+publisher:"University of KwaZulu-Natal" +contributor:("Coetzer, Theresa Helen Taillefer")

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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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

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 April 24, 2018. 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 Apr 2018.

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 2018 Apr 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. 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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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 April 24, 2018. http://hdl.handle.net/10413/9788.

MLA Handbook (7^th Edition):

Huson, Laura. “Antibody-mediated inhibition of proteases of African trypanosomes.” 2013. Web. 24 Apr 2018.

Vancouver:

Huson L. Antibody-mediated inhibition of proteases of African trypanosomes. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 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

3. [No author]. Investigation into the major surface proteases of African trypanosomes.

Degree: 2016, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/14213

► The unicellular parasite of the genus Trypanosoma infects a number of mammalian species including livestock and humans. In sub-Saharan Africa three main parasitic species cause… (more)

▼ The unicellular parasite of the genus Trypanosoma infects a number of mammalian species including livestock and humans. In sub-Saharan Africa three main parasitic species cause disease: Trypanosoma brucei, T. congolense and T. vivax. The lack of sensitive diagnosis and increased drug resistance leaves an avenue in trypanosome research for exploring novel virulence factors as diagnostic and chemotherapeutic agents. The work reported in this dissertation involved investigation of the identified virulence factor, the Major Surface Protease (MSP), of African trypanosomes. The MSPs comprise a group of metalloproteases which have been found in over 13 Leishmanian species, T. cruzi, T. brucei and T. congolense as well as other Kinetoplastids. In this study, putative M8 metalloprotease sequences were also identified in the T. vivax genome. These putative sequences were grouped into four classes of protein, TvMSP-A, -C, -D and -E by phylogenetic comparison with other MSPs. Three-dimensional modelling showed high structural identity with leishmanolysin from Leishmania major. The T. vivax MSP sequences were used, in conjunction with T. brucei and T. congolense sequences, to select immunogenic peptide regions to produce anti-peptide antibodies. Three peptides were selected with the intention to 1) detect both TbMSP-B and TcoMSP-B (peptide Tb/TcoMSP:303-314, cross-species), 2) detect only TbMSP-C (peptide TbMSP:400-412) and 3) detect only TvMSP-C (peptide TvMSP:686-697). They were used to generate two types of detection molecules: complete IgY anti-peptide antibodies and single chained fragments (scFvs), with the capability to detect the peptide in an ELISA format. Single scFv expressing E. coli colonies were successfully selected and shown to detect two (Tb/TcoMSP:303-314 and TbMSP:400-412) of the three peptides. The anti-peptide antibodies, produced in chickens, were used to successfully detect native MSP within T. brucei and T. congolense parasite lysates; however, cross-reactivity between species was seen. The T. brucei MSP-C class of protease was successfully cloned, expressed and purified, although, numerous truncated proteins and gene mutations occurred [truncated (t)TbMSP-C]. The expression constructs rTbMSP-C and rTcoMSP-C were hence synthesised. These two enzymes were successfully expressed and purified and were shown to form high molecular weight multimers. Furthermore, the enzymes were able to cleave the peptide substrate H-Suc-Leu-Tyr-AMC with acidic pH optima and activity was inhibited with metalloprotease inhibitors, EDTA and 1,10 phenanthroline. The successful detection of rTcoMSP-C by T. congolense infected cattle sera was also observed. tTbMSP-C, rTbMSP-C and rTcoMSP-C were detected with the chicken anti-peptide antibodies and it was again found that these antibodies cross-reacted with different species MSPs. The high identity shared between MSPs from all Trypanosoma species made selecting species-specific antibodies difficult. Further work to detect native MSP-C protease within infected sera or blood would… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.; Proteolyic enzymes.; Trypanosoma.

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

APA (6^th Edition):

author], [. (2016). Investigation into the major surface proteases of African trypanosomes. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/14213

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):

author], [No. “Investigation into the major surface proteases of African trypanosomes. ” 2016. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/14213.

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

MLA Handbook (7^th Edition):

author], [No. “Investigation into the major surface proteases of African trypanosomes. ” 2016. Web. 24 Apr 2018.

Vancouver:

author] [. Investigation into the major surface proteases of African trypanosomes. [Internet] [Thesis]. University of KwaZulu-Natal; 2016. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/14213.

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

Council of Science Editors:

author] [. Investigation into the major surface proteases of African trypanosomes. [Thesis]. University of KwaZulu-Natal; 2016. Available from: http://hdl.handle.net/10413/14213

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

University of KwaZulu-Natal

4. Ndlovu, Sijabulisiwe Faith. Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.

Degree: 2015, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/12249

► African Animal trypanosomosis, also known as Nagana, is a parasitic disease which affects many mammalian species, mainly livestock such as cattle, sheep and goats. The… (more)

▼ African Animal trypanosomosis, also known as Nagana, is a parasitic disease which affects many mammalian species, mainly livestock such as cattle, sheep and goats. The disease also affects humans (Human African Trypanosomosis) and in this case is referred to as sleeping sickness. Nagana is caused by the Trypanosoma parasite, which is transmitted to the host by a bite from the tsetse fly (Glossina spp). The Trypanosoma causing trypanosomosis in animals are Trypanosoma congolense, T. vivax and T. brucei brucei. Vaccine development has been unsuccessful, due to the presence of the variant surface glycoprotein on the surface of parasites which undergoes antigenic variation therefore enabling the parasite to avoid detection by vaccines. A chemotherapeutic drug such as isometamidium chloride combined with diminazene and suramin have also had little success due to the increase in drug resistance. During infection of the host, trypanosomal parasites utilise various proteolytic enzymes such as the oligopeptidases, which hydrolyse important host factors such as peptide hormones. These proteolytic enzymes are thus considered to be pathogenic factors which contribute to the manifestation of various trypanosomosis symptoms such as anaemia, fever, paralysis and disturbances in sleep cycle patterns. It is these pathogenic factors which are now being considered as drug targets in the hope to eradicate the spread or continuous advancement of trypanosomosis. Three trypanosomal pathogenic factors, which are serine oligopeptidases which belong to the prolyl oligopeptidase family of serine proteases (Clan SC in subfamily S9) were the focus of this study, namely, prolyl oligopeptidase (POP) from T. b. brucei (TbPOP) and T. congolense (TcoPOP) as well as oligopeptidase B (OPB) from T. congolense (TcoOPB). The full length TbPOP gene was cloned into pTZ57R/T cloning vector and successfully sub-cloned into pET32a expression vector and recombinantly expressed in its insoluble form at a size of approximately 100 kDa using the Escherichia coli BL21 DE3 expression system. TbPOP expression was confirmed by western blot probed with anti-His tag antibodies. Expression of TbPOP was optimised under varying temperatures and IPTG concentrations in an attempt to solubilise the inclusion bodies. However, the protein was expressed as part of inclusion bodies. Therefore, urea denaturation was used for its solubilisation. Following solubilisation, recombinant TbPOP was partially purified on a Ni2+ affinity resin. Further attempts to purify TbPOP by molecular exclusion chromatography (MEC) were unsuccessful, this could be due to aggregation of the protein during the refolding step. Therefore refolding by a Sephadex G-25 desalting column was attempted as it removes some impurities. However, further purification by MEC and ion exchange chromatography (IEC) were unsuccessful. The full-length TcoPOP gene was successfully cloned into pGEM-T® cloning vector and subsequently sub-cloned into pET32a expression vector. However, upon sequencing of the plasmid DNA, it… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Ndlovu, S. F. (2015). Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/12249

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):

Ndlovu, Sijabulisiwe Faith. “Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.” 2015. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/12249.

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

MLA Handbook (7^th Edition):

Ndlovu, Sijabulisiwe Faith. “Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.” 2015. Web. 24 Apr 2018.

Vancouver:

Ndlovu SF. Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors. [Internet] [Thesis]. University of KwaZulu-Natal; 2015. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/12249.

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

Council of Science Editors:

Ndlovu SF. Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors. [Thesis]. University of KwaZulu-Natal; 2015. Available from: http://hdl.handle.net/10413/12249

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

University of KwaZulu-Natal

5. [No author]. Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases.

