Biofumigation is an important crop protection practice that uses a plant’s natural defence mechanisms to control agricultural crop pathogens and diseases. Glucosinolates are volatile compounds found in most Brassica species and when hydrolysed, it forms a range of natural toxins including isothiocyanates that act as biofumigants. Research suggests that biofumigation is a good alternative to chemical fumigants as it is effective in controlling plant pests but with lower health and environmental risks. Several studies have confirmed the effectiveness of the breakdown products, especially isothiocyanates, as fungicidal, bactericidal and nematicidal products against a series of plant pests. However, very little information is available on the effects of glucosinolates and its breakdown products on non-target and beneficial soil organisms. Negative effects on beneficial soil organisms can have serious negative impacts on soil quality especially when essential ecosystem functions such as nutrient cycling and soil bioturbation are affected. Three biofumigants, broccoli, mustard and oilseed radish, and two chemical fumigants, metham sodium and cadusafos, were investigated for possible effects on non-target and essential soil organisms such as earthworms and the soil microbial community. Sublethal endpoints, including growth and reproductive success of the earthworms, were monitored. The genotoxicity of the biologically active compounds found in the fumigants, towards earthworms, was evaluated by means of the comet assay. The DNA damage was quantified by tail intensity parameters. Furthermore, the changes in the soil microbial community function and structure were evaluated by means of community level physiological profiling (CLPP) and phospholipid fatty acid (PLFA) analyses respectively. All exposures were done in artificial soil prepared according to the OECD standard guidelines. In the biofumigant treated soils, results varied and different effects were observed on the non-target soil organisms. Broccoli reduced cocoon production and the number of hatchlings while mustard induced more DNA strand breaks in earthworm cells compared to the control. All the biofumigants stimulated microbial growth but broccoli and oilseed radish changed the microbial functional diversity. Mustard had no lasting effect on the functional diversity but altered the microbial community structure. The chemical fumigants had a marked negative impact on the survival, growth, reproduction and the genotoxicity of the earthworms with metham sodium causing greater harm than cadusafos. The effects on the microbial community varied. Both chemicals had an inhibitory effect on the microbial growth in terms of the viable biomass determined by PLFA and the average well colour development in the Biolog™ Ecoplates. No lasting effects were observed in the community structure. Overall, cadusafos had a more pronounced effect on the microbial community functional diversity than metham sodium. Results indicated that each bioindicator species illustrates effects at their own level of organisation / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Master of Science / Department of Agronomy / P. V. Vara Prasad / Grain sorghum (Sorghum bicolor L. Moench) is the fourth most important cereal crop grown throughout the semi-arid regions of the world. It is a staple food crop in Africa and Asia, while it is an important feed crop in the United States (US). More recently it is increasingly becoming important as a potential bioenergy feedstock crop around the world. The state of Kansas is the largest producer of grain sorghum in the US and contributes 40% of the total production. Drought is one of the major environmental factors limiting sorghum production in the semi-arid regions of the US, Asia and Africa. It is estimated that global crop losses due to drought stress exceed $10 billion annually. In crop production, drought stress can be classified into pre- or post-flowering. Even though the world collections of sorghum contain over 35,000 accessions, the genetic base currently used in breeding programs is very small (about 3%). Thus, it is important to identify diverse breeding lines for crop improvement. The diversity (association) panel consisting of 300 sorghum lines from all over the world was assembled for trait evaluation and association mapping. In this research these lines were grouped into the five major races (Figure 1) and 10 intermediate races of sorghum. The objectives of the research are to: (i) quantify the performance of the diversity panel under field conditions in Kansas, (ii) identify critical physiological traits affected by drought at both pre- and post-flowering stages of sorghum development, (iii) identify the most sensitive stage to drought stress during the reproductive phase of sorghum development and, (iv) test the feasibility of using a chlorophyll fluorescence assay (CVA) as a tool for identifying stay-green lines in grain sorghum during early stages of crop development. Field experiments were conducted in 2006 and 2007 in two locations in Kansas (Manhattan and Hays) under rain fed and irrigated conditions for the association panel. Objectives (iii) and (iv) were achieved with controlled environment experiments conducted in the greenhouse at the agronomy department, Kansas State University in 2006 and 2007. Results showed that there was large genetic variability among and within different races in the diversity panel for growth, physiological traits and yield components. Some genotypes showed yield stability across the different environments that were investigated. Drought significantly decreased seed number and harvest index across genotypes and races. In grain sorghum the period prior to flowering (panicle initiation) was the most sensitive stage to drought stress, in terms of its effect on seed-set, during reproductive development. A cell viability assay showed that there were significant differences in the loss of cell viability between leaf sample of stay green and non-stay green genotypes when leaf samples are collected in the morning and subjected to high respiratory demand. Therefore the chlorophyll fluorescence assay has potential as a tool for stay green trait screening at early stages of growth in grain sorghum.

