subject:(biochemistry)

University of KwaZulu-Natal

1. Bartlett, Cara-Lesley. Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis.

Degree: MS, Biochemistry, 2010, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/4768

► African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is… (more)

▼ African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is one of the main trypanosome species affecting cattle and causes the disease known as nagana. Control of animal African trypanosomiasis currently relies on chemotherapy and vector control methods, neither of which has proven satisfactory. An effective vaccine against trypanosomiasis would be the most cost effective solution to control the disease; however, due to the phenomenon of antigenic variation, intrinsic to the parasite’s outer coat of variable surface glycoprotein, this has not yet been achieved. Recent vaccine efforts have been centred on identification of invariant parasite antigens for use as vaccine candidates. Trypanosome cytoskeleton components have in recent years been shown to be capable of providing a protective immune response against trypanosome infection. These include tubulin proteins, which form the main components of the cytoskeleton, as well as microtubule associated proteins (MAPs) and paraflagellar rod proteins. In the present study α- and β-tubulin from T. congolense were recombinantly expressed and their immuno-protective potential in mice assessed. Amplification of both α- and β-tubulin ORFs from T. congolense genomic DNA was followed by cloning of the amplicons into the T-vector pTZ57R/T, and thereafter sub-cloning into the bacterial expression vector, pET238a and the yeast expression vector pPICZαA28. Only the α-tubulin amplicon was successfully sub-cloned into pICZAαA28; however, no protein expression was achieved upon transfection of the methylotrophic yeast, Pichia pastoris, with this construct. Subcloning of both α- and β-tubulin inserts into pET28a was successful. Expression of recombinant α- and β-tubulin as fusion proteins with a histidine tag, both at a size of 55 kDa, was achieved in Escherichia coli host BL21 (DE3). Recombinant proteins were successfully purified using nickel chelate chromatography under denaturing conditions. Refolding was first attempted by dilution of purified denatured proteins in a refolding buffer followed by reconcentration, but was largely unsuccessful. A second, more successful refolding method was performed wherein denatured proteins were refolded by application of a decreasing gradient of urea, while bound to a nickel chelate column. Native tubulin from cultured T.congolense procyclics was successfully purified and renatured using a polymerisation/depolymerisation method for use as a control for immunisation. Mice were immunised separately with refolded recombinant α- and β-tubulin, native tubulin or an irrelevant protein VP4AA expressed in the same way as the tubulins. ELISA analysis confirmed the production of antibodies against each protein. Parasitaemia developed in all mice following challenge with T. congolense. Only the group immunised with β-tubulin recorded no deaths during the monitoring period despite the presence of parasitaemia, with 60% of mice immunised… Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Bartlett, C. (2010). Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/4768

Chicago Manual of Style (16^th Edition):

Bartlett, Cara-Lesley. “Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis.” 2010. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/4768.

MLA Handbook (7^th Edition):

Bartlett, Cara-Lesley. “Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis.” 2010. Web. 24 Jun 2019.

Vancouver:

Bartlett C. Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2010. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/4768.

Council of Science Editors:

Bartlett C. Recombinant expression and evaluation of a- and b- tubulin from Trypanosoma congolense as vaccine candidates for African trypanosomiasis. [Masters Thesis]. University of KwaZulu-Natal; 2010. Available from: http://hdl.handle.net/10413/4768

University of KwaZulu-Natal

2. Ndlovu, Hlumani Humphrey. Structural studies aimed at improving the antigenicity of congopain.

Degree: MS, Biochemistry, 2009, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/4774

► African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The… (more)

▼ African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were… Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor), Boulange, Alain F. V (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ndlovu, H. H. (2009). Structural studies aimed at improving the antigenicity of congopain. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/4774

Chicago Manual of Style (16^th Edition):

Ndlovu, Hlumani Humphrey. “Structural studies aimed at improving the antigenicity of congopain.” 2009. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/4774.

MLA Handbook (7^th Edition):

Ndlovu, Hlumani Humphrey. “Structural studies aimed at improving the antigenicity of congopain.” 2009. Web. 24 Jun 2019.

Vancouver:

Ndlovu HH. Structural studies aimed at improving the antigenicity of congopain. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2009. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/4774.

Council of Science Editors:

Ndlovu HH. Structural studies aimed at improving the antigenicity of congopain. [Masters Thesis]. University of KwaZulu-Natal; 2009. Available from: http://hdl.handle.net/10413/4774

University of KwaZulu-Natal

3. Kalundi, Erastus Mulinge. Molecular analysis of the congopain gene family.

Degree: MS, Biochemistry, 2008, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/4140

► Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns… (more)

▼ Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The… Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor), Boulange, Alain F. V (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Kalundi, E. M. (2008). Molecular analysis of the congopain gene family. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/4140

Chicago Manual of Style (16^th Edition):

Kalundi, Erastus Mulinge. “Molecular analysis of the congopain gene family.” 2008. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/4140.

MLA Handbook (7^th Edition):

Kalundi, Erastus Mulinge. “Molecular analysis of the congopain gene family.” 2008. Web. 24 Jun 2019.

Vancouver:

Kalundi EM. Molecular analysis of the congopain gene family. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2008. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/4140.

Council of Science Editors:

Kalundi EM. Molecular analysis of the congopain gene family. [Masters Thesis]. University of KwaZulu-Natal; 2008. Available from: http://hdl.handle.net/10413/4140

University of KwaZulu-Natal

4. Driscoll, Scott Robert Ellery. p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations.

Degree: MS, Biochemistry, 2011, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10357

► The mammalian cell cycle and its points-of-entry are well characterized pathways. These points-of-entry are normally regulated via mitogens and include, amongst others, the ERK, JNK… (more)

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Driscoll, S. R. E. (2011). p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10357

Chicago Manual of Style (16^th Edition):

Driscoll, Scott Robert Ellery. “p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations.” 2011. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10357.

MLA Handbook (7^th Edition):

Driscoll, Scott Robert Ellery. “p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations.” 2011. Web. 24 Jun 2019.

Vancouver:

Driscoll SRE. p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2011. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10357.

Council of Science Editors:

Driscoll SRE. p38 MAPK and the C2C12 cell cycle : in vitro and in silico investigations. [Masters Thesis]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/10357

University of KwaZulu-Natal

5. Sibanda, Wanani Nonhlanhla. The effect of antiretrovirals on myoblast proliferation : migration and differentation.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10551

► Successful antiretroviral (ARV) treatment is associated with suppression of HIV viral load and the reduction of clinical disease progression. Despite marked improvements in ARV medication,… (more)

▼ Successful antiretroviral (ARV) treatment is associated with suppression of HIV viral load and the reduction of clinical disease progression. Despite marked improvements in ARV medication, side effects from long-term treatment, such as loss of muscle mass do occur. The mechanism by which ARVs affect muscle mass is unclear, however, published in vitro data suggests a negative effect on myoblast fusion during differentiation. The objective of this study was therefore to determine the effect of ARVs on processes required for successful myogenesis; these included proliferation, migration during wound repair, and differentiation. C2C12 mouse skeletal myoblasts and human primary culture skeletal (HSk) myoblasts were incubated with Zidovudine (nucleoside reverse transcriptase inhibitor-NRTI), Tenofovir (nucleotide reverse transcriptase inhibitor-NtRTI) or Ritonavir (protease inhibitor-PI) at a concentration range of 0.01 μM to 10 μM. Proliferation was determined using crystal violet and migration was analyzed using a 2D wound healing assay. The commitment of myoblasts into the myogenic lineage was assessed via the expression of the transcription factor Pax7. Differentiation was measured by assessing the fusion index of multinucleated myotubes. C2C12 myoblast proliferation was observed to increase significantly in response to Tenofovir (1 μM and 10 μM). In HSk cells however, proliferation was observed to decrease significantly in response to Tenofovir (1 μM). Zidovudine had no consistent effect on C2C12 proliferation at any dose tested, but caused a decrease in HSk myoblast proliferation (0.01 μM and 0.1 μM); however this was statistically non-significant. A small dose-dependent increase in C2C12 and HSk cell number, although not significant, was seen in response to Ritonavir. Wound closure results revealed both dose-dependent and time-dependent effects of Tenofovir and Zidovudine on human myoblast migration, with significant decreases in the rate of wound closure (4-7 hours) noted at 0.1 μM and 0.01 μM doses respectively. Zidovudine had no significant effect on migration while Ritonavir (0.01 μM) was observed to significantly increase percentage wound closure of human myoblasts, suggesting an increased ability to migrate during wound repair. Differentiation results indicated a decrease in myoblast fusion in response to all three ARVs. However only Ritonavir was shown to negatively affect myosin heavy chain expression. Further research into the exact mechanism of decreased fusion is required. To our knowledge, this study is the first to suggest that selected ARVs may significantly influence myoblast regeneration capabilities by modulating myoblast proliferation, migration, differentiation and fusion, and thereby decrease their myogenic capability. Extended human myoblast studies on differentiation could confirm this hypothesis. Advisors/Committee Members: Niesler, Carola U (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Sibanda, W. N. (2013). The effect of antiretrovirals on myoblast proliferation : migration and differentation. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10551

Chicago Manual of Style (16^th Edition):

Sibanda, Wanani Nonhlanhla. “The effect of antiretrovirals on myoblast proliferation : migration and differentation.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10551.

