Characterization of Trypanosoma brucei Sti1 and its interactions with Trypanosoma brucei Hsp83 and human Hsp90
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
Innovation in ecological restoration techniques: Enhancing Portulacaria afra survivorship in degraded arid thicket
- Authors: Norman, Yondela Masande
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/419276 , vital:71630
- Description: The Albany Thicket Biome has undergone extensive degradation over the past century, particularly from overstocking of livestock in the arid types of thicket. The degradation of the biome, coupled with little to no natural recovery, prompted the South African Government to implement the Subtropical Thicket Restoration Programme (STRP) in the early 2000s to rehabilitate degraded thicket using unrooted Portulacaria afra truncheons. The STRP also sought to create job opportunities, bring about social upliftment, promote biodiversity conservation and incentivise farmers and landowners in the region to promote carbon trading and farming of P. afra using carbon credits. However, the survivorship of P. afra planted using the STRP planting protocol has been less than ⁓30% because they are planted in degraded areas, where the truncheons are subjected to harsh biophysical conditions such as hard, capped soil, high soil temperatures, drought and herbivory. The aim of this study, therefore, was to improve the survivorship of P. afra planted in degraded thicket to at least 30%, using modified planting methods. In this study, four treatments were applied in 210 pondings (or micro-dams), each with P. afra planted in them (the first three treatments used rooted cuttings). The first treatment involved watering regimes where pondings were watered at varying frequencies. The second treatment involved planting companion species alongside P. afra while the third treatment involved planting P. afra underneath a nurse canopy. In the fourth treatment, unrooted P. afra truncheons were planted inside pondings. The mean survivorship of the P. afra cuttings was assessed 18 months after planting. The watering regime treatments, including the control, all yielded a survivorship of over 75%, with the significantly highest levels of survivorship being displayed in the weekly watering treatment (90.9 ± 6.8%) (p < 0.01). The companion plant treatment also produced a high mean survivorship of P. afra (94.9 ± 3.6%), significantly higher than that of the nurse plants (87.5 ± 6.3%) (p < 0.01). Among the unrooted truncheons it was found that untreated truncheons had the highest survivorship (76.2 ± 17.6%), with the lowest mean being found in truncheons that were both pruned and scarified (70.8 ± 20.8%), suggesting that this treatment, out of all of them, is the least successful under the prevailing environmental conditions in the study area. However, there was no significant differences among the unrooted truncheon treatments and their respective survivorship values to further substantiate this assertion (p = 0.26). Findings in this study also suggest that planting P. afra cuttings under a nurse plant was ideal for P. afra survivorship due to the nurse plant’s ability to ameliorate the microclimate under which the cuttings can establish and grow. Despite the soil under the nurse canopy having a significantly lower mean soil water potential (-160.9 ± 200.5 kPa), compared to the open areas (-73.4 ± 55.7 kPa) (p = 0.04), the pondings under the nurse canopy still had a lower mean soil temperature (31.4 ± 5.25°C) than the treatments in the open areas (38.5 ± 2.7°C), during the harsh midday sun. These favourable characteristics are reflected in the P. afra cuttings under a nurse canopy having a higher chlorophyll fluorescence (0.76 ± 0.06). compared to those planted in open areas (0.73 ± 0.13), suggesting that the P. afra cuttings in the former treatment had a higher photosynthetic efficiency compared to those in the latter, despite there being no statistically significant difference in chlorophyll fluorescence between the treatments (p = 0.14). Although these findings suggest that applying these modifications to planting P. afra, having achieved its objective of increasing survivorship to well over 50%, is likely to produce favourable results not only in overall survivorship, but also in fast-tracking arid thicket rehabilitation and restoration, further research on these rehabilitation techniques and their effectiveness is required. Furthermore, the downside to these treatments is that they are costly and time consuming, which puts the feasibility of large-scale programmes using these applications into question. Further investigation is required to determine ways in which the cost-effectiveness of these applications can be enhanced. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Norman, Yondela Masande
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/419276 , vital:71630
- Description: The Albany Thicket Biome has undergone extensive degradation over the past century, particularly from overstocking of livestock in the arid types of thicket. The degradation of the biome, coupled with little to no natural recovery, prompted the South African Government to implement the Subtropical Thicket Restoration Programme (STRP) in the early 2000s to rehabilitate degraded thicket using unrooted Portulacaria afra truncheons. The STRP also sought to create job opportunities, bring about social upliftment, promote biodiversity conservation and incentivise farmers and landowners in the region to promote carbon trading and farming of P. afra using carbon credits. However, the survivorship of P. afra planted using the STRP planting protocol has been less than ⁓30% because they are planted in degraded areas, where the truncheons are subjected to harsh biophysical conditions such as hard, capped soil, high soil temperatures, drought and herbivory. The aim of this study, therefore, was to improve the survivorship of P. afra planted in degraded thicket to at least 30%, using modified planting methods. In this study, four treatments were applied in 210 pondings (or micro-dams), each with P. afra planted in them (the first three treatments used rooted cuttings). The first treatment involved watering regimes where pondings were watered at varying frequencies. The second treatment involved planting companion species alongside P. afra while the third treatment involved planting P. afra underneath a nurse canopy. In the fourth treatment, unrooted P. afra truncheons were planted inside pondings. The mean survivorship of the P. afra cuttings was assessed 18 months after planting. The watering regime treatments, including the control, all yielded a survivorship of over 75%, with the significantly highest levels of survivorship being displayed in the weekly watering treatment (90.9 ± 6.8%) (p < 0.01). The companion plant treatment also produced a high mean survivorship of P. afra (94.9 ± 3.6%), significantly higher than that of the nurse plants (87.5 ± 6.3%) (p < 0.01). Among the unrooted truncheons it was found that untreated truncheons had the highest survivorship (76.2 ± 17.6%), with the lowest mean being found in truncheons that were both pruned and scarified (70.8 ± 20.8%), suggesting that this treatment, out of all of them, is the least successful under the prevailing environmental conditions in the study area. However, there was no significant differences among the unrooted truncheon treatments and their respective survivorship values to further substantiate this assertion (p = 0.26). Findings in this study also suggest that planting P. afra cuttings under a nurse plant was ideal for P. afra survivorship due to the nurse plant’s ability to ameliorate the microclimate under which the cuttings can establish and grow. Despite the soil under the nurse canopy having a significantly lower mean soil water potential (-160.9 ± 200.5 kPa), compared to the open areas (-73.4 ± 55.7 kPa) (p = 0.04), the pondings under the nurse canopy still had a lower mean soil temperature (31.4 ± 5.25°C) than the treatments in the open areas (38.5 ± 2.7°C), during the harsh midday sun. These favourable characteristics are reflected in the P. afra cuttings under a nurse canopy having a higher chlorophyll fluorescence (0.76 ± 0.06). compared to those planted in open areas (0.73 ± 0.13), suggesting that the P. afra cuttings in the former treatment had a higher photosynthetic efficiency compared to those in the latter, despite there being no statistically significant difference in chlorophyll fluorescence between the treatments (p = 0.14). Although these findings suggest that applying these modifications to planting P. afra, having achieved its objective of increasing survivorship to well over 50%, is likely to produce favourable results not only in overall survivorship, but also in fast-tracking arid thicket rehabilitation and restoration, further research on these rehabilitation techniques and their effectiveness is required. Furthermore, the downside to these treatments is that they are costly and time consuming, which puts the feasibility of large-scale programmes using these applications into question. Further investigation is required to determine ways in which the cost-effectiveness of these applications can be enhanced. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2023
- Full Text:
- Date Issued: 2023-03-31
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