Water is considered the most vital resource on earth and its quality is deteriorating. Not all residents living in South Africa‘s rural areas have access to treated drinking water, and use water from rivers, dams, and wells. The quality of these resources is unknown, as well as the effects of the bacteria in the water on human health. The heterotrophic plate count (HPC) method is a globally used test to evaluate microbial water quality. According to South African water quality guidelines, water of good quality may not contain more than a 1 000 coliforming units (CFU)/mℓ. There is mounting evidence that HPC bacteria may be hazardous to humans with compromised, underdeveloped, and weakened immune systems. In this study the pathogenic potential of HPC bacteria was investigated. Samples were collected from boreholes in the North West Province and HPCs were enumerated with a culture-based method. Standard physico-chemical parameters were measured for the water. Different HPC bacteria were isolated and purified and tested for α- or β-haemolysis, as well as the production of extracellular enzymes such as DNase, proteinase, lecithinase, chondroitinase, hyaluronidase and lipase, as these are pathogenic characteristics. The isolates were identified with 16S rRNA gene sequencing. The model for the human intestine, Hutu-80 cells, were exposed to the potentially pathogenic HPC isolates to determine their effects on the viability of the human cells. The isolates were also exposed to different dilutions of simulated gastric fluid (SGF) to evaluate its effect on the viability of bacteria. Antibiotic resistant potential of each isolate was determined by the Kirby-Bauer disk diffusion method. Three borehole samples did not comply with the physico-chemical guidelines. Half of the samples exceeded the microbial water quality guideline and the greatest CFU was 292 350 CFU/mℓ. 27% of the isolate HPC bacteria were α- or β- haemolytic. Subsequent analysis revealed the production of: DNase in 72%, proteinase in 40%, lipase and lecithinase in 29%, hyaluronidase in 25% and least produced was chondroitinase in 25%. The HPC isolates identified included: Alcaligenes faecalis, Aeromonas hydrophila and A. taiwanesis, Bacillus sp., Bacillus thuringiensis, Bacillus subtilis, Bacillus pumilus, Brevibacillus sp., Bacillus cereus and Pseudomonas sp. All the isolates, except Alcaligenes faecalis, were toxic to the human intestinal cells to varying degrees. Seven isolates survived exposure to the most diluted SGF and of these, four isolates also survived the intermediate dilution but, only one survived the highest SGF concentration. Some isolates were resistant to selected antibiotics, but none to neomycin and vancomycin. Amoxillin and oxytetracycline were the least effective of the antibiotics tested. A pathogen score was calculated for each isolate based on the results of this study. Bacillus cereus had the highest pathogen index with declining pathogenicity as follows: Alcaligenes faecalis > B. thuringiensis > Bacillus pumilus > Pseudomonas sp. > Brevibacillus > Aeromonas taiwanesis > Aeromonas hydrophila > Bacillus subtilis > Bacillus sp. The results of this study prove that standard water quality tests such as the physico-chemical and the HPC methods are insufficient to provide protection against the effects of certain pathogenic HPC bacteria. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post-transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species.