MLA Handbook (7^th Edition):

Sibanda, Wanani Nonhlanhla. “The effect of antiretrovirals on myoblast proliferation : migration and differentation.” 2013. Web. 24 Jun 2019.

Vancouver:

Sibanda WN. The effect of antiretrovirals on myoblast proliferation : migration and differentation. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10551.

Council of Science Editors:

Sibanda WN. The effect of antiretrovirals on myoblast proliferation : migration and differentation. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10551

University of KwaZulu-Natal

6. Lin, Chien-Yu. Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells.

Degree: MS, Biochemistry, 2010, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10730

► Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells… (more)

▼ Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells to participate in myogenesis decreases with age and as a result, muscle repair and maintenance in an aging organism is characterized by fibrosis, lipid accumulation and atrophy, a process known as sarcopenia. Recent parabiotic studies have shown that satellite cells with reduced myogenic capability in aging muscle can be rejuvenated to undergo effective myogenesis when exposed to a young environment. Further analysis has suggested that the Wnt family of signaling proteins identified in serum is pivotal in regulating cell fate, proliferation and differentiation, during aging. Wnt3a is known to regulate fibrogenensis, Wnt10b adipogenesis and Wnt7 myogenesis. In the current study, we aim to determine the cytosolic and secreted expression profiles of the three Wnt isoforms, Wnt3a, 7 and 10b, during myogenesis of early and late passage C2C12 myoblasts. We then extend our analysis to determine whether conditioned media could improve the myogenic capacity of late passage cells. Late passage C2C12 cells had elevated Wnt3a cytosolic levels along with reduced differentiation capacity and a rapidly declining Wnt7 levels, in comparison to early passage cells. The elevated Wnt3a suggests an elevated fibrogenic predisposition, whereas the declining Wnt7 cytosolic levels, a decrease in myogenic capacity. Furthermore, analysis of the secreted vs. cytosolic ratio in Wnt7 levels revealed a more rapid decline in late vs. early passage cells during differentiation, supporting the observed decreased myogenic ability. Moreover, late passage cells also showed lower Wnt10b levels compared to early passage cells. This low level of Wnt10b is likely associated with an increase in adipogenic predisposition. The results obtained in the cross-over experiments indicated that conditioned media from early passage cells did not improve the differentiation of late passage cells by the low levels of Myogenin and MHC. However, early passage cells treated with conditioned media from late passage cells surprisingly showed a marginal increase in both Myogenin and MHC levels. Interestingly, cytosolic Wnt3a and 7 in late passage cells treated with ‘young media’ were increased compared to control whereas early passage cells treated with ‘old’ media showed significantly decreased levels of Wnt3a and 7. Furthermore, early passage cells acquired a declining expression when treated with ‘young’ media whereas late passage cells had an increasing level when treated with ‘old’ media. This indicates a possible improvement in differentiation in late passage cells. Taken together, our results support a role for Wnt7 and Wnt10b in promoting myogenesis while Wnt3a may decrease myogenesis. With the increase in passage numbers, the reduced myogenic predisposition is regulated by reduced Wnt10b, 7 and elevated Wnt3a levels, respectively. Moreover, we speculate that the lack of myogenic improvement in the… Advisors/Committee Members: Niesler, Carola U (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Lin, C. (2010). Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10730

Chicago Manual of Style (16^th Edition):

Lin, Chien-Yu. “Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells.” 2010. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10730.

MLA Handbook (7^th Edition):

Lin, Chien-Yu. “Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells.” 2010. Web. 24 Jun 2019.

Vancouver:

Lin C. Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2010. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10730.

Council of Science Editors:

Lin C. Expression profile of Wnt isoforms during differentiation of aging C2C12 myoblast cells. [Masters Thesis]. University of KwaZulu-Natal; 2010. Available from: http://hdl.handle.net/10413/10730

University of KwaZulu-Natal

7. Crouch, Candice Julie. Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy.

Degree: MS, Biochemistry, 2014, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10769

► Membrane type I-matrix metalloproteinase (MT1-MMP), a member of the highly active extracellular matrix (ECM)-degrading matrix metalloproteinases (MMPs), is known to be involved in connective tissue… (more)

▼ Membrane type I-matrix metalloproteinase (MT1-MMP), a member of the highly active extracellular matrix (ECM)-degrading matrix metalloproteinases (MMPs), is known to be involved in connective tissue remodelling and embryogenesis, as well as tumour invasion and metastasis. Positioned on the leading edge of the invading cell, its proteolytic activity is enhanced by activation of proMMP-2 in a complex with tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). The aim of this study was to attempt to produce highly specific and immunoinhibitory antibodies against human MT1-MMP and to test whether such antibodies are able to stop invasion by blocking MT1-MMP activity. As various MMP domains are highly conserved across species, and within specific MMP groups, and human cancer cells were to be used in invasion assays, sequence alignment of human MT1-MMP domains was used to identify the most variant and, hence, the optimal laboratory species for antibody production, and the chicken was subsequently selected. A hemopexin-like or collagen-binding domain, together with the catalytic domain (PEXcat), the hemopexin-like domain (PEX) and the propeptide and catalytic domain (PROcat) were selected as target domains for antibody production. Escherichia coli or Pichia pastoris expressed PEXcat and PEX domains, respectively, were obtained collaboratively, and an E. coli expression system was used to express the PROcat domain. Urea and β-mercaptoethanol successfully solubilised PROcat inclusion body protein, and Q- and S-Sepharose ion exchange chromatography, removed majority of the E. coli contaminating proteins, yielding >200 mg/litre expressed PROcat protein. Alum adjuvant and unrenatured soluble PEXcat and PEX proteins, or the less soluble, S-Sepharose purified PROcat protein was used for inoculations of chickens. The PROcat antigen, also injected as a homogenised band in acrylamide, proved to be inferior to S-Sepharose-purified PROcat antigen in alum, as it failed to induce an antibody response. The S-Sepharose-purified PROcat antigen, in alum adjuvant, produced the highest overall response, purified anti-PROcat IgY recognising recombinant forms of MT1-MMP (33 kDa and 50 kDa) and a 63 kDa protein in human blood, concluded to be either latent, soluble MT1-MMP or a non-specific protein. These antibodies, however, failed to detect native human and murine MT1-MMP (43 kDa) in cell line homogenates, suggesting that they possibly did not recognise the zinc-binding site of the catalytic domain in the 43 kDa processed MT1-MMP. In contrast to purified IgY, crude anti-PROcat IgY preparations recognised renatured PROcat MT1-MMP (29 kDa), indicating possible binding and removal of anti-human MT1-MMP antibodies during PEG purification. Despite this, the purified IgY resulted in higher immunoinhibition of the renatured PROcat antigen than the crude IgY. Anti-PEXcat antibodies had low titre, recognising native MT1-MMP in human cell (43 kDa) and mouse macrophage homogenates, but did not recognise the original recombinant PEXcat MT1-MMP antigen or… Advisors/Committee Members: Elliott, Edith (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Crouch, C. J. (2014). Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10769

Chicago Manual of Style (16^th Edition):

Crouch, Candice Julie. “Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy.” 2014. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10769.

MLA Handbook (7^th Edition):

Crouch, Candice Julie. “Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy.” 2014. Web. 24 Jun 2019.

Vancouver:

Crouch CJ. Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2014. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10769.

Council of Science Editors:

Crouch CJ. Membrane type-1 matrix metalloproteinase (MT1-MMP) as a target in cancer therapy. [Masters Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/10769

University of KwaZulu-Natal

8. Snyman, Celia. Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells.

Degree: PhD, Biochemistry, 2009, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10793

► Effect of c-Ha-ras(V12) on cathepsin trafficking in invasive breast cancer cells. Various mutations of Ha-ras together with lack of p53-related control over cell cycle progression,… (more)

