An evaluation of the cytotoxic activities of novel artemisinin derivatives: towards targeted therapies for triple-negative breast cancers (TNBC)
- Authors: Kajewole, Deborah Ifeoluwa
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163329 , vital:41029 , doi:10.21504/10962/163329
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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Analysis of the regulation of HSP90α expression upon differentiation of C2C12 cells
- Authors: Holm, Nathan Christopher
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163318 , vital:41028
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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Comparative analysis of the known Hop1b and the novel Hop1a isoforms of the Hop gene
- Authors: Makhubu, Portia
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164311 , vital:41108 , doi:10.21504/10962/164311
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
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Elucidation of a novel role for HSP70/HSP90 organising protein (Hop) in mRNA processing
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2020
- Language: English
- Type: thesis , text , Doctoral , Ph.D
- Identifier: http://hdl.handle.net/10962/59173 , vital:27449 , doi:10.21504/10962/59173
- Description: Thesis (PhD.)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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Investigating the relationship between Heat Shock Proteins and HIV Transactivator of Transcription
- Authors: Flax, Lili Marie
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163307 , vital:41027
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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Multiplexed Mass Spectrometry: Single, On-Bead, Detection Analysis Using MALDI-TOF MS
- Authors: Twala, Busisiwe Victoria
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/164693 , vital:41155 , doi:10.21504/10962/164693
- Description: Thesis (PhD)--Rhodes University, Biochemistry and Microbiology, 2020
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The novobiocin-induced turnover of fibronectin via low density lipoprotein receptor-related protein 1 alters matrix morphology with physiological consequences on cell growth and migration
- Authors: Boёl, Natasha Marie-Eraine
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/114778 , vital:34034 , 10.21504/10962/114778
- Description: Fibronectin (FN), an extracellular matrix protein, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion thereby contributing to the metastatic potential of cancer cells. Previous studies have shown the direct binding of Heat Shock Protein 90 kDa (Hsp90) and FN in vitro, and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. Low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitous receptor known to bind both Hsp90 and FN. Using an LRP1 expressing Hs578T breast cancer cell line and an isogenic mouse embryonic fibroblast (MEF) model system of differential LRP1 expression we demonstrate that LRP1 is involved in turnover of FN in response to C-terminal Hsp90 inhibition. The first objective of this study was to identify the mechanism of NOV-induced LRP1-mediated FN turnover. Our data show that NOV-mediated FN turnover via LRP1 did not require the activity of matrix metalloproteinases (MMPs), which play an important role in processing and degradation of the extracellular matrix and FN. In addition, the levels of the main FN receptor responsible for its extracellular assembly, β1-integrin, did not change in response to NOV. LRP1 is known to undergo regulated intramembrane proteolysis (RIP) which generates smaller fragments that may translocate to the nucleus and modulate gene transcription. Using inhibitors of LRP1 cleavage and nuclear fractionation we determined that LRP1 processing was not required for the NOV-induced FN response suggesting that a mechanism unrelated to LRP1 RIP is involved. A possible mechanism may be in altered Hsp90-LRP1 cell signalling as we observed disruption of the FN-Hsp90-LRP1 complex at the cell surface in NOV treated cells. How this affects downstream eHsp90-LRP1 signalling is still to be determined but may be related to a significant increase in phospho-AKT and loss of phospho-ERK upon NOV-treatment; two key signalling proteins involved in FN matrix regulation and which are downstream of LRP1 signalling. The second objective of this study was to determine the physiological consequences associated with FN turnover in response to NOV treatment. Using migration assays we demonstrated that levels of insoluble matrix-associated FN and FN concentration are not solely responsible for migratory capacity of cells on decellularized extracellular matrices, but rather that structural composition and integrity of the matrix plays a bigger role. Using confocal and scanning electron microscopy, we identified NOV treated matrices to be flatter, less mature and contain thicker, rope-like FN fibrils to which cells adhered better but were generally less proliferative. Comparatively, cells adhered less to the more mature and 3-dimensional untreated matrices but exhibited increased spreading and cell growth, which may in part be due to the thinner fibrils and web-like matrix. In summary, this study substantiates the role of LRP1 in NOV-mediated FN turnover, and provides new insights into the possible mechanisms of the Hsp90-LRP1 mediated loss of FN matrix. This is the first study to demonstrate some of the functional consequences related to FN turnover by NOV at the ECM level. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
