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Item An investigation of the effects of donor age on some haematological characteristics of the Wistar rat (Rattus Norwegicus)(University of the Western Cape, 1986) Wesso, Iona; Van der Horst, G.Knowledge of haematological 'normdata', of experimental animals, and the biological variables that affect it is essential in order to recognise variations from the normal. In addition, the haemopoietic system may be regarded in principle as good material for studies of the cellular events associated with ageing. These considerations, together with the well documented effects of age on various physiological processes, prompted an investigation into the effects of donor age on several blood parameters. Review of the literature revealed that age-related changes in blood parameters have been reported for several species, but the documentation thereof is incomplete, inconsistent and inconclusive in many respects. Blood samples from male Wistar rats of nine different biological ages, ranging from birth to 96 weeks of age, were analysed for haematological and biochemical parameters. These included the blood cell counts, erythrocytic indices, haemoglobin concentration, haematocrit, erythrocytic 2,3-diphosphoglycerate and adenosine triphosphate levels, and erythrocytic glucose 6-phosphate dehydrogenase and pyruvate kinase activities. Data was obtained which demonstrates that all blood parameters measured underwent significant, although not al~ays regular, age-related changes. These changes were found to be more marked during the first month of life than at any other period. Evidence is also presented to show that the depressed haemoglobin concentration during the early postnatal life may not imply a condition of 'physiologic anaemia' as was previously thought. Since the blood profile exhibits only slight changes from about 24 weeks of age, it does not seem that the haemopoietic system of the old rat deteriorates significantly as to constitute a limiting factor for the animal's life. However, the importance of taking an animal's age into account when blood parameters constitute experimental results is emphasised. The second phase of this study involved a detailed investigation of the effect of the animal's age on erythrocytes in particular. These cells have limited life-spans, and are often used as models in studies of cellular ageing. Special emphasis was therefore placed on comparing the relative effects of host and cellular ageing on the properties of these cells. Erythrocytes from rats between one and 48 weeks of age were separated into two populations by a modification of the conventional density gradient centrifugation technique. The two populations were assumed to differ in mean cell age and were analysed for erythrocytic indices, phosphate ester concentrations and the activities of glucose 6-phosphate dehydrogenase and pyruvate kinase. Evidence is presented to show that ageing rat erythrocytes exhibit a decrease in volume, phosphate ester content and enzyme activities while the cellular haemoglobin concentration increases. Differences in the mean cell age however, does not seem to account for the donor-age-related effects observed in the whole blood parameters. Rather, the significant differences found in the characteristics of similarly aged red cells, between variously aged donors, demonstrate that the biological age of the organism influences the red cells and probably the ageing thereof in vivo. The contribution of the changing status of the erythrocyte's environment of progressively older animals, to alterations which take place in the ageing red cell is discussed.Item The effect of maternal nicotine exposure on rat lung tissue morphology. ' a light and electron microscopic study(University of the Western Cape, 1991) Woolward, Keryn Miles; Maritz, G. S.The infants of women who smoke during pregnancy have a lower birth mass than those born of women who abstain. Animal studies reveal that reduced growth due to maternal nicotine exposure during gestation is accompanied by lung hypoplasia. Biochemical analysis suggests that these lungs contain more cells which implies that lung damage occurs. In this study we examined the in vivo effects of maternal nicotine exposure (lmg/Kg/day), the equivalent of 32 cigarettes per day, on the following parameters of fetal and neonatal Wistar rat lung:(i) the content and distribution of glycogen in fetal and neonatal lung (ii) the status of connective tissue in neonatal lung (iii) the cell composition of the alveoli in neonatal lung. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Transmission electron microscope (TEM) techniques were employed to investigate the characteristics and composition of the alveolus The results show clearly that maternal nicotine exposure elevates pulmonary alveolar macrophage numbers'(PAM's) and lung glycogen levels. The quantity of elastic fibres in 1 day old neonates was significantly reduced but no changes in the quantity of reticulin and collagen fibres was observed. As a result of this change in connective tissue status, emphysema-like lesions and alveolar collapse was evident in the lungs of nicotine-exposed pups. TEM investigations revealed that changes to the composition of alveoli occurred. These included increased numbers of type II pneumocytes with high numbers of lamellar bodies with degenerative changes. Thickening of the blood-air barrier was also observed. The effect of maternal nicotine exposure has been documented in this study. However, it has not been possible to pinpoint the mechanisms involved but explanations have been proposed. Further research is required to elucidate the mechanisms by which nicotine produces these effects. Information thus obtained could help prevent the harmful effects to the fetus and neonate caused by smoking during pregnancy.Item The effect of diet on the mucus histochemistry and adjacent histology of the digestive tract in Vervet monkeys.