3. Electronic Theses and Dissertations (ETDs) - All submissions

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A study of C2 and V2 pathogenicity genes of Tomato curly stunt virus variants in tomato and Nicotiana benthamiana
(2022) Siviya, Lebogang Edith
Tomatoes are the second most important agricultural crop in the world. Due to selection and inbreeding, genetic variation in tomato is restricted, rendering the crop susceptible to disease and pest outbreaks. Tomatoes have been shown to be infected by a number of viruses. Tomato production is largely affected by begomovirus infections, with tomato yellow leaf curl virus (TYLCV) being the most destructive virus. Tomato yellow leaf curl disease (TYLCD) can have a massive impact on fruit yield, reducing the value of commercial tomato products. In 1997 a disease emerged in tomatoes that were planted in the Onderberg region of South Africa. Plants showed symptoms that were similar to those caused by tomato yellow leaf curl virus (TYLCV), namely leaf curling, yellowing and plant stunting. A new begomovirus species was found to cause these symptoms and the virus was named tomato curly stunt virus (ToCSV) Infection of tomato plants with ToCSV can lead to yield loses of up to 100% thus making ToCSV a major concern to the South African economy. A later study described two variants of ToCSV in South Africa, ToCSV_V30 (severe) and ToCSV_V22 (mild). Both variants have a genomic sequence identical to the Old World monopartite begomovirus with six Open Reading Frames(ORFs): V1 (CP), V2 (pre-coat protein), C1 (Rep), C2 (TrAP), C3 (REn), and C4 (REn). The aim of this study was to determine the importance of pathogenicity determinates C2 and V2 encoded by either ToCSV_V30 or ToCSV_V22 variants and their contribution to symptom phenotype or severity in tomato and Nicotiana benthamiana. To investigate if C2 and/or V2 are crucial for viral replication and viral DNA accumulation in tomato cv. Rooikhaki, mutational studies were carried out to determine the impact of mutations in the C2 and V2 ORFs of ToCSV variants. A chimeric infectious clone was produced by substituting the entire V2 region of ToCSV_V30 with that of the ToSCV_V22. Tomato cv. Rooikhaki was subsequently agroinfiltrated with the infectious clone, symptom scores (according to the disease severity index (DSI)) and viral load (using qPCR) were assessed. Plants inoculated with the chimeric infectious clone containing the V2 swap showed no significant differences in symptom severity when compared with symptoms induced by ToCSV_V30. However, when compared to wild-type ToCSV V30 and ToCSV V22, plants infected with the chimeric infectious clone, ToCSV_V30(V22V2aa1-116) (V2 swap), had higher levels of viral DNA. Relative viral load analyses indicated that the viral load of the swap was 5-fold and 3-fold higher at 21 dpi when compared to ToCSV_V30 and ToCSV_V22, respectively. These results suggest that disease severity is coupled to viral DNA levels in tomato plants infected with the V2 swap. The results indicated that the V2 swap did affect the levels of DNA replicated when compared to ToCSV_V22 and ToCSV_V30. In order to determine the function of C2 in pathogenicity, three C2 mutants were produced by substituting the amino acids (aa) in the severe ToCSV_V30 with the corresponding ones from the mild V22 variant; Mut 10 (ToCSV_V30(V22C2aa46)), Mut 9 (ToCSV_V30(V22C2aa48)), and Mut 7 (ToCSV_V30(V22C2aa109)). Plants infected with the C2 mutants developed symptoms that were similar to those produced by ToCSV_V30. Relative viral load analyses showed that the C2 mutants accumulated higher levels of DNA at 21 dpi when compared to the wild-type virus variants. Relative viral load of mut 7, mut 9 and mut 10 was 2.3-, 2.7-, and 2-fold, respectively, higher than ToCSV_V22 at 21 dpi, whereas the relative viral load change for the mutants were 4.38, 4,4 and 3.7-fold higher than ToCSV_V30. This suggests that the specific amino acids in the C2 region studied did affect viral DNA replication. By overexpressing C2 using a PVX expression vector, pGR107, the putative function of C2- encoded TrAP in pathogenicity by both mild and severe variants was studied in N. benthamiana. Plants inoculated with PXV expressing C2_V22 enhanced viral symptoms in inoculated leaves at 7dpi, however, plants inoculated with PVX expressing C2_V30 did not alter the symptoms induced by PVX. These results indicated that C2_V22 is a pathogenicity determinant in N. benthamiana. Intriguingly, plants inoculated with C2 expressed in PVX from both V30 and V22 showed symptom recovery in systemic leaves at 14 dpi and 28 dpi. RNA silencing has been found to be involved in the recovery of geminiviral symptoms. Therefore, to investigate if indeed ToCSV C2 was a suppressor of RNA silencing, the C2 of both severe and mild variants were cloned into pTRV VIGS vector and co-inoculated with GFP into transgenic N. benthamiana expressing GFP. Results indicated that ToCSV C2 weakly suppresses local RNA silencing in inoculated leaves at 4 dpi, however, C2 was unable to suppress systemic silencing in leaves at 14 and 28 dpi. These results have given an insight into the putative roles of C2 and V2 genes encoded by ToCSV variants showing different symptom severity and phenotype in N. benthamiana and tomato plants.