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The role of the Hop co-chaperone in the formation of Hsp90 complexes: chaperone link to glycolysis
- Authors: Maharaj, Shantal
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/163593 , vital:41051 , doi:10.21504/10962/163593
- Description: Thesis (PhD)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
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Analysis of the human HSP70-HSP90 organising protein (HOP) gene - characterisation of the promoter and identification of a novel isoform
- Authors: Mattison, Stacey
- Date: 2018
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62821 , vital:28296
- Description: Expected release date-April 2020
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Identification of SNPs within the CYP2A6 enzyme of TNBC cell lines and the resulting change in activity
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64349 , vital:28536
- Description: Expected release date-May 2019
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Regulation of cell biology by extracellular species of the Hsp90- Hsp70 organising protein (Hop)
- Authors: Höft, Maxine Allison
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59199 , vital:27465
- Description: Expected release date-April 2019
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The relationship between OCT4 and an aggressive phenotype in triple negative breast cancer (TNBC)
- Authors: Jackson, Hayley Claire
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59209 , vital:27477
- Description: Expected release date-April 2019
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Identification of potential novel roles for Hsp70/Hsp90 organising protein (Hop) using proteomic analysis in human cells
- Authors: Wingate, Ianthe
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64758 , vital:28598
- Description: Expected release date-May 2018
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Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
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Biotechnology from bench to market: the design, scale-up and commercialisation strategy development of a disruptive bioprocess for potable ethanol production
- Authors: Dhanani, Karim Colin Hassan
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55863 , vital:26750
- Description: The capacity of research institutions to engage in technology transfer activities has important implications on both economic development and technological advancement. This thesis explores the developmental and commercialisation processes involved in the transfer of a potentially disruptive bioprocessing technology for beverage alcohol production. Ethanolic fermentation strategies are of interest due to their global economic importance and their potential to produce clean renewable fuels in the future. Currently used methods are both energetically wasteful and economically inefficient. To this end more effective bioprocessing methods and implementation strategies are required to enable commercially viable decentralised small-scale ethanol production. Perfusion reactors have a number of advantages over batch and other continuous fermentation strategies. This study aimed to develop and study the fermentative efficiency of a perfusion tower bioreactor system at the bench scale, and subsequently through a scale up process to a low level commercial capacity. An HPLC method was developed for the Simultaneous quantification of common fermentation analytes; this was used to determine bench scale fermentation efficacies over an operational period. At steady state the ethanol volumetric productivity of the bench scale bioreactor system was 3.40 g. L-1.h-1, the average yield of ethanol to consumed sugar was 0.467 g.g -1, with an average sugar conversion percentage of 96%. Results showed that the tower perfusion bioreactor was appropriate for high performance ethyl alcohol fermentations. This reactor design was then scaled up to pilot scale and then commercial scale ca pacity. Similar efficienCies were achieved with these larger systems. Based on the process performance data obtained, a commercialisation strategy was developed and market performance was projected. It was found that productivity rates per unit volume were favourable, and the bioreactor system was determined to be very cost effective for a decentralised ethanolic beverage manufacturing model.
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Characterisation of the HSP70-HSP90 organising protein gene and its link to cancer
- Authors: Weeks, Stacey
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56006 , vital:26764
- Description: HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.