(University of the Western Cape, 1993) Woodroof, Colin William; van der Horst, G.There is a need for defined models of human nutritional disorders partly because serious misconceptions about models are common amongst researchers. Historically a large variety of species has been used including primates, pigs, rats, lagomorphs. Advantages various small carnivores and and disadvantages are not well known and availability is a major factor. In 1753 John Hunter used pigs to study bone growth in one of the first scientifically controlled nutrition experiments (Kobler 1960). Rats were most likely the first animals to be bred specifically for scientific purposes and there is evidence that they were used in nutrition experiments during the late eighteenth century (Kobler 1960). Experience with carcinogenesis in animals has shown the great diversity of results which may possibly be obtained from different species (Lave et al. 1988). This is pertinent to nutritional research as there is an established link between diet and cancer. The selection of a suitable substitute to attempt to model possible human response to a variety of procedures is dependent upon criteria among which the following are possibly the more important. Availability; this is of great importance in Southern Africa where the cost of importation of exotic species. must be taken into account. Du Plessis (1981) referred to the fact that our indigenous primates were a valuable resource. A second consideration must be the cost the selected animal in a scientifically acceptable environment. Keeping animals of maintaining and ethically for research purposes in an uncontrolled environment could well lead to erroneous conclusions being made. Thirdly the cost of a research program in which animals are used may be increased if there is insufficient knowledge of the model selected. A paucity of knowledge available about an animal may affect the viability of an experiment. The need for precise information regarding the effects of extended term dietary supplementation of experimental animals has been noted by Fincham et. al. (1987) . Additionally the selected animal should preferably have similar dietary requirements to man, and have a life span which will enable extended term investigations.Item The influence of nicotine exposure on the male reproductive system(University of the Western Cape, 1993) Naidu, Thulasimala; van der Horst, GerhardIt is well documented that cigarette smoking and nicotine exposure create widespread physiological disorders in humans and animals. The primary tobacco constituent that is responsible for the toxicological consequences associated with the effects of tobacco smoke is nicotine (Van Lancker 1977). After maternal nicotine exposure, the fetal gonads and lungs are the principle sites of nicotine damage (Szuts et al. 1978, Mosier & Jansons 1972). Whilst the fetal lung has received widespread attention in this regard (Maritz 1988), the testis has never been studied. Therefore, I have chosen to explore the effects of maternal nicotine exposure on the testis of male offspring by evaluating various aspects of the male reproductive tract. It is believed that, in adult male smokers (Rosenberg 1987, Handelsman et al. 1984) and sexually mature animals (Mattison 1982) that are exposed to nicotine, male fertility may be compromised. However, these studies provide conflicting data on single parameters. It was therefore my objective to identify the effect of nicotine exposure on the male reproductive tract and to establish possible sites through which these effects may be mediated in adult male rats. The influence of nicotine was then investigated in male offspring after maternal nicotine treatment (MNT), and in sexually mature adult males after direct adult nicotine treatment (ANT). In the former experiment (MNT), 7 day pregnant rats were exposed to Img nicotine/kg body weight/day. Therefore, these offspring were indirectly exposed to nicotine during fetal development and early neonatal development. After weaning the animals were divided into two groups. One group did not receive further treatment (withdrawn group), whilst the other group was continually treated till adulthood (nicotine group), after which both groups were sampled together with the control. In the latter experiment (ANT), the animals were treated daily for 3 weeks and were sampled as above (MNT animals). The fundamental parameter investigated in both experiments to assess reproductive status was sperm quality (motility and morphology). Thereafter, it was necessary to establish a possible site where the effects of nicotine would occur. Testicular growth, epididymal structure, and plasma testosterone content were measured as probable localities of nicotine's effect. The results signify that maternal nicotine exposure poses a greater threat to the male reproductive system than adult nicotine exposure. In the MNT experiment, progressive sperm motility of the nicotine and withdrawn groups were 1.7% and 3.4% respectively. The proportion of abnormal sperm was 72% in each of the above groups. The lower quality sperm that is evident after nicotine exposure implies that the fertilizing ability of the animals may be impaired during adulthood. The data on testicular growth indicates that nicotine exposure during early development results in slower testicular development until maturity. The epididymal lining of these animals also increased after nicotine exposure, indicating increased cellular activity. However, these results from testicular and epididymal studies are inconclusive and need further work. In the ANT experiment, progressive sperm motility of the nicotine group was 1.2%, whilst the proportion of abnormal sperm was 58%. No other parameter was affected after nicotine exposure to adult animals. From the above data it is evident that nicotine exposed animals were subject to greater nicotine damage after maternal nicotine exposure during early development. Moreover, within the maternal nicotine treated experiment, the withdrawal of nicotine after weaning did not appear to reverse the injurious effects of nicotine that were established during early development. These effects were evident since the nicotine and withdrawn groups showed similar levels of damage in all instances. The most profound effects after adult nicotine exposure and maternal nicotine exposure were on sperm quality. The probable site of sperm impairment appears to be via retarded testicular growth and possibly, structural status of the epididymis after maternal nicotine exposure. The results from adult nicotine exposure however, suggest that sperm cells may be directly affected by nicotine exposure. An epidemiological survey was included to validate the basic conclusions established in animal research when compared to clinical data from human patients. No statistically significant changes were observed in this study between the patient's spermiogram results versus his smoking habits, and, that of his mother. From the level of significance it was concluded that cigarette smoking does not appear to be a cause of impaired fertility in already infertile patients. However, the data does suggest that cigarette smoking may well be a precipitating agent in male infertility. Experimentally, nicotine exposure impairs the male reproductive system to some extent. The effects of which are irreversible after indirect exposure (MNT) during development and may begin with poor testicular development. The effects of adult nicotine exposure implies that nicotine exposure in mature animals (ANT) acts directly on sperm cells to incapacitate them. It is well advised that cigarette smoking should be curbed in pregnant women and in adult males to alleviate contributing effects to male infertility.Item Cloning and sequence analysis of the gene coding for a Leuconostoe Bacteriocin(University of the Western Cape, 1994) Felix, Joseline V.; Hastings, J.W; Smith, A.APrevious studies have shown that Leuconostoc (Lc.) carnosum Tal la produces a bacteriocin