Degree: Biochemistry, 2011, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/8688

► African animal trypanosomosis or nagana is a disease in livestock caused by various species of protozoan parasites belonging to the genus Trypanosoma particularly T. congolense,… (more)

▼ African animal trypanosomosis or nagana is a disease in livestock caused by various species of protozoan parasites belonging to the genus Trypanosoma particularly T. congolense, T. vivax and T. b. brucei. Nagana is the most important constraint to livestock and mixed crop-livestock farming in tropical Africa. Trypanosomes undergo part of their developmental life in their insect vector, the tsetse fly and part in their mammalian host. Measures for eradicating the continent of the tsetse fly vector include insecticidal spraying, targeting and trapping. Vaccine development has been hampered by the generation of an inexhaustible collection of variant surface glycoproteins that trypanosomes possess and allow for evasion of the host immune system. Anti-disease vaccines aimed at reducing the symptoms of the disease rather than killing the parasite itself have been demonstrated as an alternative approach. Trypanotolerant cattle are able to protect themselves from the disease-associated symptoms. They are able to mount a better antibody response to the CATL-like cysteine peptidase, TcoCATL, compared to trypanosusceptible breeds. Bovine trypanosomosis, however, continues to be controlled primarily by trypanocidal compounds such as isometamidium chloride, homidium and diaminazene that have been developed more than 50 years ago and consequently drug resistance is widespread. Trypanosomal cysteine peptidases have also been proven to be effective targets for chemotherapeutics. TcrCATL, inhibited by the vinyl sulfone pseudopeptide inhibitor K11777, was effective in curing or alleviating T. cruzi infection in preclinical proof-of-concept studies and has now entered formal preclinical drug development investigation. Understanding enzymatic as well as structural characteristics of pathogenic peptidases is the first step towards successful control of the disease. To date no such characterisation of the major cysteine peptidases from T. vivax has been conducted. Although the major cysteine peptidase from T. vivax, TviCATL, has not been proven as a pathogenic factor yet, its high sequence identity with the pathogenic counterparts such as TcrCATL and TcoCATL hold much speculation for TviCATLs role in pathogenocity. In the present study, native TviCATL was isolated from T. vivax Y486, purified and characterised. TviCATL showed to have a general sensitivity to E-64 and cystatin and has a substrate specificity defined by the S2 pocket. TviCATL exhibited no activity towards the CATB-like substrate, Z-Arg-Arg-AMC but was able to hydrolyse Z-Phe-Arg-AMC, the CATL-like substrate. Leu was preferred in the P2 position and basic and non-bulky hydrophobic residues were accepted in the P1 and P3 positions respectively. Similar findings were reported for TcoCATL. The substrate specificity of TviCATL and TcoCATL does argue for a more restricted specificity compared to TcrCATL. This was based on the Glu333 in TcrCATL substituted with Leu333 in TviCATL and TcoCATL. In the case of TcrCATL, the Glu333 allows for the… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: African trypanosomiasis.; Trypanosomiasis in animals – Immunotherapy.; Cysteine proteinases – Structure.; Biochemistry.

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

APA (6^th Edition):

author], [. (2011). Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/8688

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):

author], [No. “Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases. ” 2011. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/8688.

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

MLA Handbook (7^th Edition):

author], [No. “Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases. ” 2011. Web. 24 Apr 2018.

Vancouver:

author] [. Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases. [Internet] [Thesis]. University of KwaZulu-Natal; 2011. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/8688.

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

Council of Science Editors:

author] [. Enzymatic and crystallisation studies of CATL-like trypanosomal cysteine peptidases. [Thesis]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/8688

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

University of KwaZulu-Natal

6. [No author]. Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes.

Degree: Biochemistry, 2011, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/8708

► African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei… (more)

▼ African trypanosomosis is a parasitic disease in man and animals caused by protozoan parasites of the genus Trypanosoma. T. congolense, T. vivax and T. brucei brucei cause nagana in cattle. The variable nature of the parasite surface coat has hindered the development of an effective vaccine. An option for developing vaccines and chemotherapeutic agents against trypanosomosis is to target pathogenic factors released by the parasite during infection, namely an “anti-disease” approach. Two pathogenic factors released during infection are oligopeptidase B (OPB) and TcoCATL (congopain). TcoCATL, a major lysosomal cysteine peptidase, is a member of the papain family C1 cysteine peptidases. RNA interference (RNAi) was used to down-regulate the expression of TcoCATL in T. congolense IL3000 TRUM183:29-13 parasites in vivo during mouse infections. TcoCATL RNAi was monitored in infected mouse blood by comparing the hydrolysis of Z-Phe-Arg-AMC and parasitaemia between mice in which RNAi was induced and control mice. Mice infected with parasites induced for TcoCATL RNAi had lower parasitaemia when compared to control mice. An attempt was also made at deleting the entire CATL gene array in both T. congolense IL3000 and T. brucei 427 Lister strains. The second pathogenic factor studied, OPB, is a cytosolic trypanosomal peptidase that hydrolyses peptides smaller than 30 amino acid residues, C-terminal to basic residues. In order to evaluate the role that OPB play during disease, RNAi was also applied to knock-down the expression levels of OPB in T. brucei T7T and T. congolense IL3000 TRUM183:29-13 strains (TbOPB and TcoOPB respectively). Oligopeptidase B null mutant strains (Δopb) were also generated in T. brucei brucei Lister 427. An attempt was also made to generate OPB null mutants in T. congolense IL3000 parasites. Western blot analysis of the knock-down experiments using chicken anti-TcoOPB peptide IgY showed that only TbOPB levels were reduced in T. brucei T7T parasites induced for RNAi when compared to TcoOPB RNAi induced cultures. Quantitative assessment of a fourteen day induction experiment for OPB RNAi in T. brucei showed an 87% reduction in TbOPB levels when compared to levels on day one. There was no growth effect observed in T. brucei parasites cultured in vitro and induced for TbOPB RNAi. It was concluded that TbOPB is not necessary for the in vitro survival of T. brucei parasites, thus making the generation of OPB null mutants possible. Δopb T. brucei parasites were successfully generated and grew normally in vitro and were as virulent as wild type strains during infection in mice. Immunohistopatholgy of infected mouse testes revealed Δopb parasites in extra vascular regions showing that T. brucei OPB (TbOPB) is not involved in assisting T. brucei parasites to cross microvascular endothelial cells. Gelatin gel analysis of Δopb null mutants and wild type strains showed an increase in cysteine peptidase activity. Enzymatic activity assays were carried out to identify how closely related oligopeptidases are affected by… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Trypanosomiasis in animals.; Trypanosoma.; African trypanosomiasis.; Cysteine proteinases.; Biochemistry.

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

APA (6^th Edition):

author], [. (2011). Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/8708

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):

author], [No. “Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes. ” 2011. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/8708.

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

MLA Handbook (7^th Edition):

author], [No. “Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes. ” 2011. Web. 24 Apr 2018.

Vancouver:

author] [. Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes. [Internet] [Thesis]. University of KwaZulu-Natal; 2011. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/8708.

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

Council of Science Editors:

author] [. Gene disruption of TcoCATL (Congopain) and oligopeptidase B, pathogenic factors of African trypanosomes. [Thesis]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/8708

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

University of KwaZulu-Natal

7. [No author]. Infectious bursal disease virus receptor identification with anti-peptide antibodies.

Degree: Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9900

► Infectious bursal disease virus (IBDV) has a tropism for the lymphoid tissue of poultry and infects actively dividing and differentiating B-lymphocytes in the bursa of… (more)

▼ Infectious bursal disease virus (IBDV) has a tropism for the lymphoid tissue of poultry and infects actively dividing and differentiating B-lymphocytes in the bursa of Fabricius. This results in a high mortality rate and severe immunosuppression. These immunodepressed chickens are highly susceptible to secondary infections and have a reduced capacity to respond to vaccination. The principal method to control IBDV is through extensive vaccination using either attenuated live or inactivated IBDV vaccines. However, in recent years due to the emergence of new virulent strains, risk of reversion to pathogenicity, cost considerations and intervention by maternal antibodies, the effectiveness of these vaccines in the veterinary field is being reduced. An alternative approach to prevent infection is by interfering with the binding of IBDV to its receptor protein on the surface of bursal cells. Hence this study was undertaken on the characterisation of a possible IBDV receptor on bursal membranes. Infectious bursal disease virus was isolated from infected bursal tissue using CsCl density gradient centrifugation and visualised with Tris-Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy. Following purification of double stranded RNA from infected bursal tissue and commercially available live IBDV vaccines, a polymerase chain reaction (PCR)-based diagnostic assay based on sequences from the highly conserved viral protein (VP2) region was performed. The presence of the virus was demonstrated by the amplification of a 150 bp band in 2% agarose and 15% nondenaturing PAGE gels. The correctness of this product was confirmed byrestriction digestion with a specific restriction endonuclease (BamHI) that resulted in the predicted digestion fragments of 93 and 57 bp. Following preparation of bursal membrane proteins from uninfected bursal tissue, using sucrose density gradient centrifugation, isolation of IBDV receptor protein was carried out by immobilising IBDV on a Sepharose 4B chromatography matrix. After affinity purification, two prominent protein bands around 40 kDa were visualised using a silver stained Tris-Tricine SDS-PAGE gel. Previous work in this laboratory identified two possible IBDV receptor proteins on bursal membranes of 32 and 40 kDa. Antibodies against peptide sequences derived from the 32 kDa receptor protein were raised in rabbits in the present study. These anti-IBDV receptor peptide antibodies recognised the affinity purified native 40 kDa IBDV receptor proteins in an enzyme-linked immunosorbent assay (ELISA). However, due to the possible epitope denaturation by the reducing treatment buffer prior to Tris-Tricine SDS-PAGE such as SDS and 2-mercapthethanol or detergent (Na-deoxycholate) used during the affinity purification of the IBDV receptor protein, the anti-IBDV receptor peptide antibody did not recognise the receptor protein on a western blot. An inhibition assay was performed in an ELISA format by coating the 40 kDa IBDV receptor protein to… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Poultry – Virus diseases.; Chickens – Diseases.; Immunoglobulins – Analysis.; Biochemistry.

