List of Titles

An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues

- Mkabayi, Lithalethu

Authors: Mkabayi, Lithalethu
Date: 2021-04
Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
Language: English
Type: thesis , text , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
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Date Issued: 2021-04

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Interaction of catechol O-methyltransferase with gold and silver nanoparticles

- Usman, Aminu

Authors: Usman, Aminu
Date: 2018
Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
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Date Issued: 2018

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The evaluation of potential dietary media, measurement parameters and storage techniques for use in forensic entomotoxicology

- Mbatha, Erica Isabel Tavares Da Silva

Authors: Mbatha, Erica Isabel Tavares Da Silva
Date: 2018
Subjects: Blowflies -- Feeding and feeds , Blowflies -- Larvae , Blowflies -- Physiology , Blowflies -- Collection and preservation , Poisons -- Analysis , Death -- Causes , Forensic pathology , Forensic entomology , Forensic entomotoxicology
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/63323 , vital:28393
Description: The term forensic entomotoxicology was coined by Pounder and is used to describe the process of using insects to determine the presence or absence of toxicants in decomposing corpses. Forensic entomotoxicology is most applicable when the orthodox sources of evidence (i.e. blood and urine) are no longer available for testing due to the degree of putrefaction as a result of the decomposition process. As the field is relatively new, various authors have conducted studies to determine the effects of different toxicants on different insects. These studies have all been conducted in the absence of a standardised protocol and we hypothesise that this has led to conflicting results (i.e. two different authors will conduct a study using the same toxicant and model insect and the effects on the insects will differ significantly). The aim of this thesis was to identify the areas which might have led to the artefacts in the results and identify ways in which to standardise them. The three areas selected were the feeding substrates and the measures taken to quantify growth rate, as well as the preservation techniques that should be used for preserving larval flies. The recommendation from the literature review was that artificial diets would be the most appropriate dietary media to use for entomotoxicological studies. An artificial diet was selected and modified for potential used in entomotoxicological studies. Four different diets (no meat treatment, fish, beef and pork artificial diets) were used to rear Chrysomya chloropyga larvae and their growth rates were measured using length and width. The fly larvae reared on the fish and no meat treatment diets did not reach pupation stage. The beef and pork diets produced the largest larvae and the flies in these treatments reached adult stage. The recommendation was that the beef and pork treatments be tested with various toxicants to establish their stability in the matrix and the diet that provides the toxicants with the most stability should be used for future entomotoxicological studies. The two other factors selected for standardisation were the parameters used to quantify growth rate, as well as the preservation techniques used to store empty Chrysomya chloropyga pupal casings and Calliphora croceipalpis third instar larvae. Previous authors have suggested that width be used as an alternative to length to quantify growth rate. The results from this thesis show that length should continue to be used as the standard parameter because the incremental change in length is much larger than the change in width, and these larger increments allow for greater resolution when estimating the age of the larvae. Various authors have also suggested that pupal casings should be stored without any preservative, whereas fly larvae should be stored in concentrations of ethanol >70%. The results in this thesis have shown that the concentration of ethanol does not make any significant difference to the proportional change of length and width of the empty pupal casings and the third instar larvae. The recommendation is that when selecting the preservation technique, the integrity of the specimen for examination of other evidence (i.e. DNA or toxicological extraction) should take precedence. Although this thesis has not completely standardised the protocol for forensic entomotoxicology, it has indicated the areas that need to be focused on in order for standardisation to occur. Future studies should focus on standardisation, as this makes studies more comparable and ultimately makes entomotoxicological evidence admissible in the court of law.
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Date Issued: 2018

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Composition and fate of triclosan in the sludge from wastewater treatment in Grahamstown, South Africa and Tiaret, Algeria

