Browsing by Author "Du Preez, Jacques"

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    Assessing the m-Government readiness within the provincial government Western Cape
    (Stellenbosch : University of Stellenbosch, 2009-03) Du Preez, Jacques; Burger, Naomi; University of Stellenbosch. Faculty of Economic and Management Sciences. School of Public Management and Planning.
    m-Government or mobile-Government is seen as part of e-Government and an additional channel for the delivery of public services and information to the citizen. This study critically examines and evaluates the extent to which the Provincial Government Western Cape has adopted m-Government and implemented related services. A survey conducted by Kirsten (2006) on the adoption and readiness of mobile technology by businesses in South Africa was used as the foundation of this study to determine the level of readiness in the Provincial Government Western Cape. Managers and technical staff within the Province’s information and communication technology component, the Centre for e-Innovation, were interviewed and asked to complete the survey. The study found that, although there is a relatively high degree of adoption with regard to various aspects of mobile and wireless technology, there are many obstacles and barriers that need to be overcome in order to achieve a higher level of m-Government maturity or readiness. The study makes various suggestions on how to overcome these barriers. The most important suggestion is to develop a holistic approach to the adoption of m-Government. Plans for adoption should be incremental to ensure that small victories that can be built on are achieved; the involvement of key stakeholders is also essential.
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    The construction of an infectious clone of grapevine virus A (GV A)
    (Stellenbosch : University of Stellenbosch, 2005-04) Du Preez, Jacques; Burger, J. T.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA- mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.
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    The development and characterisation of grapevine virus-based expression vectors
    (Stellenbosch : University of Stellenbosch, 2010-03) Du Preez, Jacques; Burger, J. T.; Goszczynski, D. E.; Stephan, D.; University of Stellenbosch. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future.
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    A study of reconfigurable manufacturing systems with computer simulation
    (Stellenbosch : Stellenbosch University, 2011-12) Du Preez, Jacques; Bekker, James F.; Stellenboch University. Faculty of Engineering. Dept. of Industrial Engineering.
    ENGLISH ABSTRACT: Reconfigurable Manufacturing Systems (RMSs) have the ability to reconfigure hardware and control resources at all of the functional and organizational levels. This allows for quick adjustment of production capacity and functionality in response to sudden changes in market or in regulatory requirements. This study evaluates the characteristics and operation of automated reconfigurable assembly lines using discrete event simulation. The assembly line uses a conveyor system which transports pallets to various machines to perform the assembly process. Different conveyor configurations are developed for the same assembly process using Simio simulation software. A part family consisting of five variants are assembled on the same assembly line with a large variation in the production quantities for each product. This requires the assembly system to be able to quickly adjust its functionality and capacity. Multi-objective optimization is performed on the models through the use of a Pareto exhaustive search experiment. The two contradicting objectives used are the throughput rate of the system and the average work in progress, with the aim of maximizing the former and minimizing the latter. From the Pareto exhaustive search experiment, a Pareto front is constructed showing which configuration is preferred under certain operation conditions. However it is concluded that the Pareto front can be tailored to fit the specific needs of the decision maker, depending on what the decision maker is willing to pay. An experiment that evaluates the effect of changing the conveyor speed is performed. It is established that under certain operating conditions, increasing the conveyor speed higher than the ceiling value will not improve the performance of the system. A production scenario was also developed which include different order sizes for each of the five parts of the part family. The configurations have to alter their capacities based on the order sizes to test which system performs the best under these operating conditions. For this experiment, the ramp-up time was of interest but the best system was chosen based on the combination of throughput rate and the average work in progress. From the results of the different experiments, it is recommended to first determine the maximum capacity and the operating logic before choosing one of the configurations. Once this is decided, the information gathered from the experiments can then be tailored for the decision maker to establish the best operating conditions for the chosen con guration. The developed simulation models are used as a Decision Support System for future research on the topic. It is recommended for future research to focus on using Automated Guided Vehicles (AGVs) instead of a conveyor system as transportation method.