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

APA (6^th Edition):

author], [. (2013). Infectious bursal disease virus receptor identification with anti-peptide antibodies. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9900

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):

author], [No. “Infectious bursal disease virus receptor identification with anti-peptide antibodies. ” 2013. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/9900.

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

MLA Handbook (7^th Edition):

author], [No. “Infectious bursal disease virus receptor identification with anti-peptide antibodies. ” 2013. Web. 24 Apr 2018.

Vancouver:

author] [. Infectious bursal disease virus receptor identification with anti-peptide antibodies. [Internet] [Thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/9900.

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

Council of Science Editors:

author] [. Infectious bursal disease virus receptor identification with anti-peptide antibodies. [Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9900

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

University of KwaZulu-Natal

8. [No author]. Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis.

Degree: 2014, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/12255

► For the effective control of African animal trypanosomiasis, there is a great need for the development of point of care diagnostic tests that are affordable… (more)

▼ For the effective control of African animal trypanosomiasis, there is a great need for the development of point of care diagnostic tests that are affordable to the end-users. Parasite cysteine proteases are involved in the pathogenesis of African trypanosomiasis, hence serve as a good target for the development of new chemotherapeutics and diagnostics. The aim of this study was to evaluate the potential of the cysteine protease TviCATL from Trypanosoma vivax as a chemotherapeutic agent and diagnostic target for African animal trypanosomiasis. The catalytic domain, TviCATL, was recombinantly expressed in Pichia pastoris, auto-catalytically activated by low pH removal of the proregion, purified by three phase partitioning and molecular exclusion chromatography to homogeneity and its identity confirmed by western blot analysis. Endoglycosidase H treatment of TviCATL indicated that the protease is Asn-glycosylated. The pH optimum was determined to range between 6.5 and 7.5 suggesting that the enzyme would be active in the host bloodstream following parasite lysis. The protease was able to degrade the host proteins: bovine serum albumin, bovine haemoglobin, gelatin, type I collagen and the endogenous protease inhibitor, bovine α2-macroglobulin in vitro at neutral pH. The peptidolytic specificity of the protease was determined by considering the active site binding pocket S2- substrate -P2 interaction. TviCATL showed high endopeptidase specificity for Z-Phe-Arg-AMC and H-D-Ala-Leu-Lys-AMC, suggesting that the hydrophobic residues Phe or Leu are favoured at the P2 position in the presence of basic Arg or Lys at P1 position. The TviCATL peptidolytic activity was inhibited by E-64, iodoacetate, leupeptin, antipain, Z-Gly-Leu-Phe-CMK (albeit at a reduced level) and iodoacetamide inhibitors and this indicated that TviCATL is a cysteine protease. Satisfactory anti-TviCATL antibody levels were produced in chickens as evidenced by good signals in the ELISA and western blot analysis. The specificity and affinity of chicken anti-TviCATL IgY antibodies for TviCATL antigen was improved by affinity purification of these antibodies using a TviCATL-affinity column. The serodiagnostic potential of the TviCATL antigen and cross-reactivity with anti-T. congolense antibodies (in sera) was evaluated in an antibody inhibition ELISA format. These antibodies were able to discriminate between non-infected cattle sera and T. congolense-infected sera, thus suggesting some degree of cross-reactivity between TviCATL antigen and anti-T. congolense antibodies. As an alternative to animal-based antibody production, the Nkuku® phage library was used to select for single-chain variable fragment (scFvs) antibodies by panning against TviCATL antigen. As evaluated by polyclonal and monospecific phage enzyme-linked immunosorbent assay (ELISA), after the third round of panning, the TviCATL-scFvs binders were enriched. However, only one clone gave a promising ELISA signal suggesting that further optimisation of panning conditions are required in order to… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: African trypanosomiasis – Diagnosis.; Trypanosoma brucei.; Immunoglobulins.; Biochemistry.

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

APA (6^th Edition):

author], [. (2014). Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/12255

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):

author], [No. “Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis. ” 2014. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/12255.

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

MLA Handbook (7^th Edition):

author], [No. “Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis. ” 2014. Web. 24 Apr 2018.

Vancouver:

author] [. Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis. [Internet] [Thesis]. University of KwaZulu-Natal; 2014. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/12255.

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

Council of Science Editors:

author] [. Recombinant expression, purification and characterisation of TviCATL for antibody production and diagnosis of African animal Trypanosomiasis. [Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/12255

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

University of KwaZulu-Natal

9. [No author]. Vivapain : a cysteine peptidase from Trypanosoma vivax.

Degree: Biochemistry, 2010, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/4775

► African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the… (more)

▼ African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the trypanosome parasite from the tsetse fly vector to a mammalian host. There are three African trypanosomes namely Trypanosoma vivax, T. congolense and T. brucei brucei that are the causative agents responsible for this disease in African cattle. This disease is serious since it not only affects livestock but also has a negative impact on the sub-Saharan African economy. There is, therefore, a great demand for better control methods of the disease and suitable diagnostic methods. Current control measures such as the use of trypanocidal drugs, tsetse fly eradication methods and trypanotolerant cattle have become inadequate. The defence mechanism of the trypanosome to continuously change its surface coat by a process of antigenic variation has made it impossible to produce a suitable vaccine. Therefore, chemotherapy is still one of the key approaches for control of this wasting disease. The long existence of the current trypanocidal drugs has allowed the development of drug resistance. The development of new chemotherapeutic drugs is focused on targeting the pathogenic factors such as parasite cysteine peptidases that contribute to the disease. Vivapain is the main cysteine peptidase of T. vivax and shares high sequence identity with congopain, the main cysteine peptidase of T. congolense, which was previously shown to be a pathogenic factor contributing to trypanosomosis. Vivapain, thus, has potential as a target for chemotherapeutic drug design. Hence, the first part of this study involved the recombinant expression and enzymatic characterisation of vivapain for future production of new synthetic inhibitors for the use in new trypanocidal drugs. The catalytic domain of vivapain (Vp) was recombinantly expressed in the Pichia pastoris yeast expression system and enzymatically characterised. The main finding from this study was that Vp was only able to hydrolyse a substrate if the P2 position was occupied by either a hydrophobic Phe or Leu residue. Vp was also found to be active close to physiological pH and was inhibited by the reversible cysteine peptidases, leupeptin, antipain and chymostatin and the irreversible cysteine peptidases L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64), iodoacetic acid (IAA) and iodoacetamide (IAN). A further important aspect of controlling trypanosomosis is the diagnosis of the disease. Clinical, parasitological, molecular and serological techniques have been applied and used to diagnose trypanosomosis. One of the most promising serological techniques has proven to be the enzyme-linked immunosorbent assay (ELISA), more specifically the antibody and antigen detection ELISAs. The main requirement for this technique is a readily available and reproducible antigen such as that produced by recombinant expression. While there are recombinant antigens that are available to be used to detect T. congolense, T. brucei… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Trypanosomiasis in animals – Chemotherapy.; Peptidase.; Trypanosoma.; Drug development.; Trypanosomiasis in animals – Serodiagnosis.; Biochemistry.

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

APA (6^th Edition):

author], [. (2010). Vivapain : a cysteine peptidase from Trypanosoma vivax. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/4775

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):

author], [No. “Vivapain : a cysteine peptidase from Trypanosoma vivax. ” 2010. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/4775.

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

MLA Handbook (7^th Edition):

author], [No. “Vivapain : a cysteine peptidase from Trypanosoma vivax. ” 2010. Web. 24 Apr 2018.

Vancouver:

author] [. Vivapain : a cysteine peptidase from Trypanosoma vivax. [Internet] [Thesis]. University of KwaZulu-Natal; 2010. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/4775.

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

Council of Science Editors:

author] [. Vivapain : a cysteine peptidase from Trypanosoma vivax. [Thesis]. University of KwaZulu-Natal; 2010. Available from: http://hdl.handle.net/10413/4775

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

University of KwaZulu-Natal

10. [No author]. Epitope mapping of a trypanosomal cysteine proteinase.

Degree: Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/10108

► Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in… (more)

▼ Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Trypanosoma brucei.; Cysteine proteinases.; Trypanosomiasis in cattle – Immunological aspects.; Antigenic determinants.; Biochemistry.

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

APA (6^th Edition):

author], [. (2013). Epitope mapping of a trypanosomal cysteine proteinase. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10108

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):

author], [No. “Epitope mapping of a trypanosomal cysteine proteinase. ” 2013. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10108.

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

MLA Handbook (7^th Edition):

author], [No. “Epitope mapping of a trypanosomal cysteine proteinase. ” 2013. Web. 24 Apr 2018.

Vancouver:

author] [. Epitope mapping of a trypanosomal cysteine proteinase. [Internet] [Thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10108.

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

Council of Science Editors:

author] [. Epitope mapping of a trypanosomal cysteine proteinase. [Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10108

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

University of KwaZulu-Natal

11. Bizaaré, Lorelle Claire. Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis.

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

URL: http://hdl.handle.net/10413/10916

► The protozoan parasite Trypanosoma congolense is one of the aetiological agents of African animal trypanosomiasis that is transmitted by the tsetse fly. The parasite causes… (more)

▼ The protozoan parasite Trypanosoma congolense is one of the aetiological agents of African animal trypanosomiasis that is transmitted by the tsetse fly. The parasite causes nagana in animals and affects livestock throughout sub-Saharan Africa. The toxicity of available drugs and the emergence of drug resistant parasites have affected the treatment of trypanosomiasis. Control of the disease has also been difficult due to ineffective vector control and the potential of trypanosomes to express hundreds of antigenetically distinct proteins on their surface. Vaccination against trypanosomiasis has been thought to be a possible control method. Since a vaccine based on variable surface proteins of the parasite is unlikely, research has been directed towards the identification of invariant pathogenic factors of the parasite as potential targets for therapy. Congopain, the major cysteine protease of T. congolense has been implicated in the pathology of the disease. Antibodies against congopain are known to contribute to the mechanisms of natural resistance to trypanosomiasis known as trypanotolerance by neutralising the pathogenic effects of the enzyme. Oligopeptidase B (OpdB), a trypanosomal serine protease has also been associated as a pathogenic factor of the disease. It is released into the host’s circulation by dead or dying parasites and retains its catalytic activity since it is insensitive to host serum inhibitors. In the present study, the catalytic domain of congopain (C2) and the use of alpha-2-macroglobulin (α2M) as an adjuvant were investigated for their potential use in an anti-disease vaccine. α2-Macroglobulin has been used to varying degrees to target different antigens to cells of the immune system and enhance their immunogenicity. A previous study showed that antibodies raised in rabbits against C2 complexed to α2M gave a higher percentage inhibition than antibodies made using C2 mixed with Freund’s adjuvant. In the present study, goats were immunised with C2 complexed with α2M to confirm the enhanced immunogenicity of C2 and the production of anti-C2 antibodies with superior inhibitory properties. Following immunisation, goats were challenged with T. congolense (strain IL 1180) and showed sustained antibody production during the two month infection period. Goat antibodies made using C2 in complex with α2M inhibited the hydrolysis of hide powder azure by C2 by 96%. Maximum inhibition of the hydrolysis of azocasein was observed to be 63% and hydrolysis of Z-Phe-Arg-AMC by C2 was inhibited by 73%. In order to determine the vaccine potential of OpdB, protein was recombinantly expressed as a glutathione-S-transferase fusion protein in the pGEX expression system and purified by glutathione agarose affinity chromatography and molecular exclusion chromatography. Since a small yield of protein necessitated several rounds of expression and extensive purification, OpdB was subsequently expressed as a His-tagged fusion protein in the pET bacterial expression system. Recombinant protein was easily purified using nickel… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Bizaaré, L. C. (2008). Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10916

Chicago Manual of Style (16^th Edition):

Bizaaré, Lorelle Claire. “Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis.” 2008. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10916.

MLA Handbook (7^th Edition):

Bizaaré, Lorelle Claire. “Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis.” 2008. Web. 24 Apr 2018.

Vancouver:

Bizaaré LC. Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2008. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10916.

Council of Science Editors:

Bizaaré LC. Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis. [Masters Thesis]. University of KwaZulu-Natal; 2008. Available from: http://hdl.handle.net/10413/10916

University of KwaZulu-Natal

12. Pillay, Davita. Identification and characterisation of novel pathogenic factors of Trypanosoma congolense.

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

URL: http://hdl.handle.net/10413/10942

► Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for… (more)

▼ Trypanosoma congolense is a major causative agent of the bovine disease trypanosomosis which has a considerable economic impact on sub-Saharan Africa. Current control methods for trypanosomosis are unsatisfactory and vaccine development has been hampered by antigenic variation. An anti-disease vaccine is based on the idea that disease is caused by the pathogenic factors released by the parasite, rather than by the parasite itself. Therefore, if these pathogenic factors could be neutralised by antibodies produced by vaccination, the disease could be circumvented. The method used here for identification of novel pathogenic factors is based on the concept that trypanotolerant cattle are able to mitigate the disease by generating a specific immune response against a few key antigens (pathogenic factors). Two immuno-affinity columns were therefore prepared: one containing IgG from noninfected sera and a second column containing IgG from trypanotolerant N’Dama cattle serially infected with T. congolense. The differential binding of antigens to the two columns allowed identification of antigens specifically recognised by the immune system of a trypanotolerant animal, i.e. potential pathogenic factors. The most promising antigens identified included several variant cathepsin L-like cysteine peptidases (CPs) and the Family M1 Clan MA aminopeptidases (APs). For the CPs, a study of the genetic organisation was conducted in order to further understand the variability present in this gene family. To this end, two different mini-libraries of cathepsin L-like genes were prepared: one in which genes as different as possible from congopain (the major CP of T. congolense) were selected, and a second which contained all possible genes present in the congopain array. Analysis of the sequences obtained in these two mini-libraries showed that there was significant variability of the genes within the congopain array. Two variants of CPs, chosen for differences in their catalytic triads, were cloned for expression. The recombinantly expressed CP variants differed in substrate preferences from one another and from C2 (the recombinant truncated form of congopain), and surprisingly, all enzymes were active at physiological pH. The two APs were cloned and expressed as insoluble inclusion bodies in an E. coli system, and subsequently refolded. The refolded APs showed a substrate preference for H-Ala-AMC, an optimum pH of 8.0, localisation to the cytoplasm and inhibition by puromycin. The two APs were not developmentally regulated and present in procyclic, metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro. Immunisation of BALB/c mice with the APs did not provide protection when challenged with T. congolense. For an anti-disease vaccine to be protective, it would possibly have to include all pathogenic factors, including the two APs and at least one CP described in the present study. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Biochemistry.

Record Details Similar Records Cite « Share

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

APA (6^th Edition):

Pillay, D. (2010). Identification and characterisation of novel pathogenic factors of Trypanosoma congolense. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10942

Chicago Manual of Style (16^th Edition):

Pillay, Davita. “Identification and characterisation of novel pathogenic factors of Trypanosoma congolense.” 2010. Doctoral Dissertation, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10942.

MLA Handbook (7^th Edition):

Pillay, Davita. “Identification and characterisation of novel pathogenic factors of Trypanosoma congolense.” 2010. Web. 24 Apr 2018.

Vancouver:

Pillay D. Identification and characterisation of novel pathogenic factors of Trypanosoma congolense. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2010. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10942.

Council of Science Editors:

Pillay D. Identification and characterisation of novel pathogenic factors of Trypanosoma congolense. [Doctoral Dissertation]. University of KwaZulu-Natal; 2010. Available from: http://hdl.handle.net/10413/10942

University of KwaZulu-Natal

13. Baiyegunhi, Omolara O. Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis.

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

URL: http://hdl.handle.net/10413/11043

► Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a… (more)

▼ Accurate diagnosis of the presence of an infectious organism is very important for therapeutic interventions and consequently the recovery of the individual. There is a need for identifying new diagnostic antigens for the serological diagnosis of trypanosomiasis, a disease of humans and animals in Africa caused by protozoa belonging to the genus Trypanosoma. Invariant surface glycoproteins (ISGs) are present in most strains of the parasite and have the potential to replace the variable surface glycoproteins as diagnostic antigens. In order to avoid the challenges of in vivo culturing of bloodstream form (BSF) trypanosomes in laboratory animals, ISG65 and ISG75, the two most common ISGs were heterologously expressed in Escherichia coli and Pichia pastoris expression systems. The extracellular domains of ISG65 and ISG75 of both T. b. brucei and T. b. gambiense were amplified by PCR from genomic DNA using appropriate primers to give inserts of 1121 bp and 1342 bp sizes. These were sub-cloned into the pGEX-4T1 and pET28a expression vectors. Chemically competent E. coli BL21 (DE3) were transformed using the resultant plasmids and the transformed E. coli cells were used for heterologous protein expression. The expressed proteins were purified by three phase partitioning (TPP), nickel or glutathione affinity and molecular exclusion chromatography and analysed by reducing SDS-PAGE. The glycosylation status of ISG65 and ISG75 expressed in the M5 strain of P. pastoris, which has an engineered N-glycosylation pathway that produces glycosylated proteins similar to what is obtained in trypanosomes, was determined. The enzymatic action of Endoglycosidase H resulted in a shift in the electrophoretic migration of ISG65 but not ISG75 on SDS-PAGE, confirming N-glycosylation. Anti-ISG65 and anti-ISG75 antibodies were produced in chickens and affinity purified using the respective recombinant proteins immobilised on affinity matrices. The antibodies recognised native ISG65 and ISG75 respectively in western blots of lysates of T. b. brucei parasites cultured in vitro. Similar recognition of the native ISGs by the anti-recombinant ISG antibodies was also obtained using immunofluorescence microscopy of fixed T. b. brucei parasites. The results obtained demonstrate the potential of application of the recombinant ISG65 and ISG75 and their respective antibodies in the diagnosis of African trypanosomiasis. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Baiyegunhi, O. O. (2014). Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/11043

Chicago Manual of Style (16^th Edition):

Baiyegunhi, Omolara O. “Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis.” 2014. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/11043.

