Shade tree incorporation is beneficial in coffee cropping systems under sub-optimal conditions. This study was performed in lowland Costa Rica, at a 12-year-old experimental coffee farm. The main objective was to compare the effect of a nitrogen fixing shade tree, Erythrina poeppigiana, on nutrient availability in the rhizosphere of coffee under conventional fertilization. Accumulation of nutrients (mineral N, available P, and exchangeable base cations) in rhizosphere relative to bulk soil was greater under shade than full sun. Low nitrate availability in rhizosphere soil of full sun coffee was explained by root-induced acidification relative to bulk soil, as abundance of ammonia-oxidizing bacteria (AOB), which mediate nitrification, were positively correlated with pH. Organic fertilization enhanced AOB abundance and altered soil bacterial community structure relative to conventional fertilization. This study indicates clear effects of shade-tree presence on nutrient availability at the micro-scale, management of which is critical for stability of coffee agroforestry systems.

Shade tree incorporation is beneficial in coffee cropping systems under sub-optimal conditions. This study was performed in lowland Costa Rica, at a 12-year-old experimental coffee farm. The main objective was to compare the effect of a nitrogen fixing shade tree, Erythrina poeppigiana, on nutrient availability in the rhizosphere of coffee under conventional fertilization. Accumulation of nutrients (mineral N, available P, and exchangeable base cations) in rhizosphere relative to bulk soil was greater under shade than full sun. Low nitrate availability in rhizosphere soil of full sun coffee was explained by root-induced acidification relative to bulk soil, as abundance of ammonia-oxidizing bacteria (AOB), which mediate nitrification, were positively correlated with pH. Organic fertilization enhanced AOB abundance and altered soil bacterial community structure relative to conventional fertilization. This study indicates clear effects of shade-tree presence on nutrient availability at the micro-scale, management of which is critical for stability of coffee agroforestry systems.

Ongoing work in the laboratory of Professor Chris Backhouse is aimed at developing a portable and inexpensive lab on a chip instrument. A system capable of molecular biology protocols including sample preparation (SP), polymerase chain reaction (PCR), and melting curve analysis (MCA) would meet the requirements for point of care genetic analysis. The SP, PCR, and MCA modules were designed and tested on a standalone basis and then integrated for analysis of raw clinical samples. An automated XY stage was developed for magnetic bead-based DNA purification. In addition, a LED/CCD-based optical detection module was employed for real time PCR and MCA. Data analysis algorithms and protocols were implemented to remove noise and interpret data. This work culminated in proof of principle on-chip SP-PCR-MCA to detect ß2m DNA from human buccal cells in a modular and inexpensive system. / Biomedical Engineering

The use of PCR as a tool for the diagnostic virology and viral research laboratories has greatly increased in recent years, however the use of conventional PCR and amplicon detection systems can be a complex and relatively slow process that increases the risk of amplicon carry-over contamination. Many conventional PCR systems are unsuited for, or unable to perform as accurate diagnostic and quantitative tools because viruses are present in such a diverse variety of patient tissues and in a broad range of concentrations. Traditional viral culture, while still the gold standard for the detection of many viruses, is lengthy, expensive and often subjective. In addition, successful isolation of infectious virus is variable and dependent upon appropriate cell lines, lengthy incubations and careful transport and storage of clinical specimens. Many of the disadvantages arising from the use of traditional assays for the detection of viruses have been overcome by the development of real-time PCR. The technology has continued to develop due to the introduction of several commercial thermal cycling platforms and the appearance of numerous specific and non-specific fluorogenic chemistries. For the purpose of this thesis, human virology was sectioned into three diagnostic divisions containing the synthetic viruses, the well characterised viruses and the new or emerging viruses. This thesis proposes the hypothesis that real-time PCR could greatly improve upon traditional techniques for the detection, quantitation and characterisation of the members of these three divisions in both research and diagnostic environments. Conventional competitive quantitative PCR assays and a non-oligoprobe real-time PCR assay were constructed to detect novel synthetic gene therapy vectors developed from retroviruses. When compared to oligoprobe-based real-time PCR, it was clear that conventional molecular assays, whilst improving upon traditional methods of viral culture and immunofluorescence, were slower, more complex, less versatile and were hindered by a limited dynamic range. Synthetic control templates were developed and an improved method of assaying these template preparations was devised. The controls were used to precisely optimise each assay, create quality assurance reagents and to construct external standard curves permitting the absolute quantitation of viral templates. Real-time PCR achieved several significant goals during the studies performed for this thesis. The new assays detected human enterovirus (HEV) and the emerging pathogen, human metapneumovirus (hMPV) which were both responsible for seasonal outbreaks of serious disease that would otherwise have gone undiagnosed. These data led to the first description of hMPV outside of the Netherlands, as well as the first description of two validated rapid diagnostic RT-PCR assays which permitted the definitive classification of hMPV as a global pathogen of children and adults. Building upon its detection, an extensive molecular epidemiological study permitted the description of subtle differences between Australian and the more recently described international hMPV strains resulting in the classification of two distinct types of hMPV (A and B) and within these, four subtypes (A1, A2, B1 and B2). Real-time PCR rapidly detected, quantitated and genotyped herpes simplex viruses in a single reaction and determined the successful delivery of human and non-human genes by novel retroviral vectors in less time than any other phenotype detection assay. Additionally, these studies produced quantitative data which permitted the rapid calculation of transduction efficiency. Real-time PCR was able to quickly assess the efficiency of the PCR either in response to the titration of individual reaction components or as a result of amplification modifiers present within specimen extracts. The use of nucleotide sequencing studies ideally complemented earlier diagnostic studies of HEV and permitted the discrimination of pathogenic enterovirus 71. This thesis demonstrated that real-time PCR is more able to accommodate the demanding aspects of viral research and diagnostics than any other single method, and is now in a position to replace many of the traditional techniques still used by laboratories unfamiliar with the benefits of real-time PCR. The assays, techniques, reagents and publications resulting from these studies have benefited several areas of viral research and diagnostics and have improved the understanding of the role of real-time PCR in virology and of the technique in general, among the greater scientific community whilst successfully addressing the proposed hypothesis.

