The spatial evolution of the chemotaxis proteins of the Bacillus subtilis group
- Authors: Yssel, Anna Elizabeth Johanna
- Date: 2011
- Subjects: Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4027 , http://hdl.handle.net/10962/d1004087 , Chemotaxis , Bacillus subtilis , Bacillus (Bacteria) , Homology (Biology) , Plants -- Microbiology
- Description: The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.
- Full Text:
- Date Issued: 2011
Quantifying crop damage by Grey crowned crane balearica regulorum regulorum and evaluating changes in crane distribution in the North Eastern Cape, South Africa
- Authors: Van Niekerk, Mark Harry
- Date: 2011
- Subjects: Cranes (Birds) -- South Africa -- Eastern Cape , Cranes (Birds) -- Geographical distribution -- South Africa -- Eastern Cape , Cranes (Birds) -- Behavior -- South Africa -- Eastern Cape , Cranes (Birds) -- Food -- South Africa -- Eastern Cape , Corn -- Effect of grazing on -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5749 , http://hdl.handle.net/10962/d1005436 , Cranes (Birds) -- South Africa -- Eastern Cape , Cranes (Birds) -- Geographical distribution -- South Africa -- Eastern Cape , Cranes (Birds) -- Behavior -- South Africa -- Eastern Cape , Cranes (Birds) -- Food -- South Africa -- Eastern Cape , Corn -- Effect of grazing on -- South Africa -- Eastern Cape
- Description: Complaints of crop damage by cranes on planted maize in the North Eastern Cape, South Africa, have been increasing since the mid-1990‘s, and in some instances severe losses have been reported. Crop damage by the Grey Crowned Crane Balearica regulorum regulorum near the town of Maclear (31º04´S 28º22´E), has been quantified over two growing seasons, and assessed relative to losses caused by foraging Cape Crows Corvus capensis and other feeding damage assumed to be caused by insects. Twelve fields were selected based on previous patterns of crop depredation. Maize seed in seven of the fields was treated with the chemical ‗Gaucho‘ and five fields were planted with untreated maize. In order to determine the source of losses, twenty quadrats (4 m x 4 m) randomly distributed within each field were visited on average every second day, for a period of up to twenty eight days. Results indicate that seed treatments do act as a deterrent to feeding by both cranes and crows, however crane damage is generally insignificant compared to other sources of damage. My study also reviewed past sightings data of the Grey Crowned Crane in an effort to determine if the conversion of former grassland to plantations in this region may have increased foraging activity in maize fields. The data did not allow for clear-cut conclusions regarding changes in distribution or population trends. Conclusions provide direct input into the management of agricultural areas by enabling landowners to take steps to mitigate crop damage. These mitigation measures may either involve the application of seed treatments, or the planting of low risk crops in high risk areas. Future studies should consider the ppossible detrimental effects of chemical seed treatments on crane biology.
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- Date Issued: 2011
The design and synthesis of multidentate N-heterocyclic carbenes as metathesis catalyst ligands
- Authors: Truscott, Byron John
- Date: 2011
- Subjects: Carbenes (Methylene compounds) , Heterocyclic compounds , Ligands , Ligands -- Design , Metathesis (Chemistry) , Catalysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4304 , http://hdl.handle.net/10962/d1004962 , Carbenes (Methylene compounds) , Heterocyclic compounds , Ligands , Ligands -- Design , Metathesis (Chemistry) , Catalysis
- Description: This study has focused on the design and preparation of bi– and tridentate N–Heterocyclic Carbene (NHC) ligands in order to investigate the effect of a multidentate approach to the formation, stability and catalytic activity of coordination complexes. Chapters 1 – 3 provide background information of relevant catalysis, carbene and coordination chemistry, followed by previous work performed within our research group. In Chapter 4 attention is given to the synthetic aspects of the research conducted, comprising two distinct approaches to the preparation of unsymmetrical saturated and unsaturated NHCs. Firstly, an investigation of the saturated NHC ligands yielded three novel, unsymmetrical pro–ligands, viz., two halopropyl imidazolinium salts and a bidentate hydroxypropyl imidazolinium salt. Secondly, eight imidazolium salts have been generated, including a hydroxypropyl analogue and novel decyl and tridentate malonyl derivatives. These compounds were prepared using microwave–assisted methodology for the alkylation of N– mesitylimidazole – an approach that drastically reduced reaction times (from 8 hours – 7 days to ca. 0.5 – 2 hours) and facilitated isolation of the imidazolium salts. Many of the compounds prepared in this study are novel and were fully characterized using HRMS and 1– and 2–D NMR analysis. Coordination studies using a selection of the prepared pro–ligands afforded an alkoxy–NHC silver derivative and four novel Ru–complexes, viz., Grubbs II–type Ru–complexes containing:– chloropropyl imidazolinylidene; propenyl imidazolylidene; and bidentate alkoxypropyl imidazolylidene ligands. Furthermore, a well–defined benzyl mesitylimidazolylidene Ru–complex has been isolated, which exhibited good stability in air. DFT–level geometry–optimization studies, using the Accelrys DMol3 package have given valuable insights into the likely geometries of the prepared and putative catalysts.
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- Date Issued: 2011
Investigation into the extended capabilities of the new DPS-4D ionosonde
- Authors: Ssessanga, Nicholas
- Date: 2011
- Subjects: Ionosondes , Ionosphere , Ionosphere -- Observations -- South Africa -- Hermanus (Cape of Good Hope) , Ionosphere -- Research -- South Africa -- Hermanus (Cape of Good Hope)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5472 , http://hdl.handle.net/10962/d1005257 , Ionosondes , Ionosphere , Ionosphere -- Observations -- South Africa -- Hermanus (Cape of Good Hope) , Ionosphere -- Research -- South Africa -- Hermanus (Cape of Good Hope)
- Description: The DPS-4D is the latest version of digital ionosonde developed by the UMLCAR (University of Massachusetts in Lowell Center for Atmospheric Research) in 2008. This new ionosonde has advances in both the hardware and software which allows for the promised advanced capabilities. The aim of this thesis was to present results from an experiment undertaken using the Hermanus DPS-4D (34.4°S 19.2°E, South Africa), the first of this version to be installed globally, to answer a science question outside of the normally expected capabilities of an ionosonde. The science question posed focused on the ability of the DPS-4D to provide information on day-time Pc3 pulsations evident in the ionosphere. Day-time Pc3 ULF waves propagating down through the ionosphere cause oscillations in the Doppler shift of High Frequency (HF) radio transmissions that are correlated with the magnetic pulsations recorded on the ground. Evidence is presented which shows that no correlation exists between the ground magnetic pulsation data and DPS-4D ionospheric data. The conclusion was reached that although the DPS-4D is more advanced in its eld of technology than its predecessors it may not be used to observe Pc3 pulsations.