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Characterization of the co-chaperones of Hsp70 and Hsp90 in Trypanosoma brucei and their potential partnerships
- Authors: Mokoena, Fortunate
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54543 , vital:26583
- Description: African Trypanosomiasis, which is caused by Trypanosoma brucei, is one of the crippling agents of social and economic development in Africa. T. brucei cycles between the cold-blooded insect vector, the tsetse fly (Glossina spp), and warm-blooded mammalian hosts. T. brucei, T. cruzi and L. major are mammal infecting kinetoplastid parasites that are collectively referred to as TriTryps. These parasites experience extreme environments as they move between their warm-blooded mammalian hosts and cold-blooded insect vectors which trigger extensive morphological transformations during the life-cycle of the parasite. Molecular chaperones have been implicated in parasite differentiation. TriTryps display significant expansions and diversity in the gene complements encoding molecular chaperones, especially J-proteins. Generally, J-proteins function as co-chaperones of Hsp70s, forming part of vital protein homeostasis processes. Hsp70s show a high degree of conservation, while J-proteins appear to be an extreme case of taxonomic radiation. Although several studies have focused on the molecular and cell biology of Hsp70s in some kinetoplastid parasites, knowledge is still lacking pertaining to J-proteins and their partnerships with Hsp70s. This thesis focused on the classification of kinetoplastid Jproteins into the four types by examining the domain organizations using T. brucei as a guide. The potential partnership of J-proteins and Hsp70s were postulated based on predicted subcellular localization. Kinetoplastid parasites, particularly T. brucei, have evolved an expanded and specialized J-protein machinery, likely to be a consequence of an evolutionary fitness/trait to adapt to diverse environment present in hosts and vectors. These analyses will yield insight into the process of parasite differentiation as well as provide new leads for chemotherapeutic treatments. The presence of the STI1 mediated Hsp90 hetero-complex formation has not been confirmed in T. brucei. To this end, in silico and biochemical techniques were used to characterize the role of TbSTI1, as an adaptor protein of Hsp70 and Hsp90. Through domain architecture analysis, sequence alignments, phylogenetic analysis and three-dimensional structure prediction, TbSTI1 was demonstrated to be the most conserved TPR containing co-chaperone of Hsp70 and Hsp83 in T. brucei and also shown to be highly similar to its eukaryotic homologues. Recombinant TbSTI1 was overproduced and purified in E.coli cells and subsequently shown to associate with TcHsp70 in a concentration dependent manner and associate weakly with TbHsp70.4. TbSTI1 and TbHsp83 were also demonstrated to be expressed and upregulated upon exposure to heat shock at the bloodstream stage of parasite development. In conclusion, this study is the first to report the interaction of TbSTI1 with a chaperone. Interactions between TbSTI1 and Hsp70s were demonstrated and therefore, the formation of the hetero-complex is predicted based the similarity of TbSTI1 to other STI1 proteins.
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Human FN1 is regulated by the heat-shock response
- Authors: Dhanani, Karim Colin Hassan
- Date: 2015
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/193487 , vital:45336
- Description: Heat shock protein 90 (Hsp90) and heat shock factors (HSFs) are known to be involved in the epigenetic regulation of several fundamental oncogenic genes. Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein which plays key roles in cell adhesion and migration. Hsp90 binds directly to FN and Hsp90 inhibition has been shown to regulate FN protein levels and matrix formation. Where inhibition of Hsp90 with a C-terminal inhibitor (novobiocin) induced the loss of FN matrix, treatment with an N-terminal inhibitor (geldanamycin) increased FN matrix levels. GA treatment induced a strong dose and time dependent increase in FN1 promoter activity and increased total FN mRNA respectively. By contrast, NOV showed no increase in the promoter activity and no change in the expression of FN mRNA. As GA is known to induce the stress response, we investigated the relationship between the cell stress machinery and the transcriptional regulation of FN. Three putative heat shock elements (HSEs) were identified in the FN1 promoter. The loss of two of the three identified putative HSEs resulted in a loss in the basal transcriptional activity of the FN1 promoter in our reporter model. This was in addition to the loss of the induction of transcriptional activity with GA treatment observed with the full-length promoter. Binding of HSF1 to one of the putative HSEs, which was identified as potentially functional from the truncation analysis, was confirmed using ChIP. The occupancy of this HSE by HSF1 was shown to increase with GA treatment. These data support the hypothesis that FN1 is a functional HSF1 target gene. The 5' promoter regions of seven additional ECM protein encoding genes were analysed and mRNA levels were detected by quantitative RT-PCR upon treatment with GA. Collagen 4 _2 and laminin _3 mRNA were found to increase in the presence of GA, whereas collagen 4 _3 and osteopontin showed no change. Similarly to FN1, these data indicate that a subset of ECM genes may be under the regulation of the HSF1 mediated heat-shock response. This may have implications for our understanding of ECM dynamics in cancer, where the clinical application of Hsp90 inhibitors is intended. Additionally, our data provide a poten- tial underpinning for the role of the HSF1 mediated heat-shock response in several fibrotic and metabolic stress related pathologies. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