▼ Effect of c-Ha-ras(V12) on cathepsin trafficking in invasive breast cancer cells. Various mutations of Ha-ras together with lack of p53-related control over cell cycle progression, result in an immortal, tumorigenic phenotype in 50% human epithelial cancers. Unmutated Ha-Ras transiently mediates external growth factor-related signaling, initiating downstream kinase activity that is normally terminated by p53. This protects the cell from immortalization, i.e. uncontrolled proliferation. An MCF10A breast epithelial cell line, derived from a fibrocystic breast mastectomy specimen, spontaneously immortalized in culture, due to a chromosomal deletion (9p21-/-). This gave rise to a non-malignant and non-invasive cell line in which the effects of deletion of upstream control of both p53 and the cell cycle and c-Haras( V12) transfection may be studied. Transfection of this cell line with the c-Haras( V12) oncogene gave rise to the invasive MCF10AneoT premalignant derivate, in which distribution of cathepsin B (CB), cathepsin L (CL) and cathepsin D (CD), membrane-type 1 matrix metalloprotease (MT1-MMP), a membrane-bound collagenase, is altered. The possible role of these proteases in the premalignant invasive phenotype, as well as the role of the V12 mutation and the effect of p53 on vesicle trafficking, was explored. In the MCF10AneoT cell line lack of negative feedback by p53 and other Ha-Ras effectors such as Rac, Rho and CDC42, seems to result in lack of control over the cytoskeleton and thus cell polarity during growth stimulus-related migration. Luminal alkalinization, especially of vesicles distant from the perinuclear region, as well as degradative efficiency seem affected, possibly as functional assembly of the acidifying vacuolar-ATPase proton pump on these vesicles is compromized. In normal cells CB and CD seem discretely located, while a spread of proteases was noted in transfected cells, from a perinuclear position to along the basal plane. Increased association of CB with lysosome-associated membrane protein-2 (LAMP- 2), and of CD with an acidic juxta-nuclear structure (JNS) was also noted, while this structure was observed in two sites in transfected cells, compared to only one in normal cells. In invasive cancers increased levels of both CB and MT1-MMP have been found to correlate with accelerated pathological degradation and invasion of the underlying basement membrane (BM) barrier and extracellular matrix (ECM). MT1- MMP is known to regulate BM turnover, while the manner in which the association of CB with the plasma membrane (PM) supports such turnover, ECM degradation and migration, is not yet clear. The current investigation showed altered distribution of PM-associated CB and MT1-MMP in transformed cells, compared to normal. This phenotype seems explained in terms of the effects of the mutationally activated c-Ha- Ras(V12) on its downstream effectors, Rac and PI3K and their effectors, on cytoskeletal organization and vesicle trafficking, increased calcium and, via Rho, cytoplasmic alkalinization due to… Advisors/Committee Members: Elliott, Edith (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Snyman, C. (2009). Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10793

Chicago Manual of Style (16^th Edition):

Snyman, Celia. “Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells.” 2009. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10793.

MLA Handbook (7^th Edition):

Snyman, Celia. “Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells.” 2009. Web. 24 Jun 2019.

Vancouver:

Snyman C. Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2009. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10793.

Council of Science Editors:

Snyman C. Effect of c-Ha-ras(V12) on protease trafficking in invasive breast cancer cells. [Doctoral Dissertation]. University of KwaZulu-Natal; 2009. Available from: http://hdl.handle.net/10413/10793

University of KwaZulu-Natal

9. Marais, Johan Pieter. Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum).

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9632

► The factors affecting the accumulation of nitrate in kikuyu grass pastures and the effect of elevated nitrate levels upon digestion in the ruminant were investigated.… (more)

▼ The factors affecting the accumulation of nitrate in kikuyu grass pastures and the effect of elevated nitrate levels upon digestion in the ruminant were investigated. A high potassium level in the soil seems to be the major factor stimulating the accumulation of excessive amounts of nitrate in kikuyu grass, when the nitrate content of the soil is also high. The continuous elongation of kikuyu grass tillers allows constant exposure of high nitrate containing stem tissue to the grazing ruminant. Digestibility studies in vitro showed that nitrite, formed during the assimilatory reduction of nitrate to ammonia, reduces cellulose digestion, but the degree of reduction also depends upon the presence of readily available carbohydrates and protein in the digest. Studies in vivo showed that the microbial population can adapt to metabolise high concentrations of nitrate (500 mg% N, m/m) in fresh kikuyu grass, without the accumulation of nitrite in the rumen. However, introduction into the rumen of nitrite in excess of the capacity of the nitrite reducing microbes, causes nitrite accumulation. Nitrite has no direct effect upon rumen cellulase activity. Due to the affinity of rumen carbohydrases for the substrate, attempts to isolate these enzymes by means of isoelectric focusing and other separation techniques met with limited success. Nitrite strongly reduces the xylanolytic, total and cellulolytic microbial numbers with a concomitant decrease in xylanase and cellulase activity of the digest. Decreased microbial numbers could not be .attributed to a less negative redox potential of the digest in the presence of nitrite, nor could the effect upon the cellulolytic microbes be attributed to an effect of nitrite on branched chain fatty acid synthesis required for cellulolytic microbial growth. A study of the effect of nitrite upon the specific growth rate of pure cultures of the major cellulolytic bacteria, Ruminococcus flavefaciens strain FDI, Butyrivibrio fibrisolvens strain Ce 51, Bacteroides succinogenes strain S 85 and Ruminococcus albus strain 22.08.6A and the non-cellulolytic bacterium Selenomonas ruminantium strain ATCC 19205 revealed the extreme sensitivity to nitrite of some of these bacteria and the relative insensitivity of others. Growth inhibition seems to depend primarily upon the extent to which these microbes derive their energy from electron transport-mediated processes. Advisors/Committee Members: Dennison, Clive (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Marais, J. P. (2013). Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum). (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9632

Chicago Manual of Style (16^th Edition):

Marais, Johan Pieter. “Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum).” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9632.

MLA Handbook (7^th Edition):

Marais, Johan Pieter. “Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum).” 2013. Web. 24 Jun 2019.

Vancouver:

Marais JP. Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum). [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9632.

Council of Science Editors:

Marais JP. Effect of nitrate upon the digestibility of kikuyu grass (Pennisetum clandestinum). [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9632

University of KwaZulu-Natal

10. Dehrmann, Frieda Marie. Properties of Cathepsin L in relation to a role in invasive cancer.

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9777

► Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a… (more)

▼ Cathepsin L, which has been implicated in many tissue degradative pathologies by virtue of its ability to degrade extracellular matrix components, was isolated by a novel, scaled-up protein purification method and purified to homogeneity in the single-chain form. In addition, the high molecular weight variant of cathepsin L covalently complexed with stefin B was isolated. Both cathepsin L and the complex were stable, in respect of their proteolytic activity, to the chaotropic agent urea, both showing enhanced activity in the presence of urea. Urea did not dissociate the complex. The suitability of cathepsin L for a purported extracellular role was addressed by investigating its pH optimum and pH stability. Cathepsins L and B are affected by ionic strength and so buffers of constant ionic strength (rather than constant molarity, and therefore varying ionic strength) were used in determining their pH optima and stability. Cathepsins L and B had apparent pH optima of pH 6.5 and 7.5, respectively, (measured with synthetic substrates) and, contrary to the previous belief, were substantially stable at physiological pH. In Hanks' balanced salt solution, a model of the extracellular fluid, they were shown to be active and stable, cathepsin L having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumour pH). It was also shown that prior reductive activation of these enzymes increased their stability to extracellular conditions, supporting the hypothesis that the active site thiolate-imidazolium ion pair contributes to their stability. The nature of the bond between cathepsin L and stefin B in the covalent complex was examined, using CNBr cleavage, HPLC and amino acid sequencing. Stefin B was shown to be associated with residues 1-137 of cathepsin L via a reduction sensitive linkage which was deduced to be a thioester bond betwen Asp-71 of cathepsin L and Cys-3 of stefin B. Polyclonal antibodies to cathepsin L and stefin B-complexed cathepsin L were raised in rabbits and chickens, and characterised with respect to their suitability for immunocytochemical localisation of these forms of cathepsin L. Advisors/Committee Members: Dennison, Clive (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Dehrmann, F. M. (2013). Properties of Cathepsin L in relation to a role in invasive cancer. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9777

Chicago Manual of Style (16^th Edition):

Dehrmann, Frieda Marie. “Properties of Cathepsin L in relation to a role in invasive cancer.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9777.

MLA Handbook (7^th Edition):

Dehrmann, Frieda Marie. “Properties of Cathepsin L in relation to a role in invasive cancer.” 2013. Web. 24 Jun 2019.

Vancouver:

Dehrmann FM. Properties of Cathepsin L in relation to a role in invasive cancer. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9777.

Council of Science Editors:

Dehrmann FM. Properties of Cathepsin L in relation to a role in invasive cancer. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9777

University of KwaZulu-Natal

11. Pillay, Ché Sobashkar. Redox properties of cathepsin B in relation to its activity in vivo.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9781

► The main site for protein degradation along the endosomal pathway is believed to be the late endosome. Lysosomes are thought to be storage organelles that,… (more)

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Pillay, C. S. (2013). Redox properties of cathepsin B in relation to its activity in vivo. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9781

Chicago Manual of Style (16^th Edition):

Pillay, Ché Sobashkar. “Redox properties of cathepsin B in relation to its activity in vivo.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9781.

MLA Handbook (7^th Edition):

Pillay, Ché Sobashkar. “Redox properties of cathepsin B in relation to its activity in vivo.” 2013. Web. 24 Jun 2019.

Vancouver:

Pillay CS. Redox properties of cathepsin B in relation to its activity in vivo. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9781.

Council of Science Editors:

Pillay CS. Redox properties of cathepsin B in relation to its activity in vivo. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9781

University of KwaZulu-Natal

12. Taylor, Kerry Lyn. Methods for serotype classification of Haemophilus paragallinarum field isolates.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9782

► Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program… (more)

▼ Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished. Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor), Horner, Roger F (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Taylor, K. L. (2013). Methods for serotype classification of Haemophilus paragallinarum field isolates. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9782

Chicago Manual of Style (16^th Edition):

Taylor, Kerry Lyn. “Methods for serotype classification of Haemophilus paragallinarum field isolates.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9782.

MLA Handbook (7^th Edition):

Taylor, Kerry Lyn. “Methods for serotype classification of Haemophilus paragallinarum field isolates.” 2013. Web. 24 Jun 2019.