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- Date Issued: 2011
Neogene fluvial deposits along the south-west coast of South Africa understanding the palaeoclimate through proxies
- Authors: Sciscio, Lara
- Date: 2011
- Subjects: Alluvium , Paleoclimatology -- South Africa , Paleoecology -- Miocene -- South Africa , Paleobotany -- Miocene -- South Africa , Paleogeography -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4981 , http://hdl.handle.net/10962/d1005593 , Alluvium , Paleoclimatology -- South Africa , Paleoecology -- Miocene -- South Africa , Paleobotany -- Miocene -- South Africa , Paleogeography -- South Africa
- Description: Branched glycerol dialkyl glycerol tetraether (GDGTs) membrane lipids have been used as a new proxy for the reconstruction of terrestrial palaeoclimates. These biomarkers (or molecular ‗fossils‘) in conjunction with palynology, have been effective in the novel analysis of Miocene organic-rich sediments from three South African west coast sites at Rondeberg, Noordhoek and Langebaanweg. Lastly, a Quaternary south coast site at Rietvlei, South Africa, was also studied to further elucidate the extent of use of this new proxy. The fluvial peat and organic-rich deposits of the Elandsfontyn Formation (Sandveld Group) were investigated at Noordhoek, Langebaanweg and Rondeberg to provide new evidence for the climate and vegetation patterns during Miocene in this region. Drill-core and quarry samples from all four sites were freeze-dried, powered, and prepared for biogeochemical and palynological analyses. The methylation index of branched tetraethers (MBT) and cyclisation ratio of branched tetraethers (CBT) proxies were used to calculate the mean annual air temperature (MAAT) and pH values of the organic-rich horizons at time of deposition. The Branched versus isoprenoid index of tetraethers (BIT) was used to assess the relative contributions of marine archaeal and terrestrial bacterial tetraethers, and thereby assess the validity of the MBT, CBT and calculated palaeoenvironmental factors. The results presented in this thesis suggest that the use of the MBT/CBT proxy has significant potential in southern Africa, and may complement previously attempted palaeoclimatic and palaeoecological studies of Neogene-aged South African sediments. This type of research has the capacity to provide palaeoenvironmental information where other proxies may be absent. Results indicate that all sites yielded branched tetraether membrane lipids with the exception of Rondeberg, where GDGTs were below detection as a result of poor preservation conditions. Palynological investigation confirmed proxy derived temperatures. Furthermore palynomorph analyses supplemented earlier studies of the Noordhoek site and were piloted for the Rondeberg site, reaffirming alternating sequences of tropical and subtropical palynofloras. The MAATs, likewise, show variability and pronounced trends through time at the Langebaanweg and Noordhoek sites, generally corresponding with the variation and diversity of the pollen population. The terrestrial MAAT results appear to compliment Southern Hemisphere sea level changes associated with Antarctic glaciations. Additionally, this data shows a pattern similar to the Southern and Northern Hemisphere marine isotope records of relative fluctuations in the global climate and sea level change from the early to middle Miocene. The application of these past climate change indicators have been proved to be useful in the reconstruction of South Africa Miocene palaeoclimates, and may aid in understanding the consequences of climate change in the Cape region.
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- Date Issued: 2011
Bluetooth audio and video streaming on the J2ME platform
- Authors: Sahd, Curtis Lee
- Date: 2011 , 2010-09-09
- Subjects: Bluetooth technology , Mobile communication systems , Communication -- Technological innovations , Communication -- Network analysis , Wireless communication systems , L2TP (Computer network protocol) , Computer network protocols , Streaming audio , Streaming video
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4633 , http://hdl.handle.net/10962/d1006521 , Bluetooth technology , Mobile communication systems , Communication -- Technological innovations , Communication -- Network analysis , Wireless communication systems , L2TP (Computer network protocol) , Computer network protocols , Streaming audio , Streaming video
- Description: With the increase in bandwidth, more widespread distribution of media, and increased capability of mobile devices, multimedia streaming has not only become feasible, but more economical in terms of space occupied by the media file and the costs involved in attaining it. Although much attention has been paid to peer to peer media streaming over the Internet using HTTP and RTSP, little research has focussed on the use of the Bluetooth protocol for streaming audio and video between mobile devices. This project investigates the feasibility of Bluetooth as a protocol for audio and video streaming between mobile phones using the J2ME platform, through the analysis of Bluetooth protocols, media formats, optimum packet sizes, and the effects of distance on transfer speed. A comparison was made between RFCOMM and L2CAP to determine which protocol could support the fastest transfer speed between two mobile devices. The L2CAP protocol proved to be the most suitable, providing average transfer rates of 136.17 KBps. Using this protocol a second experiment was undertaken to determine the most suitable media format for streaming in terms of: file size, bandwidth usage, quality, and ease of implementation. Out of the eight media formats investigated, the MP3 format provided the smallest file size, smallest bandwidth usage, best quality and highest ease of implementation. Another experiment was conducted to determine the optimum packet size for transfer between devices. A tradeoff was found between packet size and the quality of the sound file, with highest transfer rates being recorded with the MTU size of 668 bytes (136.58 KBps). The class of Bluetooth transmitter typically used in mobile devices (class 2) is considered a weak signal and is adversely affected by distance. As such, the final investigation that was undertaken was aimed at determining the effects of distance on audio streaming and playback. As can be expected, when devices were situated close to each other, the transfer speeds obtained were higher than when devices were far apart. Readings were taken at varying distances (1-15 metres), with erratic transfer speeds observed from 7 metres onwards. This research showed that audio streaming on the J2ME platform is feasible, however using the currently available class of Bluetooth transmitter, video streaming is not feasible. Video files were only playable once the entire media file had been transferred.
