeficientes de determinación bajos aunque se mostró que la proteína cruda, la fibra en detergente neutro, la fibra en detergente ácido, la hemicelulosa y la celulosa son los componentes que más afectan la digestibilidad. Las ecuaciones simples y múltiples para la energía digestible también presentaron bajos coeficientes de determinación lo cual muestra nuevamente que la proteína cruda, la fibra en detergente neutro, la fibra en detergente ácido, la hemicelulosa y la celulosa son los componentes químicos más importantes en la predicción de la energía digestible.Gamba grass (Andropogon gayanus), known locally as Ca.rimagua.1 grass, presents good agronomic characteristics and high capacity to grow in acid, low fertility soils and during long dry periods. This is the case of the western plains of Colombia known as the Uanos Orientales. The present study was conducted to determine the nutritive value of Carimagua-1 grass on samples taken every two weeks in four paddocks under continous grazing condition with two animals/Ha. The chemical components changed during the year with great variations between seasons. The nutritive value was expressed as the crude protein, NDF, ADF, and lignin contents. The in vitro digestibility and the digestible energy showed low values in wet and dry seasons without differences between periods. This means that the quantity of grass and the selectivity of green parts with high nutritive value can bring to the animals the opportunity to gain weight in wet seasons and get enough nutrient for maintenance in dry seasons. The lineal and the multiple regressions between chemical components and dry matter digestibility were significant and showed that the crude protein, ADF and cellulose are the principal components that affected the digestibility. The same trend was found with the digestible energy.Pastos y forraje

Fluctuaciones minerales en pastos tropicales :II Andropogon gayanus en los Llanos Orientales.

Experimento realizado en La Libertad, Centro Regional de Investigación ICA, Departamento del Meta, 400 m.s.n.m., 2989 mm de precipitación y 26 grados centigrados de temperatura, zona de suelos ácidos deficientes en P y períodos definidos de lluvía (abril-noviembre) y sequía (diciembre-marzo). El pasto fué sembrado en septiembre de 1979, fertilizado con cal dolomítica (350 kg/ha), superfosfato triple (67 kg/ha) y cloruro de potasio (67 kg/ha), se emplearon 8 kg/ha de semilla con lo cual se obtuvo una población de 12 a 14 plantas por m2. Las muestras de pastos para análisis fueron recolectadas durante 12 meses, de dos potreros con carga de 2 U.G.G./ha en época de lluvia y 1 U.G.G./ha en época de sequía. Se encontró que el Andropogon gayanus suministró 6.8 por ciento de proteína en invierno y 5.3 por ciento en época de sequía, diferencia que fué significativa (P menor que 0.05), sin embargo, estos valores estuvieron por debajo del nivel normal (8 por ciento). Los valores medios anuales encontrados para los demás minerales fueron: Ca 0.47 por ciento, Fe 178 ppm, P 0.13 por ciento, Mg 0.17 por ciento, K 1.40 por ciento, Na 0.045 por ciento, Mn 336 ppm, Cu 3.88 ppm y Zn 15.4 ppm. No se encontraron diferencias estadísticas para las dos épocas. El Andropogon gayanus puede suministrar Fe, Mg y K en cantidades suficientes para los requerimientos animalesPastos y forraje

Prognostic significance of infarct size and location: The case of insular stroke

The prognostic relevance of strokes in different locations is debated. For example, insular strokes have been associated with increased mortality, but this association could reflect their greater severity. In two independent cohorts of patients with supratentorial ischemic stroke (n = 90 and 105), we studied the prognostic consequences of lesion size and location using voxel-based lesion-symptom mapping before and after volume control, which better accounts for total lesion volume. Strokes affecting the insula were larger than non-insular strokes (28 vs 2cc and 25 vs 3cc, p < 0.001 in both cohorts). A number of supratentorial areas (mainly in the left hemisphere), including the insula, were associated with poor functional outcome in both cohorts before (4014 voxels) and after volume control (1378 voxels), while the associations with death were greatly reduced after volume control (from 8716 to 325 voxels). Exploratory analyses suggested that the method of lesion volume quantification, the National Institutes of Health Stroke Scale hemispheric bias and patient selection can result in false associations between specific brain lesions and outcomes. In conclusion, death in the first months after stroke is mainly explained by large infarct volumes, whereas lesions of specific supratentorial structures, mostly in the left hemisphere, also contribute to poor functional outcomes