MLA Handbook (7^th Edition):

Vancouver:

Baiyegunhi OO. Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2014. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/11043.

Council of Science Editors:

Baiyegunhi OO. Heterologous expression of invariant surface glycoproteins, ISG75 of Trypanosoma brucei brucei and T.b. gambiense, for antibody production and diagnosis of African Trypanosomiasis. [Masters Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/11043

University of KwaZulu-Natal

14. Eyssen, Lauren Elizabeth-Ann. Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis.

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

URL: http://hdl.handle.net/10413/11165

► The lack of a vaccine candidate due to antigenic variation by trypanosomal parasites, the causative agents of human and animal African trypanosomiasis, requires the disease… (more)

▼ The lack of a vaccine candidate due to antigenic variation by trypanosomal parasites, the causative agents of human and animal African trypanosomiasis, requires the disease to be controlled by surveillance, diagnosis and appropriate treatment schedules. Due to the non-specific symptoms along with the toxicity and side effects of the current trypanocides, diagnosis needs to be accurate, cost effective and applicable to active case finding in mostly rural settings. Trypanosomal proteases have been identified as virulence factors as they are essential to the parasites‟ survival. Here the diagnostic potential of previously described virulence factors, oligopeptidase B (OPB), pyroglutamyl peptidase (PGP) and the full length and catalytic domain of the cathepsin L-like peptidases (CATLFL and CATL respectively) from T. congolense (Tc) as well as OPB and CATL from T. vivax (Tv), was determined. These antigens were recombinantly expressed, purified and used to generate antibodies in chickens. The purified recombinant antigens were tested in an inhibition and indirect ELISA format using two separate blinded serum panels consisting of sera from non-infected and experimentally infected cattle, one each for T. congolense and for T. vivax. The tested sera were diluted 1:10 for the TcCATLFL, TcCATL antigens whilst the TvCATL antigen used a 1:100 serum dilution. The TcCATLFL, TcCATL and TvCATL antigens had the highest diagnostic potential in the indirect ELISA format with a 90.91, 92.21% accuracy at the second cut-off and a 77.22% accuracy at the third cut-off along with 0.8084, 0.7785 and 0.8813 area under curve (AUC) values respectively. These antigens show potential for development of lateral flow tests to detect T. congolense and T. vivax infections in cattle. The recently discovered metacaspases (MCAs) have been implicated in caspase-like activity and differentiation in T. b. brucei, T. cruzi and L. major and are considered to be virulence factors. The putative metacaspase 5 gene from T. congolense (TcMCA5) was successfully cloned, expressed within inclusion bodies, resolubilised and refolded using immobilised metal affinity chromatography. Recombinant TcMCA5 was successfully refolded as evident by the hydrolysis of the synthetic peptide substrate, Z-Gly-Gly-Arg-AMC. Autocatalytic processing was observed within the inclusion bodies and the products were purified along with the full length recombinant protein. Anti-TcMCA5 IgY antibodies, raised in chickens, were able to detect the native TcMCA5 along with the autocatalytic processed products within the lysate of the procyclic T. congolense (strain IL 3000) parasites. The diagnostic potential of TcMCA5 still requires verification. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Eyssen, L. E. (2014). Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/11165

Chicago Manual of Style (16^th Edition):

Eyssen, Lauren Elizabeth-Ann. “Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis.” 2014. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/11165.

MLA Handbook (7^th Edition):

Eyssen, Lauren Elizabeth-Ann. “Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis.” 2014. Web. 24 Apr 2018.

Vancouver:

Eyssen LE. Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2014. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/11165.

Council of Science Editors:

Eyssen LE. Studying trypanosomal peptidase antigen targets for the diagnosis of animal African trypanosomiasis. [Masters Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/11165

University of KwaZulu-Natal

15. Vukea, Phillia Rixongile. Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.

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

URL: http://hdl.handle.net/10413/10356

► Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV… (more)

▼ Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used.… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Vukea, P. R. (2011). Characterisation of infectious bursal disease virus (IBDV) polyprotein processing. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10356

Chicago Manual of Style (16^th Edition):

Vukea, Phillia Rixongile. “Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.” 2011. Doctoral Dissertation, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10356.

MLA Handbook (7^th Edition):

Vukea, Phillia Rixongile. “Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.” 2011. Web. 24 Apr 2018.

Vancouver:

Vukea PR. Characterisation of infectious bursal disease virus (IBDV) polyprotein processing. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2011. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10356.

Council of Science Editors:

Vukea PR. Characterisation of infectious bursal disease virus (IBDV) polyprotein processing. [Doctoral Dissertation]. University of KwaZulu-Natal; 2011. Available from: http://hdl.handle.net/10413/10356

16. Scholfield, Nicola Gillian. VP4 : a putative protease encoded by infectious bursal disease virus.

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

URL: http://hdl.handle.net/10413/10269

► Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry… (more)

▼ Infectious bursal disease virus (IBDV) causes an acute and highly contagious disease affecting young chickens, which is responsible for significant losses in the poultry industry world-wide. The virus specifically infects and destroys B-cell precursors within the bursa of Fabricius, an avian lymphoid organ, leading to immunosuppression. IBDV has a bi-segmented, double-stranded RNA genome. The larger segment encodes a 110-kDa precursor polyprotein, designated NH₂-VPX-VP4-VP3-COOH, in a single open reading frame. The autocatalytic processing of this precursor into mature proteins is a critical step in viral replication and VP4 is the putative protease responsible for this cleavage. This study concerns the development of a strategy to clone and express recombinant VP4 and describes the use of VP4 as a marker for rapid and effective detection of IBDV. VP4 cDNA was produced and amplified by optimisation of a reverse transcription coupled to the polymerase chain reaction (RT-PCR), providing a clear and sensitive assay. Anti-peptide antibodies were raised against a selected peptide from VP4 and were used to probe homogenates of infected bursae for the native protein to assess their potential for immunological detection. These antibody-related results are promising though inconclusive, due to the complex nature of the assayed sample. Amplified VP4 cDNA from KwaZulu-Natal strains of IBDV isolated from 1989 to 1997 was also examined by restriction fragment length polymorphism (RFLP) analysis to determine the relatedness of local IBDV to global strains. All KwaZulu-Natal samples produced identical patterns, which were most similar to one of ten international strains examined, namely, the British strain UK661. Samples infected with IBDV were also probed for VP4 activity. Double basic amino acid cleavage sites have been proposed for the putative protease and infected samples were assayed for activity against the fluorogenic peptide Cbz-Arg-Arg-AMC. Demonstrably higher activity was found in infected versus uninfected samples, although the origin of this activity is unclear. The findings in this study suggest that VP4 warrants further attention, both as a marker for infectious bursal disease, and as a novel viral protease. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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):

Scholfield, N. G. (2013). VP4 : a putative protease encoded by infectious bursal disease virus. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10269

Chicago Manual of Style (16^th Edition):

Scholfield, Nicola Gillian. “VP4 : a putative protease encoded by infectious bursal disease virus.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10269.

MLA Handbook (7^th Edition):

Scholfield, Nicola Gillian. “VP4 : a putative protease encoded by infectious bursal disease virus.” 2013. Web. 24 Apr 2018.

Vancouver:

Scholfield NG. VP4 : a putative protease encoded by infectious bursal disease virus. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10269.

Council of Science Editors:

Scholfield NG. VP4 : a putative protease encoded by infectious bursal disease virus. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10269

University of KwaZulu-Natal

17. 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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Horner%2C%20Roger%20F%22%29&pagesize-30">Horner, Roger F (advisor).

Subjects/Keywords: Biochemistry.

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

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 April 24, 2018. 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 Apr 2018.

Vancouver:

Taylor KL. Methods for serotype classification of Haemophilus paragallinarum field isolates. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 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

18. Bridgemohan, Roshini. Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal.

Degree: M.Med.Sci, Haematology, 2006, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/2055

► Hereditary Spherocytosis (HS) is a common inherited haemolytic anaemia with variable clinical expression. Fifty subjects with HS from KwaZulu-Natal were studied with the aim of… (more)

▼ Hereditary Spherocytosis (HS) is a common inherited haemolytic anaemia with variable clinical expression. Fifty subjects with HS from KwaZulu-Natal were studied with the aim of providing further information on the protein abnormalities of the red blood cell (RBC) membrane and their relationship with clinical presentations. Haematological and biochemical tests were performed by routine procedures. Mean Corpuscular Haemoglobin Concentration ( MCHC) in the HS group was 35.1g /dl. This was significantly higher than in normal control subjects (33.6g /dl) (p value < 0.001); indicating its usefulness for the screening of HS. Mean Red Cell Distribution Width (RDW) was also significantly higher in subjects with HS (p<0.001); thus providing an additional screening tool. Erythrocyte membrane proteins from 21 subjects were analysed by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. The most common abnormality was a deficiency of band 3 (10 subjects), followed by a combined spectrin and ankyrin deficiency in five subjects. One subject had increased band 6 and in five cases no abnormality was detected. A decreased ratio of protein 4.1a / 4.1b on the Laemmli SDS PAGE correlated with an increased reticulocyte count. The degree of haemolysis and clinical findings did not correlate with the type of red cell membrane protein defect. In this study red cell membrane analysis did not contribute further to the initial laboratory diagnosis. In addition it did not influence clinical management. The presence of red cell membrane abnormalities, either single or multiple, did not correlate with disease severity. Red cell membrane analysis, however, will play an important role for future management such as gene therapy. Red cell membrane analysis is also useful as a research tool to determine the underlying molecular defect and to assess racial or ethnic differences. It is also of value as a differential diagnostic tool in cases where the clinical and laboratory findings are not conclusive for HS. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Jogessar%2C%20Vinod%20B%22%29&pagesize-30">Jogessar, Vinod B (advisor), Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Haematology.

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

APA (6^th Edition):

Bridgemohan, R. (2006). Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/2055

Chicago Manual of Style (16^th Edition):

Bridgemohan, Roshini. “Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal.” 2006. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/2055.

MLA Handbook (7^th Edition):

Bridgemohan, Roshini. “Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal.” 2006. Web. 24 Apr 2018.

Vancouver:

Bridgemohan R. Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2006. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/2055.

Council of Science Editors:

Bridgemohan R. Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal. [Masters Thesis]. University of KwaZulu-Natal; 2006. Available from: http://hdl.handle.net/10413/2055

University of KwaZulu-Natal

19. [No author]. Methods for serological and PCR detection of Salmonella enteritidis in chickens.

Degree: Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9937

► Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently one of the leading causes of human food poisoning in the world.… (more)

▼ Salmonella enteritidis (S. enteritidis) is a bacterial pathogen of chickens, and is currently one of the leading causes of human food poisoning in the world. It is believed that contaminated poultry products, especially eggs and egg products, have been responsible for the dramatic increase in the incidence of this Salmonella serotype. Detection of S. entertidis has conventionally involved bacteriological examination of samples, yet these procedures are time-consuming which could lead to the rapid spread of S. enteritidis through commercial flocks and potentially cause a human health risk. A number of alternative detection techniques, mostly based on serological methods, have been reported as effective diagnostic assays. However, some of these reports have not been supported by representations of SDS-PAGE gels or Western blots. The objective of this study was the evaluation of these serological techniques as well as a PCR amplification technique, which has been reported to show promising results as a diagnostic method. The techniques discussed in these reports were evaluated with regards to how rapid they were, their specificity and their potential for use in local diagnostic laboratories. Antigens from the outer surface of S. enteritidis were purified by several methods and their antigenicity was tested by separating the antigens by means of SDS-PAGE, followed by Western blotting using sera of chickens infected with S. enteritidis. A high degree of cross reactivity was observed with many of the antigens tested, especially the lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) which had previously been reported as containing antigens which could be used for specific detection of S. enteritidis. This cross-reactivity could be explained by the conserved nature of many of the LPS and OMP antigens among the Salmonella serotypes tested. A fimbrial antigen, SEF14, which has been reported as a novel antigen, was seen as a prominent band at 14.3 kDa and was found to react with antibodies against S. enteritidis, yet not to the specificity levels described in previous reports. PCR amplification of the sefA gene sequence, which encodes for the SEF14 fimbrial antigen, was found to give a predicted product of 310 bp when using a previously described oligonucleotide primer pair. This amplified product was found to be specific for S. enteritidis and other serogroup D Salmonella serotypes that are not poultry pathogens The cross-reactivity observed with many of the serological techniques used in this study, meant that detection of S. enteritidis infection in chickens was considerably hindered. However, the identification of further novel antigens by serological means, could result in the development of new vaccines. The specificity and speed afforded by PCR amplification indicated that this technique showed excellent potential for use in local diagnostic laboratories. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Horner%2C%20Roger%20F%22%29&pagesize-30">Horner, Roger F (advisor).

Subjects/Keywords: Salmonella enteritidis.; Chicken – Diseases.; Poultry – Infections.; Polymerase chain reaction – Methodology.; Pathogenic bacteria.; Biochemistry.

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

APA (6^th Edition):

author], [. (2013). Methods for serological and PCR detection of Salmonella enteritidis in chickens. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9937

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):

author], [No. “Methods for serological and PCR detection of Salmonella enteritidis in chickens. ” 2013. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/9937.

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

MLA Handbook (7^th Edition):

author], [No. “Methods for serological and PCR detection of Salmonella enteritidis in chickens. ” 2013. Web. 24 Apr 2018.

Vancouver:

author] [. Methods for serological and PCR detection of Salmonella enteritidis in chickens. [Internet] [Thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/9937.

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

Council of Science Editors:

author] [. Methods for serological and PCR detection of Salmonella enteritidis in chickens. [Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9937

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

University of KwaZulu-Natal

20. Mkhize, Phumzile. Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings.

Degree: Plant pathology, 2014, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/11230

► Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus… (more)

▼ Fusarium circinatum is a fungal pathogen that has had a serious impact on pine production throughout the world. It attacks most Pinus species including Pinus elliottii, Pinus patula and Pinus radiata. Infections in South Africa (SA) are largely on seedlings, and result in fatal seedling wilt. Accurate and quick detection systems suitable for field use are needed to monitor the spread of the disease and optimize fungicide applications. Detection of F. circinatum is currently based on visual observations of typical symptoms. However, symptoms are not unique to the pathogen and can be caused by other biotic and abiotic stress factors. Nucleic acid-based identification techniques using PCR are available for different fungal species. These are sensitive and accurate, but they are expensive and require skilled biotechnologists to conduct the assays. In this study an enzyme-linked immunosorbent assay (ELISA) was developed to identify F. circinatum in infected seedlings. This optimized ELISA is able to discriminate between F. circinatum and two other fungi that frequently affect pine. This method has advantages over other assays because of its ease of operation and sample preparation, sensitivity and the ability to run multiple tests simultaneously. Mycelium-soluble antigens from Diplodia pinea (=Sphaeropsis sapinea), F. circinatum and F. oxysporum were prepared in nutrient broth. Analysis of these antigens on SDS-PAGE indicated the presence of common antigens between the different fungal pathogens. Some antigens were expressed more by some isolates than by others. Separate groups of chickens were immunised with mycelium-soluble antigens from D. pinea, F. circinatum and F. oxysporum and exo-antigen from F. circinatum prepared in nutrient broth. A 34 kDa protein purified from SDS-PAGE specific for D. pinea was also used for immunisation. Five sets of antibodies were obtained including anti-D. pinea, anti-F. circinatum, anti-F. oxysporum, anti-F. circinatumexo and anti-D. pinea 34 kDa antibodies, respectively. Reactivity of these antibodies was evaluated against antigens prepared in nutrient broth using western blotting and ELISA. Western blot analysis indicated that immuno-dominant antigens for F. circinatum were larger than 34 kDa and their reactivity was not the same between different isolates. Each of the antibodies prepared using mycelium-soluble antigens showed increased reactivity when detecting its own specific pathogen, but cross-reactivity was observed. Anti-D.pineaantibodies showed minimal cross-reactivity with antigens from F. circinatum and F. oxysporum. Anti-F. circinatum antibodies cross-reacted with antigens from F. oxysporum but showed little cross-reactivity with D. pinea antigens. Anti-F. oxysporum antibodies showed more cross-reactivity towards antigens from F. circinatum than those from D. pinea. No reactivity was observed when anti-F. circinatum-exo antigen and anti-D. pinea 34 kDa antibodies were used in immuno-blotting analysis. Evaluation of antibody reactivity using indirect ELISA showed patterns… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Laing%2C%20Mark%20D%22%29&pagesize-30">Laing, Mark D (advisor), Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Plant pathology.