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- Date Issued: 2011
Universal approximation properties of feedforward artificial neural networks.
- Authors: Redpath, Stuart Frederick
- Date: 2011
- Subjects: Neural networks (Computer science) , Artificial intelligence -- Biological applications , Functional analysis , Weierstrass-Stone Theorem , Banach-Hahn theorem
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5430 , http://hdl.handle.net/10962/d1015869
- Description: In this thesis we summarise several results in the literature which show the approximation capabilities of multilayer feedforward artificial neural networks. We show that multilayer feedforward artificial neural networks are capable of approximating continuous and measurable functions from Rn to R to any degree of accuracy under certain conditions. In particular making use of the Stone-Weierstrass and Hahn-Banach theorems, we show that a multilayer feedforward artificial neural network can approximate any continuous function to any degree of accuracy, by using either an arbitrary squashing function or any continuous sigmoidal function for activation. Making use of the Stone-Weirstrass Theorem again, we extend these approximation capabilities of multilayer feedforward artificial neural networks to the space of measurable functions under any probability measure.
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- Date Issued: 2011
The genetic integrity of Labeo capensis and L. umbratus (Cyprinidae) in South Africa in relation to inter-basin water transfer schemes
- Authors: Ramoejane, Mpho
- Date: 2011
- Subjects: Cyprinidae -- South Africa -- Darlington Dam , Cyprinidae -- Namibia -- Hardap Dam , Labeo -- South Africa -- Darlington Dam , Labeo -- Namibia -- Hardap Dam , Fishes -- Hybridisation -- South Africa -- Darlington Dam , Fishes -- Hybridisation -- Namibia -- Hardap Dam , Fishes -- Anatomy -- South Africa -- Darlington Dam , Fishes -- Anatomy -- Namibia -- Hardap Dam , Water transfer -- South Africa-- Darlington Dam , Water transfer -- Namibia -- Hardap Dam
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5367 , http://hdl.handle.net/10962/d1013049
- Description: The Orange-Fish and Cookhouse tunnels that are part of a major inter-basin water transfer scheme (IBT) act as a pathway for several fish species from the Orange River system to enter the Great Fish and Sundays River systems in South Africa. These include Labeo capensis and L. umbratus. Labeo capensis was restricted to the Orange River system before the inter-basin water transfer scheme. Labeo umbratus occurred naturally in the Orange River and in southern flowing river systems. Previous studies showed that the two species hybridise in Hardap Dam, located in a tributary of the Orange River system in Namibia. There are also unconfirmed reports of hybrids from Darlington Dam on the Sundays River system. The aim of the thesis was to confirm hybridisation in Hardap Dam, assess whether hybridisation between L. capensis and L. umbratus has occurred in Darlington Dam and to gain a better understanding of the diversity of these two species. Morphology (morphometrics and meristics), a nuclear S7 intron and the mitochondrial cytochrome ♭ gene were used to assess for hybridisation. A total of 275 specimens were analysed from across the geographical range of the two species. The two species could be distinguished using morphometrics (dorsal fin base, interorbital width and operculum to eye distance) and meristics (lateral line, origin of the dorsal fin to lateral line, origin of the pelvic fin to lateral line and caudal peduncle scale counts) characters. Hybrids from Hardap and Darlington dams were placed between the two species clusters. Labeo umbratus from the Orange River and southern flowing rivers formed a single cluster. The two species could also be distinguished from each other with six nuclear DNA mutations and hybrids were heterozygous at such sites in both dams. Labeo umbratus populations from the Orange River and southern flowing rivers (Gouritz, Gamtoos, Sundays, Bushmans, Great Fish and Nahoon) formed a single lineage. Analysis of mitochondrial DNA, however, revealed that L. umbratus populations from the Orange River and southern flowing rivers were two lineages that differ from each other by 5 mutations. Labeo capensis could be differentiated from both these lineages. Being maternally inherited, mitochondrial DNA did not reveal hybridisation, but ten specimens with L. capensis haplotypes were found in the Darlington Dam. In Hardap Dam, however, it appears that only L. capensis mitochondrial DNA haplotypes persist, despite morphological and nuclear DNA analysis suggesting that both morphs and hybrids of the two species occur. The genetic integrity of these Labeo species has therefore been compromised in at least Hardap and Darlington dams. The Great Fish and Sundays populations are considered to be under threat of complete introgression. The Kat River and Slagboom Dam populations that were isolated before the IBTs have to remain isolated to protect the genetic integrity of the southern lineage of L. umbratus in these two systems.
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- Date Issued: 2011
An ion imprinted polymer for the determination of Ni (II) ions from mine tailing samples
- Authors: Rammika, Modise
- Date: 2011
- Subjects: Imprinted polymers , Metal ions , Polymerization , Mineral industries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4323 , http://hdl.handle.net/10962/d1004981 , Imprinted polymers , Metal ions , Polymerization , Mineral industries -- Waste disposal
- Description: A Ni(II)-dimethylglyoxime ion imprinted polymer {Ni(II)-DMG IIP} was synthesized by the trapping method using the bulk polymerisation format. The structures of the imprinted and non-imprinted polymer were evaluated by infrared spectroscopy and the morphology was observed by scanning electron microscopy. The Ni(II)-DMG IIP was optimised for pH, mass, time and by the uniform design experimental method for the molar ratios of monomer to crosslinker to porogen and template to ligands as well as keeping these parameters constant and varying the quantities of initiator, 2,2'-azobisisobutyronitrile (AIBN). The optimum pH was 8.5, optimum mass was 50 mg, optimum time was 1 min and the optimum molar ratios of crosslinker to monomer, monomer to template and nickel(II) sulfate hexahydrate (NiSO₄.6H₂O) to 4-vinylpyridine to dimethylglyoxime were found to be 3.3:1.0, 0.6:1.0 and 1.0:0.6:3.6 respectively with 30 mg and 8 mL as the optimum amounts of initiator and porogen respectively. Through this optimisation, recovery of Ni(II) was increased from 98 to 100%. Selectivity of the ion imprinted polymer was evaluated by analysing, using an inductively coupled plasma-optical emission spectrometer, for Ni(II) ions that were spiked with varying concentrations of Co(II), Cu(II), Zn(II), Pd(II), Fe(II), Ca(II), Mg(II), Na(I) and K(I) in aqueous samples. Selectivity studies also confirmed that the ion imprinted polymer had very good selectivity characterised by % RSD of less than 5 %. Co(II) was the only ion found to slightly interfere with the determination of Ni(II). The limits of detection and quantification were found to be 3x10⁻⁴ μg/mL and 9x10⁻⁴ μg/mL respectively. The method was evaluated by a custom solution of ground water certified reference material (SEP-3) and sandy soil reference material (BCR-142R) and the concentrations of Ni(II) obtained were not significantly different to the certified ones. The Ni(II)-DMG IIP was then evaluated in aqueous and soil samples where recoveries of 93 to 100% and 98 to 99% respectively were obtained with enrichment factors ranging from 2 to 18 in aqueous and 27 to 40 in soil samples. Finally, the Ni(II)-DMG IIP was used to analyse mine tailings samples and Ni(II) recovery of 99% was obtained with an enrichment factor of 2.