that has been designated leucocin B-Tal la [Papathanasopoulos, 1993, M.Sc thesis, University of the Witwatersrandl. Leucocin B-Talla is active against Listeria nnnocytogenes and several lactic acid bacteria. An 8.9 MDa plasmid in Leuconostoc carnosum Tal1a hybridised to a 36-meroligonucleotide probe (JF-1) that is homologous to the amino-terminal sequence of the leucocin A-UAL187 structural gene. A library of Lc. carnosum Talla plasmid DNA was constructed by partial digestion of DNA with ^lar3A and ligation into the BamHl site of pUC1l8. A plasmid (pJF8.1), containing a 4.9 kb insert was identified by Southern blotting and hybridisation to JF-l. A subclone of this plasmid, with a 2.3 kb insert (pJF5.5), was generated by internal deletion of a 2.6 kb Xbal fragment and religation of the plasmid. Sequence analysis of pJF8.1 and pJF5.5 revealed the presence of two open reading frames (ORF). ORFI codes for a protein of 61 amino acid residues. This protein product is proposed to be the prepeptide of a 37 amino acid bacteriocin, leucocin B-Ta11a, by virtue of DNA sequence homology to leucocin A-UALI87 [Hastings et al., 1991. J. Bacteriol 173:749L-7500]. The 24 amino acid residue amino-terminal extension, possibly cleaved during processing of the prepeptide may function as a leader peptide. The aminoterminal extension of leucocin B-Talla differed trom the similar region in Ieucocin A-UAL187 by seven residues. The predicted protein of the ORF2 consists of 113 amino acids and is identical to the amino acid sequence of the cognate ORF of the leucocin A-UAL187 operon. Expression of leucocin B- Talla was attempted in Escheichia coli JMl03 transformed with pJF8.1 and pJF5.5. Results of inhibition studies with various cell fractions of the transformed strains showed that no bacteriocin was produced by these transformants.Item An assessment of the virulence of listeria spp. isolated from raw milk and raw chicken(University of the Western Cape, 1994) Howard, R.L; Hastings, J.W; Gouws, PListeria a f~rn__e pathogen is the etiological agent of the deadly disease listeriosis. Although, the virulence traits of international isolates of Listeria are well studied, nothing is known about the virulence traits of Listeria found in raw milk and raw chicken in the Western Cape, South Africa. Therefore, the aim of this study was to isolate Listeria from raw milk and raw chicken in the Western Cape, South Africa, in order to determine the virulence nature of such Listeria isolatesItem Science text: Facilitating access to physiology through cognition-based reading intervention(University of the Western Cape, 1995) Wesso, Iona; Sinclair, A. J. L.Reading and understanding science text is the principal means by which students at tertiary level access scientific information and attain scientific literacy. However, understanding and learning from science texts require cognitive processing abilities which students mayor may not have. If students fail to understand scientific text, their acquisition of subject knowledge and expertise will be impeded and they will fail to develop into thinking and independent learners, so crucial for academic progress and achievement. A major assumption in this study is thus that in order to increase access to science subjects there is a need to explicitly teach the thinking abilities involved in learning science from text. A review of the literature showed that while reading to learn from scientific text poses special challenges to students faced with this unfamiliar genre, little is known about reading (and thinking) for science learning. A synthesis of current research which describes the neglected interface between science learning, science reading and cognition is given in the literature review of this study. This synthesis highlights, in particular, the parallel developments in research into science learning and reading; the lack of integration of research in these areas; the absence of investigations on science reading located within the cognitive domain; and the absence of research into reading as it affects cognition and cognition as it affects reading in subject-specific areas such as physiology Possibilities for improving students' cognitive performance in reading to learn through intervention were considered from a cognitive perspective. From this perspective, students' observable intellectual performance can be attributed to their underlying knowledge, behaviour, and thought processes. Accordingly, the mental processes involved in comprehending scientific concepts from text and the cognitive processes which the students bring to the learning situation become highly relevant to efforts to improve cognitive skills for learning science Key questions which were identified to serve as a basis for intervention included: a) What cognitive abilities are needed for competent reading comprehension as demanded by physiology text?; b) How adequate is the cognitive repertoire of students in dealing with physiology text? With regard to these questions a catalogue of cognitive functions as formulated by Feuerstein et al (1980) was identified as optimally suited for establishing the cognitive match between reading tasks and students. Micro-analyses of the cognitive demands of students' textbook material and the cognitive make-up of second-year university students revealed a profound mismatch between students and their learning material. Students lacked both comprehension fostering and comprehension monitoring abilities appropriate to the demands of the learning task. The explication of the cognitive requirements which physiology text demands served as a basis for systematically designing instruction whereby appropriate intellectual performance for scientific comprehension from text may be attained Subsequent intervention was based on the explicit teaching of thinking abilities within the context of domain-specific (physiology) knowledge. An instructional framework was developed that integrated cognitive learning theories and instructional prescriptions to achieve an effective learning environment and improve students' cognitive abilities to employ and extend their knowledge. The objective was that the instructional model and resultant instructional methods would ensure that students learn not only the desired kinds of knowledge by conceptual change, but also the thought processes embedded and required by reading scientific material for appropriate conceptual change to take place. Micro-analysis of the cognitive processes intrinsic to understanding physiology text illuminated cognitive demands such as, for example, the ability to: transform linearly presented material into structural patterns which illuminate physiological relationships; analyse conceptually dense text rich in "paradoxical jargon"; activate and retrieve extensive amounts of topic-specific and subject-specific prior knowledge; to visualise events; and contextualise concepts by establishing an application for it. Within the above instructional setting, the