Assessment of fluctuating asymmetry as an indicator of water quality stress in South Africa
- Authors: Holland, Alexandra Jennifer
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/430 , vital:19958
- Description: South Africa’s freshwater resources are facing numerous water quality challenges and need to be protected from degradation and pollution by appropriate management strategies as they are a limited and shared resource. The South African Scoring System (SASS5), which assesses macroinvertebrate communities at family level, is used in routine monitoring of riverine ecosystems in South Africa. Assessing the condition of these ecosystems is limited as SASS5 does not allow for changes at lower levels of biological organisation to be detected. Fluctuating asymmetry (FA) - small random deviations from perfect symmetry - is considered a direct measure of developmental instability. This phenotypical response results from numerous internal and external factors and has a low level of heritability. FA is based on sound scientific principles, easy to measure, biologically robust and cost-effective. It reflects synergistic interactions between stressors and provides an integrated measure of several anthropogenic stresses, which strengthens the assertion that FA is an environmental indicator of water quality stress, and can potentially be used to detect stress in populations before irreversible effects manifest. FA responses were investigated by (1) exposing freshwater shrimp to increasing concentrations of cadmium chloride in a long-term experiment and (2) comparing FA responses to water quality changes and macroinvertebrate community responses in two case studies (Kwazulu-Natal and Limpopo Province) in South Africa. Although no consistent concentration-response curve could be established, this study suggests that FA responses can be used as a sublethal endpoint in exposure experiments. Determining water quality parameters causing FA responses was not possible in field collected freshwater shrimp in either case study. Although FA did not specifically respond to any of the measured water quality parameters identified in the case studies, it has potential as a general indicator of water quality stress in freshwater shrimp. This study shows that FA responses are potentially more sensitive than macroinvertebrate community responses to pollution since it is not affected by habitat. Since FA has the potential to be a general indicator of population quality, particularly where there are natural habitat differences, it can be useful at the level of biomonitoring required for routine basic river status assessments in South Africa. However, in order for FA to become a robust tool that can be used routinely in conjunction with SASS5 more research is required.
- Full Text:
- Date Issued: 2016
- Authors: Holland, Alexandra Jennifer
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/430 , vital:19958
- Description: South Africa’s freshwater resources are facing numerous water quality challenges and need to be protected from degradation and pollution by appropriate management strategies as they are a limited and shared resource. The South African Scoring System (SASS5), which assesses macroinvertebrate communities at family level, is used in routine monitoring of riverine ecosystems in South Africa. Assessing the condition of these ecosystems is limited as SASS5 does not allow for changes at lower levels of biological organisation to be detected. Fluctuating asymmetry (FA) - small random deviations from perfect symmetry - is considered a direct measure of developmental instability. This phenotypical response results from numerous internal and external factors and has a low level of heritability. FA is based on sound scientific principles, easy to measure, biologically robust and cost-effective. It reflects synergistic interactions between stressors and provides an integrated measure of several anthropogenic stresses, which strengthens the assertion that FA is an environmental indicator of water quality stress, and can potentially be used to detect stress in populations before irreversible effects manifest. FA responses were investigated by (1) exposing freshwater shrimp to increasing concentrations of cadmium chloride in a long-term experiment and (2) comparing FA responses to water quality changes and macroinvertebrate community responses in two case studies (Kwazulu-Natal and Limpopo Province) in South Africa. Although no consistent concentration-response curve could be established, this study suggests that FA responses can be used as a sublethal endpoint in exposure experiments. Determining water quality parameters causing FA responses was not possible in field collected freshwater shrimp in either case study. Although FA did not specifically respond to any of the measured water quality parameters identified in the case studies, it has potential as a general indicator of water quality stress in freshwater shrimp. This study shows that FA responses are potentially more sensitive than macroinvertebrate community responses to pollution since it is not affected by habitat. Since FA has the potential to be a general indicator of population quality, particularly where there are natural habitat differences, it can be useful at the level of biomonitoring required for routine basic river status assessments in South Africa. However, in order for FA to become a robust tool that can be used routinely in conjunction with SASS5 more research is required.
