Isolation, expression and purification of the hydantoin hydrolysing enzymes of agrobacterium tumefaciens
- Authors: Clark, Sally-Ann
- Date: 2003
- Subjects: Agrobacterium tumefaciens , Amino acids Hydantoin Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4140 , http://hdl.handle.net/10962/d1016233
- Description: The production of enantiomerically pure amino acids is of industrial importance as they are used in the synthesis of a number of pharmaceuticals, insecticides and herbicides and biologically active peptides and hormones. A number of microorganisms have been identified which possess hydantoin hydrolysing enzymes that stereoselectively convert racemic hydantoins into anantiomerically pure amino acids. Consequently these microorganisms and their enzymes are sought after as biocatalysts for the production of amino acids. The isolation of novel hydantoin hydrolising enzymes with unique or improved biocatalytic characteristics is of importance for the development of potential biocatalysts to be used in the production of enantiomerically pure amino acids. The genes encoding an N-carbamoyl-amino acid amidohydrolase, an enzyme involved in the hydrolysis of hydantoin, was isolated by screening a genomic DNA library of Agrobacterium tumefacience RU-AE01. Nucleotide sequence analysis of the region upstream of this gene revealed a fragment of a gene encoding the hydantoinase enzyme. I this study, a DNA probe consisting of the gene encoding the N-carbamoyl amino acid amidohydrolase, on a large enough fragment of the genomic DNA library which would allow for the simultaneous isolation the hydantoinase gene located upstream. Recombinant expression of the genes encoding hydantoin hydrolysing enzymes has been used to facilitate the production and purification of these enzymes for their use as biocatalysts. Two genes (ncaR1 and ncaR2) encoding different N-carbamoyl-amino acid amidohydrolases with distinct nucleotide and deduced amino acid sequences were isolated from the genome of A, tumefaciens RU-OR. In this study, the heterologous expression of ncaR1 and ncaR2 was explored. Investigation into the optimisation of the heterologous expression of ncaR1 showed that reducing the growth temperature of the recombinant E. coli producing NcaR1 resulted in a two-fold increase in N-carbamoyl-amino acid amidohydrolase activity and solubility. Furthermore, NcaR1 was produced with a C-terminal 6xHis tag, but NcaR1-6xHis did not possess N-carbamoyl amino acid amidohydrolase activity. Furthermore, purification of NcaR-6xHis under native conditions using affinity chromatography performed, and used for the production of antibodies.
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- Date Issued: 2003
Mesozooplankton community structure and grazing impact in the polar frontal zone of the Southern Ocean
- Authors: Bernard, Kim Sarah
- Date: 2003
- Subjects: Zooplankton -- Antarctic Ocean , Copepoda -- Feeding and feeds
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5731 , http://hdl.handle.net/10962/d1005417 , Zooplankton -- Antarctic Ocean , Copepoda -- Feeding and feeds
- Description: Mesozooplankton community structure and grazing impact in the Polar Frontal Zone (PFZ) of the Southern Ocean were investigated during two cruises of the South African National Antarctic Programme (SANAP), the Marion Offshore Ecosystem Variability Study I & II (MOEVS). During the first cruise (MOEVS I), a meso-scale oceanographic grid survey was conducted in the upstream region of the Prince Edward Islands (PEI) in austral autumn (April) 2001. Mesozooplankton samples, collected using a Bongo net (fitted with 200 and 300µm mesh nets) at depths between 200 and 300 m, were separated into three size fractions: 200-500 µm; 500-1000 µm; 1000-2000 µm by reverse filtration. Total surface (depth <5 m) chlorophyll-a (chl-a) concentration (measured fluorometrically) during the study ranged between 0.11 and 0.34 µg 1^(-1) and was always dominated by picophytoplankton (<2.0 µm). Total mesozooplankton abundance and biomass during the survey ranged between 49 and 1512 ind. m^(-3) and between 0.7 and 25 mg Dwt. m^(-3), respectively. Throughout the survey, the 200-500 µm class numerically dominated the mesozooplankton community, comprising an average of ~ 69% (SD = ± 12.3%). The dominant species in the 200-500 µm size fraction were the copepods Oithona similis, Calanus simillimus and Metridia lucens and the pteropod, Limacina retroversa. However, in terms of biomass, the 1000-2000 µm group was predominant, with dry weight values constituting an average of ~ 66% (SD = ± 10.2%). Biomass was dominated by carnivorous zooplankton, particularly the euphausiids, Euphausia vallentini and Thysanoessa vicina and the chaetognaths, Sagitta gazellae and Eukrohnia hamata. Three distinct groupings of stations were identified by multivariate analysis. The different station groupings identified reflect changes in the relative contributions of the rather than different species assemblages. During the second cruise (MOEVS II), conducted in April 2002 (austral autumn), mesozooplankton community structure and grazing impact were investigated at 13 stations in the west Indian sector of the PFZ. Total integrated chl-a biomass ranged between 11.17 and 28.34 mg chl-a m^(-2) and was always dominated by nano- and picophytoplankton (<20 µm). Throughout the study, small