study shows that the notion of explicitly teaching the cognitive processes intrinsic to physiology text is possible. By translating the cognitive processes into cognitive strategies such as assessing the situation, planning, processing, organisation, elaboration, monitoring and reflective responses, the heuristic approach effectively served to guide students through various phases of learning from text. Systematic and deliberate methods of thought that would enhance students problem-solving and thinking abilities were taught. One very successful strategy for learning from physiology text was the ability to reorganise the linearly presented information into a different text structure by means of the construction of graphic organisers. The latter allowed students to read systematically, establish relationships between concepts, identify important ideas, summarise passages, readily retrieve information from memory, go beyond the given textual information and very effectively monitor and evaluate their understanding In addition to teaching appropriate cognitive strategies as demanded by physiology text, this programme also facilitated an awareness of expository text conventions, the nature of physiological understanding, the value of active strategic involvement in constructing knowledge and the value of metacognitive awareness. Also, since the intervention was executed within the context of physiology content, the acquisition of content-specific information took place quite readily. This overcame the problem of transfer, so often experienced with "content-free" programmes. In conclusion, this study makes specific recommendations to improve science education. Inparticular, the notion of teaching the appropriate cognitive behaviour and thought processes as demanded by academic tasks such as reading to learn physiology seems to be a particularly fruitful area into which science educational research should develop and be encouraged.Item Adenine nucleotides and selected chemical Parameters as potential markers for fertility assessment(University of the Western Cape, 1997) Burger, D J G; Van der Horst, GDiagnostic andrology with its descriptive approach to the diagnosis of defective sperm for the last 40 years has focused mainly on the macro- and microscopic appearance of the ejaculate. More emphasis has recently been placed on the quantification of biochemical components in semen and sperm. In this regard, substances can be measured more accurately and in some cases as multi-components in the same run. The value of bioenergetics of sperm as a marker for fertility assessment is a controversial topic in the literature. What complicates this issue is the relatively new information on how adenosine triphosphate (ATP), as the major high energy molecule, can be synthesized from other sources which c/as, until recently, unknown. This study attempts to investigate the role of adenine nucleotides as potential markers in semen for fertility assessment using the Tygerberg strict criteria. In this study a reverse phase liquid chromatography (RPLC) technique was developed which measures all the nucleotides in a single run. Because the cycle of energy yielding and energy consuming processes operate simultaneously, it is difficult to assess the energy status of spermatozoa by only measuring the ATP concentrations as suggested in the literature. In this study all the adenine nucleotides and their relative ratios were measured. The results did not show significant differences using the Tygerberg strict criteria of morphology evaluation. This is in line with some workers who indicate that there is no value in measuring ATP levels and correlating this with fertilization. This study furthermore indicates that the breakdown products of ATP and the different adenine nucleotide ratios do not correlate positively with morphology and fertilization in the IVF and GIFT procedures using the Tygerberg strict criteria of morphology. As an application of the technique preliminary experiments indicated that the duration of incubating semen or spermatozoa is important when measuring nucleotides. An unknown product, that relates to motility, is formed during the incubation period of 18 hours (absent after 40 minutes). It was also shown in this preliminary study that the ATP/ADP ratio is the most sensitive biochemical parameter for changes in motility and that the older concept of the adenylate energy charge (AEC) is not a good indicator of the energy status of spermatozoa. The experiments with the energy related enzymes Lactate dehydrogenase and Creatine kinase versus the morphological groups, have been proved not to be predictive. The sperm membrane enzyme y-glutamyl transferase which also correlates negatively with the Tygerberg morphological groups could be more informative if the number of the P Pattern group (<4o/o) were increased. This membrane enzyme could be of more value than previously realized, since it may relate to the lipid peroxidation of sperm which are extremely susceptible to oxidative stress.Item What constitutes morphological normal and abnormal human sperm heads(University of the Western Cape, 1998) Janse van Rensburg, Tholoana 'M'atahleho Leubane; van der Horst, G.The percentage morphologically normal sperm appears to be of predictive value in the in vitro fertilization laboratory. However, the methodology used in this context is technically inaccurate and imprecise (subjective) and needs to be improved. Nevertheless, most laboratories continue to use such techniques. It is therefore not surprising to find that three different sperm morphology classification systems are in use. Some of the published methods include, the World Health Organization system (WHO), the Tygerberg strict criteria (TSC) and the Dusseldorf criteria (DC) to define morphologically normal sperm. Each of these methods use different criteria and different cut-off values to define sperm morphology normality in patients. For WHO it is >30%, for TSC it is >14% and for DC it is >30%. Consequently, there is no objective morphological criteria for defining normal spermatozoa in human semen at present. Such criteria can be established only on the basis of extensive studies that assess the morphometric characteristics of spermatozoa. Therefore, there is a great need to standardize methodology in this context. The second problem lies with the training of technicians. Unfortunately implementation of visual semen analysis often differs between laboratories. Moreover, few laboratories systematically train their technicians by one standard method, and monitor within and between technician variability. Accurate and precise visual semen analysis will only be achieved by implementing a program of international standardization and technician training and proficiency testing. The third problem is that specimen handling and preparation for evaluation of sperm morphology are not standardized and this needs to be done to the highest degree possible. ln this investigation