- Full Text:
- Date Issued: 2016
The characterization of DNAJC3: elucidating the function of the TPR domains
- Mutsvunguma, Lorraine Zvichapera
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
- Full Text:
- Date Issued: 2014
- Authors: Mutsvunguma, Lorraine Zvichapera
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/55874 , vital:26751
- Description: DNAJC3 is a novel member of the DNAJ family with two domains linked to co-chaperone functions, namely the tetratricopeptide repeat (TPR) and J domain. Out of the two domains, the TPR domains are the least characterized. Therefore, the aim of this study was to characterize and elucidate additional functions of DNAJC3 TPR domains through in silico, in vitro and ex vivo approaches. Through multiple sequence and structural alignment as well as electrostatic potential analysis, DNAJC3 TPR domain were found to be most similar to TPR-containing proteins with Hsp90 or Hsp70 independent functions. In vitro pull down assays illustrated that DNAJC3 TPR domains did not interact with either cytosolic Hsp90 and Hsp70 or Grp78 and Grp94 directly, however a potential indirect interaction with Grp94 and Hsp90 was observed in mammalian lysates, via pull down assays; suggesting the formation of a complex between the proteins mediated by a specific substrate. DNAJC3 TPR domains were found to bind indiscriminately to both native and heat denatured substrates in a dose dependent manner. DNAJC3 TPR domains bound to β-galactosidase with greater affinity than malate dehydrogenase (MDH), suggesting that DNAJC3 TPR domains might exhibit substrate specificity that has not been reported before. Preliminary ex vivo analysis of DNAJC3 in mammalian cells showed that induced stress conditions did not alter the cytosolic or endoplasmic reticulum (ER) localization, or levels of DNAJC3 protein, suggesting that the protein is not stress inducible. However, protein levels of DNAJC3 were dramatically reduced by Hsp90 inhibitor novobiocin at 500 μM. Transient knockdown DNAJC3 did not change the protein levels of either Grp78 or Grp94, but decreased the protein levels of Hsp70/Hsp90 organizing protein HOP. On the other hand, protein levels of DNAJC3 were increased in HOP depleted cells. In conclusion, this study was the first to experimentally demonstrate that DNAJC3 TPR domains do not interact directly with Hsp90, Hsp70, Grp78 or Grp94, and therefore DNAJC3 is unlikely to participate in traditional co-chaperone interactions with those proteins via its TPR domain. However, the J domain is known to interact with Grp78. The discovery that DNAJC3 TPR domains resemble that of TPR-containing proteins with functions independent of Hsp90 or Hsp70 suggests that DNAJC3 might link the Hsp70/Grp78 chaperone machinery to non co-chaperone related functions, which requires further analysis.
- Full Text:
- Date Issued: 2014
Development of molecularly imprinted polymer based solid phase extraction sorbents for the selective cleanup of food and pharmaceutical residue samples
- Authors: Batlokwa, Bareki Shima
- Date: 2012
- Subjects: Sorbents -- Research Nanofibers -- Research Aflatoxins -- Research Electrospinning -- Research Extraction (Chemistry) -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4309 , http://hdl.handle.net/10962/d1004967
- Description: This thesis presents the development of chlorophyll, cholic acid, aflatoxin B1 molecularly imprinted polymer (MIP) particles and cholic acid MIP nanofibers for application as selective solid phase extraction (SPE) sorbents. The particles were prepared by bulk polymerization and the nanofibers by a novel approach combining molecular imprinting and electrospinning technology. The AFB1 MIP particles were compared with an aflatoxin specific immunoextraction sorbent in cleaning-up and pre-concentrating aflatoxins from nut extracts. They both recorded high extraction efficiencies (EEs) of > 97 % in selectively extracting the aflatoxins (AFB1, AFB2, AFG1 and AFG2). High reproducibility marked by the low %RSDs of < 1% and low LODs of ≤ 0.02 ng/g were calculated in all cases. The LODs were within the monitoring requirements of the European Commission. The results were validated with a peanut butter certified reference material. The chlorophyll MIP on the other hand selectively removed chlorophyll that would otherwise interfere during pesticide residue analysis (PRA) from > 0.6 to <0.09 Au in green plants extracts. The extracted chlorophyll was removed to far below the level of ≥ 0.399 Au that is usually associated with interference during PRA. Furthermore, the MIP demonstrated better selectivity by removing only chlorophyll (> 99%) in the presence of planar pesticides than the currently employed graphitized carbon black (GCB) that removed both the chlorophyll (> 88%) and planar pesticides (> 89%). For the interfering cholic acid during drug residue analysis, cholic acid MIP electrospun nanofibers demonstrated to be more sensitive and possessing higher loading capacity than the MIP particles. 100% cholic acid was removed by the nanofibers from standard solutions relative to 80% by the particles. This showed that the nanofibers have better performance than the micro particles and as such have potential to replace the particle based SPE sorbents that are currently in use. All the templates were optimally removed from the prepared MIPs by employing a novel pressurized hot water extraction template removal method that was used for the first time in this thesis. The method employed only water, an environmentally friendly solvent to remove templates to ≥ 99.6% with template residual bleeding of ≤ 0.02%.