copepods, mainly Oithona similis and Ctenocalanus vanus, numerically dominated the mesozooplankton community comprising up to 85% (range 30 to 85%) of the total abundance. Grazing activity of the four most abundant copepods (O. similis, C. vanus, Calanus simillimus and Clausocalanus spp.), which comprised up to 93% of total mesozooplankton abundance, was investigated using the gut fluorescent technique. Results of gut fluorescence analyses indicated that C. simillimus, Clausocalanus spp. and Ctenocalanus vanus exhibited diel variability in gut pigments, with maximum values at various stages of the night. In contrast, O. similis did not demonstrate diel variation in gut pigment contents. Ingestion rates of the four copepods ranged from 23.23 to 1462.02 ng (pigm.) ind^(-1) day^(-1), depending on the species. The combined grazing impact of the four copepods, ranged between 1 and 36% of the phytoplankton standing stock per day, with the highest daily impact (~ 35.86%) occurring at stations in the vicinity of the Antarctic Polar Front. Among the copepods, O. similis and C. vanus were generally the most important consumers of phytoplankton biomass; together they were responsible for up to 89% (range 15 to 89%) of the total daily grazing impact. Carbon specific ingestion rates of the copepods varied between 42 and 320% body carbon per day, depending on the species. The study highlights the importance of small copepods in terms of both their significant contribution to total mesozooplankton numbers and their grazing impact on the phytoplankton standing stocks in the PFZ during austral autumn.
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- Date Issued: 2003
Morphometrics and reproduction of Terebrasabella heterouncinata (Polychaeta:Sabellidae), infesting abalone (Haliotis midae) from different culture environments
- Authors: Gray, Michael
- Date: 2003
- Subjects: Polychaeta , Sabellidae , Abalones -- Physiology , Abalones -- Nutrition , Abalone culture
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5384 , http://hdl.handle.net/10962/d1018231
- Description: In the late 1980's abalone culturalists noticed reduced growth rate and shell deformities in some abalone stocks. These problems were the result of infestations by a shell boring polychaete, Terebrasabella heterouncinata. Under intensive abalone culture conditions the level of infestation can reach epidemic proportions and there are often severe consequences for the host abalone. Heavy sabellid infestation levels have placed the economic viability of several South African farms under threat. This study formed part of an ongoing project that is aimed at investigating the basic biology of Terebrasabella heterouncinata. The majority of abalone farmers in South Africa feed their abalone either naturally occurring kelp (Ecklonia maxima) or the formulated abalone feed, Abfeed. Farmers have suggested that the use of Abfeed is associated with higher sabellid infestation levels and changing the abalone diet from Abfeed to kelp helps reduce sabellid infestation. Speculation has arisen indicating that older, slower growing abalone are more susceptible to sabellid infestation. The effect of host abalone diet history and their growth on sabellid settlement success, morphometries and reproduction was quantified. To better understand the plasticity of the expression of life history traits the variability of morphometric and reproductive characteristics was compared between different farm environments. And more...
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- Date Issued: 2003
Novel approaches to the monitoring of computer networks
- Authors: Halse, G A
- Date: 2003
- Subjects: Computer networks , Computer networks -- Management , Computer networks -- South Africa -- Grahamstown , Rhodes University -- Information Technology Division
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4645 , http://hdl.handle.net/10962/d1006601
- Description: Traditional network monitoring techniques suffer from a number of limitations. They are usually designed to solve the most general case, and as a result often fall short of expectation. This project sets out to provide the network administrator with a set of alternative tools to solve specific, but common, problems. It uses the network at Rhodes University as a case study and addresses a number of issues that arise on this network. Four problematic areas are identified within this network: the automatic determination of network topology and layout, the tracking of network growth, the determination of the physical and logical locations of hosts on the network, and the need for intelligent fault reporting systems. These areas are chosen because other network monitoring techniques have failed to adequately address these problems, and because they present problems that are common across a large number of networks. Each area is examined separately and a solution is sought for each of the problems identified. As a result, a set of tools is developed to solve these problems using a number of novel network monitoring techniques. These tools are designed to be as portable as possible so as not to limit their use to the case study network. Their use within Rhodes, as well as their applicability to other situations is discussed. In all cases, any limitations and shortfalls in the approaches that were employed are examined.
