Next generation of sequencing (NGS) technologies have taken life science research into a new era. With the rapid advances in these technologies and the associated reduction in overall costs, the sequencing and assembly of genomes have come within reach of most laboratories. Studies related to the evolution, ecology and biology of an organism now rely heavily on genomic data and obtaining a genome sequence has become an essential resource for the rapid progress and success of these studies. Pantoea ananatis is recognised as an emerging but rather unconventional pathogen capable of infecting a wide range of different hosts. Numerous plants of agricultural and economic importance including maize, rice, onion, pineapple, melon, sudan grass and Eucalyptus trees have been affected. With the outbreak of P. ananatis in a South African Eucalyptus nursery in 1998, it was realised that very little is known about this pathogen. A better understanding of the pathogenicity, metabolism and ecology of the bacterium is required to develop strategies for the control of the disease. During this study, the genome sequence of P. ananatis strain LMG 20103 was obtained using the Roche 454 technology. To aid in the assembly of this Eucalyptus pathogen’s genome sequence, the type strain of P. ananatis LMG 2665 was also sequenced using Illinima’s Genome Analyzer (GA). A draft assembly of P. ananatis LMG 20103, consisting of 117 contigs, was generated after optimization of the Newbler assembly parameters and comparison with other genome assemblies and genomes. This study demonstrated that the assembly could be completed using both in-vitro, and in-silico approaches such as contig scaffolding, gap closure with conventional PCR reactions and sequencing, manual curation and automated genome annotation. The final complete genome consisted of a 4 386 227 bp chromosome and a 317 146 bp mega-plasmid. With the complete genome sequence available, the reconstruction of metabolic network of P. ananatis LMG 20103 was attempted using two pathways reconstruction pipelines namely, Pathway Tools and Model SEED. It was found that missing metabolic reactions and incomplete pathways in the draft metabolic networks were mainly caused by incorrect gene annotations or bioinformatic errors during the automated network reconstruction. These two pipelines differed substantially in the way network reconstruction is undertaken. Performing a comparison between the two proposed networks, annotation errors could be detected and corrected. Although some improvement could be made to the predicted network further experimental data is still required to improve the accuracy of the draft metabolic network. Despite the amount of effort and cost, it is believed that the complete genome and a draft metabolic network of P. ananatis LMG 20103 will be a valuable resource for many subsequent studies to investigate the evolution and biology of this emerging plant pathogen. This information will be essential for the development of strategies to predict and control future disease outbreaks associated with this pathogen. / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

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The aim of this study was to validate the two presumptive blood tests LMG, LCV and the three visualising blood methods Bluestar Forensics, Lumiscene and the Ruhoff method. The methods’ sensitivity, durability, matrices effects, false positive results and the methods effect on subsequent DNA analysis were studied. DNA analyses were also performed to assess the detection limit of the forensic DNA analysis. Drops of diluted blood were applied on different absorptive matrices and the sensitivity was investigated. The solutions were also placed under different conditions to investigate the durability of the solutions. The solutions were applied upon panels using different chemicals and materials and the false positive results were studied. The DNA analyses were performed by diluting the blood with Bluestar Forensics, the hydrogen peroxide method, the Ruhoff method and deionised water. The study showed that the LMG with a 3 % H2O2 concentration performs the best and it is suited for practical casework. The positive results of LMG was easier to interpret than those of LCV, this is probably due to the fixative agent of the used LCV solution. Bluestar Forensics and Lumiscene did perform similar on the different matrices tested, but the Lumiscene solution had a slightly higher durability. The results strongly indicate that the Ruhoff method can be used without luminol, hence only as a hydrogen peroxide solution (the hydrogen peroxide method). All three visualising blood methods decreases chances of retrieving a positive DNA profile, however the visualising blood methods could be used if the blood cannot be found in any other way. A DNA profile was obtained from the one blood sample analysed at dilution of 1:256 in deionized water.

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<p>Radiotherapy is used in many clinics to deliver a sufficient and uniform dose to the cancerous tumours while the dose to normal tissues is minimized. However, there is a possibility of missing the target volume due to patient set up/motion errors, or any fluctuation in treatment delivery. Therefore, accurate dose verification tools are essential to evaluate the delivered dose distribution of the designed treatment plan under realistic treatment conditions.</p> <p>Current research is focused on developing 3D dose verification tools to record the complex dose distributions for quality assurance purposes and the evaluation of new treatment techniques. New and novel materials and read-out techniques suitable for use in hospitals are desirable. The objective of this research is to fabricate a transparent radiochromic gel dosimeter that may be used as quality assurance tool. Also, the fabricated gel must be analyzed using a simple optical read-out technique.</p> <p>Gel dosimeters are gels that undergo some chemical changes upon irradiation as a function of absorbed dose. The absorbed dose may be recorded in three dimensions depending on the type of gel dosimeter. Radiochromic gels are dosimeters that change colour upon irradiation. A radiosensitive dye, leucomalachite green (LMG) is dissolved in a matrix material to record the dose distribution in 3D. LMG changes its colour upon irradiation, and has an absorbance band of 629nm.</p> <p>In this research two different matrix materials were investigated: poly (vinyl alcohol) and gelatin. PVA was studied as the primary agent due to its adjustable mechanical strength and high transparency. PVA has also been studied to have a low diffusion rate when it was used as the matrix material in Fricke gel dosimeters [41]. Even though PVA had all the desired characteristics, fabricating a PVA based radiochromic dosimeter was not successful. Consequently, gelatin was used as the matrix material to fabricate a gelatin-based radiochromic dosimeter.</p> <p>Using gelatin, highly transparent radiosensitive gels were successfully fabricated. The absorbencies of the irradiated gels were measured as a function of absorbed dose, using a 1D set up. After, the gels were formed into 5mm thick films and used as two-dimensional dose verification tools. The relationship between absorbance and absorbed dose for 1D measurement was obtained to be 0.00241± 0.00004 , and 0.0022 ± 0.00007 for 2D gels scaled to a thickness of 1 cm.</p> <p>In all of the experiments the absorbance-dose relationships were similar in slopes, but there was an offset between different batches. The offset was 20% between the different experiments. Moreover, there was less than 5% error associated with the physical set up; the major source of error was due to the production and handling of the mixture, possibly due to the effects of inconsistent heating and UV light exposure.</p> <p>The 2D gels were used to verify the dose distribution for the purpose of quality assurance. Six different complicated beams were delivered to the gels and their dose distributions were compared to their respective Pinnacle Calculated Planar (PCP) dose maps. The difference was found to be about 35% at worst; however, this error may be reduced by utilizing more sophisticated data processing methods. Nevertheless, the images were quite similar above 20Gy. Furthermore, the dose distributions recorded by the gels are qualitatively and quantitatively similar to the (PCP) dose map. Although the fabricated gel dosimeters show some promise as future tools for quality assurance purposes, they must go through many more stages of research to be used clinically.</p> / Master of Science (MSc)

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