four microscopic techniques were used to study sperm morphology. The purpose of this part of the investigation was to test whether Papanicolaou stained (PAP) sperm smears studied by means of bright field microscopy represents a reliable method to study sperm morphology when compared to more sophisticated microscopic techniques. Consequently Bright field microscopy (PAP staining), Normaski differential interference microscopy (NDIM), Scanning electron microscopy (SEM) and Confocal microscopy of normal and abnormal sperm types were compared. However, a new technique had to be developed for preparing sperm for confocal microscopy. For this purpose both unfixed and fixed sperm were embedded in agarose to avoid motion artifacts during confocal imaging. Both groups of unfixed and fixed sperm were placed in PBS buffer containing O.874mM dihexaoxacarbocyanine iodide (D!OC6(3)) at room temperature. Sperm were pre-loaded for 30 minutes with Tetramethyl rhodamine methylester (TMRM). DIOC6(3) is a lipophilic fluorescent dye, and it has been found that the fluorescent intensity of sperm increased over time. For Laser Scanning Confocal Microscopy 3D sperm morphology was reconstructed from a series of 20 - 30 optical sections taken at 0.2:m intervals through each sperm. ln order to examine 3D morphology a series of projected (rotated) views were reconstructed to allow visualization of a 3D animation set. All forms of abnormal sperm could be clearly identified by all four microscopic techniques. Despite the fact that human sperm structure can be visualized and studied more comprehensively by means of both NDIM and confocal microscopy than with bright field microscopy, the latter technique of PAP stained smears is adequate to identify abnormal sperm morphology on a routine basis in the clinical laboratory. However, confocal microscopy of human sperm reveals that sperm may be scored normal/abnormal on the basis of its orientation. Only slight rotation of a 3D constructed image of a spermatozoon by means of confocal microscopy can change the classification of a sperm from normal to abnormal. ln the second part of the investigation bright field microscopy of PAP stained smears were used to test the reliability/repeatability of scoring among three technicians from three different laboratories using the Tygerberg Strict Criteria. All three technicians scored the same 77 patients. While two technicians scored within a relatively close range, the third technician varied more when TSC was employed. One technician classified 43"/o of the samples in a different TSC category when compared to a second technician. A fourth technician scored the same sperm smears but used the WHO criteria (1992). The scores for TSC and WHO were consequently compared. It was found that a 3-5% TSC range of abnormal sperm corresponded to a 6-30"/" WHO range.Item Listeria in food and water in the Western Cape(University of the Western Cape, 1998) Leonard, Carmen Myra; Brozel, V. S.Listeria are glam positive non-spong rods that were found in diverse environments including water, seafood, dairy products and vegetables. Outbreaks of listeriosis due to the consumption of foods contaminated L.monocytogenes, coupled with the high mortality rate and the nsk posed to immuno compromised individuals have resulted in more focus being placed on Listeria-free products. Although most recorded Listeria outbreaks have been linked to contaminated food, most of these foods have a previous history of soil and / or water contact but little is known regarding its prevalence in soil and water. The aim of the work reported here was to ascertain the prevaluice and diversity of Listeria and bacteria that could be regarded as Listeria in various foods and in natural and irrigation waters in order to develop a reliable and conclusive detection method. Various methods including conventional culturing selective techniques, immunological methods and a variety of polymerase cham reaction methods have been developed for the detection of Listeria. In this study, preminary physiological tests were utilized to detect Listeria from 108 natural and irrigation water samples, 34 dairy samples, 33 raw and processed fish samples and 30 vegetable samples. After these preliminary tests, one-hundred and twelve isolates were found to be gram positive non-sporing rods that could hydrolyse aescuhn. These isolates were further tested using both genotypical (PCR with primers directed at the 165 rRNA gene and the invasive associated protein gene ) and phenotypical (BIOLOG and whole-cell protein analysis). The whole protein profiles divided the various isolates into two main divisions namely, Listeria and ListeriaJike organisms. A diverse selection of Listeria and, Listerio-like strarns, some of which are very distant from currently accepted members of the genus, were isolated. Dalry products (especially soft cheeses), seafoods and water isolates clustered in separate niches within the Listeria division. The majority of vegetables, however, were mainly contaminated by Listeria-like organisms and not true Listeria. The presence of these Listeria in a Iocal river was of some concernsince the local children used the river as a swimming facility. Some isolates from the river, although related to the type strains, were separate from typical Listeria. Thus, the possibility exists that this group could be a new species or subspecies. The development of a l65 rDNA primer set (CLis2 and CLis4) for the detection is reported. All isolates that contained the invasive associated protein (iap) gene resulted in a PCR product of l.5kb and those that contained the 165 rRNA gene resulted in a product of 0.68kb Although these primer sets could detect most Listeria, they could not detect all. The development of more selective media for the preliminary detection of Listeria and the reduction in the number of physiologrcal tests, were recommended as well as the optimisation of the l65 rRNA PCR for the direct detection of Listeria rn food products.Item The identification of regulatory Elements in pseudomonas aeruginosa and p. putida responsive to specific heavy metals.