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- Date Issued: 2003
Over-expression, purification and biochemical characterisation of trypanosomal heat shock protein
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
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- Date Issued: 2003
Pharmaceutical analysis and aspects of the quality control of St. John's Wort products
- Authors: Wild, Tracy Joy
- Date: 2003
- Subjects: Hypericum perforatum , Hypericum perforatum -- Analysis , Hypericum perforatum -- Therapeutic use , Hypericum perforatum -- Physiological effect , Flavonoids -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3804 , http://hdl.handle.net/10962/d1003282 , Hypericum perforatum , Hypericum perforatum -- Analysis , Hypericum perforatum -- Therapeutic use , Hypericum perforatum -- Physiological effect , Flavonoids -- Analysis
- Description: Most complementary medicines contain a multitude of chemical components, some of which are claimed to contribute to the biological activity of such products. Use of complementary medicines for preventative and therapeutic purposes is increasing rapidly worldwide. Unfortunately, although control of these products is essential to ensure quality, safety, and efficacy, the quality control of most herbal preparations is currently poor to non-existent, with little or no safety and efficacy data required to support the marketing and use of these products. The objective of this study was therefore to develop suitable analytical methods to qualitatively and quantitatively analyse the relevant components (rutin, isoquercitrin, hyperoside, quercitrin, quercetin, kaempferol, hypericin, pseudohypericin and hyperforin) in St John's Wort dosage forms for quality control purposes. A gradient HPLC method using a Luna 5·mC₁₈(2) 150 x 2.00mm internal diameter (i.d.) column and UV detection, was developed for the separation of six of the relevant flavonoid compounds in St John's Wort, namely rutin, isoquercitrin, hyperoside, quercitrin, quercetin and kaempferol. The development process involved a systematic investigation of gradient conditions, flow rate, and temperature. This method was subsequently applied to assay selected commercially available St John's Wort products. This system provided the necessary accuracy, precision and reproducibility and was associated with several advantages when compared to using standard bore (4.60 mm i.d.) HPLC columns. The method developed is currently the only known method that separates all six relevant flavonoids in a reasonable run time (less than 20 minutes). It is also one of the few methods that has sufficient separation between rutin, isoquercitrin and hyperoside. A qualitative method for the fingerprinting of flavonoid components was also developed, using capillary electrophoresis (CE). CE is a rapidly growing powerful analytical technique for the separation of charged compounds. Micellar electrokinetic chromatography (MEKC) is a very powerful electrophoretic technique that is capable of selectively resolving both neutral and ionic solutes in a single run. A MEKC method suitable for the separation and determination of various flavonoid constituents used as marker compounds in Hypericum perforatum was developed. Investigations into the effect of pH, ionic strength, applied voltage and capillary dimensions on separation were performed. The optimised method was then applied to qualitatively analyse various St John's Wort products on the market. This method was found to be advantageous in that it was simple, cost-effective, required minimal sample preparation and utilised very small quantities of sample. Due to the vast differences in chemical properties between the various marker and active components in St John's Wort, it was necessary to develop separate analytical methods for the flavonoids and for the other three relevant compounds (hypericin, pseudohypericin and hyperforin). An isocratic HPLC method using a Luna 5·mC₁₈(2) 150 x 2.00mm (i.d.) column and UV detection was developed for the separation of hypericin, pseudohypericin and hyperforin. The development process involved a systematic investigation of buffer molarity, mobile phase composition, pH, flow rate, and temperature. This method was subsequently applied to assay selected commercially available St John's Wort products on the South African market. This system also provided the necessary accuracy, precision and reproducibility, as well as the advantages associated with the use of a narrow bore column as opposed to the use of the more commonly used wider bore columns. This method was validated and used to quantitate these three compounds in various commercial St John's Wort products. By applying this method to liquid chromatography – tandem mass spectrometry (LC-MS-MS), qualitative analyses of the same products was performed to obtain confirmation of the quantitative HPLC results. Mass spectrometry is a powerful detection tool that is more selective and specific than many detection systems used with HPLC. Natural medicines usually constitute a multitude of constituents with much potential interference. In this regard LC-MS-MS is a powerful tool, with its ability to unequivocally identify target analytes regardless of the presence of interferences or complex matrices. ESI-MS-MS was used for the qualitative analysis of the content of the naphthodianthrones and hyperforin in the respective tablet products assayed with HPLC. LC-MS-MS analyses were performed in order to identify the constituents and to verify the specificity of the HPLC method. High inter-product and inter-batch variability was observed for all nine compounds assayed. These quantitative results were confirmed with the respective qualitative analyses. This study confirms the need for strict quality control of herbal medicinal products commercially available to consumers.