- Ncube, Mbonisi

Authors: Ncube, Mbonisi
Date: 2017
Subjects: Sewage sludge , Sewage Purification South Africa Grahamstown , Sewage Purification Algeria Tiaret , Sewage sludge as fertilizer , Anti-infective agents
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/65156 , vital:28697
Description: Physicochemical properties such as pH, specific surface area (SSA), cationic exchange capacity (CEC), loss on ignition (LOI), pathogens, plant nutrients (nitrates, ammonium and phosphates), and heavy metals (manganese, copper, lead and cadmium) were determined for sewage sludge from Grahamstown and Tiaret. The values obtained were log transformed thereafter a t-test at 5 % level of significance was used to test for the difference in each parameter for both sludges. The pH of sludge was determined in 1:3 water, 16 water, 1:3 0.01 M calcium chloride and 1:3 1 M potassium chloride. The pH for Grahamstown and Tiaret sludge were in the ranges of 6.66-7.11 and 7.88-8.18 respectively. The SSA values for Grahamstown and Tiaret were 218 ± 108 and 261 ± 99.9 m2/g, and the CEC values were 119 ± 2.09 and 136 ± 6.03 mEq/100, respectively. The LOI values obtained were 1.33 ± 0.03 and 1.48 ± 0.11 % for Grahamstown and Tiaret, respectively. E. coll and heterotrophic bacteria were the pathogens determined, and were extracted from sludge using sterile saline and nutrient broth. The concentration of E. coll in Grahamstown and Tiaret sludge were 468 ± 7.63 and 7769 ± 1268 CFU/g d.w and for heterotrophic bacteria were 1.17x109 ± 7.42x108 and 1.43x109 ± 9.11 x108 CFU/g d.w. For Grahamstown sludge, the concentration of nitrates, ammonium and phosphates were 55.61 ± 55.20 mg/g d.w, 6.60 ± 2.36 mg/g d.w and 1.40 ± 0.30 mg/g d.w, respectively. For Tiaret sludge, the concentration of nitrates, ammonium and phosphates were 2.56 ± 2.90 mg/g d.w, 0.64 ± 0.45 mg/g d.w and 0.24 ± 0.19 mg/g d.w, respectively. The concentration of Mn, Cu, Pb and Cd in Grahamstown sludge were 423 ± 101, 353 ± 92, 40.2 ± 20 and 0.0 mg/kg d.w respectively, and for Tiaret sludge, the corresponding concentrations were 358± 295, 549±50, 1427± 1352 and 1.54 ± 0.61 mg/kg d.w. Sewage sludge was found to contain Triclosan, and solubility studies of the compound were conducted using sodium deoxycholate and sodium lithocholate. The apparent solubilities and rate constants indicated in brackets of TCS at 37 °C were 35.4 ± 1.21 mg/L (1.28 ± 0.36 Hr-) and 14.4 ± 0.34 mg/L (0.99 ± 0.17 Hr-) in sodium lithocholate and sodium deoxycholate, respectively. The apparent solubilities and rate constants indicated in brackets of TCS at 15 °C were 32.3 ± 0.88 mg/L (2.16 ± 0.80 Hr-) and 14.2 ± 0.39 mg/L (1.02 ± 0.17 Hr-) in sodium lithocholate and sodium deoxycholate, respectively. Triclosan was extracted from sludge using 1 g/L sodium deoxycholate and the determined concentration were 142 ± 33.5 gg/g d.w for Grahamstown sludge and 0-12 gg/g d.w for Tiaret sludge. Finally plant growth studies were conducted on radish and garden cress plants using Grahamstown sludge at 0, 20, 40, 80 and 100 % treatments. Statistical analysis (t-test and Kruskal-Wallis) at 5 % level of significance was done to compare growth parameters between control and different sludge treatments. For radish plants, the values for plant height, root length, number of leaves, leaf length and dry mass were 28.4-80-7 mm, 4.3-44.7 mm, 3.3-17.0 mm, 2.3-4.0 leaves and 6.3-15.3 %, respectively. For garden cress, the values for plant height, root length, number of leaves, leaf length and dry mass were 13.7-25.0 mm, 7.7-20.3 mm, 5.7-8.3 leaves, 3.0-8.3 mm and 8.8-15.0 %, respectively. Twenty percent (20 %) sludge treatment gave the best results in radish and garden cress plants with respect to plant height, root length, number of leaves and dry mass. Triclosan concentration in radish and garden cress plants was below the detection limit of 32.4 gg/g d.w. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
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Date Issued: 2017

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Molecular cloning and expression of equine CYP1A2 in Escherichia coli

- Mkabayi, Lithalethu

Authors: Mkabayi, Lithalethu
Date: 2017
Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/4830 , vital:20734
Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
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Date Issued: 2017

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Sorptive and microbial properties of low-cost adsorbents used in the extraction of ciprofloxacin and isoniazid from aqueous solution

- Dube, Cyril Simbarashe

Authors: Dube, Cyril Simbarashe
Date: 2017
Language: English
Type: text , Thesis , Masters , MSC
Identifier: http://hdl.handle.net/10962/59178 , vital:27450
Description: This work describes how coal fly ash (FA), kaolinite, perlite, talc and vermiculite were used to remove ciprofloxacin and isoniazid from aqueous solutions. The adsorptive features of the adsorbents were evaluated for ciprofloxacin and isoniazid with regards to the effects of contact time, pH, solid/liquid ratio and antibiotic concentration. All adsorbents were sterilised by dry heat before use to avoid the proliferation of antimicrobial resistance by the bacteria present on the adsorbents during experiments. The regression correlation coefficients indicate that the linearised form of the Langmuir isotherm gives the best fit for the sorption of both antibiotics onto FA and talc, ciprofloxacin onto kaolinite, and isoniazid onto perlite and vermiculite with R2 values ranging from 0.908 - 0.999. The linearised form of the Freundlich isotherm best describes the sorption of ciprofloxacin onto vermiculite and isoniazid onto kaolinite with R2 values of 0.999 for both. The linearised form of the Temkin isotherm best describes the sorption of ciprofloxacin onto perlite with an R2 = 0.997. The values of the Freundlich exponent, 1/n, range from 0.221 - 0.998, indicating a favourable adsorption of ciprofloxacin and isoniazid onto the adsorbents. The heat of sorption, B, calculated from the Temkin plots has values ranging from 0.018 - 10.460 J/mol, indicating a physical adsorption process (physisorption). Adsorption equilibrium on all adsorbents was achieved after 30 min for both antibiotics and the kinetic data obtained conforms best to the pseudo-second order equation with R2 values ranging from 0.998 - 0.999. The removal of ciprofloxacin and isoniazid by all adsorbents except FA was strongly influenced by the pH suggesting that electrostatic interactions play a major role in the adsorption processes. All adsorbents except FA removed showed excellent adsorption of ciprofloxacin from aqueous solutions with all of them achieving removals ranging from 80 - 99%. The adsorbents were less efficient in removing isoniazid and kaolinite gave the highest removal of 55 %. Furthermore, the microbial quality of the adsorbents was investigated and the results revealed that kaolinite, talc, perlite and vermiculite were heavily contaminated with microorganisms. FA was sterile. The fungi isolated from the mineral adsorbents were in concentrations ranging from 2.13 x 106 to 1.25 x 107 CFU/g and were mostly moulds; Penicillium spp., Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Cladosporium spp. and Rhizopus oryzae. One yeast was isolated and was identified as Candida albicans. The bacteria identified were in concentrations ranging from 4.96 x 106 - 1.19 x 109 CFU/g. E. coli, Enterobacter cloacae, Exiguobacterium spp., Pseudomonas aeruginosa, Bacillus spp. and Serratia liquefaciens. The leachability index (LI) values obtained for adsorbents indicated that it is highly unlikely that microorganisms could be leached out of the adsorbents by rain. Heat inactivation of the microorganisms at a 105 °C was totally unsuccessful. However, it was established that a dry heat dose of 160 °C for at least 15 min was sufficient to eradicate all microorganisms present in the adsorbents. The D-values for coliform bacteria from all samples were very similar ranging from 1.7-2.2 min indicating homogeneity in heat resistance by the microorganisms. The Pseudomonas aureginosa isolated had a D-value of 2.2 min. The fungi isolated from the samples had D-values ranging from 2.1-3.2 min.
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Date Issued: 2017

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Identification of novel SNPSTRs by 454 sequencing in Nguni and Sotho-Tswana populations

- Laurence, Jo-Anne Elizabeth

Authors: Laurence, Jo-Anne Elizabeth
Date: 2015
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/55885 , vital:26752
Description: DNA profiling is currently performed by analysis of the electropherogram that results following the amplification of a panel of Short Tandem Repeat (STR) loci. A need has arisen, however, for the development of a typing method that generates results which are compatible and comparable with existing databases, but that have a higher discrimination power by supplying sequence data as well as repeat-number data. Recent studies that explore these alternative typing methodologies have revealed the existence of a number of STR variants. There is, however, little information about the exact nature and prevalence of these sub-alleles. There have also been limited population studies of the genetic profiles of sub-Saharan African populations, despite the fact that evidence suggests that there is greater genetic structure and genetic diversity in these populations. In this study, a processing protocol for the generation of 454 sequencing-ready amplicons of vWA, D2S441, D3S1358, D13S317, D21S11 and D7S820 loci was developed. This protocol was applied to buccal swabs collected from 144 individuals of the Nguni and Sotho-Tswana population groups. A total of 145 485 reads were obtained from the sequencing of these amplicons, of which 97 400 and 48 085 reads were obtained for the Nguni and Sotho-Tswana populations respectively. The proportional representation for each locus ranged from 8-20%, and the allele calls and observed frequencies of these alleles suggested a high degree of relatedness between population groups. The sequencing results, furthermore, enabled the identification of a number of previously undescribed STR variants and SNPSTRs; with allele 13´ for D13S317 representing a SNP that may be predictive of Nguni-ancestry. The results also demonstrated the usefulness of next generation sequencing for increasing the number of discernible alleles for STR profiling.
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Date Issued: 2015

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Polyunsaturated fatty acid metabolism and effects on colon cancer cell biology in vitro.

- Bulcao, Candice

Authors: Bulcao, Candice
Date: 2013
Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
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Date Issued: 2013

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The development of an in vitro system for the production of drug metabolites using microsomal enzymes from bovine liver

- Morrison, Roxanne

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

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Molecular and biochemical analysis of the diet of the black rhinoceros

- Kgopa, Ananias Hodi

Authors: Kgopa, Ananias Hodi
Date: 2009 , 2013-07-15
Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
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Date Issued: 2009

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