(University of Western Cape, 1999) Africander, Nolan Lindsay; Brozel, Volker SHeavy metals constitute a group of about sixty-five elements having a density greater than five (Gadd and Griffiths, 1978). Some metals, for example lead, copper, and nickel are extremely precious and profitable to man due to its widespread application in industry. As there has been an increase in heavy metal demand, necessary development of elegant and efficient methods for their isolation were also required. These methods, which led to an increase in industrial development, also resulted in an increase in the complexity and variety of heavy metals polluting the environment (Morby,1996Item New Algorithms for EST clustering(University of the Western Cape, 2000) Ptitsyn, Andrey; Hide, WinstonExpressed sequence tag database is a rich and fast growing source of data for gene expression analysis and drug discovery. Clustering of raw EST data is a necessary step for further analysis and one of the most challenging problems of modem computational biology. There are a few systems, designed for this purpose and a few more are currently under development. These systems are reviewed in the "Literature and software review". Different strategies of supervised and unsupervised clustering are discussed, as well as sequence comparison techniques, such as based on alignment or oligonucleotide compositions. Analysis of potential bottlenecks and estimation of computation complexity of EST clustering is done in Chapter 2. This chapter also states the goals for the research and justifies the need for new algorithm that has to be fast, but still sensitive to relatively short (40 bp) regions of local similarity. A new sequence comparison algorithm is developed and described in Chapter 3. This algorithm has a linear computation complexity and sufficient sensitivity to detect short regions of local similarity between nucleotide sequences. The algorithm utilizes an asymmetric approach, when one of the compared sequences is presented in a form of oligonucleotide table, while the second sequence is in standard, linear form. A short window is moved along the linear sequence and all overlapping oligonucleotides of a constant length in the frame are compared for the oligonucleotide table. The result of comparison of two sequences is a single figure, which can be compared to a threshold. For each measure of sequence similarity a probability of false positive and false negative can be estimated. The algorithm was set up and implemented to recognize matching ESTs with overlapping regions of 40bp with 95% identity, which is better than resolution ability of contemporary EST clustering tools This algorithm was used as a sequence comparison engine for two EST clustering programs, described in Chapter 4. These programs implement two different strategies: stringent and loose clustering. Both are tested on small, but realistic benchmark data sets and show the results, similar to one of the best existing clustering programs, 02_cluster, but with a significant advantage in speed and sensitivity to small overlapping regions of ESTs. On three different CPUs the new algorithm run at least two times faster, leaving less singletons and producing bigger clusters. With parallel optimization this algorithm is capable of clustering millions of ESTs on relatively inexpensive computers. The loose clustering variant is a highly portable application, relying on third-party software for cluster assembly. It was built to the same specifications as 02_ cluster and can be immediately included into the STACKPack package for EST clustering. The stringent clustering program produces already assembled clusters and can apprehend alternatively processed variants during the clustering process.Item New algorithms for EST clustering(University of the Western Cape, 2000) Ptitsyn, Andrey; Hide, Winston; Davidson, Sean; Dept. of Microbiology; Faculty of ScienceSummary: Expressed sequence tag database is a rich and fast growing source of data for gene expression analysis and drug discovery. Clustering of raw EST data is a necessary step for further analysis and one of the most challenging problems of modem computational biology. There are a few systems, designed for this purpose and a few more are currently under development. These systems are reviewed in the "Literature and software review". Different strategies of supervised and unsupervised clustering are discussed, as well as sequence comparison techniques, such as based on alignment or oligonucleotide compositions. Analysis of potential bottlenecks and estimation of computation complexity of EST clustering is done in Chapter 2. This chapter also states the goals for the research and justifies the need for new algorithm that has to be fast, but still sensitive to relatively short (40 bp) regions of local similarity. A new sequence comparison algorithm is developed and described in Chapter 3. This algorithm has a linear computation complexity and sufficient sensitivity to detect short regions of local similarity between nucleotide sequences. The algorithm utilizes an asymmetric approach, when one of the compared sequences is presented in a form of oligonucleotide table, while the second sequence is in standard, linear form. A short window is moved along the linear sequence and all overlapping oligonucleotides of a constant length in the frame are compared for the oligonucleotide table. The result of 85 comparison of two sequencesis a single figure, which can be compared to a threshold. For each measure of sequence similarity a probability of false positive and false negative can be estimated. The algorithm was set up and implemented to recognize matching ESTs with overlapping regions of 40bp with 95% identity, which is better than resolution ability of contemporary EST clustering tools. This algorithm was used as a sequence comparison engine for two EST clustering programs, described in Chapter 4. These programs implement two different strategies: stringent and loose clustering. Both are tested on small, but realistic benchmark data sets and show the results, similar to one of the best existing clustering programs, D2_cluster, but with a significant advantage in speed and sensitivity to small overlapping regions of ESTs. On three different CPUs the new algorithm run at least two times faster, leaving less singletons and producing bigger clusters. With parallel optimization this algorithm is capable of clustering millions of ESTs on relatively inexpensive computers. The loose clustering variant is a highly portable application, relying on third-party software for cluster assembly. It was built to the same specifications as D2_cluster and can be immediately included into the ST ACKPack package for EST clustering. The stringent clustering program produces already assembled clusters and can apprehend alternatively processed variants during the clustering process.Item Parameters affecting the production of fumonisin B1 by fusarium verticillioides in culture(University of the Western Cape, 2001) Keyser, Zanephyn; Klaasen, J.AFi1sarium verticillioides is a very important mycotoxin-produeing fungus associated with maize. Fverticillioides produces a group of mycotoxins known as fumonisins under suitable environmental conditions. A series of studies was designed to provide information regarding some of the factors associated with the production offumonisin B1 (FB1) in maize patties and MYRO liquid medium. Our investigation together with previous studies have detailed the important influence of several factors on the production of fumonisins by F verticillioides strains. To understand why these strains are able to produce these toxins, an investigation into the complex interaction that occurs between biotic and abiotic parameters and their impact on fumonisin production was necessary. The results reflect the interacting