Vancouver:

Taylor KL. Methods for serotype classification of Haemophilus paragallinarum field isolates. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9782.

Council of Science Editors:

Taylor KL. Methods for serotype classification of Haemophilus paragallinarum field isolates. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9782

University of KwaZulu-Natal

13. Tosomba, Omalokoho Médard. Studies on acid phosphatases of Trypanosoma congolense.

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9783

► Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present… (more)

▼ Bloodstream forms of African trypanosomes, which endocytose macromolecules exclusively through their flagellar pockets, contain an acid phosphatase (AcP) activity in this organelle. In the present thesis, AcP activity was demonstrated cytochemically in some intracellular vesicles and on the surface of Trypanosoma congolense as well as in the flagellar pocket. Unlike other trypanosomatids such as Leishmania spp. and Trichomonas spp., these trypanosomes, while viable, did not release this enzyme into the surrounding medium. In contrast to mammalian cells, the AcP in T. congolense was shown by cell fractionation to be a non-lysosomal enzyme. The enzyme was mostly recovered in the microsomal and cytosolic fractions which had 52.7% and 44.4% of the total activity, respectively. Further separation of the microsomal fraction showed an association of AcP activity with vesicles derived from the plasma membrane, Golgi apparatus and endoplasmic reticulum. After ammonium sulfate precipitation and chromatography on a succession of columns containing Sephacryl S-300, DEAE-cellulose and Sephadex G-75, two acid phosphatases (AcPi and ACP2) were produced from the cytosolic fraction. A membrane-bound acid phosphatase (ACP3) was isolated from the microsomal pellets extracted with Triton X-l 14 and subjected to the above chromatographic procedures. The molecular mass of AcP 1 was higher than 700 kDa. It had an isoelectric point of 4.7. AcP2 (pi 5.3) and AcP3 (pi 6.5) had molecular masses of 33 and 320 kDa, respectively. AcPi and ACP3 were strongly inhibited by vanadate while ACP2 was strongly inhibited by p-chloromercuribenzoate. None of the enzymes was inhibited by tartrate but all were inhibited by NaF. The Km values for each of the various substrates differed widely between the three AcPs indicating that the binding site of each enzyme was distinct. The best of all the substrates tested was para-nitrophenyl phosphate. On non-denaturing gels the enzymes exhibited very high molecular masses but on denaturing SDS-PAGE, two similar bands of activity, localised at 62 and 65 kDa, were observed in all three AcP preparations. Thus the three isolated enzymes may be derived from the same base 62 and 65 kDa units. Differences between enzymes may be derived from differential processing of the isoenzymes for different functions at different locations. Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor), Lonsdale-Eccles, John D (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Tosomba, O. M. (2013). Studies on acid phosphatases of Trypanosoma congolense. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9783

Chicago Manual of Style (16^th Edition):

Tosomba, Omalokoho Médard. “Studies on acid phosphatases of Trypanosoma congolense.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9783.

MLA Handbook (7^th Edition):

Tosomba, Omalokoho Médard. “Studies on acid phosphatases of Trypanosoma congolense.” 2013. Web. 24 Jun 2019.

Vancouver:

Tosomba OM. Studies on acid phosphatases of Trypanosoma congolense. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9783.

Council of Science Editors:

Tosomba OM. Studies on acid phosphatases of Trypanosoma congolense. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9783

University of KwaZulu-Natal

14. Goredema, Wadzanayi Patience. An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9779

► Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic… (more)

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Goredema, W. P. (2013). An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9779

Chicago Manual of Style (16^th Edition):

Goredema, Wadzanayi Patience. “An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9779.

MLA Handbook (7^th Edition):

Goredema, Wadzanayi Patience. “An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species.” 2013. Web. 24 Jun 2019.

Vancouver:

Goredema WP. An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9779.

Council of Science Editors:

Goredema WP. An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9779

University of KwaZulu-Natal

15. Huson, Laura. Antibody-mediated inhibition of proteases of African trypanosomes.

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9788

► The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis… (more)

▼ The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less… Advisors/Committee Members: Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Huson, L. (2013). Antibody-mediated inhibition of proteases of African trypanosomes. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9788

Chicago Manual of Style (16^th Edition):

Huson, Laura. “Antibody-mediated inhibition of proteases of African trypanosomes.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9788.

MLA Handbook (7^th Edition):

Huson, Laura. “Antibody-mediated inhibition of proteases of African trypanosomes.” 2013. Web. 24 Jun 2019.

Vancouver:

Huson L. Antibody-mediated inhibition of proteases of African trypanosomes. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9788.

Council of Science Editors:

Huson L. Antibody-mediated inhibition of proteases of African trypanosomes. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9788

University of KwaZulu-Natal

16. Ramnath, Manilduth. Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9794

► Leucocin A is a class 11a bacteriocin produced by Leuconostoc mesenteroides TA33a that was previously shown to inhibit Listeria monocytogenes. A spontaneous resistant mutant of… (more)

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Ramnath, M. (2013). Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9794

Chicago Manual of Style (16^th Edition):

Ramnath, Manilduth. “Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9794.

MLA Handbook (7^th Edition):

Ramnath, Manilduth. “Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A.” 2013. Web. 24 Jun 2019.

Vancouver:

Ramnath M. Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9794.

Council of Science Editors:

Ramnath M. Molecular analysis of a Listeria monocytogenes strain that is resistant to leucocin A. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9794

University of KwaZulu-Natal

17. Moodley, Thunicia. Apoptosis, redox stress and cancer.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9810

► Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves… (more)

▼ Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed. Advisors/Committee Members: Elliott, Edith (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Moodley, T. (2013). Apoptosis, redox stress and cancer. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9810

Chicago Manual of Style (16^th Edition):

Moodley, Thunicia. “Apoptosis, redox stress and cancer.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9810.

MLA Handbook (7^th Edition):

Moodley, Thunicia. “Apoptosis, redox stress and cancer.” 2013. Web. 24 Jun 2019.

Vancouver:

Moodley T. Apoptosis, redox stress and cancer. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9810.

Council of Science Editors:

Moodley T. Apoptosis, redox stress and cancer. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9810

University of KwaZulu-Natal

18. Myende, Pride Siyanda. Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10065

► Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis… (more)

▼ Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis of TB is impeded by slow growth of an organism with a generation time of 21 days. The emergence of multidrug-resistant TB isolates which are resistant to rifampicin and isoniazid worsened the treatment programme. Furthermore, surfacing of extensively drug-resistant TB isolates especially in HIV positive patients has led to a treatment failure. Currently available diagnostic methods are time consuming and laborious. Polymerase chain reaction-based assay proved to have a better resolution for TB strain discrimination, nevertheless are costly and cannot be routinely employed in resource poor settings. As a result there is an urgent need of cheap, cost effective and rapid diagnostic methods that will reduce a turnaround time. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry potentially offers an alternative rapid and cheaper method for discrimination of TB isolates. Proper discrimination of TB isolates depends on the sample preparation method that is capable of yielding high protein content. Conventional formic/ethanol sample preparation was investigated for mycobacteria MALDI-TOF mass spectrometric analysis. Poor quality of spectra was obtained due to a complex cell wall structure of mycobacteria which released less amounts of proteins. Further attempts were made to optimize the sample preparation method by introducing glass beads for maximum cell wall disruption. Non-consistent spectra were obtained in some mycobacterial strain; therefore it was not used as a method of choice. Introduction of delipidation step using chloroform/methanol (1:1, v/v) before formic/ethanol sample preparation step, led to a generation of reproducible and consistent spectra. This newly developed method was selected to extract protein content from large number of clinical TB isolates. With MALDI-TOF MS and chloroform/methanol-based method, all mycobacterial isolates used in the proof-of-concept were correctly identified and clustered. Fifty six of sixty clinical TB isolates were correctly identified using Biotyper software. Four were incorrectly identified; it might be possible that they carry mutations in unknown regions in their genome which led to a translation of proteins that affected the overall spectra profile. MALDI-TOF MS showed the potential to be used in the clinical laboratories for discrimination of TB isolates at lower costs. Advisors/Committee Members: Govender, Patrick (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Myende, P. S. (2013). Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10065

Chicago Manual of Style (16^th Edition):

Myende, Pride Siyanda. “Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10065.

MLA Handbook (7^th Edition):

Myende, Pride Siyanda. “Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS.” 2013. Web. 24 Jun 2019.

Vancouver:

Myende PS. Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10065.