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- Date Issued: 2003
Physiological, perceptual and performance responses during cricket activity
- Authors: King, Gregory Allen
- Date: 2003
- Subjects: Cricket , Sports -- Psychological aspects , Sports -- Physiological aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5131 , http://hdl.handle.net/10962/d1005210 , Cricket , Sports -- Psychological aspects , Sports -- Physiological aspects
- Description: The present study sought to determine the influence of environmental conditions and protective clothing on physiological, perceptual and performance responses during batting activity. The investigation examined mean skin temperature, average heart rate, estimated sweat rate, rating of perceived exertion, thermal sensation rating, average sprint time and pre-post choice reaction time. Twenty-five cricketers (18-22 yr, 73.1 ± 9.6 kg, 1768 ± 75 mm, 12.6 ± 3.1% body fat, 1.89 ± 0.16 m2) performed a work-bout consisting of a seven-Over batting period, during which time they faced deliveries from a bowling machine and performed two shuttle runs every third ball to total four sprints per Over. Trials were carried out under High-stress (23.8 ± 2.2 °C) and Low-stress (13.3 ± 1.9 °C) environmental conditions (WBGT). Within each environmental condition subjects performed the test wearing full protective batting gear and no protective gear. Thus, four specific conditions were examined; high full-gear (HFG), high no-gear (HNG), low full-gear (LFG) and low no-gear (LNG). Two-way ANOVAs were calculated to determine whether there were differences between environmental conditions and whether differences existed between the clothing conditions. One-way ANOVAs were utilised to compute differences between the four specific conditions combining clothing and environment. High environmental stress and wearing protective clothing caused batsmen to experience significant physiological strain. The environment was the greatest stressor, with the protective gear exacerbating these effects. However, when padding covered skin areas directly, this was the primary skin temperature stressor, particularly later in the activity. For skin temperature and heart rate, the strain was the most pronounced at the end of the trials. Perceptual responses indicated that the protective gear had no influence on effort sense, thermal sensation or thermal comfort. However, environmental conditions had an effect, and High-stress conditions resulted in significantly higher perceptions of effort, elevated sensations of heat and greater thermal discomfort. Effort was perceived to be greatest towards the end of the trial. There were mixed findings for performance factors. In general sprint performance was not hindered by environmental stress, but protective clothing caused a reduction in several sprint times. Choice reaction times were for the most part unaffected by either environment or clothing and few differences were observed between pre and post times. It is contended that intense short duration batting activity, likely encountered during one-day participation, imposes a stress on batsmen. The stress is greater when conditions are warmer and protective padding is worn, although it is not sufficient to impede choice reaction time. However, protective gear did have a deleterious effect on sprint performance.
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- Date Issued: 2003
Progress towards the development and implementation of an unambiguous copper wire fingerprinting system
- Authors: Poole, Martin
- Date: 2003
- Subjects: Electroplating , Copper , Telecommunication , Theft -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5484 , http://hdl.handle.net/10962/d1005270 , Electroplating , Copper , Telecommunication , Theft -- Prevention
- Description: The Telecommunications industry in Southern Africa is faced with the problem of theft of the signal carrying copper wire, both from the ground and from telephone poles. In many cases, if the offenders are caught, the prosecuting party has no way of proving that the wire is the property of any one Telecommunication company, as any inked markings on the insulating sheaths have been burned off along with the insulation and protective coatings themselves. Through this work we * describe the problem, * specify the necessary and preferred technical properties of a viable solution, * report the preliminary investigations into the devising of an unambiguous "fingerprinting" of the 0.5 mm wires, including some of those solutions that, upon investigation, appear non-viable, * describe the development and implementation of an electrochemical marker with detection mechanism which has shown in proof-of-principle to work, * outline the road-map of necessary future work.
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- Date Issued: 2003
Spatial and temporal occurrence of forensically important South African blowflies (Diptera: Calliphorida)
- Authors: Williams, Kirstin Alexa
- Date: 2003
- Subjects: Blowflies -- South Africa , Forensic entomology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5617 , http://hdl.handle.net/10962/d1003217 , Blowflies -- South Africa , Forensic entomology
- Description: Forensic entomology is an emergjng field in South Africa. Little is known about South African blowflies and factors that affect their use in a forensic context. This work provides a review and synthesis of previous work in South Africa and supplements some of the background and basic knowledge required for forensic entomology in South Africa. The seasonal occurrence of eight forensicaIIy important blowfly species was quantified by fortnightly trapping in Grahamstown, South Africa. The spatial distribution of each species was related to seasonal occurrence and habitat preference. Seasonal distributions of blowflies in carcasses in South Africa were obtained from the literature and compared to the seasonal trapping. By mapping South African locality records of forensicaIIy important blowflies and analyzing these records in a modified Principal Components Analysis of climatic data, the potential geographic distributions of each fly species was modeIIed. Most species were widespread, but Calliphora croceipalpis, Jaennicke, 1867, was found in cold places. This information is important for determining where certain species are likely to occur in forensic investigations. Nocturnal oviposition was examined in both field and laboratory experiments. Lucilia species could oviposit nocturnaIIy in the field, while Lucilia species, Chrysomya chloropyga, (Weidemann, 1818) and C. putoria (Weidemann, 1830) could oviposit nocturnaIIy in the laboratory. These findings are important factors in affecting the precision of estimates of a post mortem interval (PM!) by up to 12 hours. The thermophysiological ranges of four species of adult blowflies were determined by measuring onset temperatures of four significant behaviours: onset of neural activity; onset of coordinated movement; shade-seeking and death. There was a sexual size dimorphism in Lucilia species, Chrysomya chloropyga and Calliphora croceipalpis with females being larger than males. Chrysomya megacephala (Fabricius, 1794) had an unexpectedly high death threshold, while Calliphora croceipalpis had the lowest death threshold of the flies tested. These points were related to the seasonal and geographic occurrence of each species, to nocturnal activity and placed in a forensic context.