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

APA (6^th Edition):

Mkhize, P. (2014). Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/11230

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):

Mkhize, Phumzile. “Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings.” 2014. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/11230.

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

MLA Handbook (7^th Edition):

Vancouver:

Mkhize P. Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings. [Internet] [Thesis]. University of KwaZulu-Natal; 2014. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/11230.

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

Council of Science Editors:

Mkhize P. Development of an enzyme-linked immunosorbent assay (ELISA) for field detection and discrimination of Fusarium circinatum from Fusarium oxysporum and Diplodia pinea in pine seedlings. [Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/11230

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

University of KwaZulu-Natal

21. 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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Boulange%2C%20Alain%20F.%20V%22%29&pagesize-30">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 April 24, 2018. 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 Apr 2018.

Vancouver:

Ndlovu HH. Structural studies aimed at improving the antigenicity of congopain. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2009. [cited 2018 Apr 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

22. 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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Boulange%2C%20Alain%20F.%20V%22%29&pagesize-30">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 April 24, 2018. http://hdl.handle.net/10413/4140.

MLA Handbook (7^th Edition):

Kalundi, Erastus Mulinge. “Molecular analysis of the congopain gene family.” 2008. Web. 24 Apr 2018.

Vancouver:

Kalundi EM. Molecular analysis of the congopain gene family. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2008. [cited 2018 Apr 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

23. 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: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Lonsdale-Eccles%2C%20John%20D%22%29&pagesize-30">Lonsdale-Eccles, John D (advisor).

Subjects/Keywords: Biochemistry.

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

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 April 24, 2018. http://hdl.handle.net/10413/9783.

MLA Handbook (7^th Edition):

Tosomba, Omalokoho Médard. “Studies on acid phosphatases of Trypanosoma congolense.” 2013. Web. 24 Apr 2018.

Vancouver:

Tosomba OM. Studies on acid phosphatases of Trypanosoma congolense. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 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

24. 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: Natal%22%20%2Bcontributor%3A%28%22Boulange%2C%20Alain%20F.%20V%22%29&pagesize-30">Boulange, Alain F. V (advisor), Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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 April 24, 2018. 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 Apr 2018.

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 2018 Apr 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

25. 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: Natal%22%20%2Bcontributor%3A%28%22Boulange%2C%20Alain%20F.%20V%22%29&pagesize-30">Boulange, Alain F. V (advisor), Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">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 April 24, 2018. 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 Apr 2018.

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 2018 Apr 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

26. [No author]. A serine oligopeptidase from African Trypanosomes.

Degree: Biochemistry, 2013, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/9778

► Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and… (more)

▼ Protozoan parasites of the genus Trypanosoma are responsible for chronic and widespread disease in livestock and humans in Africa. This study describes the purification and characterisation of a serine oligopeptidase from Trypanosoma brucei brucei and from T. congolense. Serine peptidase activity has previously been described for T. b. brucei although the responsible enzyme was not purified to electrophoretic homogeneity. In the present study this enzyme was purified from bloodstream-form T. b. brucei by a combination of three-phase partitioning, ion-exchange, affinity and molecular exclusion chromatography. Characterisation of the enzyme revealed that it closely resembled a bacterial serine oligopeptidase, Escherichia coli oligopeptidase B, in terms of cleavage-site specificity, inhibition characteristics and molecular mass. Its overall properties indicate that it is probably a serine oligopeptidase and we have called it OP-Tb (oligopeptidase from Trypanosoma brucei). Antibodies to OP-Tb were prepared in chickens. These antibodies were used in the purification of a similar enzyme, designated OP-Tc, from T. congolense. OP-Tc closely resembled OP-Tb in its enzymatic properties. OP-Tb appears to be monomeric, with an apparent molecular mass of 80 kDa. Activity is optimal between pH 8.0 and 10.0, and is enhanced in the presence of reducing agents. Inhibition by 4-(2-aminoethyl)benzenesulfonylfluoride, 3,4-dichloroisocoumarin and diisopropylfluorophosphate indicates that the enzyme may be classified as a serine protease. While various natural and synthetic fluorogenic peptide substrates were hydrolysed by OP-Tb, larger potential substrates (proteins) were not. Studies of the digestion of naturally occurring bioactive peptides suggested that substrates were restricted to peptides smaller than approximately 4 or 5 kDa. These peptides were cleaved at the carboxy side of basic amino acid residues such as arginine and lysine. This is characteristic of a trypsin-like specificity. Because the enzyme is known to be readily released from the parasites, and because it was possible to detect OP-Tb-like activity in the blood of T. b. brucei-infected mammalian hosts, it appears that the enzyme is released into the host bloodstream where it remains uninhibited by endogenous protease inhibitors. Indeed, OP-Tb was not inhibited by mammalian plasma serpins or 012-macroglobulin in vitro. This, and the degradation of host peptide regulatory hormones in vitro, suggests that OP-Tb may have secondary, but important, extracellular roles in the pathogenesis of African trypanosomiasis. A variety of serine protease inhibitors, including inhibitors of OP-Tb were tested for their potential as trypanocidal agents. The results from both in vitro and in vivo studies, suggest that inhibitors of trypanosome oligopeptidases are promising new lead targets for drug development. Furthermore, data presented here also shows that OP-Tb is efficiently inhibited by several of the currently employed trypanocidal drugs. Thus, OP-Tb may already… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Lonsdale-Eccles%2C%20John%20D%22%29&pagesize-30">Lonsdale-Eccles, John D (advisor), Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor).

Subjects/Keywords: Trypanosomiasis – Control.; Proteolytic enzymes.; Serine proteinases – Inhibitors.; Oligopeptides – Purification.; Biochemistry.

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APA (6^th Edition):

author], [. (2013). A serine oligopeptidase from African Trypanosomes. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/9778

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):

author], [No. “A serine oligopeptidase from African Trypanosomes. ” 2013. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/9778.

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

MLA Handbook (7^th Edition):

author], [No. “A serine oligopeptidase from African Trypanosomes. ” 2013. Web. 24 Apr 2018.

Vancouver:

author] [. A serine oligopeptidase from African Trypanosomes. [Internet] [Thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/9778.

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

Council of Science Editors:

author] [. A serine oligopeptidase from African Trypanosomes. [Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/9778

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

27. Edwards, Thomas Jonathan. Identification of possible infectious bursal disease virus receptors.

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

URL: http://hdl.handle.net/10413/10259

► Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system… (more)

▼ Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Horner%2C%20Roger%20F%22%29&pagesize-30">Horner, Roger F (advisor).

Subjects/Keywords: Biochemistry.

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APA (6^th Edition):

Edwards, T. J. (2013). Identification of possible infectious bursal disease virus receptors. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10259

Chicago Manual of Style (16^th Edition):

Edwards, Thomas Jonathan. “Identification of possible infectious bursal disease virus receptors.” 2013. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10259.

MLA Handbook (7^th Edition):

Edwards, Thomas Jonathan. “Identification of possible infectious bursal disease virus receptors.” 2013. Web. 24 Apr 2018.

Vancouver:

Edwards TJ. Identification of possible infectious bursal disease virus receptors. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10259.

Council of Science Editors:

Edwards TJ. Identification of possible infectious bursal disease virus receptors. [Masters Thesis]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10259

28. Lomo, Peter Onyimbo. Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.

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

URL: http://hdl.handle.net/10413/10278

► Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic… (more)

▼ Subcellular fractionation (together with immunocytochemical localisation studies) showed that the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb). This complex is predominantly cytosolic but some activity is also present in the nuclear fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells (MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion chromatography on Sephacryl S-300 and glycerol density gradient sedimentation. The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc. Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12 different polypeptide components compared to the 28 different polypeptide components of MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not have any obvious sequence homology with the subunits of proteasomes from other cells. Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct. The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes. MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes. Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell types are activated by endogenous high molecular mass complexes such as the bovine 19S complex called PA700. These complexes form end-on associations with the 20S proteasome. However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited unique end-on associations between individual units forming long (up to 200 nm) ribbon-like chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism for controlling their proteasome activity. Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Lonsdale-Eccles%2C%20John%20D%22%29&pagesize-30">Lonsdale-Eccles, John D (advisor).

Subjects/Keywords: Biochemistry.

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

APA (6^th Edition):

Lomo, P. O. (2013). Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei. (Doctoral Dissertation). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/10278

Chicago Manual of Style (16^th Edition):

Lomo, Peter Onyimbo. “Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.” 2013. Doctoral Dissertation, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/10278.