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- Date Issued: 2011
Solid-phase extraction based sample preparation for the determination of drug and organic pollutant residue
- Authors: Pule, Bellah Oreeditse
- Date: 2011 , 2011-02-08
- Subjects: Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4406 , http://hdl.handle.net/10962/d1006711 , Food contamination , Drugs -- Analysis , Pharmaceutical chemistry , Extraction (Chemistry) , Sorbents
- Description: This thesis presents solid phase extraction (SPE) methodologies based on mixed-mode polymeric sorbents; a mixed mode strong anion exchanger (Agilent SampliQ SAX) and a mixed mode strong cation exchanger (Agilent SampliQ SCX). Furthermore, dispersive-SPE based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) method was assessed for applicability in the determination of drug residues. The mixed-mode polymeric sorbents were evaluated for the simultaneous fractionation of drugs that exhibit diverse polarities with acidic, basic and neutral functionalities in biological matrices (plasma and urine). The polymeric skeleton of these sorbents entails an exchanger group and therefore provides two retention mechanisms, strong cation or anion exchange retention mechanisms with hydrophobic interactions. It was demonstrated that with a sequential elution protocol for sample clean-up analytes were fractionated into acidic, basic and neutral classes. The SAX was employed for analysis of ketoprofen, naproxen (acidic drugs), nortriptyline (basic) and secobarbital (neutral) from urine sample. The SCX was used for fractionating phenobarbital, p-toluamide (acidic), amphetamine, m-toluidine (basic) and acetaminophen (neutral drug) from plasma sample. QuEChERS method was employed for quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) from fish fillets and soil; 9 sulfonamides (SAs) from chicken muscles and acrylamide (AA) in cooking oil. The analyte recoveries ranged from 79.6 - 109% with RSDs ranging from 0.06 - 1.9% at three different fortification levels. Good linearity (r2 > 0.9990) was attained for most analytes. The limits of detection and quantification ranged from 0.03 - 0.84 μg/ml and 0.81 - 1.89 μg/ml respectively for analytes in biological samples. LODs and LOQs for analytes in food and environmental samples ranged from 0.02 to 0.39 and 0.25 to 1.30 ng/g respectively.
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- Date Issued: 2011
Investigations of the assessment of bioequivalence of topical clotrimazole products using a dermatopharmacokinetic approach
- Authors: Parfitt, Natalie Rae
- Date: 2011 , 2010-07-05
- Subjects: Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3840 , http://hdl.handle.net/10962/d1007659 , Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Description: The specialised nature of the stratum corneum makes it an efficient barrier to foreign substances, including drug molecules. Therefore, cutaneous drug absorption is a slow and complex process of which stratum corneum penetration is the rate limiting step. The rate and extent of stratum corneum penetration by a drug compound depends greatly on the presence of penetration enhancing/retarding excipients and therefore the clinical outcomes of a product rely greatly on the components and quality of the formulation. Hence, establishing bioequivalence between topical products is crucial to ensure that patients receiving multisource drug products are assured of the same efficacy and safety as the brand product. Since locally acting topical formulations do not target the systemic circulation, conventional methods of assessing bioequivalence using plasma levels are not appropriate. Consequently, the current regulatory guidelines require comparative clinical trials to be carried out to show bioequivalence between topical products. As these studies are very expensive and time consuming, the development of a more direct and relatively rapid and inexpensive method for determining bioequivalence between topical products is required. Clotrimazole is an anti-fungal agent where the target site of action is in the stratum corneum. In this work, tape stripping, which involves the sampling of stratum corneum, was investigated as a tool for the determination of bioequivalence between topical clotrimazole products. The tape stripping method involved the analysis of each tape strip individually and standardization of stratum corneum thickness between subjects was carried out using TEWL measurements. This approach provided detailed information regarding the amount of clotrimazole present in the stratum corneum as well as the extent of drug penetration. Prior to the tape stripping studies an HPLC method was developed for the quantitative analysis of clotrimazole from the tape strip samples. This method was shown to be accurate and reproducible across the required range. It was also shown to be selective for clotrimazole in the presence of possible interfering substances such as those present in the tape adhesive and also skin components. The bioequivalence studies were conducted using a single “uptake” time point. In order to determine an appropriate dose duration for these studies a novel approach was employed, involving a preliminary dose duration study. For the bioequivalence investigations, Canesten® Topical cream was used as both test and reference products to determine if the method was capable of showing bioequivalence. Subsequently, Canesten® Topical cream was also compared to a 1% gel formulation to determine if the method could detect formulation differences. The conventional BE limits of 0.8 – 1.25 were used for the assessment of BE, however, the clinical relevance of using these limits for dermal studies is debatable since they are derived from oral pharmacokinetic studies. Therefore, the data from the tape stripping investigations were also assessed using more realistic limits of 0.75 – 1.33 and even 0.7 – 1.44. In addition to the tape stripping studies a novel method of determining the amount of drug present in the stratum corneum, the “Residual Method”, was investigated. This method involved assaying the amount of clotrimazole found in the residual formulation after a specified dose duration had elapsed and subtracting that amount from the amount of clotrimazole initially applied. The results of tape stripping investigations showed that, if the study is sufficiently powered, tape stripping may be used to determine bioequivalence according to the conventional limits, as well as possibly detect formulation differences between different clotrimazole products. Bioequivalence assessment using the widened intervals showed that fewer subjects were required to achieve a sufficient statistical power. The variability associated with this method was acceptable and tape stripping may therefore have the potential to be used as a BE tool in a regulatory setting for clotrimazole or other antifungal topical formulations. The “Residual Method” also showed promising results as a bioequivalence tool, but further investigation and extensive validation of this method is required before it can be suggested as a regulatory method. The results of these studies have clearly indicated that tape stripping has the potential to be used as an alternative to comparative clinical trails for the assessment of bioequivalence between clotrimazole formulations and also to assess bioequivalence between other antifungal products.