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- Date Issued: 2003
Symplasmic pathway in phloem loading and unloading in source and sink leaves of Zea mays L. as evidenced under normal and elevated CO₂ conditions
- Authors: Nogemane, Noluyolo
- Date: 2003
- Subjects: Phloem , Plant translocation , Plant cells and tissues , Corn -- Metabolsim
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4254 , http://hdl.handle.net/10962/d1007813
- Description: Zea mays plants kept at ambient (ca 375ppm) and elevated CO₂ (ca 650 to 700ppm) were used to examine the possibility of a symplasmic loading, unloading and transport pathway in dark-adapted and illuminated (200μmolm⁻²sec⁻¹ ) sink and source leaves. 5,6-carboxyfluorescein diacetate was introduced into the mesophyll cells and symplasmic transfer observed 3h after application. In sink and source leaves exposed to ambient CO₂ and illuminated at 200 molm-2sec-1, the fluorescence front was observed approximately 3cm from the point of application, while in dark-adapted plants, the fluorescence front was observed approximately 1cm from the point of application. Under elevated CO₂ conditions the fluorescence front in illuminated plants appeared to transport faster moving approximately 5cm from the point of application, and in dark-adapted plants, only 3cm from the point of application. Based on the increase in 5,6-CF accumulation under elevated CO₂ conditions, the present study suggests that there was an increase in capacity for assimilate loading and transport under elevated CO₂ conditions. In source leaves, 5,6-CFDA was taken up into the mesophyll cells, loaded symplasmically and transported basipetally. In sink leaves 5,6- CFDA was taken up from basal mesophyll and after symplasmic loading, was transported acropetally where it was offloaded into the younger immature sink region. Transport in the sieve tubes was confirmed by using aniline blue, which was applied 3h after 5,6-CF transport. Aniline blue coupled with 5,6-CF transport studies showed that the sieve tubes of both cross and longitudinal veins are involved in symplasmic unloading, loading and transport processes in sink and source leaves. Apoplasmic uptake of 5,6-CFDA by cut leaves showed that after apoplasmic transport via the transpiration stream, 5,6-CFDA was offioaded to the xylem parenchyma where it was metabolically cleaved , releasing fluorescent 5,6-CF into the xylem parenchyma. Transverse sections cut after 3h of uptake were observed after 120 and 180 min suggesting that a retrieval of solutes occurs from the xylem to the xylem parenchyma, bundle sheath, phloem parenchyma and to the th in-walled sieve tubes. It was not possible to determine if the thick-walled sieve tubes were involved or if they took up 5,6-CF. Given the available data on loading and offioading of assimilates in sink and source leaves respectively, this study demonstrated that a slow symplasmic pathway exists from the mesophyll to the phloem, and that offloading from the phloem in sink leaves can occur via a symplasmic route.
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- Date Issued: 2003
Synthesis of zinc phthalocyanine derivatives for possible use in photodynamic therapy
- Authors: Matlaba, Pulane Maseleka
- Date: 2003
- Subjects: Photochemotherapy , Electrochemistry , Phthalocyanines , Zinc
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4374 , http://hdl.handle.net/10962/d1005039 , Photochemotherapy , Electrochemistry , Phthalocyanines , Zinc
- Description: The synthesis of symmetrically and unsymmetrically substituted zinc phthalocyanines (ZnPc) derivatives is done according to reported procedures. The unsymmetrical ZnPc derivatives are synthesized by ring expansion of sub-phthalocyanine complexes. Ring substitution is effected with tert-butyl phenol, naphthol, and hydroxybenzoic acid. Comparison of the redox potentials for the complexes substituted with varying numbers of tert-butyl phenol: 1, 2, 3, 6 and 8 show that the complex with the highest number of substituents are more difficult to oxidize and easier to reduce. Water soluble sulphonated ZnPc (ZnPcSn) was prepared. The possibility of using axial ligation to increase the solubility and the photochemical activity of sulphotnated ZnPc in aqueous solutions was investigated. Pyridine, aminopyridyl and bipyridyl were used as axial ligands. When bipyridyl was used as the axial ligand, solubility of the ZnPcSn increased, shown by the increase in the Q-band of the monomer species in solution and the singlet oxygen quantum yields was relatively higher than that of the unligated ZnPcSn. The singlet oxygen quantum yields by the various complexes in DMF using diphenylisobenzofuran as a chemical quencher for organic solvent were determined. Singlet oxygen quantum yields for the unsymmetrically ring substituted complexes range from 0.22 to 0.68. Photobleaching quantum yields are in the order of 10-5, which means that the complexes are relatively photostable.
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- Date Issued: 2003
The bioaccumulation of platinum (IV) from aqueous solution using sulphate reducing bacteria: role of a hydrogenase enzyme
- Authors: Rashamuse, Konanani Justice
- Date: 2003
- Subjects: Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4003 , http://hdl.handle.net/10962/d1004063 , Sulfur bacteria , Bioremediation , Enzymes -- Metabolism , Platinum , Platinum compounds , Reduction (Chemistry) , Hydrogenation
- Description: The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.
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- Date Issued: 2003
The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules
- Authors: Mosisili, Kekeletso Mpho Thakane
- Date: 2003
- Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
- Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
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- Date Issued: 2003
The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
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- Date Issued: 2003
The hydrolysis of primary sewage sludge under biosulphidogenic conditions
- Authors: Molwantwa, Jennifer Balatedi
- Date: 2003
- Subjects: Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3961 , http://hdl.handle.net/10962/d1004020 , Sewage sludge , Hydrolysis , Sewage -- Purification -- Activated sludge process
- Description: The potential for using readily available and cost-effective complex carbon sources such as primary sewage sludge for a range of environmental remediation processes, including biological sulphate reduction, biological nutrient removal and the bioremediation of acid mine drainage, has been constrained by the slow rate of solubilization and low yield of soluble products, which drive the above mentioned processes. Previous work conducted by the Environmental Biotechnology Group at Rhodes University indicated that the degradation of primary sewage sludge was enhanced under sulphate reducing conditions. This was proven in both laboratory and pilot-scale (Reciprocating Sludge Bed Reactor) systems, where the particulate matter accumulated in the sludge bed and the molecules in smaller flocs were rapidly solubilized. The current study was aimed at investigating in more detail the factors that govern the enhanced hydrolysis under sulphate reducing conditions, and to develop a descriptive model to explain the underlying mechanism involved. The solubilization of primary sewage sludge under sulphate reducing conditions was conducted in controlled flask studies and previously reported findings of enhanced hydrolysis were confirmed. The maximum percentage solubilization obtained in this study was 31% and 63% for the methanogenic and sulphidogenic systems respectively, and this was achieved over a period of 10 days. A rate of reducing sugar production and complex molecule breakdown of 51 mg. L⁻¹.hr⁻¹ and 167 mg.L⁻¹.hr⁻¹ was observed for the methanogenic and sulphidogenic systems respectively. The flask studies revealed that during hydrolysis of primary sewage sludge under sulphidogenic conditions there was enhanced production of soluble products, specifically carbohydrates (reducing sugars) and volatile fatty acids, compared to methanogenic conditions. The rate at which these products were utilized was also found to be more rapid under sulphidogenic as compared to methanogenic conditions. A study of the distribution of volatile fatty acids indicated that acetate was utilized preferentially in the methanogenic system, and that propionate, butyrate and valerate accumulated with time. The converse was found to occur in the sulphidogenic system. The descriptive model developed from the results of this study was based on the fact that a consortium of bacteria, composed of hydrolytic, acidogenic and acetogenic species, carries out the solubilization of complex carbon sources. Furthermore, it is essential that equilibrium between product formation and utilization is maintained, and that accumulation of soluble end products impacts negatively on the rate of the hydrolysis step. It is therefore proposed that the relatively poor utilization of VFA and reducing sugars in the methanogenic system activates a negative feedback inhibition on the hydrolytic and/ or acidogenic step. This inhibition is reduced in the sulphidogenic system where the utilization of end products is higher.
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- Date Issued: 2003
The potential of abalone stock enhancement in the Eastern Cape Province of South Africa
- Authors: Godfrey, Brian Peter
- Date: 2003
- Subjects: Abalones -- South Africa -- Eastern Cape , Abalone culture , Abalone fisheries -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5312 , http://hdl.handle.net/10962/d1005157 , Abalones -- South Africa -- Eastern Cape , Abalone culture , Abalone fisheries -- South Africa -- Eastern Cape
- Description: The largest abalone in South Africa, the perlemoen, Haliotis midae, occurs along approximately two-thirds of the country's coastline, but has only been the target of an extensive commercial fishery in the south-western part of South Africa. Large-scale illegal fishing has however proliferated throughout its entire range over the last 10–15 years, which has had serious effects on stock abundance and once productive populations are facing economic collapse. Abalone stock enhancement has been put forward as an addition or alternative to traditional fisheries management practices, which can potentially rehabilitate overfished abalone populations and enhance natural production. The aim of this project was to investigate the potential of abalone stock enhancement for managing an area in the Eastern Cape Province, which was being subjected to intense illegal fishing pressure. A research approach was adopted to investigate the scale and effects of this poaching and to investigate the survival of artificially cultured abalone seed in the natural environment. A model of a commercial-scale ranching operation was investigated to assess the economic feasibility of such a scheme. Analysis of poaching cases and research samples from Cape Recife indicated high levels of fishing effort that appeared to be causing the observed declines in emergent abalone abundance and average size. Poaching cases from Cape Recife contributed 32% of the total number of cases of known origin in the Eastern Cape Province from 1998–2002, with the majority of the cases (82%) originating from within the Port Elizabeth metropole. There was an exponential increase in the number of poaching cases in the Eastern Cape Province (r²=0.967) and Port Elizabeth over this period but actual annual total catch stabilized, particularly at Cape Recife, where estimates of CPUE declined significantly from 2000–2002 (p<0.001). The annual proportion of emergent abalone under the MLS from Cape Recife was always >85% in confiscations and research collections, and did not change significantly over the period of examination. Comparison of illegal catches with an adjacent site indicated that the emergent abalone population at Cape Recife had a significantly higher proportion of undersized emergent animals (p<0.001) and they were significantly smaller than the abalone from the closest site, Noordhoek, and other areas in Port Elizabeth from 2000–2002 (p<0.001). The apparent declines in emergent abalone abundance indicated by the poaching data were corroborated by sampling of emergent abalone abundance at Cape Recife. Numbers of abalone declined significantly from 125.6 to 53.8 per 20min count (p<0.001), from April 1998–October 2001 and density declined from 1.3 to 0.8m⁻² over a similar period (p<0.001). Juvenile density did not change over this period, although there were significant differences in density observed between two different habitats (p<0.001). Initial releases of cultured juvenile abalone showed that they could be distinguished from their wild counterparts for at least a year after release, by their different shell colouration. Short-term, small-scale trials (7–10 days) using animals between 17 and 30mm SL had mean survival rates of 64–82% (mean 70.4%) and the effect of size on survival was not significant in most cases, although the power to detect differences was low. Attempts to measure the effect of habitat on survival were not conclusive, although refuges under sea urchins appeared to be favoured by both seed and wild abalone, although urchins were not an absolute requirement for survival. Similar medium-term trials (31–74 days) in sheltered sites yielded mean returns of 53.1% using 25mm SL animals and similar trials in a more exposed area had more variable survival rates of between 18.4 and 73.6%, after 25–27 days. A comparison between careful hand-seeding and surface-scattering seed release methods in the open-ocean habitat showed no significant differences in survival rate after 41 days. Larger seed survived significantly better than the smaller seed when scattered on the surface (p<0.0001) and the smaller seed survived significantly better when seeded carefully by hand onto the substrate (p<0.028). Further medium-term releases of large numbers of seed abalone, released using the surface-scattering method in open-ocean habitat, gave mean survival rates of 32.8% over periods of 83–114 days. Assessment of a seeding trial using release modules to seed juvenile animals in the open-ocean was hampered by poor sea conditions and difficult to search substrates, and a mean recovery rate of 3.1% was obtained for the sites that were sampled. Movement of seed was low in the more sheltered sites, and also appeared to be affected by the amount of available habitat. Growth of seed was measured using changes in shell colouration and there were significant differences between areas. An average growth of 1.6mm.month⁻¹ shell length was calculated for all areas. The potential for commercial scale abalone ranching to be used to enhance a territorial user right fishery was investigated using a model of economic feasibility. Internal rates of return of 30.3 and 36.9% were obtained from two harvest regimes using different harvest sizes. There was a high degree of risk associated with these figures owing to a lack of consistent seed survival rate estimates, and the input parameters, which are subject to variation, showed a significant effect on profitability. The operation of this type of ranching scheme is probably not economically feasible as a stand-alone operation but could probably be operated effectively in conjunction with an existing abalone farm. By adding a small wild catch component the profitability of a ranching scheme could be significantly improved. The results of the present study indicate that high levels of poaching at Cape Recife have led to declines in emergent abalone abundance, which will probably lead to stock collapse in the near future. This implies that fisheries managers have failed to fulfil the provisions of fisheries policy in South Africa. The present results from seeding trials show that cultured juveniles can survive and make a contribution to overall stock abundance. Furthermore, while there are obstacles to economic feasibility, commercial ranching has the potential to be a valuable addition to current abalone management strategy.
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- Date Issued: 2003
The structure of ant communities and their impact on soil-pupating pests in citrus orchards in the Grahamstown area of the Eastern Cape
- Authors: Bownes, Angela
- Date: 2003
- Subjects: Ants -- South Africa -- Eastern Cape , Pests -- Control -- South Africa , Homoptera -- Control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5775 , http://hdl.handle.net/10962/d1005463 , Ants -- South Africa -- Eastern Cape , Pests -- Control -- South Africa , Homoptera -- Control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- South Africa -- Eastern Cape
- Description: Two ant species, Pheidole megacephala (Fabricius) and Anoplolepis custodiens (Smith) reach pest status in citrus orchards through precipitating outbreaks of homopterous pests. However, predacious ants, including these two ant species, play an important role in pest suppression in agroecosystems and are therefore beneficial to these systems. If A. custodiens and P. megacephala are important natural control agents in citrus, using ant bands to break the mutualism between the ants and the Homoptera as a method of ant control is preferable to poisoning. Ant communities were sampled by pitfall trapping in three experimental subunits of 2-, 4-, 15- and 30-year-old citrus orchards, in the Grahamstown area of the Eastern Cape. In one subunit in each orchard, populations of P. megacephala and A. custodiens were suppressed by poison applications. In a second subunit, trees were banded with trunk barriers so that ants were prevented from foraging in the trees and a third subunit served as the untreated control. Bait pupae of bollworm, false codling moth and fruit fly were planted in bait trays in all of the subunits to investigate predation on these citrus pests in the relative absence of predacious ants and where they were excluded from the trees. Pheidole megacephala dominated exclusively in all of the plots. Community composition did not change dramatically with increasing age of the trees, but species diversity and species abundance did. Rank-abundance curves showed that community diversity was highest in the 2-year-old plots and lowest in the 30-year-old plots. The Simpson and Shannon-Wiener diversity indices and their evenness measures indicated that diversity and equitability were highest in the poisoned subunits and lowest in the banded subunits. Principle component analysis revealed that the poisoned subunits were similar and distinct in species composition, that there was significant monthly variation in species composition and that community stability increases with an increase in orchard age. The presence of P. megacephala was significantly negatively correlated (rs = -0.293; p < 0.001) with pest pupal survival. Pupal survival was significantly higher for bollworm (p < 0.001), FCM (p < 0.001) and fruit fly (p < 0.001) in the poisoned subunits, than in the banded and control subunits. There was a general trend for survivorship to increase with an increase in the age of the trees. A significant difference (p < 0.001) was found between the months in which the trials were carried out. Pupal survival was significantly lower (p < 0.001) for FCM than for bollworm and fruit fly. In citrus orchards, ant communities are organised by ecological processes and interactions and are influenced by methods of ant control. Ant bands are preferable to poisoning as a method of ant control, so that beneficial species are left on the ground to prey on pests that pupate in the soil. Maintaining high ant species diversity in citrus orchards would be beneficial as predation on the pupae was more effective where ant species diversity was higher.
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- Date Issued: 2003
The suitability of Alagoasa extrema Jacoby (Coleoptera: Chrysomelidae: Alticinae), as a biological control agent for Lantana camara L. in South Africa
- Authors: Williams, Hester Elizabeth
- Date: 2003
- Subjects: Lantana camara , Lantana camara -- South Africa , Biological pest control agents -- South Africa , Chrysomelidae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5783 , http://hdl.handle.net/10962/d1005471 , Lantana camara , Lantana camara -- South Africa , Biological pest control agents -- South Africa , Chrysomelidae
- Description: Lantana camara Linnaeus (Verbenaceae), commonly known as lantana, is a highly invasive weed in many parts of the world. In South Africa it is naturalized in several provinces where it invades pastures, riverbanks, mountain slopes and valleys and commercial and natural forests, forming dense, impenetrable thickets. Chemical and mechanical control methods are expensive, labour intensive and provide only temporary relief as cleared areas are rapidly reinfested by seedlings and coppice growth. A biological control programme was initiated in South Africa in the 1960s, but despite the establishment of 11 agent species, it was considered to have had limited success. Several factors are thought to restrict the impact of the biocontrol agents. Firstly, L. camara occurs in a range of climatic regions, some of which are unsuitable for the establishment of agent species of tropical and subtropical origin. Secondly, L. camara is the result of hybridization between several Lantana species, forming a complex of hybridized and hybridizing varieties in the field, which match none of the Lantana species in the region of origin. This causes partial insect-host incompatibility, displayed as varietal preference. Thirdly, parasitism appears to have significantly reduced the effectiveness of several natural enemies. In spite of all these constraints, biological control has reduced invasion by L. camara by 26%. However, the weed is still very damaging and additional natural enemies are required to reduce infestations further. A flea-beetle species, Alagoasa extrema Jacoby (Coleoptera: Chrysomelidae), was collected from several sites in the humid subtropical and tropical regions of Mexico, and imported into quarantine in South Africa and studied as a potential biocontrol agent for L. camara. Favourable biological characteristics of this beetle included long-lived adults, several overlapping generations per year, and high adult and larval feeding rates. Observations from the insect’s native range and studies in South Africa suggest that A. extrema would probably be more suited to the subtropical, rather than the temperate areas in South Africa. Laboratory impact studies indicated that feeding damage by A. extrema larvae, over a period spanning the larval stage (16 to 20 days), reduced the above-ground biomass of L. camara plants by up to 29%. Higher larval populations resulted in a higher reduction of biomass. Varietal preference and suitability studies indicated that A. extrema exhibits a degree of varietal preference under laboratory conditions, with one of the white pink L. camara varieties proving the most suitable host. This variety is one of the most damaging varieties in South Africa and is particularly widespread in Mpumalanga Province. Although A. extrema proved to be damaging to L. camara, laboratory host range trials showed it to be an oligophagous species, capable of feeding and developing on several non-target species, especially two native Lippia species (Verbenaceae). The host suitability of these species was marginally lower than that of L. camara and the potential risk to these indigenous species was deemed to be too high to warrant release. It was therefore recommended that A. extrema not be considered for release in South Africa.
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- Date Issued: 2003