- Full Text:
- Date Issued: 2012
- Authors: Batlokwa, Bareki Shima
- Date: 2012
- Subjects: Sorbents -- Research Nanofibers -- Research Aflatoxins -- Research Electrospinning -- Research Extraction (Chemistry) -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4309 , http://hdl.handle.net/10962/d1004967
- Description: This thesis presents the development of chlorophyll, cholic acid, aflatoxin B1 molecularly imprinted polymer (MIP) particles and cholic acid MIP nanofibers for application as selective solid phase extraction (SPE) sorbents. The particles were prepared by bulk polymerization and the nanofibers by a novel approach combining molecular imprinting and electrospinning technology. The AFB1 MIP particles were compared with an aflatoxin specific immunoextraction sorbent in cleaning-up and pre-concentrating aflatoxins from nut extracts. They both recorded high extraction efficiencies (EEs) of > 97 % in selectively extracting the aflatoxins (AFB1, AFB2, AFG1 and AFG2). High reproducibility marked by the low %RSDs of < 1% and low LODs of ≤ 0.02 ng/g were calculated in all cases. The LODs were within the monitoring requirements of the European Commission. The results were validated with a peanut butter certified reference material. The chlorophyll MIP on the other hand selectively removed chlorophyll that would otherwise interfere during pesticide residue analysis (PRA) from > 0.6 to <0.09 Au in green plants extracts. The extracted chlorophyll was removed to far below the level of ≥ 0.399 Au that is usually associated with interference during PRA. Furthermore, the MIP demonstrated better selectivity by removing only chlorophyll (> 99%) in the presence of planar pesticides than the currently employed graphitized carbon black (GCB) that removed both the chlorophyll (> 88%) and planar pesticides (> 89%). For the interfering cholic acid during drug residue analysis, cholic acid MIP electrospun nanofibers demonstrated to be more sensitive and possessing higher loading capacity than the MIP particles. 100% cholic acid was removed by the nanofibers from standard solutions relative to 80% by the particles. This showed that the nanofibers have better performance than the micro particles and as such have potential to replace the particle based SPE sorbents that are currently in use. All the templates were optimally removed from the prepared MIPs by employing a novel pressurized hot water extraction template removal method that was used for the first time in this thesis. The method employed only water, an environmentally friendly solvent to remove templates to ≥ 99.6% with template residual bleeding of ≤ 0.02%.
- Full Text:
- Date Issued: 2012
Molecular analysis of genetic diversity in dometicated pigeonpea (Cajanus cajan (L.) Millsp.) and wild relatives
- Authors: Kassa, Mulualem Tamiru
- Date: 2011
- Subjects: Pigeon pea -- Variation Cajanus -- Variation Pigeon pea -- Genetics Cajanus -- Genetics Biodiversity Pigeon pea -- Phylogeny Cajanus -- Phylogeny Plant hybridization Plant diversity Transgenic plants
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4204 , http://hdl.handle.net/10962/d1003773
- Description: Cajanus cajan (L.) Millsp. (Pigeonpea) belongs to the Leguminosae genus Cajanus which is composed of 34 species. Pigeonpea is the only cultivated member of the genus, while the remaining species are wild relatives belonging mainly to the secondary gene pool. DNA sequence data from the nuclear ITS region and the chloroplast trnL-F spacer were utilized to investigate the phylogenetic relationships between Cajanus and five other allied genera in the subtribe Cajaninae. This study revealed the non-monophyly of Cajanus and Rhynchosia and supported the monophyly of Eriosema and Flemingia, but more sampling ,especially from the large genera of Rhynchosia and Eriosema, is recommend to adequately test the hypothesis of generic monophyly. The phylogenetic relationships within the genus Cajanus resolved Cajanus scarabaeoides (L.) Thouars as the most basal species in the Cajanus clade. The study also utilized Single Nucleotide Polymorphism (SNP) markers derived from low copy orthologous genes and genotyped using the high throughput SNP-OPA Illumina golden gate assay. The aim was to understand phylogenetic and domestication history, genetic structure, patterns of genetic diversity, gene flow and historical hybridization between Cajanus cajan (pigeonpea) and wild relatives. The neighbor-joining tree