Council of Science Editors:

Myende PS. Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10065

University of KwaZulu-Natal

19. Mucache, Hermogenes Neves. Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9915

► Trypanosoma congolense is a protozoan parasite transmitted by tsetse flies. It causes bovine trypanosomosis, the major disease for livestock in sub-Saharan Africa. Control methods include… (more)

▼ Trypanosoma congolense is a protozoan parasite transmitted by tsetse flies. It causes bovine trypanosomosis, the major disease for livestock in sub-Saharan Africa. Control methods include trypanocidal drugs and vector control, but none is fully satisfactory, due to resistance and environmental issues. A method that would have the greatest impact on controlling the disease is vaccination. However, development of a conventional vaccine has been hampered by the mechanism of antigenic variation, which allows the parasite to evade the host’s immune system. An alternative strategy in vaccine design is to target the bioactive compounds released by dead and dying trypanosomes. This approach is termed ‘‘anti-disease’’, and does not affect the survival of the parasite but targets the pathogenic factors released by the trypanosomes. The development of a successful anti-disease vaccine necessitates knowledge of all pathogenic factors involved in the disease process. Several macromolecules, primarily peptidases, have been implicated in the pathogenesis of trypanosomosis. Pyroglutamyl peptidase type I (PGP) was shown to be involved in abnormal degradation of thyrotropin- and gonadotropin-releasing hormones in rodents infected with T. brucei, but to date no data are available on the T. congolense PGP. Molecular cloning and expression in E. coli of the coding sequence of T. congolense PGP, as well as the enzymatic characterisation of the recombinant protein, are reported here, completed by the development of reverse genetics tools for studies of gene function. A 678 bp PCR fragment covering the complete open reading frame of PGP was cloned and sequenced. The deduced amino acid sequence showed 52% and 29% identity with the T. brucei and Leishmania major enzymes respectively. The catalytic residues Glu, Cys and His described in Bacilus amyloliquefaciens PGP are conserved in the T. congolense sequence. PGP was expressed in bacterial systems as a soluble active, 26 kDa enzyme. The recombinant enzyme showed activity specific for the fluorescent substrate pGlu-AMC, with a kcat/Km of 1.11 s-1μM. PGP showed activity in the pH 6.5-10 range, with maximal activity at pH 9.0. The enzyme was strongly inhibited by sulfhydryl-blocking reagents such as iodoacetic acid and iodoacetamide with a kass of 125 M-1 s-1 and 177 M-1 s-1 respectively. Antibodies raised in chickens against the recombinant enzyme allowed the detection of native PGP in both procyclic and bloodstream T. congolense developmental stages, and displayed complete inhibition of the enzyme in vitro at physiological concentrations. To get insight into the role of PGP in parasite biology and trypanosomosis progression, two types of vectors for reverse genetics studies were developed. For RNA interference, a 400 bp 3′ end segment of the PGP open reading frame was cloned into the plasmid p2T7Ti, that will allow PGP gene down-regulation upon integration into the genome of an engineered tetracycline-inducible strain such as TRUM:29-13. For gene knock-out, several rounds of molecular… Advisors/Committee Members: Boulange, Alain F. V (advisor), Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Mucache, H. N. (2013). Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9915

Chicago Manual of Style (16^th Edition):

Mucache, Hermogenes Neves. “Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9915.

MLA Handbook (7^th Edition):

Mucache, Hermogenes Neves. “Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools.” 2013. Web. 24 Jun 2019.

Vancouver:

Mucache HN. Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9915.

Council of Science Editors:

Mucache HN. Functional expression of Trypanosoma congolense pyroglutamyl peptidase type 1 and development of reverse genetics tools. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9915

University of KwaZulu-Natal

20. Xulu, Bongiwe Ziphelele. Assessment of hypoxoside and its derivatives as anti-cancer drugs.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9911

► Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from… (more)

▼ Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea (African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and premalignant, invasive breast epithelial cells (MCF10A-NeoT). Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a C-18 reverse phase column and the compound examined by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity. This was also the case for the synthesized compounds. To assess possible selective effects (cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells (respectively). Propidium iodide staining of detached cells was carried out for flow cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic cells). After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected (MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA) post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative had a statistically significant effect on the metabolic activity of the abnormal cell line relative to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF- 10A cell line after treatment with any of the test compounds. Supravital PI staining of adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion assays and need re-investigating, though result trends were similar. Total cell counts, show that HYP and its derivatives appear to increase both cancer and normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug screening and indicates the statistically significant selective toxicity of the DAH compound at 150 and 300 μM in the MCF10A-NeoT, suggesting… Advisors/Committee Members: Elliott, Edith (advisor), Drewes, Siegfried E (advisor), Van Heerden, Fanie R (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Xulu, B. Z. (2013). Assessment of hypoxoside and its derivatives as anti-cancer drugs. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9911

Chicago Manual of Style (16^th Edition):

Xulu, Bongiwe Ziphelele. “Assessment of hypoxoside and its derivatives as anti-cancer drugs.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/9911.

MLA Handbook (7^th Edition):

Xulu, Bongiwe Ziphelele. “Assessment of hypoxoside and its derivatives as anti-cancer drugs.” 2013. Web. 24 Jun 2019.

Vancouver:

Xulu BZ. Assessment of hypoxoside and its derivatives as anti-cancer drugs. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/9911.

Council of Science Editors:

Xulu BZ. Assessment of hypoxoside and its derivatives as anti-cancer drugs. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9911

University of KwaZulu-Natal

21. Zani, Babalwa. The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10009

► Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World… (more)

▼ Effective case management of malaria is hampered by the spread of parasite resistance to nonartemisinin antimalarials. To counteract the impact of drug resistance, the World Health Organization (WHO) has endorsed artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Currently recommended ACTs are artemether-lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine, artesunate plus sulfadoxine-pyrimethamine and dihydroartemisinin-piperaquine. This study sought to review evidence of the efficacy and safety of different non-artemisinin antimalarials in combination with artesunate, artemether or dihydroartemisinin for the treatment of uncomplicated P. falciparum malaria in non-pregnant adults and children. The search for randomized controlled trials (RCTs) was conducted in the Cochrane Central Register for Controlled Trials (CENTRAL), MEDLINE, EMBASE and in ClinicalTrials.gov in January 2009. The eligibility and the methodological quality of trials were assessed and data were extracted, using standard forms. Data were captured and analyzed in Review Manager Software, versions 4.2 and 5.0. The outcomes assessed were: treatment failure, fever and parasite clearance time, calculating the relative risk (RR) and a weighted mean difference (WMD) with a 95% confidence interval and p-values, indicating statistical significance at 0.05. Thirty-seven trials with 6862 participants were included. Artesunate combined with amodiaquine had a statistically significant lower risk of treatment failure compared to the combination of artesunate with sulfadoxine-pyrimethamine (RR=0.57, 95% CI [0.33, 0.97], p=0.04, seven trials, N=1341). In addition, treatment with artesunate plus mefloquine was significantly associated with a lower risk of treatment failure compared to artesunate plus azithromycin (RR=0.04, 95% CI [0.00, 0.64], p=0.02, one trial, N=54). There was no significant difference when either mefloquine or atovaquone-proguanil were combination partners with artesunate (RR=2.6, 95% CI [0.93; 7.24], p=0.07, one trial, N=1066). When artesunate was combined with chloroquine, primaquine or azithromycin and compared with artesunate monotherapy, there was no statistically significant difference in the risk of unadjusted treatment failure. Each of these comparisons had one trial each. Artesunate plus chloroquine was quicker at clearing fever compared to artesunate plus sulfadoxinepyrimethamine (WMD= -7.20, 95% CI [-12.53, -1.87], one trial, N=132). Few trials adequately reported adverse events. There was no significant difference observed in the risk of adverse events between artesunate plus amodiaquine compared with artesunate monotherapy, however, adverse events were significantly less in artesunate plus amodiaquine compared to artesunate plus methylene-blue. Artesunate plus amodiaquine on the other hand had significantly more adverse events reported compared to artesunate plus sulfadoxine-pyrimethamine. The findings of this study support the implementation of… Advisors/Committee Members: Goldring, J. P. Dean (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Zani, B. (2013). The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10009

Chicago Manual of Style (16^th Edition):

Zani, Babalwa. “The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10009.

MLA Handbook (7^th Edition):

Zani, Babalwa. “The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review.” 2013. Web. 24 Jun 2019.

Vancouver:

Zani B. The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10009.

Council of Science Editors:

Zani B. The efficacy and safety of artemisinin-based combination therapy for the treatment of uncomplicated Plasmodium falciparum malaria in non-pregnant adults and children : a systematic review. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10009

University of KwaZulu-Natal

22. Price, Brendon. Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role.

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10014

► At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and… (more)

▼ At the beginning of this study, the granule localisation and regulation of release of human neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and thought to be relevant in invasion and inflammation, had been established while that of their inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not. Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly located in a distinct oval, electron translucent organelle, a little larger than azurophil granules. A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored proteins, established using granule fractionation and immunolabelling to be markers for the secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal nature of this organelle. Density gradient cofractionation with the least dense secretory vesicle population and some pleiomorphism of the organelle suggested that it is a "vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and termination differentiation, but before secretory vesicle synthesis. Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that specific and azurophil granules and a small number of proMMP-8-containing granules (a specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium regulated. However, studies using the calcium ionophore, ionomycin, and monitoring extracellular granule marker protein release upon addition of increasing levels of extracellular calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated. This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be studied further, but results achieved to date may explain the observed differential mode of release of TIMP-1 relative to proMMP-9. The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a novel reverse zymography method. The role and relevance of… Advisors/Committee Members: Elliott, Edith (advisor), Tschesche, Harald (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Price, B. (2013). Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10014

Chicago Manual of Style (16^th Edition):

Price, Brendon. “Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10014.

MLA Handbook (7^th Edition):

Price, Brendon. “Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role.” 2013. Web. 24 Jun 2019.