factors and the intraspecific differences between strains, which may also be present in field conditions. The parameters that were varied under a predetermined set of culture conditions, included initial moisture content of maize patty cultures, temperature, initial pH and the addition of the fumonisin precursors, L-alanine and L-methionineto the cultures. Investigations into the three-way interactions of initial maize patty moisture content (30 ml water to 30g of maize), L-methionine (0.3 %) and temperature (25°C), resulted in the highest yield ofFB1 (5777.26 μgig) produced by MRC 4316. In contrast, MRC 826 was negatively affected, producing lower levels ofFB1 (3492.24 μg/g), compared to MRC 4316 at an initial moisture content (20 ml water to 30 g maize), L-methionine (0.3 %) and 25 °C. An American strain of F verticillioides MRC 7424 (= NRRL 13616), produced the highest levels of FB, (116 μg/ml), while the South African isolates, MRC 4316 and MRC 826, produced lower FB1 levels (93 and 62 μg/ml, respectively) in MYRO liquid medium. In general, FB1 production in maize patty cultures far exceeded levels obtained in liquid shake cultures. It appears that not only the ability of a particular strain of F. verticillioides, but the interaction of a variety of physiological and nutritional factors and the culture medium, are important in the production of FB,. Thus, variation of a single factor such as temperature under field conditions due to seasonal change, may therefore have a major effect on fomonisin production. A chain reaction may occur when changes in moisture, pH, etc. take place, which may influence fumonisin production further. Lyophilisation of fungal cultures proves to be an excellent method to preserve a wide range of fungi over long periods of time. It is, however, necessary to determine the viability of conidia stored in lyophilised vials at 4 ° Con a regular basis. At present, plate count methods remain the most valid technique for the detection of the viability of lyophilised conidia. Membrane-permeant nucleic acid-binding dyes (FUN-I) are viability stains that are relatively new flourescent probes for assessing the viability of metabolically active yeast cells. The purpose of this study was to microscopically determine the viability oflyophilised conidia of Fusarium and A lternaria species, using the yeast, Saccharomyces cerevisiae, as a control. FUN-1 viability stain was compared to two other staining methods, i.e. ethidium bromide (EB) and methylene blue (MB) and the viability of the conidia was compared to colony-forming units (CFU) on solid media as a control. For the purpose of determining or screening for percentage viability in a specific inoculum, results indicate that EB can be used in the case of lyophilised conidia, and MB in the case of freshly harvested conidia. Although FUN-I are recommended as a good way to determine the cell viability of a fungus, it needs relatively complicated procedures and has a time limit in which the stain can be used. The result of this study emphasize that the use of dyes to determine viability of lyophilised conidia require a critical definition of protocols for a specific fungal species, and that a good correlation with CFU needs to be demonstrated. The findings of this study could find useful applications in various studies on living and dead conidial populations. The diverse toxicological effects of fumonisins m animals and plants raised the possibility that fumonisins may also inhibit the growth of filamentous fungi. This study investigated the antifungal activity of FB1 to some h1sariu111 and other fungal species. The sensitivity of these fungi was tested by an agar-diffusion method on PDA plates. FB1 inhibited the myceliaJ growth of five of the nine fungi tested. The FB1-producing Fusarium species isolated from maize, i.e. F verticil/ioides, F glohosum and F proliferatum were resistant to FB1 even though a small inhibition zone at the highest FB1 concentration of 40mM was noted in the case of F. proliferatum. However, amongst two non-producing Fusarium spp. also isolated from maize, one (F subglutinans) was resistant and one (F graminearum) was sensitive. The most sensitive fungi tested were non-producing species not isolated from maize, i.e. A lternaria alternata, Botrytis cinerea and Penicillium expansum. The minimum inhibitory concentration ofFB1 ranged between 0.25-0.SmM for A. alternata, 1-SmM for P. expansum and B. cinerea and 5-1 OmM for F. graminearum, while the other fungi tested showed no sensitivity to FB1. This is the first report on the antifungal activity ofFB1 to filamentous fungi. Another study investigated the effect of FB1 on the germination of freshly harvested conidia of Fusarium and some other fungal species. The FB1 -producing F'usarium species isolated from maize, i. e. F vertici llioides, F. globosum and F. prolifer alum showed a decrease in germ tube length with an increase in FB1 concentrations. This indicated that these fungi can tolerate their own toxic metabolite to a ce11ain extent. However, amongst the two non-fumonisin producing Fi1sarium spp. examined, i.e. F. subglutinans and F. graminearum, isolated from maize, F. subglutinans was induced to genninate faster in the presence ofFB1 but soon developed stunted germ tubes, while F graminearum developed shorter germ tubes compared to the control cultures. The most sensitive fungi tested were species not isolated from maize, i.e. A. alternata, B. cinerea and P. expansum, which did not germinate at higher FB1 concentrations at all. Statistical analyses showed that the inhibiting effect of FB1 was highly significant (P <0.001). The conidial germination bioassay was more sensitive in the detection of the antifungal activity ofFB1 than the petri dish bioassay. The minimum inhibitory concentrations of FB1 for visible mycelial growth were closely comparable to those obtained from conidial germination. Results of these studies provide considerable information on the parameters affecting the production of FB1 and will be of great benefit in further studies focussing on fumonisin prodnction.Item The molecular characterisation of a baculovirus isolated from trichoplusia ni(University of the Western Cape, 2001) Fielding, Burtram Clinton; Davison, S.In South Africa there are more than 106 insects pests which attack a wide variety of crops. The top ten or twenty of these can seriously limit successful production on the farm. Costs involved in controlling these pests are considerable, often higher than the value of the crop itself. Trichoplusia ri (common name: cabbage looper) is a pest that can cause considerable damage to a wide variety of economically important crops. Although Trichoplusia ni has successfully been controlled with synthetic chemical pesticides, awareness about the negative impact of these control measures on the environment has necessitated the development of safer alternatives. Additionally, cabbage looper resistance to the commonly used pesticides has been