MLA Handbook (7^th Edition):

Lomo, Peter Onyimbo. “Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.” 2013. Web. 24 Apr 2018.

Vancouver:

Lomo PO. Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei. [Internet] [Doctoral dissertation]. University of KwaZulu-Natal; 2013. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/10278.

Council of Science Editors:

Lomo PO. Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei. [Doctoral Dissertation]. University of KwaZulu-Natal; 2013. Available from: http://hdl.handle.net/10413/10278

29. Mnkandla, Sanele Michelle. Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense.

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

URL: http://hdl.handle.net/10413/11042

► Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in… (more)

▼ Human African Trypanosomiasis (HAT), also known as sleeping sickness is one of the many life threatening tropical diseases posing a serious risk to livelihoods in Africa. The disease is restricted to the rural poor across sub–Saharan Africa, where tsetse flies that transmit the disease, are endemic. Sleeping sickness is known to be caused by protozoan parasites of the genus Trypanosoma brucei, with the two sub-species: T. b. gambiense and T. b. rhodesiense, responsible for causing infection in humans. The disease develops in two stages, firstly, the infection is found in the blood and secondly, when the parasites cross the blood-brain barrier entering the nervous system. To date, no vaccines have been developed, however, there is a range of drugs and treatments available which depend on the type of infection (T. b. gambiense or T. b. rhodesiense) as well as disease stage. The trypanosome parasites have a two-host life cycle i.e. in the mammalian host as well as the tsetse fly vector. Throughout the cycle, the parasite undergoes changes, one of them being the acquisition of a variable surface glycoprotein (VSG) coat prior to entry into the human host bloodstream. Once in the host, the infection progresses and through a phenomenon known as antigenic variation, the parasite expresses a different VSG coat periodically, enabling the parasites to constantly evade the host’s immune response, facilitating their survival. The VSG genes coding for the proteins are activated by an intricate process involving the encoding of a gene which is kept silent, until activated in one of several expression sites. Despite the constant switching of VSG surface coats, there are VSG forms that are predominantly expressed in T. b. gambiense namely VSGs LiTat 1.3, LiTat 1.5 and LiTat 1.6 which are used in diagnostic tests, as antigens to detect antibodies in infected sera of HAT patients. The acquisition of these VSG antigens is, however, of high risk to staff handling the parasites, and so the first part of the study was aimed at cloning, recombinantly expressing and purifying the two VSGs known to be recognised by all gambiense HAT patients: LiTat 1.3 and LiTat 1.5, for possible use as alternative antigens in diagnostic tests. The genes encoding both VSGs, LiTat 1.3 and LiTat 1.5, were first amplified from either genomic or complementary DNA (gDNA or cDNA), cloned into a pTZ57R/T-vector and sub-cloned into pGEX or pET expression vectors prior to recombinant expression in E. coli BL21 DE3 and purification by Ni-affinity chromatography. Amplification and subsequent cloning yielded the expected 1.4 kb and 1.5 kb for the LiTat 1.3 and LiTat 1.5 genes respectively. Recombinant expression in E. coli was only successful with the constructs cloned from cDNA, i.e. the pGEX4T-1-cLiTat 1.3 and pET-28a-cLiTat 1.3 clones. Purification of the 63 kDa cLiTat 1.3His protein following solubilising and refolding did not yield pure protein and there were also signs of protein degradation. For comparison, expression was also carried out in P. pastoris and… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Vukea%2C%20Phillia%20Rixongile%22%29&pagesize-30">Vukea, Phillia Rixongile (advisor).

Subjects/Keywords: Biochemistry.

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

APA (6^th Edition):

Mnkandla, S. M. (2014). Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense. (Masters Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/11042

Chicago Manual of Style (16^th Edition):

Mnkandla, Sanele Michelle. “Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense.” 2014. Masters Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/11042.

MLA Handbook (7^th Edition):

Vancouver:

Mnkandla SM. Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense. [Internet] [Masters thesis]. University of KwaZulu-Natal; 2014. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/11042.

Council of Science Editors:

Mnkandla SM. Recombinant expression of, and characterisation of antibodies against variable surface glycoproteins : LiTat 1.3 and LiTat 1.5 of Trypanosoma brucei gambiense. [Masters Thesis]. University of KwaZulu-Natal; 2014. Available from: http://hdl.handle.net/10413/11042

30. Groenink, Shaun Reinder. A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay.

Degree: Biochemistry, 2015, University of KwaZulu-Natal

URL: http://hdl.handle.net/10413/12013

► African horse sickness (AHS) is a viral disease that afflicts all equine species and has a 90% mortality rate in unvaccinated horses. The disease has… (more)

▼ African horse sickness (AHS) is a viral disease that afflicts all equine species and has a 90% mortality rate in unvaccinated horses. The disease has a devastating effect on the national herd of South Africa each year and affects both the sport and racehorse industries, including the export of prized bloodstock, as well as the rural and subsistence economies that depend on animal traction. Transmitted by the Culicoides spp. of biting midge, the virus belongs to the Orbivirus genus of the Reoviridae family with nine known serotypes and ten genome segments. Segment 2 (which encodes VP2) is responsible for serotype determination while segment 10 (which encodes NS3) is merely serotype-divergent. Knowledge of the seroprevalence of the virus is poor. The increasing reluctance of horse owners to use the registered vaccine due to perceived inefficacy is of concern. As a means to increase knowledge output in this regard, and potentially provide a service to horse owners, a rapid serotyping assay is sought based on High Resolution Melt (HRM) analysis. HRM analysis is a powerful tool that is based on the release of a DNA intercalating dye from polymerase chain reaction products through gradual and controlled heating. The dye is released at a specific point that is dependent on the unique sequence of the amplicon. It can thus be used to distinguish, very sensitively, differences in divergent amplicons. Using a range of freely available bioinformatics software, such as Clustal X2, Primaclade, Treeview and BLAST analysis, primers were designed based on segment 2 that sought to differentiate the individual serotype from previously defined clades based on a pair of segment 10 primers. Reference and field isolates of the AHS virus were obtained from the National Institute of Communicable Disease and the Onderstepoort Veterinary Institute, South Africa, and were propagated on Vero cell monolayers. Total RNA was extracted using guanidine-thiocyanate and verified as containing AHSV genomic material using primers recognised by the World Organisation for Animal Health that target the genome segment encoding VP7. Variable amounts of total RNA did not influence the downstream analysis as individual serotypes were easily distinguished using HRM despite wide ranging template concentrations. Through testing the primers designed in the present study, various serotype anomalies were discovered with regard to the isolates obtained from the Onderstepoort Veterinary Institute. Serotype-specific primers and the segment 10 primers were used to interpret the serotype anomalies through High Resolution Melt analysis. Sequencing confirmed the anomalies: serotype 2 isolates were serotype 6 isolates and a serotype 5 isolate was serotype 8. A proposed protocol for a rapid serotyping assay was investigated. This involved an initial PCR to determine into which clade of segment 10 the sample fits. Following this, the serotype was elucidated for each clade using segment 2 clade-specific primers. These reactions were performed in the Corbett Rotor-Gene™ 6000 and… Advisors/Committee Members: Natal%22%20%2Bcontributor%3A%28%22Coetzer%2C%20Theresa%20Helen%20Taillefer%22%29&pagesize-30">Coetzer, Theresa Helen Taillefer (advisor), Natal%22%20%2Bcontributor%3A%28%22Young%2C%20Marion%20Belinda%22%29&pagesize-30">Young, Marion Belinda (advisor), Natal%22%20%2Bcontributor%3A%28%22Watson%2C%20Gregory%20M.%20F%22%29&pagesize-30">Watson, Gregory M. F (advisor).

Subjects/Keywords: Biochemistry.

…support during the course of my post-graduate career. The University of KwaZulu-Natal is…

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APA (6^th Edition):

Groenink, S. R. (2015). A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay. (Thesis). University of KwaZulu-Natal. Retrieved from http://hdl.handle.net/10413/12013

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):

Groenink, Shaun Reinder. “A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay.” 2015. Thesis, University of KwaZulu-Natal. Accessed April 24, 2018. http://hdl.handle.net/10413/12013.

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

MLA Handbook (7^th Edition):

Groenink, Shaun Reinder. “A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay.” 2015. Web. 24 Apr 2018.

Vancouver:

Groenink SR. A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay. [Internet] [Thesis]. University of KwaZulu-Natal; 2015. [cited 2018 Apr 24]. Available from: http://hdl.handle.net/10413/12013.

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

Council of Science Editors:

Groenink SR. A study of the African horse sickness virus using High Resolution Melt, multivariate and phylogenetic analyses for a potential serotyping assay. [Thesis]. University of KwaZulu-Natal; 2015. Available from: http://hdl.handle.net/10413/12013

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

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