Vancouver:

Price B. Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10014.

Council of Science Editors:

Price B. Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10014

University of KwaZulu-Natal

23. Hopkins, Cheryl. The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents.

Degree: PhD, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10181

► In traditional forage breeding programmes, breeders have spent decades improving the agronomic characteristics of grasses, such as herbage yield, persistence and resistance to diseases, without… (more)

▼ In traditional forage breeding programmes, breeders have spent decades improving the agronomic characteristics of grasses, such as herbage yield, persistence and resistance to diseases, without considering the nutrient requirements of the grazing animal. In an attempt to improve the nutritive value of Italian ryegrass, which is widely utilised for intensive dairy, lamb and beef production in South Africa, Enhancer ryegrass was developed from predominantly Italian types of Lolium multiflorum, with a minor Westerwolds component, by selecting for a higher concentration of total nonstructural carbohydrate (TNC) and lower moisture content than that currently available in commercial cultivars. The nutritional value of Enhancer was compared with Midmar ryegrass in a controlled environment study and in a grazing trial with weaned lambs; and with Dargle ryegrass in a grazing trial with Holstein dairy cows. Neutral detergent fibre, acid detergent fibre, lignin, nitrogenous compounds, mineral content and in vitro digestibility were also investigated as parameters of nutritive value. The anatomical features of Enhancer and Midmar were studied to determine possible structural differences. Weaned lambs grazed Enhancer and Midmar in an eight-paddock rotational grazing system, with 3.5 days spent in each paddock, allowing a 24.5 day regrowth period for the pastures. Holstein dairy cows grazed Enhancer and Dargle which were established on 16 and 19 hectare pastures, respectively. The n-alkane technique was used to estimate dry matter intake (DMI) in both grazing trials. Results from the controlled environment study suggest that the differences in the dry matter and TNC concentration of Enhancer are not positively linked to anti-quality factors associated with forage species, but can be attributed to genetic differences between the two grasses. Despite the significantly higher (P < 0.01) DMI of weaned lambs grazing Midmar compared with Enhancer, the lambs on Enhancer outperformed those on Midmar in terms of liveweight gain and carcass quality. The superior animal performance on Enhancer is likely due to an improvement in the readily digestible energy to protein ratio as a result of its significantly higher (P < 0.001) concentration of TNC compared with Midmar. Milk yield for cows grazing Enhancer in period 1 of the cross-over study was significantly higher (P < 0.05) than for cows grazing Dargle, despite the significantly lower (P < 0.05) DMI of animals on Enhancer. The higher TNC concentration relative to the true protein content of Enhancer would suggest that the protein metabolism in the rumen can be enhanced. Advisors/Committee Members: Marais, Johan Pieter (advisor), Dennison, Clive (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hopkins, C. (2013). The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10181

Chicago Manual of Style (16^th Edition):

Hopkins, Cheryl. “The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10181.

MLA Handbook (7^th Edition):

Hopkins, Cheryl. “The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents.” 2013. Web. 24 Jun 2019.

Vancouver:

Hopkins C. The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10181.

Council of Science Editors:

Hopkins C. The nutritive value of Italian ryegrass (Lolium multiflorum) selected for high dry matter and nonstructural carbohydrate contents. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10181

University of KwaZulu-Natal

24. Hadebe, Sabelo Goodman. Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10192

► African animal trypanosomiasis is a major threat to African agriculture causing a loss estimated to 4.5 billion US$ per annum. Trypanosoma congolense is the major… (more)

▼ African animal trypanosomiasis is a major threat to African agriculture causing a loss estimated to 4.5 billion US$ per annum. Trypanosoma congolense is the major causative agent in African animal trypanosomiasis and is transmitted by tsetse flies of the Glossina spp. Congopain, a major cathepsin L-like cysteine peptidase in T. congolense is associated with trypanotolerance in N‘Dama cattle and is a target for an anti-disease vaccine. It is suggested that trypanotolerant cattle control the disease by antibody mediated neutralisation of congopain, and that immunisation of cattle against congopain can mimic trypanotolerance resulting in minimised disease pathology. Susceptible cattle immunised with recombinant catalytic domain of congopain, C2, produced high levels of anti-congopain IgG specific antibodies against congopain, maintained weight and exhibited less severe anaemia. However, there was no effect on the establishment of T. congolense infection and acute anaemia development in trypanosusceptible cattle. It has been suggested that failure of congopain to give full protection of the host may be due to poor presentation to the immune system by conventional adjuvants used in previous studies. The aim of the present study was to improve the presentation of the catalytic domain of congopain (C2) to the immune system, by linking it to the proposed molecular adjuvant, BiP, an ER localised HSP70. A further aim was to localise the domain(s) of BiP where the adjuvant properties reside. BiP consists of an ATPase domain (ATPD), a peptide binding domain (PBD) and a C-terminal domain (C-term). Consequently, BiP69, BiP69 lacking the C-terminal domain (BiP60), BiP coding fragments (ATPD, PBD and C-term) and the C2 coding sequence were amplified by PCR from either genomic T. congolense DNA or plasmid DNA. The PCR products were each sub-cloned into a pTZ57RT vector, and C2 cloned into a pET-28a expression vector. The BiP coding fragments were inserted into the recombinant pET-28a-C2 vector, resulting in pET-28a-BiP69-C2, pET-28a-BiP60-C2, pET-28a-ATPD-C2, pET-28a-PBD-C2 and pET-28a-C-term-C2 coding chimeras. The fusion proteins were expressed in an E. coli system as insoluble inclusion bodies at the expected sizes of 96 kDa (BiP69-C2), 88 kDa (BiP60-C2), 47 kDa (PBD-C2), 34 kDa (C-term-C2) and 27 kDa (C2). However, the ATPD-C2 fusion protein was expressed at a larger and smaller size in different attempts. Protein expression was confirmed by western blots using anti-BiP antibodies and anti-congopain N-terminal peptide antibodies. Recombinantly expressed peptide binding domain (PBD)-C2, C-terminus-C2, BiP69-C2, BiP60-C2 chimeras and a BiP69 fusion protein were purified and refolded by a Ni-NTA based one-step on-column refolding method. Bacterial proteins co-purifying with BiP69-C2 and BiP60-C2 chimeras were removed by incubation with 5 mM ATP in the dissociation buffer, but poor yields resulted in using these chimeras as non-pure proteins. Immunisation of Balb/c mice with the BiP69-C2 fusion protein chimera induced a higher… Advisors/Committee Members: Boulange, Alain F. V (advisor), Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Hadebe, S. G. (2013). Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10192

Chicago Manual of Style (16^th Edition):

Hadebe, Sabelo Goodman. “Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10192.

MLA Handbook (7^th Edition):

Hadebe, Sabelo Goodman. “Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen.” 2013. Web. 24 Jun 2019.

Vancouver:

Hadebe SG. Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10192.

Council of Science Editors:

Hadebe SG. Investigation of the molecular adjuvant potential of Trypanosoma congolense BiP/HSP70 using congopain as model antigen. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10192

University of KwaZulu-Natal

25. Viljoen, Jacqueline Ethel. A study of recombinant Plasmodium falciparum PFC0760c.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10198

► Malaria is a devastating disease caused by one of the world's most pathogenic parasites, Plasmodium. Five species of Plasmodium infect humans: P. falciparum, P. vivax,… (more)

▼ Malaria is a devastating disease caused by one of the world's most pathogenic parasites, Plasmodium. Five species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum is the most pathogenic and causes the greatest numbers of deaths. To date, no licensed vaccine against malaria is available, although there are numerous vaccine candidates in various stages of development. Pca 96 is a 96 kDa Plasmodium chabaudi adami protein shown to have a protective property in mice challenged with P. chabaudi adami. Thus, a P. falciparum orthologue of Pca 96 may be useful in vaccine development. BLAST searches with the Pca 96 amino acid and nucleotide sequences revealed proteins with high sequence identity to Pca 96 including the hypothetical P. falciparum PFC0760c and P. yoelii yoelii PY05757 proteins. A peptide sequence FKLGSCYLYIINRNLKEI was found to be conserved in all homologues of Pca 96, including PFC0760c, PY05757 and in the sequences of proteins from 5 other Plasmodium species. Bioinformatic approaches were explored to attempt to find a possible role of the protein and the possible importance of the conserved sequence. The conserved sequence was predicted to be an alpha helix and to contain possible HLA-DRB1*1101 and HLA-DRB1*0401(Dr4Dw4) T-cell epitopes (GSCYLYIINRNLKEI) in addition to a possible H2-Kd T-cell epitope (CYLYIINRNL). Protein-protein interaction predictions revealed that PFC0760c was likely to interact with proteins involved with nucleic acid binding. PFC0760c was predicted to have a domain found in proteins involved in the structural maintenance of chromosomes, which may suggest the protein is involved in chromatid cohesion during mitotic chromosome condensation. PFC0760c was also predicted to be located in the nucleus by the sub cellular prediction program, SubLoc. Anti-peptide antibodies were raised against the conserved amino acid sequence and against a peptide specific for PY05757 (SDDDNRQIQDFE). Both antibodies detected native antigens with immunofluorescence microscopy. The fluorescent signal appeared throughout the parasite cytoplasm and as an intense signal in the parasite nucleus. These immunofluorescence data supports the predicted nuclear location of the protein. A 822 bp portion of PFC0760c gene was expressed as a maltose-binding protein fusion protein (Pf33-MBP). Pf33-MBP was expressed and purified. Reducing SDS-PAGE and western blotting analysis revealed the fusion protein to be expressed at low levels as four bands (79, 60, 45 and 37 kDa). The purified fusion protein was cleaved with Factor Xa. MBP and Pf33 were of similar molecular mass after cleavage. To attempt to obtain better expression and purification, the 822 bp insert from pTS822 was sub-cloned into pGEX4T1. A glutathione-S-transferase (GST)-fusion protein (Pf33-GST) was expressed. The level of expression was poor and therefore not pursued. To take the study further, potential proteins that interact with PFC0760c and Pf33 need to be identified. In addition, immunisation of mice with… Advisors/Committee Members: Goldring, J. P. Dean (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Viljoen, J. E. (2013). A study of recombinant Plasmodium falciparum PFC0760c. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10198

Chicago Manual of Style (16^th Edition):

Viljoen, Jacqueline Ethel. “A study of recombinant Plasmodium falciparum PFC0760c.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10198.

MLA Handbook (7^th Edition):

Viljoen, Jacqueline Ethel. “A study of recombinant Plasmodium falciparum PFC0760c.” 2013. Web. 24 Jun 2019.

Vancouver:

Viljoen JE. A study of recombinant Plasmodium falciparum PFC0760c. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10198.

Council of Science Editors:

Viljoen JE. A study of recombinant Plasmodium falciparum PFC0760c. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10198

University of KwaZulu-Natal

26. Jugmohan, Mayuri. Killing of mycobacteria by macrophage cathepsin D.

Degree: MS, Biochemistry, 2011, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10417

► Tuberculosis (TB) is the fifth largest cause of death in South Africa, with one in ten cases being resistant to treatment due to the development… (more)

▼ Tuberculosis (TB) is the fifth largest cause of death in South Africa, with one in ten cases being resistant to treatment due to the development of multidrug-resistance and extensively drug-resistance in the agent responsible for this disease, Mycobacterium tuberculosis. This pathogen has developed mechanisms to evade killing by immune cells such as macrophages. Mycobacterium smegmatis, a non-pathogen, that does not evade killing by the macrophage, is often used to gain a better insight into the bacteriocidal pathways used to kill mycobacteria, and those potentially blocked by M.tuberculosis. In such studies nitric oxide and “lysosomal” proteases have emerged as major bacteriocidal pathways. Studies on the role of aspartic protease, cathepsin D, in killing green fluorescent protein- (GFP-) tagged-M.smegmatis in J774 macrophages required antibodies that would not cross-react with mycobacterial antigens. These were raised in chickens, using alum and saponin as adjuvants, and porcine and human cathepsin D. Using such antibodies, quantitative colocalization analysis using ImageJ and the JACoP colocalization plugins showed a greater degree of colocalization between cathepsin D and LysoTracker Red DND-99 in M.smegmatis-infected J774 macrophages than in uninfected cells. This indicates the possible presence of active, bacteriocidal cathepsin D in acidic, and hence matured phagosomes. A higher colocalization between cathepsin D and LAMP-1 and cathepsin D and LAMP-2 in uninfected cells possibly indicates the recycling of these two markers from vesicles not containing killed bacteria. Propidium iodide (PI) labelling and loss of GFP fluorescence appeared reliable indicators of M.smegmatis death or viability, respectively, as myobacteria that took up PI also lost green fluorescence, while M.smegmatis that exhibited green fluorescence (viable) were not observed to take up propidium iodide (dead). Faint colocalization between cathepsin D, LAMP-1 and -2 with dead, and to a lesser extent with live M.smegmatis occurred. Besides intensity correlation values other colocalization programs indicate the absence of colocalization between these markers and dead M.smegmatis, but, together with in vitro killing experiments (cathepsin D, 0.0098 units/ml resulting in 59% killing in 4 h) these appear to indicate a possible role of cathepsin D in killing of M.smegmatis. Advisors/Committee Members: Elliott, Edith (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Jugmohan, M. (2011). Killing of mycobacteria by macrophage cathepsin D. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10417

Chicago Manual of Style (16^th Edition):

Jugmohan, Mayuri. “Killing of mycobacteria by macrophage cathepsin D.” 2011. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10417.

MLA Handbook (7^th Edition):

Jugmohan, Mayuri. “Killing of mycobacteria by macrophage cathepsin D.” 2011. Web. 24 Jun 2019.

Vancouver:

Jugmohan M. Killing of mycobacteria by macrophage cathepsin D. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2011. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10417.

Council of Science Editors:

Jugmohan M. Killing of mycobacteria by macrophage cathepsin D. [Masters Thesis]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/10417

University of KwaZulu-Natal

27. Bohnen, Daniel. Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors.

Degree: MS, Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10552

► Several diseases, including cancer, have been associated with high membrane type-1 matrix metalloproteinase (MT1-MMP) expression levels. MT1-MMP together with a non-membrane-bound, soluble MMP, MMP-2, also… (more)

▼ Several diseases, including cancer, have been associated with high membrane type-1 matrix metalloproteinase (MT1-MMP) expression levels. MT1-MMP together with a non-membrane-bound, soluble MMP, MMP-2, also associated with many other biological functions, have been implicated in breast cancer progression, invasion and metastasis, and poor prognosis. Researchers who ran early clinical trials that employed broad-spectrum MMP inhibitors (MMPIs) lacked understanding of the intricate physiological and patho-physiological roles that MMPs play in tissues. In addition, structural similarities between MMPs hamper selective inhibition. Selective inhibition of MT1-MMP is of particular interest as MT1-MMP is overexpressed in target cancer cells relative to normal cells and is key in signalling for invasion. The inhibition profiles of two structurally similar synthetic pyrimidine-class MMPIs, with increased bioavailability and stability compared to hydroxamates tested in earlier clinical trials, TF 17-2 and TF 22d, were assessed. TF 17-2 and TF 22d were applied to a normal MCF-10A breast epithelial cell line and its premalignant H-ras(V12)-transfected MCF-10AneoT derivative to assess their efficacy for inhibiting MT1-MMP-mediated normal and premalignant cell migration, and indirectly, invasion. Both inhibitors form a co-ordination complex with the zinc ion of the catalytic site of MT1-MMP and MMP-2 with greater affinity for MT1-MMP. The computational molecular docking package, AutoDock Vina, was used for in silico predictions of binding affinities that could potentially substitute for in vitro kinetic assays when assessing inhibitor potential for inhibiting target MMPs. The binding of the two MMPIs was assessed using AutoDock Vina and compared to established kinetic data. The AutoDock Vina program was found to be an unreliable predictor for assessing relative efficacy of inhibition. During in vitro applications, analysis of the induction of apoptosis and metabolic effects were assessed using flow cytometry and the MTS assay, respectively. These showed no significant toxicity. Effects of inhibitors on collective and single cell migration in the normal and premalignant cell model, assessed using time lapse live cell imaging, cell morphology and labelling for vinculin and F-actin (for focal adhesions, FAs) showed that the TF 17-2 and TF 22d inhibitors reduced the collective cell migration of MCF-10A cells in scratch assays. Live-cell analysis of single cell migration, however, showed that TF 22d increased cell migration rates, and reduced the size of FAs and actin stability in MCF-10AneoT cells, resulting in a predominently rounded cell morphology in the premalignant cell line. TF 17-2, on the other hand was seen to be a relatively selective inhibitor of premalignant cell migration and resulted in MCF-10AneoT cells re-establishing larger focal - v - adhesions due to more stable F-actin networks resembling those of the non-transfected MCF-10A cell line, but reduced MCF-10AneoT cell migration most markedly. FA size and velocity of… Advisors/Committee Members: Elliott, Edith (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Bohnen, D. (2013). Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10552

Chicago Manual of Style (16^th Edition):

Bohnen, Daniel. “Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/10552.

MLA Handbook (7^th Edition):

Bohnen, Daniel. “Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors.” 2013. Web. 24 Jun 2019.

Vancouver:

Bohnen D. Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/10552.