resolved well-supported clusters, which reflect the distinctiveness of species and congruence with their geographical origin. It supported the ITS based phylogeny and resolved C. scarabaeoides as basal to the Cajanus clade. The phylogenetic signal and genetic signatures revealed insights into the domestication history of pigeonpea. Our results supported Cajanus cajanifolius as the presumed progenitor of pigeonpea and we speculate that for pigeonpea there was a single major domestication event in India. Genetic admixture and historical hybridization were evident between pigeonpea and wild relatives. Abundant allelic variation and genetic diversity was found in the wild relatives, with the exception of wild species from Australia, as compared to the domesticated pigeonpea. There was a reduction of about 75% in genetic polymorphism in domesticated pigeonpea as compared to the wild relatives, indicating a severe “domestication bottleneck” during pigeonpea domestication. We discovered SNP markers associated with disease resistance (NBS-LRR) loci. The SNPs were mined in a comparison of BAC-end sequences (BES) of C. cajan and amplicons of the wild species, C. scarabaeoides. A total of ~3000 SNPs were identified from 304 BES. These SNPs could potentially be used in constructing a genetic map and for marker assisted breeding.
- Full Text:
- Date Issued: 2011
- Authors: Kassa, Mulualem Tamiru
- Date: 2011
- Subjects: Pigeon pea -- Variation Cajanus -- Variation Pigeon pea -- Genetics Cajanus -- Genetics Biodiversity Pigeon pea -- Phylogeny Cajanus -- Phylogeny Plant hybridization Plant diversity Transgenic plants
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4204 , http://hdl.handle.net/10962/d1003773
- Description: Cajanus cajan (L.) Millsp. (Pigeonpea) belongs to the Leguminosae genus Cajanus which is composed of 34 species. Pigeonpea is the only cultivated member of the genus, while the remaining species are wild relatives belonging mainly to the secondary gene pool. DNA sequence data from the nuclear ITS region and the chloroplast trnL-F spacer were utilized to investigate the phylogenetic relationships between Cajanus and five other allied genera in the subtribe Cajaninae. This study revealed the non-monophyly of Cajanus and Rhynchosia and supported the monophyly of Eriosema and Flemingia, but more sampling ,especially from the large genera of Rhynchosia and Eriosema, is recommend to adequately test the hypothesis of generic monophyly. The phylogenetic relationships within the genus Cajanus resolved Cajanus scarabaeoides (L.) Thouars as the most basal species in the Cajanus clade. The study also utilized Single Nucleotide Polymorphism (SNP) markers derived from low copy orthologous genes and genotyped using the high throughput SNP-OPA Illumina golden gate assay. The aim was to understand phylogenetic and domestication history, genetic structure, patterns of genetic diversity, gene flow and historical hybridization between Cajanus cajan (pigeonpea) and wild relatives. The neighbor-joining tree resolved well-supported clusters, which reflect the distinctiveness of species and congruence with their geographical origin. It supported the ITS based phylogeny and resolved C. scarabaeoides as basal to the Cajanus clade. The phylogenetic signal and genetic signatures revealed insights into the domestication history of pigeonpea. Our results supported Cajanus cajanifolius as the presumed progenitor of pigeonpea and we speculate that for pigeonpea there was a single major domestication event in India. Genetic admixture and historical hybridization were evident between pigeonpea and wild relatives. Abundant allelic variation and genetic diversity was found in the wild relatives, with the exception of wild species from Australia, as compared to the domesticated pigeonpea. There was a reduction of about 75% in genetic polymorphism in domesticated pigeonpea as compared to the wild relatives, indicating a severe “domestication bottleneck” during pigeonpea domestication. We discovered SNP markers associated with disease resistance (NBS-LRR) loci. The SNPs were mined in a comparison of BAC-end sequences (BES) of C. cajan and amplicons of the wild species, C. scarabaeoides. A total of ~3000 SNPs were identified from 304 BES. These SNPs could potentially be used in constructing a genetic map and for marker assisted breeding.
- Full Text:
- Date Issued: 2011
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