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- Date Issued: 2011
Neuronal nitric oxide synthase : a biomarker for Alzheimers disease : interaction of neuronal nitric oxide synthase with beta-amyloid peptides in the brain
- Authors: Padayachee, Eden Rebecca
- Date: 2011 , 2013-07-19
- Subjects: Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4086 , http://hdl.handle.net/10962/d1007677 , Alzheimer's disease , Nitric-oxide synthase , Biochemical markers , Amyloid beta-protein , Peptide hormones
- Description: High levels of the amino acid arginine and low levels of the product citrulline in the cerebrospinal fluid of Alzheimer's patients could mean that there is a decrease in the enzymes that metabolize this amino acid. One such enzyme is neuronal nitric oxide synthase (nNOS). In this study, neuronal nitric oxide synthase (nNOS), sourced from bovine brain was extracted and concentrated using two methods of precipitation: poly (ethylene glycol) 20 000 (PEG) and ammonium sulphate [(NH₄)₂S0₄). These two techniques gave no increase in yield nor fold purification and hence were abandoned in favour of ion exchange chromatography by DEAE-Sepharose. The enzyme was then successfully purified by anion-exchange and after dialysis produced a 38% yield and three fold purification and yielded the highest specific activity of 2.27 U/mg. Neuronal nitric oxide synthase (nNOS) was a heterodimeric protein with a total molecular mass of ± 225 kDa (95 and 130 kDa monomers). The temperature and pH optima of the enzyme were 40⁰C and 6.5, respectively. The kinetic parameters (KM and Vmax) of nNOS were 70 μM and 0.332 μmol.min⁻¹, respectively. Moreover neuronal nitric oxide synthase (nNOS) was relatively stable at 40⁰C (t½ = 3 h). It was also confirmed that β-amyloid peptides inhibited nNOS when bound to the enzyme and that nNOS behaved as a catalyst in fibril formation through association-dissociation between enzyme and β-amyloid peptide. It was further shown that Aβ₁₇₋₂₈ inhibited nNOS the most with a Ki of 1.92 μM and also had the highest Stern-Volmer value (Ksv) of 0.11 μM⁻¹ indicating tight binding affinity to nNOS and easier accessibility to fluor molecules during binding. Congo red, turbidity, thioflavin-T assays and transmission electron microscopy were successfully used to detect and visualize the presence of fibrils by studying the process of fibrillogenesis. Computerized molecular modeling successfully studied protein dynamics and conformational changes of nNOS. These results correlated with resonance energy transfer (FRET) results which revealed the distance of tryptophan residues from the arginine bound at enzyme active site. Both the aforementioned techniques revealed that in the natural state of the enzyme with arginine bound at the active site, the tryptophan residues (TRP₆₂₅ and TRP₇₂₁) were positioned at the surface of the enzyme 28 Å away from the active site. When the amyloid peptide (Aβ₁₇₋₂₈) was bound to the active site, these same two amino acids moved 14 Å closer to the active site. A five residue hydrophobic fragment Aβ₁₇₋₂₁ [Leu₁₇ - Val₁₈ - Phe₁₉ - Phe₂₀ - Ala₁] within Aβ₁₇₋₂₈ was shown by computer modeling to be critical to the binding of the peptide to the active site of nNOS.
- Full Text:
- Date Issued: 2011
Characterization of stratified L-topological spaces by convergence of stratified L-filters
- Authors: Orpen, David Lisle
- Date: 2011
- Subjects: Topology , Generalized spaces , Filters (Mathematics) , Topological spaces
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5402 , http://hdl.handle.net/10962/d1005216 , Topology , Generalized spaces , Filters (Mathematics) , Topological spaces
- Description: For the case where L is an ecl-premonoid, we explore various characterizations of SL-topological spaces, in particular characterization in terms of a convergence function lim: FS L(X) ! LX. We find we have to introduce a new axiom , L on the lim function in order to completely describe SL-topological spaces, which is not required in the case where L is a frame. We generalize the classical Kowalski and Fischer axioms to the lattice context and examine their relationship to the convergence axioms. We define the category of stratified L-generalized convergence spaces, as a generalization of the classical convergence spaces and investigate conditions under which it contains the category of stratified L-topological spaces as a reflective subcategory. We investigate some subcategories of the category of stratified L-generalized convergence spaces obtained by generalizing various classical convergence axioms.