The anuran Xenopus tropicalis tadpole is an attractive model animal in toxicological evaluation of suspected thyroid disrupting xenobiotics. Due to its reliance of a functioning hypothalamic-pituitary-thyroid (HPT) axis for normal metamorphosis, effects on the HPT axis produces apical endpoints, which are easy to measure. More sensitive endpoints of mRNA expression and histological evaluation of the thyroid gland itself provide strong indications of in vivo thyroid endocrine disruption. X. tropicalis is traditionally exposed in groups of 20 animals in four replicates for each treatment group. However, exposing tadpoles individually can provide stronger statistics and a reduction of total animal sacrifice. In this study we have developed and optimized an individual exposure system by a method development study. This method was then applied in an exposure experiment of a suspected thyroid endocrine disruptor, propylparaben (PrP). Prometamorphotic (NF stage 51) X. tropicalis tadpoles were distributed in three treatment groups (0.05, 0.5 and 5.0 mg PrP/L water) and maintained semi-statically for 14 days. Afterwards, apical measurements (body weight (BW), total body length (BL), snout to vent length (SVL) and hind limb length (HLL)) and reached developmental stage were assessed. In addition, mRNA expression of HPT axis relevant genes encoding deiodinase 2 (D2, hepatic tissue), deiodinase 3 (D3, hepatic and tail tissue) and transthyretin (Ttr, hepatic tissue) were measured by quantitative reverse transcription PCR (qRT-PCR). The PrP exposure did not affect general growth and development, but it did cause a downregulation of dio3 and ttr. The downregulation of dio3 could possibly be associated with a reduced serum content of thyroid hormone, while ttr might be connected to a previously described xenoestrogenic effect of PrP in vitro and in fish.

Regenerative medicine (RM) represents a promising enabling technology to revolutionize healthcare. This said there are still major gaps between the commercial promise and the reality of the cell therapy sector of regenerative medicine. There is consensus to develop high through-put, automated technologies for the manufacture of RM products. Imaging methods will have the capacity to contribute to this technological gap for cell therapies and are particularly attractive to provide non-destructive monitoring with high spatial and temporal resolution. This work applied an automated, non-invasive phase contrast imaging platform (Cell-IQ) to measure, analyse and ultimately quantify image derived metrics for human embryonic stem cells (hESCs) and haematopoietic stem cells (HSCs) as part of the colony forming unit (CFU) assay. This work has shown through thresholding and machine vision identification technology, imaging has the ability to improve the precision of current evaluation methods for cell culture, providing novel information regarding culture state and show image derived metrics to be predictive of future culture state. Building on this, differentiation through the addition of a growth factor cocktail highlighted how in-process monitoring enables protocol optimisation. After equilibrating the Cell-IQ incubator to a standard incubator, the progress of the CFU assay was monitored and image metrics representative of colony phenotype were analysed. Cell count, distance between cells and cell migration within individual colonies were identified to be informative and provide a degree of colony phenotype separation. Quantitative, novel, image derived metrics were identified that improve reliability through computer automation, cost by removing user verification and time by reducing the assay time from 14 days to 7 days. Non-invasive imaging provides a fantastic opportunity to create bespoke sampling frequencies to achieve desired precision for manufacturing cell therapies, this work has developed and shown improvement and a level of control to current culture process for ESCs and HSCs.

The enzyme spermidine/spermine N1-acetyltransferase (SSAT) is a candidate biomarker for various cancers as its activity in cancerous tissues is significantly increased. An artificial molecule, amantadine, is exclusively acetylated by SSAT to acetylamantadine (AcAm), levels of which in urine can serve as a proxy biomarker for malignancy. Current method of AcAm detection is laborious, time-consuming, and lacks the possibility of transforming to a point-of-care device. In this thesis, two different approaches were applied to detect AcAm in deionized water and in human urine using optical methods. The first one was fluorescence-based indicator displacement assay using cucurbit[7]uril as the receptor molecule. The second was programmed gold nanoparticle disaggregation with cucurbit[7]uril as a molecular linker. / Graduate

Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier. / text

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

<p>ABSTRACT</p><p>Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent.</p>