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels. / Singapore-MIT Alliance (SMA)

Determination of quantitative expression levels of alternatively spliced isoforms provides an important approach to the understanding of the functional significance of each isoform. Real-time PCR using exon junction overlapping primers has been shown to allow specific detection of each isoform. However, this design often suffers from severe cross amplification of sequences with high homology at the exon junctions. We used human GFRα2b as a model to evaluate the specificity of primers substituted with locked nucleic acids (LNAs). We demonstrate here that single LNA substitutions at different positions of 3’ terminus could improve the discrimination of the primers against GFRα2a template, a highly homologous isoform. While LNA substitutions of GFRα2b primer at the residues possessing different sequences as GFRα2a has limited improvement in specificity, two consecutive LNA substitutions preceding the different sequences has dramatically improved the discrimination by greater than 100,000-fold compared to the non-substituted primer. Thus, LNA when substituted at certain residues can allow the discrimination of highly homologous sequences. / Singapore-MIT Alliance (SMA)

Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998).

The aim of this study was to confirm that the polyunsaturated fatty acids n-3 (n-3 PUFA) have a positive effect on plasma lipids. These acids can reduce cholesterol by increasing gene expression Insig-1 while decreasing the expression of genes encoding Hmgcr and Ldlr. We tested in experimental rats, which were added to the feed mixture of 6 % safflower oil , 6 % fish oil or 6 % of the oil from the algae Schizochytrium. Relative gene expression was Insig-1 in the test group with addition of fish oil to 120% of controls (P<0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 170 % of control (P<0.05). These results confirm our hypothesis, only a part, as the relative expression of the gene and Hmgcr and Ldlr was in the test group with addition of fish oil 103% (P>0.05) and 101 % of control (P>0.05) and in the group with addition of oils from algae Schizochytrium the relative expression of 117% (P>0.05) and 156 % (P>0.05) compared to control. Thus, to reduce the relative expression of these genes did not. However, we have shown that n-3 PUFA contribute to a reduction in plasma cholesterol and in this case up to 20 % of control. The concentration of cholesterol in the group with addition of safflower oil was 1.35 mmol.l-1, the group with the addition of fish oil 0.98 mmol.l-1.

Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998) ... [cont.].

The transmission of human pathogens by faecally contaminated fruit and vegetables is well established, but the burden of disease caused by foodborne pathogens is unknown. Fresh produce can be contaminated through the use of polluted irrigation water or by the handling of the produce by infected individuals either pre- or post harvest. There is very little known regarding the extent of viral contamination of irrigation water and fresh produce in South Africa. Noroviruses (NoV) and hepatitis A virus (HAV) are recognized as leading causes of foodborne viral disease. These viruses are transmitted predominantly via the faecal–oral route, primarily person-to-person by direct contact with an infected person, or indirectly by ingestion of contaminated food and water. The detection of enteric viruses in food or water is problematical and complex as many foodborne viruses, including HAV and NoV, cannot be readily isolated in cell culture. The aim of this investigation was to develop and optimise simple and efficient methods for the concentration and detection of NoV GII and HAV in irrigation water and fresh produce. These methods would then be applied to field samples of irrigation water and fresh produce to try and establish a link between viral contamination detected in irrigation water and that on associated irrigated fresh produce. The efficiency of different commercial real-time reverse transcriptase-polymerase chain reaction amplification kits for the realtime detection of HAV, NoV GI and NoV GII was assessed, and standard curves for the quantitative detection of these viruses were constructed using the most appropriate kit. Using two types of fresh produce, three different elution buffers, each at two pHs, with two different elution times were compared to establish which buffer was the most efficient for the extraction of viruses from the fresh produce. The tris-glycine beef extract buffer (pH 9.5) with an elution time of 20 minutes most efficient for the extraction of the selected enteric viruses from fresh produce. From April 2008 to November 2009, 86 irrigation water and 72 fresh produce samples were collected from commercial and subsistence farms, street vendors and commercial outlets. All the irrigation water and fresh produce samples were analysed for HAV, NoV GI and NoV GII. Overall, 16.3 % (13/86) and 12.5 % (9/72) of irrigation water and fresh produce samples tested positive for one or more human pathogenic viruses, namely NoV GII and HAV, respectively. Nucleotide sequence and phylogenetic analysis of the HAV and NoV GII strains identified clinically relevant viruses in the irrigation water and on the fresh produce. A direct link between contaminated irrigation water and contamination of fresh produce could not be established, but irrigation water was identified as a possible source of contamination of the fresh produce. The results also suggested that food handlers contributed significantly to the viral contamination of the fresh produce. This study highlights the potential health risk posed by fresh produce to consumers in South Africa and highlights the need for further in depth studies to quantify the risk to consumers. This study represents new data on the occurrence of enteric viruses in food and water in South Africa and is crucial for the development of effective intervention and control strategies for food safety in South Africa. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Medical Virology / unrestricted