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- Date Issued: 2011
Application of macroinvertebrate based biomonitoring approaches to assess anthropogenic impacts in the Swartkops River, South Africa
- Authors: Odume, Oghenekaro Nelson
- Date: 2011
- Subjects: Water -- Pollution -- South Africa -- Swartkops River Water quality biological assessment -- South Africa -- Swartkops River Environmental monitoring -- South Africa -- Swartkops River Aquatic invertebrates -- Effect of water pollution on -- South Africa -- Swartkops River
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:6040 , http://hdl.handle.net/10962/d1006199
- Description: A growing human population accompanied by urbanisation and industrialisation have led to over exploitation and pollution of freshwater resources and have consequently impacted on aquatic ecosystem health. The Swartkops River in the Eastern Cape of South Africa is no exception. It drains a heavily industrialised catchment which has led to deterioration of its water quality due to pollution. Integrated water resources management (IWRM) requires the concurrent sustainable use of water resources and the protection of aquatic ecosystem health. Macroinvertebrates are well known for their ability to reflect the health of the environment in which they live, thus they were used to assess anthropogenic impacts in the Swartkops River for this study. Macroinvertebrate based biomonitoring approaches, including the South African Scoring System version 5 (SASS5); a multimetric approach involving 19 metrics; Chironomidae community assessments and screening of morphological deformities in Chironomidae larvae, were applied at four selected sampling sites to assess environmental water quality in the Swartkops River. Macroinvertebrates were sampled us ing the SASS5 protocols. Chironomidae were mounted and identified as far as practically possible using available keys. Mentum, ligula, mandible, paraligula and antenna in Chironomidae larvae were screened for deformities. Physical and chemical water quality variables were measured at each of the selected sampling sites. All data were subjected to relevant statistical analyses. Of the four sites sampled during the study period, results revealed that water quality at site 1 was the least impacted with highest SASS5 scores, average score per taxa (ASPT) values, richness, diversity, equitability and Ephemeroptera –Plecoptera-Trichoptera (EPT) richness, as well as least incidences of chironomid deformities. Water quality at site 2 was considered the next least impacted with higher SASS5 scores, A SPT values, richness, diversity and equitability, and lower incidences of deformities compared to sites 3 and 4. SASS5 scores and ASPT values revealed that both sites 3 and 4 were critically modified but the multimetric analysis, Chironomidae community assessment and incidences of deformities in Chironomidae larvae indicated that site 3 is the most impacted of the four sampling sites, with least species diversity, richness, equitability and highest incidences of deformities. The study revealed the importance of multicriteria approach to environmental biomonitoring as an integrated water resources management tool, and based on the results, site 3, as the most impacted, could be prioritised for restoration intervention.
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- Date Issued: 2011
Investigating the role of heat shock proteins (Hsps) 40, 70 and 90 in the life cycle of Theiler's murine encephalomyelitis virus (TMEV)
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2011
- Subjects: Heat shock proteins , Picornaviruses , Encephalomyelitis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3966 , http://hdl.handle.net/10962/d1004025 , Heat shock proteins , Picornaviruses , Encephalomyelitis
- Description: Introduction: Picornaviruses are a family of RNA viruses which are economically and clinically significant. Like many other viruses, picornaviruses utilise host cell machinery to facilitate their replication and assembly, including heat shock proteins (Hsps). The aim of this research was to investigate the role of Hsp40, Hsp70 and Hsp90 during picornavirus infection using the cardiovirus, Theiler’s murine encephalomyelitis virus (TMEV), as a study model. Methodology: Picornavirus VP1 capsid proteins were analysed by multiple sequence alignment and multiple structural comparisons. Protein domain architecture was used to analyse Hsp90 cellular and viral client proteins. Effects of Hsp90 inhibitors, novobiocin and geldanamycin, on TMEV growth in BHK-21 cells was observed over a 48hr period. Localisation of Hsp40, Hsp90 and Hsp70 in TMEV-infected BHK-21 cells was investigated by indirect immunofluorescence and confocal microscopy. Results and Discussion: VP1 proteins of picornaviruses are highly divergent within the family at the amino acid level, which might be linked to the protein’s function in determining virus tropism and antibody neutralisation. An eight-stranded anti-parallel beta-barrel structure was found conserved in the VP1 protein structures which might be linked to the highly conserved picornavirus capsid assembly process. Absence of a common protein domain between Hsp90 viral and cellular client proteins that might be functionally connected to Hsp90, suggests that Hsp90 most likely recognises surface features rather than sequence motifs/patterns. The Hsp90 inhibitors, novobiocin and geldanamycin, had a negative effect on virus growth as virus-induced cytopathic effect was not observed in treated cell after 48hrs. TMEV 2C protein was detected by Western analysis in infected cell lysates treated with geldanamycin but not novobiocin, suggesting novobiocin affects the translation or processing of TMEV 2C. Immunofluorescence analysis of TMEV-infected cells showed a relocalisation of Hsp40 into the nucleus during infection. Overlap of Hsp40 and TMEV P1 was observed in the perinuclear region, suggesting colocalisation between these proteins. Hsp70 converged around the replication complex during infection but did not overlap with TMEV 2C. Hsp90 concentrated in the region of the replication complex where it overlapped with TMEV 2C and this redistribution was found to be dependent on the stage of infection. The overlap between Hsp90 and TMEV 2C signals observed, suggested colocalisation between the two proteins. Conclusion: This study identified Hsp90, Hsp70 and Hsp40 as possible host factors required in TMEV replication.
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- Date Issued: 2011
A platform for computer-assisted multilingual literacy development
- Authors: Mudimba, Bwini Chizabubi
- Date: 2011
- Subjects: FundaWethu , Language acquisition -- Computer-assisted instruction , Language arts (Elementary) -- Computer-assisted instruction , Language and education , Education, Bilingual , Computer-assisted instruction , Educational technology , Computers and literacy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4600 , http://hdl.handle.net/10962/d1004850 , FundaWethu , Language acquisition -- Computer-assisted instruction , Language arts (Elementary) -- Computer-assisted instruction , Language and education , Education, Bilingual , Computer-assisted instruction , Educational technology , Computers and literacy
- Description: FundaWethu is reading software that is designed to deliver reading lessons to Grade R-3 (foundation phase) children who are learning to read in a multilingual context. Starting from a premise that the system should be both educative and entertaining, the system allows literacy researchers or teachers to construct rich multimedia reading lessons, with text, pictures (possibly animated), and audio files. Using the design-based research methodology which is problem driven and iterative, we followed a user-centred design process in creating FundaWethu. To promote sustainability of the software, we chose to bring teachers on board as “co-designers” using the lesson authoring tool. We made the authoring tool simple enough for use by non computer specialists, but expressive enough to enable a wide range of beginners reading exercises to be constructed in a number of different languages (indigenous South African languages in particular). This project therefore centred on the use of designbased research to build FundaWethu, the design and construction of FundaWethu and the usability study carried out to determine the adequacy of FundaWethu.