reported. Since a Trichoplusia ni multiple nuclear polyhedrosis virus has previously been used in the effective control of the pest, the potential of characterizing a South African baculovirus isolate showed great potential. A latent baculovirus infecting a field population of Trichoplusia ni was isolated and characterised. Initial DNA and protein characterization identified it as a novel baculovirus. The aim of this research was to characterise the baculovirus at a molecular level. This could lead to future improvement of the viral insecticidal properties The family Baculoviridae include more than 600 viruses with only nineteen receiving species status (Murphy et ol., 1995). The genome sequencing and mapping of NPVs could prove important in determining the relationships between these viruses. Additionally, it could be useful in understanding the importance of gene arrangement and the essential domains of genes' This could provide insight into the crs- and trans-regulation among genes (Jin et al., 1997). The determination of gene order and arrangement of the novel baculovirus isolated from a field population of Trichoplusia ni was presented in Chapter 2. Data were used to construct a partial functional map of the TniSNPV genome. Subsequently, the order and homology of genes identified were used as a phylogenetic marker, identi$,ing TniSNpv as a putative member of the Group II NpVs.Item Bioinformatics and polymerase chain reaction: Tools to determine the host specificity of Salmonella typhi murium(University of the Western Cape, 2002) Davids, R; Gouws, P.AThe nucleotide primer pair, Redl and Red2, was designed from the nucleotide sequence obtained from the NCBI database (Fig. 2.1). This nucleotide sequence encodes for the Salmonella typhimurium invasion gene D protein (sigD) and invasion gene E protein (sigE) genes. The Sa/monella and the non-Salmonella serovars used were subjected to PCR conditions at various annealing temperatures (T.) (Tables 2.4, 2.5,2.6). This was performed in order to optimize the PCR. Plasmid DNA PCR amplicons (350bp) detected S.braenderup and S.gallinarum at 53oC (Fig. 2.7) and at 56oC, S.braenderup at 54oC and S.gatlinarum at 55oC. PCR of the plasmid DNA did however not detect Salmonella typhimurium, the serotype it was designed to detecItem Thermal stability and defect structure of hot-wire deposited amorphous silicon(UWC, 2004) Arendse, CJ; Knoesen, DHydrogenated amorphous silicon (a-Si:H) thin films are presently used in several large-area thin-film applications. However, one major concern of a-Si:H is the fact that the stability of the material degrades when it is exposed to prolonged sunlight illumination. This effect, referred to as the Staebler-Wronski effect (SWE), is however reduced when using hot-wire (HW) deposited a-Si:H material with a low hydrogen concentration and favorable microstructure. In this thesis we report on the thermal stability of HW-deposited a-Si:H thin films, with different H-concentrations and bonding configurations, when exposed to elevated temperatures in excess of 100 "C.Item Differentiation between Quinolone Resistant and Sensitive Isolates of Campylobacter jejuni by a Multiplex PCR Assay(University of the Western Cape, 2006) Ebrahim, Nazneen; Gouws, Pieter A; Dept. of Microbiology; Faculty of ScienceItem Three-dimensional structure of a type III glutamine synthetase by single particle reconstruction(University of the Western Cape, 2007) Van Rooyen, Jason Macrae; Abratt, Val; Sewell, TrevorThis study represents the first structural investigation of any type III glutamine synthetase (GS). The GS, GlnA, from the medically important opportunistic human pathogen Bacteroides fragilis was studied with a view to better understanding its structure/functioning in relation to the extensively characterised GSIs. GSIIIs are the most recently discovered family of GSs and are the most phylogenitically distant GSs from the GSIs. Images (160) of negatively stained rGlnA, expressed in E.coli YMC11 (glnA-), were recorded at 50K magnification using a Leo 912 operating at 120kV with energy filtering coupled to a 4 megapixel CCD camera. An angular refinement based reconstruction strategy was adopted using SPIDER. A reconstruction based on an ab initio starting model, derived by a common-lines based simultaneous minimization of rotationally invariant K-mean clustered class averages, converged to the same structure as a reconstruction based on a GSI starting model to a resolution of 2.1nm as determined by Fourier shell correlation). In contrast to preliminary EM observations, which identified GlnA as a hexamer, this work has revealed a dodecameric structure, with subunits (82.8KDa) arranged in two opposing hexagonal rings with distinct handedness. This is similar to the quaternary structure of GSIs and GlnTs except that the complex is 50% longer and the two rings are not symmetrically related. They differ not only in diameter (16.5 or 15.0nm) but also the degree of separation of subunits and as such the particle possesses only C6 and not D6 symmetry. The finding that particles lie in a preferred orientation, with the larger ring in contact with the carbon support, accounted for this asymmetry, through partial staining. Hexameric views, with similar overall arrangement but larger size in comparison to GSI, were also observed. However, it was uncertain whether these were true hexamers resulting from dissociation of the dodecamers or were a consequence of partial staining. Homology modelling was also undertaken in an attempt to predict the structure of GlnA based on GSI, with a view to interpreting the low resolution EM structure. Due to the failures of state of the art algorithms in detecting the distant homologies between GS families, manual profile-based alignment strategies, incorporating structural information, were employed. Through the first full length alignments of GS sequences from all four families, conservation of all active site residues, core active site αβ barrel fold motifs, and additional previously unreported regions was demonstrated. Docking of these homology models into the 3D structure confirmed the presence of the αβ barrel fold predicted by the bioinformatic analysis of the sequences alone, thus, identifying the indentations between subunits in the volume as putative active sites. In addition to providing unequivocal proof that GlnA is a GS and confirming the presence of putative αβ barrel active site folds, this work has made steps towards understanding the regulation of this enzyme. It is hypothesised that GlnA occurs as both active hexamer and an inactive dodecamer, the interconversion of which, is thought to represent a means of reversible post-translational regulation.Item Purification and characterisation of plasmodium falciparum Hypoxanthine phosphoribosyltransferase(University of the Western Cape, 2007) Murungi, Edwin Kimathi; McIntosh, David B.; Sewell, Trevor; Faculty of ScienceMalaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates.