An investigation into the deployment of IEEE 802.11 networks
- Authors: Janse van Rensburg, Johanna Hendrina
- Date: 2007
- Subjects: Local area networks (Computer networks)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4596 , http://hdl.handle.net/10962/d1004839 , Local area networks (Computer networks)
- Description: Currently, the IEEE 802.11 standard is the leading technology in the Wireless Local Area Network (WLAN) market. It provides flexibility and mobility to users, which in turn, increase productivity. Opposed to traditional fixed Local Area Network (LAN) technologies, WLANs are easier to deploy and have lower installation costs. Unfortunately, there are problems inherent within the technology and standard that inhibits its performance. Technological problems can be attributed to the physical medium of a WLAN, the electromagnetic (EM) wave. Standards based problems include security issues and the MAC layer design. However the impact of these problems can be mitigated with proper planning and design of the WLAN. To do this, an understanding of WLAN issues and the use of WLAN software tools are necessary. This thesis discusses WLAN issues such as security and electromagnetic wave propagation and introduces software that can aid the planning, deployment and maintenance of a WLAN. Furthermore the planning, implementation and auditing phases of a WLAN lifecylce are discussed. The aim being to provide an understanding of the complexities involved to deploy and maintain a secure and reliable WLAN.
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Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4
- Authors: McNamara, Caryn
- Date: 2007
- Subjects: Heat shock proteins , Protein folding , Proteins -- Analysis , Proteins -- Structure , Plasmodium , Malaria , Molecular chaperones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4083 , http://hdl.handle.net/10962/d1007603
- Description: Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
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Combined and additive effects of assembly tasks and constrained body postures
- Authors: Skelton, Sarah Anne
- Date: 2007
- Subjects: Musculoskeletal system -- Diseases , Human engineering , Posture , Posture disorders , Work -- Physiological aspects , Occupational diseases , Manual work , Job stress
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5107 , http://hdl.handle.net/10962/d1005185 , Musculoskeletal system -- Diseases , Human engineering , Posture , Posture disorders , Work -- Physiological aspects , Occupational diseases , Manual work , Job stress
- Description: Despite extensive research into musculoskeletal disorders (MSDs) they continue to plague workers. Manual materials handling (MMH), in particular the concurrence of load manipulation and awkward body posture, has been identified as a key factor in the onset of MSDs. Only a few studies have looked at the interaction between manipulation tasks and working posture during assembly tasks and as a result their relationship has not been widely explored. Assessing the stresses resulting from individual task factors and body posture in isolation and adding them together may be too simplified to estimate an overall risk profile, since this does not take into account that there may be a non-linear interaction in strain responses when manipulation task and body posture interact. Therefore, the present study investigated biophysical, physiological and psychophysical responses to combined tasks, rather than individual tasks of body posture and manipulative tasks. The objective of the research was to establish the interactive effects of constrained body postures and manipulative tasks and to identify whether a cumulative or compensatory reaction occurs during this interaction. Nine conditions were assessed in a laboratory setting, which included combinations of three working postures (standing, sitting and stooping) and three assembly tasks (torque wrenching, precision and no task). Thirty-six subjects were required to complete all nine conditions, with each condition lasting ninety seconds. Muscle activity was recorded for seven muscles from the upper extremity, trunk and lower extremity regions and was complemented by physiological (heart rate, tidal volume, minute ventilation, oxygen consumption, energy expenditure and breathing frequency) and psychophysical (body discomfort) data. At the completion of all nine conditions subjects completed a retrospective psychophysical rating questionnaire pertaining to discomfort felt during the conditions. Responses obtained for the different task and posture combinations revealed compensatory reactions (additive > combined) for most of the conditions assessed for the biomechanical and physiological responses. In the majority of cases for muscle activity, no significant differences were found between the combined and the additive effects (p < 0.05), while for the physiological responses there were mostly significant differences observed. Psychophysical responses indicated that there was a significant difference overall between the additive and combined effects. The results of this study demonstrate that in order to identify risk areas, manipulation tasks and constrained working postures may be considered either in isolation and added together (additive) or as a combined task, since there were very few significant differences observed between these two effects. Further studies are required, however, to provide conclusive evidence.
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Conservation incentives for private commercial farmers in the thicket biome, Eastern Cape, South Africa
- Authors: Cumming, Tracey Lyn
- Date: 2007
- Subjects: Landowners -- South Africa -- Eastern Cape , Invasive plants -- South Africa -- Eastern Cape , Plant conservation -- South Africa -- Eastern Cape , Conservation of natural resources -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4745 , http://hdl.handle.net/10962/d1006953 , Landowners -- South Africa -- Eastern Cape , Invasive plants -- South Africa -- Eastern Cape , Plant conservation -- South Africa -- Eastern Cape , Conservation of natural resources -- South Africa -- Eastern Cape
- Description: This study sought opportunities to mitigate the pressures of land transformation and alien invader plants on commercial farm land in the thicket biome in the lower reaches of the Fish Kowie Corridor. It had two aims. Firstly, to determine the role incentives could play in mitigating these pressures. Secondly, to determine the characteristics of an incentive programme that would most effectively achieve this. In order to do this, an understanding of landowner activities, needs, opinions and barriers to behaviour; the nature of the pressures on thicket and the nature of the required behaviour to reduce these pressures; and current and past institutional arrangements needed to be achieved. This was done predominantly through a current literature review and personal interviews with landowners and key informants. These findings were used to make recommendations for an effective incentive programme. Landowners showed a preference towards tangible incentives, in particular management assistance, financial compensation and law enforcement. They indicated an aversion to an incentive programme implemented by a government agency, particularly district and provincial government. Rather, landowners showed a propensity towards a nongovernment organisation (NGO) or a farmers group implementing an incentive programme. It was recommended that the two major pressures, namely land transformation and alien invader plants, required different interventions by different agencies in order to be mitigated. The pressure of land transformation required a stewardship model response, with the primary drive being a non-contractual environmental extension service to landowners. The extension service should focus on promoting pro-conservation practises, raising awareness and disseminating information. It should also build a relationship of trust between landowners and the implementing agency. The pressure of alien invader plants would be most effectively addressed through the Working for Water programme. Tangible incentives must be provided to the landowner to induce the costly exercise of alien invader plant control. In particular, the high cost of labour must be addressed. The regulatory incentive of applying laws requiring landowners to control alien invader plants on their land should also be enforced.
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Constructing a low-cost, open-source, VoiceXML
- Authors: King, Adam
- Date: 2007 , 2013-07-01
- Subjects: VoiceXML (Document markup language) , Asterisk (Computer file) , Internet telephony , Open source software
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4585 , http://hdl.handle.net/10962/d1004735 , VoiceXML (Document markup language) , Asterisk (Computer file) , Internet telephony , Open source software
- Description: Voice-enabled applications, applications that interact with a user via an audio channel, are used extensively today. Their use is growing as speech related technologies improve, as speech is one of the most natural methods of interaction. They can provide customer support as IVRs, can be used as an assistive technology, or can become an aural interface to the Internet. Given that the telephone is used extensively throughout the globe, the number of potential users of voice-enabled applications is very high. VoiceXML is a popular, open, high-level, standard means of creating voice-enabled applications which was designed to bring the benefits of web based development to services. While VoiceXML is an ideal language for creating these applications, VoiceXML gateways, the hardware and software responsible for interpreting VoiceXML applications and interfacing with the PSTN, are still expensive and so there is a need for a low-cost gateway. Asterisk, and open-source, TDM/VoIP telephony platform, can be used as a low-cost PSTN interface. This thesis investigates adding a VoiceXML service to Asterisk, creating a low-cost VoiceXML prototype gateway which is able to render voice-enabled applications. Following the Component-Based Software Engineering (CBSE) paradigm, the VoiceXML gateway is divided into a set of components which are sourced from the open-source community, and integrated to create the gateway. The browser requires a VoiceXML interpreter (OpenVXI), a Text-To-Speech engine (Festival) and a speech recognition engine (Sphinx 4). The integration of the components results in a low-cost, open-source VoiceXML gateway. System tests show that the integration of the components was successful, and that the system can handle concurrent calls. A fully compliant version of the gateway can be used in the real world to render voice-enabled applications at a low cost. , KMBT_363 , Adobe Acrobat 9.55 Paper Capture Plug-in
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Determination of the botanical composition of black rhinoceros (Diceros bicornis) dung using the rbcL gene as a molecular marker, and analysis of antioxidant and phenolic content of its browse
- Authors: Bulani, Siyavuya Ishmael
- Date: 2007 , 2013-06-25
- Subjects: Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4070 , http://hdl.handle.net/10962/d1006468 , Black rhinoceros -- Food , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- Analysis -- South Africa -- Eastern Cape , Plant ecology -- South Africa -- Eastern Cape , Genetic markers , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants
- Description: The black rhinoceros remains one of the world's extremely endangered species despite a variety of policies to protect it. The black rhinoceros population at the Great Fish River Reserve (GFRR) in the Eastern Cape in South Africa has increased steadily since their re-introduction in 1986. This megaherbivore is a browser, with a diet obtained largely from the short and medium succulent thicket of the GFRR. Knowledge of the preferential diet of the black rhinoceros on the reserve is an important factor for the effective management of the land and the herbivores that compete for its resources. The dietary preferences of the black rhinoceros at the reserve have been established using backtracking methods. In this study the rbcL gene was used to establish an rbcL gene database of the plants from the GFRR and determine the botanical composition of the black rhinoceros dung from the GFRR. Due to the limited number of rbcL gene plant sequences from the GFRR deposited in the GenBank database, 18 plant species from the GFRR were sequenced. Sequence analyses between the partial rbcL gene sequences generated were able to distinguish between plants down to species level. Plant species from the family Euphorbiaceae and Fabaceae showed sequence variation at intra-specific level compared to those of Tiliaceae which were more conserved. The generated rbcL gene sequences from seasonal dung samples were compared to the rbcL gene sequenced from 18 plant species obtained from the GFRR and those from the GenBank database. A wide range of plant species were identified from the dung samples. There were no major differences in botanical composition between the dung samples, except that Grewia spp. were found to dominate in almost all seasons. The results obtained on the free radical scavenging activity of the extracts against 2,2-Diphenyl-l-picrylhydrazyl (DPPH) increased in the order of methanol > ethyl acetate > chloroform. The DPPH free radical scavenging activity of the methanol plant extracts increased in the order Brachylaena elliptica > Plumbago auriculata > Grewia robusta > Azima tetracantha. Methanol extracts on the TLC plate sprayed with Fe³⁺-2,4,6-Tri-2-pyridyl-s-triazine (TPTZ) showed that the compounds present in the extracts react differently to ferric ion, with most compounds unable to reduce ferric ion. Furthermore the methanol extracts were able to exhibit reduction potentials vs. Ag/AgCl at low concentrations. The compounds in the extracts were shown to be phenolic acids and flavonoid glycosides.
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Development and assessment of an oxytocin parenteral dosage form prepared using pluronic ® F127
- Authors: Chaibva, Faith Anesu
- Date: 2007
- Subjects: Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3747 , http://hdl.handle.net/10962/d1003225 , Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
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Eluvial chromite resources of the Great Dyke of Zimbabwe
- Authors: Musa, Caston Tamburayi
- Date: 2007
- Subjects: Dikes (Geology) -- Zimbabwe Chromite -- Zimbabwe Geology -- Zimbabwe Olivine Serpentinite Eluvium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5046 , http://hdl.handle.net/10962/d1007731
- Description: Apart from the concentrations of chromite in layers within the Great Dyke and other ultramafic complexes, chromite also occurs as interstitial grains throughout the olivine-bearing rock-types. These olivine-bearing rocks include no rites, gabbros, dunites and pyroxenites. Chromite concentration in these rocks varies from 0.48 to 3.09 per cent of the rock, usually in the form of chromite (Ahrens, 1965; Worst, 1960). A small fraction of this chromite settled to form chromitite layers whilst the remainder is retained within the rock mass as finely disseminated chromite and chromite interstitial to olivine. This retained chromite is much finer grained than layer chromite and is the primary source of eluvial chromite (Cotterill, 1981). During weathering of the serpentine rock and transportation by rainwater, the heavier chromite and magnetite grains are re-deposited along watercourses and vleis or valleys as the speed of the water is retarded sufficiently for the heavier particles to settle. The lighter serpentine material is removed and the chromite concentration in the soil is increased, thus resulting in eluvial chromite (Keech et ai, 1961; Worst, 1960; Prendergast, 1978). The concentration of chromite particles in soil can be up to 15 (or more) Cr₂O₃ %, resulting in economic and exploitable deposits, located primarily along the Great Dyke fiacks. A preliminary evaluation of the eluvials indicate that the Great Dyke could be host to up to 10 million tonnes of potential chromite concentrates which could be processed from such eluvial concentrates. These chromite-rich soils can be mined more cheaply than the traditional seams mining and processed into chromite concentrates through simple mechanical processing techniques of spirals, jigs and heavy media separators. The resultant chromite concentrates are of high quality and can be used to manufacture chromite ore briquettes, which are an alternative to lumpy chromite smelter feed. The main challenges to eluvial mining are the inevitable environmental degradation and coming up with methods that could possibly mitigate against such environmental damage. The distribution of these eluvials over vast plains as thin soil horizons, necessitate use of mobile concentrator plants and hence establishment of extensive infrastructure. These challenges, however, are not insurmountable and test mining and previous production runs have proved profitable. The eluvials are also associated with some lateritic nickel concentrations. The nickel occurs in close association with some oxide such as goethite and garnierite and is associated with iron-manganiferous soil pisolites. The analyses of these pisolites indicate high nickel grades of generally above 1.00 %Ni. Such high nickel-content of Great Dyke laterites warrant, further investigations.
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Estimating erosion of cretaceous-aged kimberlites in the Republic of South Africa through the examination of upper-crustal xenoliths
- Authors: Hanson, Emily Kate
- Date: 2007
- Subjects: Kimberlite -- South Africa , Igneous rocks -- Inclusions -- South Africa , Erosion -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4942 , http://hdl.handle.net/10962/d1005554 , Kimberlite -- South Africa , Igneous rocks -- Inclusions -- South Africa , Erosion -- South Africa
- Description: he estimation of post-emplacement kimberlite erosion in South Africa through the study of upper-crustal xenoliths is relatively unexplored; however the presence of these xenoliths has been recognized for well over 100 years. Post-emplacement erosion levels of a small number of South African kimberlite pipes have been inferred through the study of the degree of country-rock diagenesis, the depth of sill formation, the depth of the initiation of the diatreme and fission track studies. Through these studies, several estimates were proposed for the Group I Kimberley kimberlites. Although the 1400 m estimate of erosion remains widely accepted today, this estimate relies on the presence of Karoo-like basalt xenoliths in the Group I Kimberley kimberlites, as their presence proves that basalt existed in the Kimberley area when the kimberlites were emplaced. Basaltic xenoliths were described during the early stages of mining in Kimberley, though only one of these descriptions suggests that the ‘basaltic’ boulders correlate with the Karoo basalts. Because of the discrepancy between these early documentations of upper-crustal xenoliths and because the occurrence of Karoo-like basalt xenoliths in the Group I Kimberley kimberlites is under question, a re-investigation of the erosion levels and the upper crustal xenolith suites in South African, Cretaceous-aged kimberlites, including Melton Wold, Voorspoed, Roberts Victor, West End, Record Stone Quarry, Finsch, Markt, Frank Smith, Pampoenpoort, Uintjiesberg, Koffiefontein / Ebenheuyser, Monastery, Kimberley (Big Hole), Kamfersdam , Jagersfontein, Kaal Vallei, De Beers, Bultfontein, Lushof, Britstown Cluster, Hebron and Lovedale, was conducted. This study presents the analytical results for upper-crustal sandstone and basalt xenoliths collected from dumps, excavation pits and borehole core at the above-mentioned kimberlites, and demonstrates that they correlate with stratigraphic units of the Karoo Supergroup on the basis of mineral and geochemical compositions. These upper-crustal xenoliths are incorporated into kimberlites and down-rafted to levels below their stratigraphic position during kimberlite emplacement, consequently recording the broad stratigraphy into which each kimberlite is emplaced. Therefore, the Cretaceous lateral extent of the Karoo Supergroup is inferred and post-emplacement erosion estimated by reconstructing the stratigraphy based on upper-crustal xenolith suites for each kimberlite and calculating the total thickness of the now-eroded units. The distribution of sandstone xenoliths indicates that during the Cretaceous the lateral extent of the Dwyka, Ecca and Beaufort Groups encompassed all of the examined kimberlites, while the ‘Stormberg’ Group was constrained to an area outlined by the Voorspoed and Monastery kimberlites. Similarly, basalt xenoliths occur in all of the Group II and transitional (143 – 100 Ma) kimberlites but only in the Group I (90 – 74 Ma) kimberlites that lie within close proximity to the western outcrop margin of the outcrop area of the Drakensberg Group basalts (Lesotho Remnant), namely Monastery, Jagersfontein and Kaal Vallei. This trend implies an eastward-retreat of the inland erosion front of the Karoo basalts between 140 and 90 Ma and subsequent erosion of the underlying sedimentary units. It also suggests that a thicker succession of Karoo strata was present at the time of Group II and transitional kimberlite emplacement and that there has been more post-emplacement erosion in these kimberlites than the younger Group I kimberlites, except for Monastery, Jagersfontein and Kaal Vallei. Estimates are unique to each kimberlite as they are dependent on both stratigraphic location, elevation and present country rock, and range from approximately 1000 – 2500 m for the older kimberlites and less than 700 m to 1400 m for the younger kimberlites. Furthermore, the upper-crustal xenoliths found at the Group I Kimberley kimberlites and the coinciding trend of basalt erosion demonstrate that Karoo basalts were eroded from the Kimberley area by the time the Group I Kimberley kimberlites erupted (~85 Ma). Therefore, basalts are omitted from the Group I Kimberley kimberlites post-emplacement erosion estimate, and the upper Beaufort Group is considered the upper limit of the stratigraphy that was present at the time of the eruption of the Group I Kimberley pipes. Therefore, the erosion estimates decrease from a previous estimate of 1400 m down to 400 to 1100 m, where 850 m is considered a dependable intermediate estimate.
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Evaluation and application of electroanalysis for the determination of antioxidants
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
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Evaluation of fishway designs for use at the ebb and flow region of rivers in the Eastern Cape, South Africa
- Authors: Lewis, Hylton Varian
- Date: 2007
- Subjects: Fishways -- South Africa -- Eastern Cape , Fishways -- Design and construction , Aquatic ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5279 , http://hdl.handle.net/10962/d1005123 , Fishways -- South Africa -- Eastern Cape , Fishways -- Design and construction , Aquatic ecology -- South Africa -- Eastern Cape
- Description: Fishways are devices that are increasingly being used worldwide to assist aquatic biofauna to migrate through man-made barriers such as weirs and large-scale dams that are used for water storage, electricity generation etc. and have a negative impact on both the upstream and downstream movement of aquatic organisms. Fishways are usually low gradient channels with evenly placed chambers which allow such migrating aquatic species minimal stress on their passage over these barriers. Despite the existence of national and regional policies for their provision, their construction has often been inhibited through a lack of local assessment of the available designs, and use of ineffective international designs. As part of a larger national research programme, sponsored by the Water Research Commission, to develop a protocol for fishway design and implementation, this study sought to investigate the suitability of vertical slot and sloping baffle designs to assist the migrations of juvenile catadromic fish species in the ebb and flow region of rivers in the Eastern Cape, South Africa. These fishways were initially tested under controlled conditions at the Experimental Fish Farm at the Department of Ichthyology and Fisheries Science, Rhodes University using various fish species. Using the performance data thus collected they were then installed and monitored in the field for the catadromic Myxus capensis (freshwater mullet) and Monodactylus falciformis (cape moony) migrations during March and November of 2005 at the Kowie River ebb and flow weir. The laboratory tests showed that there was a significantly higher level of migration success in the vertical slot compared with the sloping baffle fishway under all conditions (F=82.157; p<0.001). There was, however, a slow decline in levels of success with increased discharge in the vertical slot fishway. This was in contrast to the sloping baffle design where success increased as discharge increased at a steep gradient (F=74.894; p<0.005). The level of success with the M. capensis was related to the size of the fish for both systems with success increasing from the small to the large fish (F=17.755; p<0.001). For the M. falciformis higher levels of success were found to occur in the vertical slot fishway compared to the sloping baffle fishway (F=11.792; p<0.00086), with no significant differences being found with an increase in discharge. Field data indicated similar trends with higher migration success using the vertical slot fishway. M. capensis were better able to negotiate both devices compared to the M. falciformis and overall levels of success were higher for both species in the field than under laboratory conditions where the migration urge may have declined.
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Evaluation of the safety and efficacy of topical mometasone furoate formulations
- Authors: Chamboko, Bernadett Vongayi
- Date: 2007
- Subjects: Adrenocortical hormones -- Physiological effect , Drugs -- Testing , Dermatopharmacology , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3748 , http://hdl.handle.net/10962/d1003226 , Adrenocortical hormones -- Physiological effect , Drugs -- Testing , Dermatopharmacology , High performance liquid chromatography
- Description: The human skin blanching assay (HSBA) is a well-researched and validated method for the bioequivalence assessment of topical corticosteroids. Traditionally, visual assessment of skin blanching has been used. Such testing methods are not conducive for interlaboratory comparisons. Regulatory bodies prefer less subjective methods of analysis. The FDA released guidelines on the assessment of bioequivalence for topical corticosteroids that recommends the use of a chromameter as a reliable method to measure skin blanching although the use of visual assessment with acceptable validation is also provided for. However, the FDA does not elucidate on the manipulation and handling of the chromameter during skin blanching measurements. The purpose of this project was several fold, which included investigations to standardize the manipulation and handling of a chromameter. In particular, measures to avoid skin whitening resulting from the effects of pressure on the skin during chromameter use were investigated. Other methods of analysis should surpass or at least be comparable to the HSBA if such methods are to be used for the assessment of topical corticosteroids. Microdialysis is a relatively new technique for assessing the rate at which drug penetrates the skin. The advantage of using this method is that there are fewer restrictions for selection of an appropriate study population unlike those required for the HSBA where one has to be both a ‘responder’ and a ‘detector’ for their results to be used in data analysis. Microdialysis was investigated by initially conducting experiments in which microdialysis probes were embedded into topical formulations containing mometasone furoate (MF) and the initial results revealed that relatively low drug was released from the formulations. These results indicated that should microdialysis be applied to measure the in vivo release of MF from such topical formulations following application to the skin, even lower concentrations of MF would likely result in the dialysate, necessitating the need for ultra-high sensitive methods of analysis. Typically, the availability of an appropriate analytical technique such as liquid chromatography coupled with mass spectrometry (LCMS) would be a pre-requisite for such in vivo studies. However, only high-pressure liquid chromatography (HPLC) and other less sensitive equipment was available in the laboratories. The study objectives were therefore focussed on in vitro assessment of the release of MF from topical formulations using microdialysis and Franz cells. In addition, the in vivo release of MF was also studied using the HSBA. Data obtained from the microdialysis experiments were compared with the data obtained from the Franz cell diffusion studies in order to provide information on the pharmaceutical availability of MF from the various topical MF dosage forms. Subsequently, pharmaceutical equivalence was investigated from the comparative pharmaceutical availability data using statistical analysis. An additional objective was to attempt to correlate in vitro with in vivo data (IVIVC) to establish a model that could be used to assess safety and efficacy of generic topical drug products. The in vivo data obtained from the HSBA were processed according to the FDA requirements and these pharmacodynamic data were subsequently compared with the microdialysis and Franz cell results. In summary the objectives of this project were: 1. To develop a system to improve the reproducibility of the use of a Minolta® chromameter and compare this with the standard/normal manipulation and handling of such instruments. 2. To develop and validate an HPLC method for the analysis of MF for use with in vitro diffusion studies using microdialysis and Franz cells. 3. To conduct a comparative HSBA on proprietary MF topical creams from two different countries in accordance with the FDA guidance. 4. To assess the pharmaceutical equivalence of topical formulations containing MF using Franz diffusion cells and in vitro microdialysis. 5. To compare the in vivo data obtained from the HSBA with those obtained in vitro using microdialysis and Franz cells.
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Extending the reach of personal area networks by transporting Bluetooth communications over IP networks
- Authors: Mackie, David Sean
- Date: 2007 , 2007-03-29
- Subjects: Bluetooth technology , Communication -- Technological innovations , Communication -- Network analysis , TCP/IP (Computer network protocol) , Computer networks , Computer network protocols , Wireless communication systems
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4637 , http://hdl.handle.net/10962/d1006551 , Bluetooth technology , Communication -- Technological innovations , Communication -- Network analysis , TCP/IP (Computer network protocol) , Computer networks , Computer network protocols , Wireless communication systems
- Description: This thesis presents an investigation of how to extend the reach of a Bluetooth personal area network by introducing the concept of Bluetooth Hotspots. Currently two Bluetooth devices cannot communicate with each other unless they are within radio range, since Bluetooth is designed as a cable-replacement technology for wireless communications over short ranges. An investigation was done into the feasibility of creating Bluetooth hotspots that allow distant Bluetooth devices to communicate with each other by transporting their communications between these hotspots via an alternative network infrastructure such as an IP network. Two approaches were investigated, masquerading of remote devices by the local hotspot to allow seamless communications and proxying services on remote devices by providing them on a local hotspot using a distributed service discovery database. The latter approach was used to develop applications capable of transporting Bluetooth’s RFCOMM and L2CAP protocols. Quantitative tests were performed to establish the throughput performance and latency of these transport applications. Furthermore, a number of selected Bluetooth services were tested which lead us to conclude that most data-based protocols can be transported by the system.
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Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
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Investigating the effect of Glomus etunicatum colonization on structure and phloem transport in roots of Eragrostis curvula (Umgeni)
- Authors: Skinner, Amy
- Date: 2007
- Subjects: Glomus (Fungi) , Phloem , Plant translocation , Weeping lovegrass , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Mycorrhizas
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4227 , http://hdl.handle.net/10962/d1003796 , Glomus (Fungi) , Phloem , Plant translocation , Weeping lovegrass , Vesicular-arbuscular mycorrhizas , Mycorrhizal fungi , Mycorrhizas
- Description: The symbiotic unit of an arbuscular mycorrhizal fungus and its host is able toachieve and maintain far higher inflow of nutrients than non-mycorrhizal roots. The colonization strategy of the mycobiont within the plant is intrinsic to the symbiosis with respect to both structural adaptations and nutrient exchange. An investigation into the effect of Glomus etunicatum colonization on the structure and phloem transport in Eragrostis curvula (Umgeni) allowed for greater insight into the dynamic of the symbiosis. The combined use of stains (such as Trypan Blue, Chlorazol Black, Safranin and Fast Green), and techniques, (such as freeze-microtome transverse sectioning and permanent slide preparations) contributed to a successful general observation of an intermediate colonization strategy using light microscopy methods. However, clarity into structural detail of mycorrhizal forms required electron microscopy studies. The SEM method used with freeze fracture was a relatively quick and simple method allowing for the observation of surface and internal features. The TEM method allowed for highresolution images providing insight into the variations in the apoplasmic compartmental form, and how this may relate to the function of the symbiosis with regard to fungal coils or arbuscules. The apoplasmic nature of mycorrhizas was substantiated and no symplasmic connections were found between symbionts. Fluorescence studies demonstrated that 5,6-carboxyfluorescein was transported through the phloem into the roots of E. curvula, but remained predominantly in the root phloem. Unloading only occurred in optimal nutrient exchange areas of meristimatic lateral or apical growth regions. It was not possible, using fluorescence techniques and related equipment available, to conclusively establish if there were symplasmic connections between the mycobiont and its host or if bidirectional transfer of nutrients occurred at the same interface.
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Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
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Isolation, propagation and rapid molecular detection of the Kalahari truffle, a mycorrhizal fungus occurring in South Africa
- Authors: Adeleke, Rasheed Adegbola
- Date: 2007 , 2013-04-03
- Subjects: Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3889 , http://hdl.handle.net/10962/d1002951 , Truffles -- Kalahari Desert , Fungi -- Identification , Mycorrhizal fungi -- South Africa , Edible fungi -- South Africa , Mushroom culture -- South Africa , Fungi -- Cultures and culture media -- South Africa , Truffles -- South Africa , Truffles -- Lifecycles , Mycorrhizal fungi -- Lifecycles
- Description: Terfezia pfeilii is an edible mycorrhizal fungus that thrives in the Kalahari Desert of southern Africa. It is best known by desert dwellers for its flavour and as a source of nutrition. Although the genus Terfezia is generally regarded as being an ectomycorrhizal mycobiont, the exact mycorrhizal type formed by T. pfeilli and its' associated host plants remains uncertain. Discovery of the host plants for T. pfeilii would first be required in order to further investigate the life cycle and cultivation of this truffle. This study focussed on the isolation of mycelia from the ascocarp, optimising the growth conditions of the mycelial cultures, rapid molecular identification of T. pfeilii, investigation of potential helper bacteria and mycorrhizal synthesis experiments. T. pfeilii ascocarps were harvested from the Spitskop Nature Reserve in Upington, South Africa. Ascocarps were successfully identified using both morphological and molecular methods. Despite the delayed growth mostly caused by contaminating microorganisms, the isolation of T. pfeilii mycelia culture was successful. Molecular techniques were used to confirm the identity of the pure culture. Further studies were conducted on ways to improve the growth conditions of the mycelial culture on Fontana medium. An optimum temperature of 32°C, the addition of Bovine Serum Albumin as a nitrogen source and a pH of 7.5 significantly improved the growth of T. pfeilii in vitro. A rapid PeR-based molecular method was developed to speed up the identification of T. pfeilii. Specific primers that can exclusively amplify the ITS region of T. pfeilii were designed and used to identify both the ascocarps and the mycelial culture. The specificity of these primers was confirmed by their inability to amplify DNA from the isolates of contamining fungi obtained during the isolation process. Molecular comparison was made to confirm the reclassification of South African samples of T. pfeilii as Kalaharituber pfeilii as proposed by Ferdman et al.,(2005). However, in this study, the name T. pfeilii has been retained. A total of 17 bacterial isolates were obtained from the fruiting bodies of T. pfeaii and these were tested for stimulation of mycelial growth in vitro, indole production and phosphate solubilising capabilities. Bacterial isolates that showed potential to be Mycorrhization Helper Bacteria (MHB) were identified as Paenibacillus sp., Bacillus sp. and Rhizobium tropici. Selected plant seedlings were inoculated with T. pfeilii cultures or ascocarp slurry in order to re-establish the mycorrhizal association. After 8 months, light microscopy observations revealed an endomycorrhizal type association between Cynodon dactylon and T. pfeilii. This was confirmed with molecular analysis using specific T. pfeilii ITS primers. After 15 months, molecular methods confirmed Acacia erioloba as another host plant. These results have provided essential information paving the way for further investigation into the life cycle and biology of the Kalahari truffle. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
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Laboratory investigation of a load carriage task observed in forestry
- Authors: Furney, Sheena Elizabeth
- Date: 2007
- Subjects: Work -- Physiological aspects , Foresters -- Workload , Human engineering , Lifting and carrying
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5145 , http://hdl.handle.net/10962/d1008188 , Work -- Physiological aspects , Foresters -- Workload , Human engineering , Lifting and carrying
- Description: The objective of the present study was to investigate and compare the human responses to two load carriage tasks performed with three different load masses and on three different gradients. The task of carrying hydrogel in one hand was observed in a silviculture industry and crude physiological and perceptual responses were measured. This task was simulated in a laboratory setting together with a suggested intervention of backpack carriage. Eighteen conditions were established which consisted of the two modes of carriage and a combination of three load masses (9kg, 12kg and 15kg) and three gradients (5%, 10% and 15%). Twenty eight Rhodes University female students comprised the sample and the experimental procedures were conducted on a Quinton treadmill. Each participant was required to complete nine of the eighteen conditions which were each four minutes in duration. Postural changes were assessed using lateral and posterior digital images taken at the second and fourth minute and compression and shearing forces were estimated with the ErgolmagerTM Physiological responses (heart rate, ventilation and metabolic responses) were measured continuously with the Quark b² and perceptual responses ('central' and 'local' RPE) were measured every minute during the experimentation and body discomfort was rated at the completion of each condition. Overall responses revealed that hand carriage (146 bt.min⁻¹ , 25.09 mIO₂. kg-l.min⁻¹) was generally found to be more physiologically stressful than backpack carriage (130 bt.min⁻¹, 22.15 mIO₂.kg⁻¹ .min⁻¹) independent of load mass and gradient. Physiological responses were higher (113 bt.min-1 to 174 bt.min⁻¹ ) in responses to increasing gradient as opposed to increasing load mass (104 bt.min-1 to 153 bt.min⁻¹ ) for both backpack and hand carriage. Categorisation using the guidelines of Sanders and McCormick (1993) allowed for classification of conditions, with respect to physiological responses, into 'moderate', 'heavy' and 'very heavy' stress. For almost all of the physiological responses the majority of conditions which were classified as 'moderate' were backpack carriage conditions and the conditions classified as 'very heavy' were mostly hand carriage conditions. In terms of postural responses hand carriage resulted in more strain and greater compression and shearing forces on the spine. In terms of the compression forces increasing gradient had a greater affect on backpack carriage (681 N to 935 N) compared to hand carriage (570N to 793N). In contrast, increasing load mass had a larger affect on hand carriage postures and compression forces (751 N to 935N) in comparison to backpack carriage (723N to 780N). Shearing forces were found to be worse in hand carriage conditions overall. Although participants generally underrated perceived exertion in relation to cardiorespiratory responses, these perceptions revealed that backpack carriage, with a mean 'central' RPE of 12 compared to 11 for hand carriage, was somewhat preferred to hand carriage and that increasing gradient was perceived to be marginally more straining than increasing load mass.
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Limiting vulnerability exposure through effective patch management: threat mitigation through vulnerability remediation
- Authors: White, Dominic Stjohn Dolin
- Date: 2007 , 2007-02-08
- Subjects: Computer networks -- Security measures , Computer viruses , Computer security
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4629 , http://hdl.handle.net/10962/d1006510 , Computer networks -- Security measures , Computer viruses , Computer security
- Description: This document aims to provide a complete discussion on vulnerability and patch management. The first chapters look at the trends relating to vulnerabilities, exploits, attacks and patches. These trends describe the drivers of patch and vulnerability management and situate the discussion in the current security climate. The following chapters then aim to present both policy and technical solutions to the problem. The policies described lay out a comprehensive set of steps that can be followed by any organisation to implement their own patch management policy, including practical advice on integration with other policies, managing risk, identifying vulnerability, strategies for reducing downtime and generating metrics to measure progress. Having covered the steps that can be taken by users, a strategy describing how best a vendor should implement a related patch release policy is provided. An argument is made that current monthly patch release schedules are inadequate to allow users to most effectively and timeously mitigate vulnerabilities. The final chapters discuss the technical aspect of automating parts of the policies described. In particular the concept of 'defense in depth' is used to discuss additional strategies for 'buying time' during the patch process. The document then goes on to conclude that in the face of increasing malicious activity and more complex patching, solid frameworks such as those provided in this document are required to ensure an organisation can fully manage the patching process. However, more research is required to fully understand vulnerabilities and exploits. In particular more attention must be paid to threats, as little work as been done to fully understand threat-agent capabilities and activities from a day to day basis. , TeX output 2007.02.08:2212 , Adobe Acrobat 9.51 Paper Capture Plug-in
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Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication
- Authors: Murray, Lindsay
- Date: 2007
- Subjects: Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4090 , http://hdl.handle.net/10962/d1007722 , Encephalomyelitis -- Genetic aspects , Amino acid sequence , Picornaviruses , Viruses -- Reproduction
- Description: The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
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