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- Date Issued: 2011
Assessment of organochlorine pesticide residues in fish samples from the Okavango Delta, Botswana
- Authors: Mpofu, Christopher
- Date: 2011 , 2011-02-28
- Subjects: Pesticides , Organochlorine compounds , Freshwater fishes -- Effect of pesticides on -- Botswana -- Okavango River Delta , Water -- Pesticide content -- Botswana -- Okavango River Delta , Catfishes -- Botswana -- Okavango River Delta , Tigerfish -- Botswana -- Okavango River Delta , Redbreast tilapia -- Botswana -- Okavango River Delta
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4404 , http://hdl.handle.net/10962/d1006707 , Pesticides , Organochlorine compounds , Freshwater fishes -- Effect of pesticides on -- Botswana -- Okavango River Delta , Water -- Pesticide content -- Botswana -- Okavango River Delta , Catfishes -- Botswana -- Okavango River Delta , Tigerfish -- Botswana -- Okavango River Delta , Redbreast tilapia -- Botswana -- Okavango River Delta
- Description: This thesis presents an evaluation of the dispersive solid-phase extraction (d-SPE) method referred to as the quick, easy, cheap, effective, rugged and safe (QuEChERS) method for the determination of four organochlorine pesticide residues in fish samples. The pesticides investigated in this study were o, p′-DDT, p, p′-DDE, aldrin and dieldrin. The combined use of Gas Chromatography with an Electron Capture Detector (GC-ECD) and sensitive Time of Flight (TOF) mass detector facilitated the identification of the target analytes. In the absence of certified reference material, the overall analytical procedure was validated by systematic recovery experiments on spiked samples at three levels of 2, 5 and 10 ng/g. The targeted compounds were successfully extracted and their recovery ranged from 76 to 96% with relative standard deviations of less than 13%. The optimum QuEChERS conditions were 2g of fish powder, 10 ml acetonitrile and 1 min shaking time. The optimal conditions were applied to assess the levels of chlorinated pesticides in blunt-tooth catfish (Clarias ngamensis), tigerfish (Hydrocynus vittatus), Oreochromis andersonii and red-breasted tilapia (Tilapia rendalli) from the Okavango Delta, Botswana. Dieldrin, p, p′-DDE and aldrin were detected in all the analysed samples with a concentration range of 0.04 – 0.29, 0.07 – 0.33, 0.04 – 0.28 and 0.03 – 0.24 ng/g per dry weight in O. andersonii, C. ngamensis, T. rendalli and H. vittatus respectively. These concentrations were below the US-EPA 0.1 μg/g allowable limit in edible fish and the Australian Maximum Residue Limit (MRL) of 50 - 1000 ng/g in fresh fish. DDT was not detected in all the fish species investigated. The mean lipid content recorded in the fish samples were 1.24, 2.16, 2.18 and 4.21% for H. vittatus, T. rendalli, O. andersonni and C. ngamensis respectively. No systematic trend was observed between fish age and pesticide levels in fish. Acetylcholinesterase (AChE) activity assays were performed to assess the effects of organochlorine pesticides in Clarias ngamensis. The enzyme activity recorded in Clarias ngamensis from the Okavango Delta and the reference site was 12.31 μmol of acetylcholine iodide hydrolysed/min/g brain tissue. The enzyme activity remained the same indicating no enzyme inhibition. The conclusions drawn from this study are that the QuEChERS method is applicable for the determination of organochlorine pesticide residues in fish matrices. The fish from the Okavango Delta are safe for human consumption.
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- Date Issued: 2011
Responses of intertidal macroalgae and associated fauna to interactive processes acting over multiple spatial scales
- Authors: Mostert, Bruce Petrus
- Date: 2011 , 2010-12-01
- Subjects: Biodiversity -- South Africa , Spatial ecology , Marine algae -- South Africa , Marine organisms -- Effect of water pollution on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5890 , http://hdl.handle.net/10962/d1013370
- Description: The decrease of biological diversity from low latitudes near the equator towards high latitudes is one of the most fundamental patterns noted in ecology. These trends have been the subject of numerous terrestrial and marine studies and have caused the proposal of several explanatory hypotheses. No single hypothesis has adequately accounted for these observed trends. Furthermore, large-scale patterns are frequently modified by processes acting at meso- to small-scales. It is imperative to understand the interaction of these processes to elucidate the mechanisms controlling the structure of intertidal assemblages. The main aim of this thesis was to test the influence of multiple processes at a range of spatial scales on biogenic engineers. Biogenic engineers, such as macroalgae, have been reported to be strongly influenced by processes such as grazing, biogeography and upwelling and subsequent effects are expected to be linked to their associated assemblages. I used infaunal assemblages associated with macroalgae as a model system to understand the interactive effects of meso-scale upwelling in conjunction with large-scale factors (regional and biogeographic). Three species of macroalgae, Hypnea spicifera, Champia lumbricalis and Bifurcaria brassicaeformis, inhabiting different but overlapping home ranges were used in this study. Smaller scale, physical attributes and the associated fauna of these algal species were compared. The effects of meso- to large-scale physical gradients on marine organisms have been investigated in many instances and resultant gradients in physical variables observed. This study was undertaken to investigate morphological trends exhibited by Hypnea spicifera and Champia lumbricalis to compare possible similarities between coastlines and to determine if inferred meso- to large-scale processes influence physical trends in a similar manner despite the coastlines being affected by different hydrodynamic processes. The macroalgae were located in two distinctly different upwelling regimes, Hypnea spicifera on the east and south coasts and Champia lumbricalis on the west coast. Upwelling (delivery of nutrients and primary production) was not found to be an important factor in determining the physical characteristics of macroalgae but there were clear patterns linked to latitude. Mean percentage cover of Hypnea spicifera was positively correlated with distance along the east and south coasts. Mean mass and mean surface area of H. spicifera were, however, negatively correlated with distance from Port St Johns. Mean frond length showed a negative trend but this relationship was not statistically significant. On the west coast, there was a significant increase in mean frond length from north to south for Champia lumbricalis. Mean surface area and mean mass of C. lumbricalis exhibited non-significant negative trends from north to south while there was a non-significant positive trend for mean percentage cover. Following the “Productivity Hypothesis”, these patterns from north to south along the coastlines of South Africa could have important implications for biodiversity associated with these algae. Many previous studies have focussed on the effects of upwelling on species (i.e. effects of nutrients and