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Identification of novel SNPSTRs by 454 sequencing in Nguni and Sotho-Tswana populations
- Authors: Laurence, Jo-Anne Elizabeth
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/55885 , vital:26752
- Description: DNA profiling is currently performed by analysis of the electropherogram that results following the amplification of a panel of Short Tandem Repeat (STR) loci. A need has arisen, however, for the development of a typing method that generates results which are compatible and comparable with existing databases, but that have a higher discrimination power by supplying sequence data as well as repeat-number data. Recent studies that explore these alternative typing methodologies have revealed the existence of a number of STR variants. There is, however, little information about the exact nature and prevalence of these sub-alleles. There have also been limited population studies of the genetic profiles of sub-Saharan African populations, despite the fact that evidence suggests that there is greater genetic structure and genetic diversity in these populations. In this study, a processing protocol for the generation of 454 sequencing-ready amplicons of vWA, D2S441, D3S1358, D13S317, D21S11 and D7S820 loci was developed. This protocol was applied to buccal swabs collected from 144 individuals of the Nguni and Sotho-Tswana population groups. A total of 145 485 reads were obtained from the sequencing of these amplicons, of which 97 400 and 48 085 reads were obtained for the Nguni and Sotho-Tswana populations respectively. The proportional representation for each locus ranged from 8-20%, and the allele calls and observed frequencies of these alleles suggested a high degree of relatedness between population groups. The sequencing results, furthermore, enabled the identification of a number of previously undescribed STR variants and SNPSTRs; with allele 13´ for D13S317 representing a SNP that may be predictive of Nguni-ancestry. The results also demonstrated the usefulness of next generation sequencing for increasing the number of discernible alleles for STR profiling.
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The development of biological tools to aid in the genetic investigation of the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros mitochondrial genomes
- Authors: Parsons, Michelle
- Date: 2015
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/56059 , vital:26769
- Description: The black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros are found in South Africa. A decline in the populations of these species has resulted due to human activities such as habitat fragmentation and poaching. This has contributed to the loss of genetic diversity amongst the black and white rhinoceros. Conservation and anti-poaching efforts are needed to help maintain genetic diversity. These efforts could be improved through the development of non-invasive techniques to examine DNA from threatened animals. The aim of this research was to develop a molecular technique which would allow for the identification of the black and white rhinoceros and to develop a molecular technique which would allow for intraspecies genetic variation to be examined. DNA extractions were performed on matched faecal and tissue samples that were collected from two regions in South Africa. Polymerase chain reaction (PCR) primer sets were designed to investigate several regions of the rhinoceros mitochondrial genome. PCR optimisation was completed for the target regions. Sequencing was conducted on all final PCR products. The cytochrome c oxidase subunit 1 (COIi) gene allowed for the rhinoceros family to be identified. This region was digested with the HindIII restriction enzyme, which allowed for the specific identification of either the black or white rhinoceros. A subsequent region of the cytochrome c oxidase subunit 1 (COIii) as well as the D-loop, hypervariable regions (HV1 and HV2), cytochrome b (cytb) and 16s rRNA regions were investigated. These regions displayed potential for establishing geographic origin for black rhinoceros samples, whereas the D-loop and HV2 show potential for the white rhinoceros. The white rhinoceros displayed sequence variation in the HV2 and cytb region, while variation was observed in the COIi and HV1 for the black rhinoceros. All investigated target regions allowed for the rhinoceros family to be identified. The COI (COIi and COIii), HV2 and cytb regions allowed for the subspecies of rhinoceros to be identified, however the D-loop was not able to identify the white rhinoceros species. The 16s rRNA and HV1 regions allowed for the correct subspecies of rhinoceros to be identified, however as the primers were only compatible for the black rhinoceros therefore a subsequent investigation is required for the white rhinoceros. The establishment of this novel PCR based technique to identify white and black rhinoceros will allow for efficient species identification in wildlife forensic cases. A biological method was established to study intraspecies variation for the white and black rhinoceros; however the investigated target regions did not yield sufficient genetic variation. The core techniques developed in this study will be valuable for future studies that wish to investigate genetic variation in mammal species.
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