Council of Science Editors:

Bohnen D. Membrane type I metalloproteinase (mt1-mmp) as a target in cancer : a study of two inhibitors. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10552

University of KwaZulu-Natal

28. Ramlucken, Uraisha. Membrane permeability of HIV-1 protease inhibitors.

Degree: Biochemistry, 2014, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/11372

► According to the 2012 UNAIDS global report, sub-Saharan Africa hosts 69% of the world’s total population living with HIV, South Africa being the most affected… (more)

▼ According to the 2012 UNAIDS global report, sub-Saharan Africa hosts 69% of the world’s total population living with HIV, South Africa being the most affected with a reported 24% incidence rate. To date, extensive research is being conducted globally, particularly involving anti-HIV treatment that targets the retroviral enzymes: reverse transcriptase, integrase and protease. The discovery of inhibitors to HIV protease which disrupts virion protein assembly has made this enzyme a prime target of anti-retroviral therapies, thus there exists a concerted research initiative to identify compounds with HIV protease inactivation potential. This study employs HIV protease that is isolated and purified from a genetically modified HIV protease overexpressing Escherichia coli strain to monitor the inhibitory capacity of new lead compounds. Optimized growth conditions for HIV protease production displayed that the use of chemically defined media resulted in higher yields of the enzyme. Recent research studies have shown that peptide-based cage and glycosylated compounds displayed HIV protease inhibitor activity in cell free enzymatic reactions that are comparable to commercially available HIV protease inhibitors. However, in contrast it has also been reported that these inhibitors are inactive in whole T-cell assays, when employing HIV infected CD4 cells. It is a well-known fact that potential new chemical entities that do not possess oral bioavailability, in terms of their absorption properties, are not successful candidates within the drug discovery industry. Following this, the current study was designed to determine if inefficient membrane permeability of these promising anti-HIV protease lead compounds could result in their inactivity in whole T-cell assays. Two different methods were considered, a cell-based method using the Madin Darby Canine Kidney strain I (MDCKI) cell line and a non-cell based method, the parallel artificial membrane permeability assay (PAMPA). MDCKI cells have been extensively used to form monolayers that mimic human intestinal membranes whilst the PAMPA utilizes an artificial lipid membrane composition on a filter support. Data from permeability assays using the novel chemically synthesized inhibitors have been compared to commercially available drugs, antipyrine, metoprolol and caffeine, which displayed efficient membrane permeability characteristics, thereby validating the assay. The results indicated that novel cage-derived and glycosylated peptide inhibitors do not possess sufficient passive diffusion properties which may explain their inactivity in whole T-cell assays. Advisors/Committee Members: Govender, Patrick (advisor), Pillay, Karen (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Ramlucken, U. (2014). Membrane permeability of HIV-1 protease inhibitors. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/11372

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16^th Edition):

Ramlucken, Uraisha. “Membrane permeability of HIV-1 protease inhibitors.” 2014. Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/11372.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7^th Edition):

Ramlucken, Uraisha. “Membrane permeability of HIV-1 protease inhibitors.” 2014. Web. 24 Jun 2019.

Vancouver:

Ramlucken U. Membrane permeability of HIV-1 protease inhibitors. [Internet] [Thesis]. University of KwaZulu-Natal; 2014. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/11372.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Ramlucken U. Membrane permeability of HIV-1 protease inhibitors. [Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/11372

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of KwaZulu-Natal

29. Moothoo-Padayachie, Anushka. Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

Degree: MS, Biochemistry, 2011, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/5839

► In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping).… (more)

▼ In clinical diagnosis and fermentation industries there is a need for a method that allows for the differentiation of yeast to the strain level (biotyping). The ideal biotyping method should be accurate, simple, rapid and cost-effective, and capable of testing a large number of yeast isolates. Matrix assisted laser desorption/ionization time of flight mass spectrometry has emerged as a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, mainly Candida. It has been found that the MALDI-TOF MS signals from yeast are harder to obtain than from bacteria. It has been reported by several research studies that a cell lysis step is required to obtain a mass spectral signal for clinical Candida strains. To date an optimized sample preparation protocol has not been devised for the biotyping of S. cerevisiae strains. Studies on the identification of yeast using MALDI-TOF MS have focused primarily on clinical Candida yeast isolates but have included very few S. cerevisiae strains. Furthermore these yeast identification studies using MALDI-TOF MS have only achieved identification to the species and not strain level. A major limiting attribute of MALDI-TOF MS for the accurate identification of microbes, is its dependency on a comprehensive mass spectral database. Bruker Daltonics is a pioneer and leader in providing innovative life science tools based on mass spectrometry thus the Bruker Daltonics mass spectral database and state-of-the-art instruments and accompanying software were selected for this study. The Bruker Daltonics mass spectral database currently holds three thousand seven hundred and forty microorganisms of which only a mere seven are S. cerevisiae strains. Initially in this study, a number of parameters of a generic ethanol/formic acid protein extraction procedure as originally described by Bruker Daltoincs were considered in the development of a sample preparation protocol that yielded characteristic and highly reproducible MALDI-TOF mass spectra. The parameters considered included cell number, alcohol fixation, matrix solution and media. It was found that using the optimized sample preparation protocol unique and highly reproducible mass spectral profiles were obtained for all three S. cerevisiae strains. Multivariate analysis confirmed that the differences between all three S. cerevisiae strains were statistically significant. For quality assurance, the spectra of the three strains were sent for evaluation by Bruker Daltonics and were deemed suitable for the purpose of biotyping. The newly created ethanol/formic acid extraction procedure was used to generate an S. cerevisiae mass spectral database comprising of forty-five S. cerevisiae strains within a local context but also of global significance. The accuracy of the mass spectral database was assessed using blind coded S. cerevisiae strains obtained from the Agricultural Research Council Infruitec-Nietvoorbij (Institute for Deciduous Fruit, Vines and Wine), Stellenbosch, South Africa. It was found that S. cerevisiae… Advisors/Committee Members: Kruger, Hendrik Gert (advisor), Maguire, Glen Eamonn Mitchel (advisor), Govender, Thavendran (advisor), Govender, Patrick (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Moothoo-Padayachie, A. (2011). Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/5839

Chicago Manual of Style (16^th Edition):

Moothoo-Padayachie, Anushka. “Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).” 2011. Masters Thesis, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/5839.

MLA Handbook (7^th Edition):

Moothoo-Padayachie, Anushka. “Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).” 2011. Web. 24 Jun 2019.

Vancouver:

Moothoo-Padayachie A. Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). [Internet] [Masters thesis]. University of KwaZulu-Natal; 2011. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/5839.

Council of Science Editors:

Moothoo-Padayachie A. Biotyping Saccharomyces cerevisiae strains using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). [Masters Thesis]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/5839

University of KwaZulu-Natal

30. Du Plessis, Lynn. The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level.

Degree: PhD, Biochemistry, 2006, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/5530

► The literature contains conflicting ideas about the effectiveness of diagrams, and their constituent symbolism as teaching and learning tools. In addition, only limited research has… (more)

▼ The literature contains conflicting ideas about the effectiveness of diagrams, and their constituent symbolism as teaching and learning tools. In addition, only limited research has been specifically conducted on the presentation and interpretation of arrow symbolism used in biology diagrams, let alone on the nature, source and remediation of student difficulties caused by arrows. On the basis of this limited research and 30 years of experience of teaching biology at secondary-level, the author suspected that students might have difficulties interpreting arrow symbolism in diagrams used as explanatory tools and decided to thoroughly investigate this issue. The hypothesis, 'Secondary-level students have difficulty with the use of arrow symbolism in biology diagrams' was formulated and the following broad research questions defined to address the hypothesis: 1. How much of a problem is arrow symbolism in diagrams? 2. How effectively is arrow symbolism used in diagrams to promote the communication of intended ideas? 3. To what extent does the design of arrow symbolism in diagrams influence students ' interpretation and difficulties? 4. How can the emerging empirical data and ideas from literature be combined to illustrate the process of interpretation of arrow symbolism? 5. What measures can be suggested for improving the presentation and interpretation of arrow symbolism in biology diagrams at secondary-level? To address Research question 1, a content analysis of all arrow symbolism in seven popular secondary-level biology textbooks was undertaken. This revealed a wide diversity of arrow styles, spatial organisations, purposes and meanings that could be confusing to students. These results suggested the need for an evaluation of the effectiveness of arrow symbolism (Research question 2). As there was no definitive set of guidelines available for specifically evaluating arrows, general guidelines from the literature on diagrams were used to develop a set of 10 criteria, to evaluate the syntactic, semantic and pragmatic dimensions of arrow symbolism, which were validated by selected educators, students and a graphic design expert. Application of the criteria (which constituted expert opinion) to the arrow symbolism used in 614 realistic, stylised and abstract diagram types, revealed a relatively high incidence (30%) of inappropriately presented arrow designs that could mislead students. To establish whether this problem could be the cause of student difficulties, and to thereby address Research question 3, a stylised and an abstract diagram were selected and evaluated according to the criteria. The results of the evaluation were compared to the responses given by 174 students to a range of written and interview probes and student modified diagrams. In this way, student performance was correlated with expert opinion. The results confirmed that students experience a wide range of difficulties (26 categories) when interpreting arrow symbolism, with some (12 categories) being attributable to inappropriately presented arrow… Advisors/Committee Members: Anderson, Trevor Ryan (advisor).

Subjects/Keywords: Biochemistry.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Du Plessis, L. (2006). The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/5530

Chicago Manual of Style (16^th Edition):

Du Plessis, Lynn. “The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level.” 2006. Doctoral Dissertation, University of KwaZulu-Natal. Accessed June 24, 2019. http://hdl.handle.net/10413/5530.

MLA Handbook (7^th Edition):

Du Plessis, Lynn. “The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level.” 2006. Web. 24 Jun 2019.

Vancouver:

Du Plessis L. The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2006. [cited 2019 Jun 24]. Available from: http://hdl.handle.net/10413/5530.

Council of Science Editors:

Du Plessis L. The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level. [Doctoral Dissertation]. University of KwaZulu-Natal; 2006. Available from: http://hdl.handle.net/10413/5530

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