temperature) but have failed to separate this effect from large scale effects such as biogeography and latitudinal gradients. I tested the influence meso-scale upwelling, large-scale biogeographic processes and latitudinal gradients with two different species of macroalgae one on the east and south coasts of South Africa and the other on the west coast. Hypnea spicifera inhabits the east and south coasts of South Africa spanning two biogeographic provinces and is generally affected by relatively weak upwelling, whereas Champia lumbricalis inhabits the west coast spanning one biogeographic province which is subjected to intense persistent upwelling year round. Within the east and west coast biogeographic provinces there are, however, regions with both upwelling and non-upwelling. On the east and south coasts, entire assemblages differed significantly among the three Regions (St Lucia, Port Alfred and Knysna, while assemblages between upwelling/non-upwelling areas were not different. Assemblages on the different shores differed significantly from each other. There were no significant effects of region or upwelling for the number of individuals of Crustacea or Polychaeta, while Mollusca showed a significant effect of region. The number of individuals of other taxa showed a significant interaction of region and upwelling. Region had a significant effect on number of species of molluscs and other taxa, while there was no effect for either region or upwelling for the Crustacea or Polychaeta. In general different factors were shown to be important (region and shore) while upwelling was rarely important. On the west coast assemblages associated with C. lumbricalis were not influenced by region or upwelling but there were significant differences between shores. Region, upwelling ad shore did not affect number of individuals. There was a significant interaction of region and upwelling for the number of species of crustaceans, while numbers of species of Mollusca, Polychaeta and other taxa showed no effects. In general, regional factors strongly influenced most organisms, while upwelling played a minor role. Hypotheses about differences between upwelling and non-upwelling regions in terms of species abundances and composition due to the input of cold nutrient rich water were not supported. A link between the effects of larger-scale biogeographic factors and their influence on habitat forming taxa and the resultant effects on associated infauna would be a likely explanation for the patterns observed in this study. Diversity of assemblages is known to be influenced by the structural complexity of a habitat, increasing complexity increases the amount of available niches therefore potentially increasing the number of species found within that habitat. Three species of macroalgae, Hypnea spicifera, Champia lumbricalis and Bifurcaria brassicaeformis were selected on the basis of being structurally similar and having their distributional ranges overlap in order to elucidate the effects of structure and macroalgal species on associated assemblages. Hypnea spicifera is different in terms of surface area and biomass from Champia lumbricalis and Bifurcaria brassicaeformis. Assemblages of species found on the three rhodophytes showed both shore and the species of alga had significant effects on composition. Only numbers of individuals of molluscs were affected by the species of alga. Crustacea and Polychaeta showed an interaction of the species of alga with shore. In the case of this study, it is likely that the three species of macroalgae mitigate biological stressors such as predation and physical stressors such as wave exposure and desiccation. In general, meso-scale upwelling is suggested to be marginalised when considering the structuring of assemblages associated with macroalgae, while large scale biogeography has more of an influence. Within shores, assemblages were also strongly influenced by smaller-scale factors such as differences in the structure and species of alga. This study indicates the importance of investigating patterns across a range of spatial scales to gain a comprehensive understanding of factors influencing intertidal organisms.
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- Date Issued: 2011
The development of an in vitro system for the production of drug metabolites using microsomal enzymes from bovine liver
- Authors: Morrison, Roxanne
- Date: 2011
- Subjects: Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4087 , http://hdl.handle.net/10962/d1007698 , Drugs -- Metabolism , Xenobiotics -- Metabolism , Metabolites , Drugs -- Testing , Toxicity testing -- In vitro , Doping in horse racing -- Control -- Research
- Description: Drug metabolism is a specialised subset of xenobiotic metabolism, pertaining to the breakdown and elimination of pharmaceutical drugs. The enzymes involved in these pathways are the cytochrome P450 family of isozymes. Metabolism is an important factor in determining the pharmacological effects of drugs. The main aim of this study was to develop a system whereby the major metabolites of drugs can be produced in vitro. An in vitro system was developed and optimised using commercially prepared microsomes from rat liver and coumarin (by monitoring its conversion to 7-hydroxycoumarin) as a model. The optimum running conditions for the incubations were 50 μM coumarin, 50 μg protein/ml microsomes, 1 mM NADP⁺, 5 mM G6P and 1U/ml G6PDH incubated for 30 minutes at 38℃. The HPLC method for the detection of coumarin and 7-hydroxycoumarin was also validated with respect to linearity, reproducibility, precision, accuracy and lower limits of detection and quantification. The system developed was then tested using microsomes prepared from fresh bovine liver on these ten drugs of interest in doping control in horse racing: diazepam, nordiazepam, oxazepam, promazine, acepromazine, chlorpromazine, morphine, codeine, etoricoxib and lumiracoxib. The bovine liver microsomes were prepared using differential centrifugation and had activity on a par with the commercial preparations. This in vitro system metabolised the drugs and produced both phase I and II metabolites, similar to those observed in humans and horses in vivo. For example, the major metabolites of the benzodiazepine drug, diazepam, nordiazepam, temazepam and oxazepam as well as the glucuronidated phase II products were all found after incubations with the bovine liver microsomes. The metabolism of the drugs was also investigated in silico using the computational procedure, MetaSite. MetaSite was able to successfully predict known metabolites for most of the drugs studied. Differences were observed from the in vitro incubations and this is most likely due to MetaSite using only human cytochrome P450s for analysis.
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- Date Issued: 2011
SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors
- Authors: Molawa, Letshego Gloria
- Date: 2011
- Subjects: Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3989 , http://hdl.handle.net/10962/d1004048 , Immobilized enzymes , Hydrolases , Hydrolysis , SphereZyme , Biosensors , Proteolytic enzymes
- Description: Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.
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- Date Issued: 2011