Antifungal activity of kigelia africana phytochemicals against cryptococcus neoformans
(2024) Kanhanda, Elsie
Cryptococcus neoformans is an opportunistic fungal pathogen which frequently causes cryptococcosis in immunocompromised individuals all over the world. Each year, approximately one million cases of cryptococcosis are reported worldwide, resulting in increased mortality. Cryptococcosis can be treated with a limited number of antifungal drugs, with amphotericin B and fluconazole being the preferred choices. The main challenge faced in the current treatment strategy for cryptococcosis is the development of antifungal resistance by C. neoformans. A different approach is therefore necessary to identify and develop novel antifungal drugs and targets. The traditional approach for antifungal drug development has been to target the growth of the pathogen but a potentially effective alternative would be to target the virulence factors. Virulence factors are the elements of a pathogen that can cause damage in the host and lead to disease progression. They are not deemed essential for cell survival and as such are exposed to less evolutionary pressures. The most recognized virulence factors of C. neoformans include the enzyme urease, synthesis of melanin, thermotolerance, and the production of a polysaccharide capsule. In a recent study, bioactive molecules were used to inhibit cryptococcal urease activity. In Chapter 1, literature based on the classification, life cycle, geographic distribution as well as pathogenicity and virulence factors of C. neoformans is reviewed. This is followed by reviewing literature on the current treatment strategies for cryptococcosis, antifungal drug resistance mechanisms of C. neoformans and the role of plant extracts as prospects in antifungal therapy. In Chapter 2 differentially extracted Kigelia africana crude extracts were screened for their antifungal activity against C. neoformans. Ethanolic extracted crude extracts were selected as the best performing bioactives. A sub-lethal concentration of the crude extracts was established and used for the treatment of C. neoformans in the enzyme and biofilm assays. Laccase and urease activity were inhibited. Biofilm formation was inhibited, and pre-formed biofilms were disrupted. Metabolic activity of cryptococcal biofilms was significantly reduced. The effect of treating C. neoformans with K. africana crude extracts on the expression of virulence associated genes was evaluated in Chapter 3. The expression of COX1, LAC1, and URE1 was upregulated, whilst the expression of CAP10 was downregulated. 4 The findings of this study reveal evidence that K. africana crude extracts indeed have antifungal and antivirulence effects against C. neoformans and can therefore be used in the development of novel antifungal drugs which can be used to treat cryptococcal infections
Biophysicochemical properties of the 28-kDa Schistosoma haematobium and pseudo-26-kDa Schistosoma haematobium/bovis glutathione transferase
(2022) Padi, Neo
Schistosomiasis is a debilitating parasitic worm-induced, neglected tropical disease with veterinary and medical concerns in areas with poor socio-economic establishments. S. haematobium is one of the species that mainly affect humans and it has a zoonotic character enabling it to form hybrids with S. bovis thus justifying their common ancestor. In schistosomes, GSTs are primary detoxification enzymes. Moreover, they are involved in host immune response which makes them attractive drug candidates. However, studies are limited by an incomplete sequence of S. haematobium and there is a drug-resistant threat that calls for a new generation of anthelmintics. In this study, we designed a pseudo-Sbh26GST to elucidate its structural and functional properties in response to potential inhibitors, praziquantel (PZQ); artemisinin (ART) and bromosulfophthalein (BSP) in comparison to the well-studied Sh28GST. Several sequence analysis tools were used to complete the sequence of S. haematobium/bovis and generate the pseudo-Sbh26GST which was overexpressed successfully in E. coli with vector, pMAL-c5x while Sh28GST was expressed in pET-11a. All proteins were in the soluble fraction and then purified to ˃95% homogeneity. Functional characterisations were based on the classical GSTs glutathione (GSH) and 1-chloro-2,4 (CDNB) conjugation assay. Sh28GST had a higher 44 µmol/min/mg activity towards CDNB relative to 13 µmol/min/mg for Sbh26GST. PZQ and ART slightly increased the activity insignificantly while BSP inhibited Sbh26GST with an IC50 of 27 µM and 0. 88 µM for Sh28GST. The mode of inhibition, determined by kinetics was found to be random and non-competitive respectively. Structural characterisations were studied through Far-UV circular dichroism which showed that both proteins have a predominantly α-helical secondary structure content. Fluorescence spectroscopy studies showed that BSP and ART disturbed the local Trp environment whereas PZQ had an insignificant effect, all in the presence and absence of co-substrate, GSH and its similar structure, S-hexylglutathione (GTX). Additionally, extrinsic 8-anilino-1- naphthalenesulfonate fluorescence proved that BSP outcompetes it, while PZQ enhances it thus showing competition for the dimer interface and hydrophobic binding site. Thermodynamics parameters obtained by isothermal titration calorimetry indicate that the interaction between dimeric Sbh26GST and Sh28GST with one molecule of BSP is spontaneous and enthalpically driven, with additional entropy support in Sbh26GST but entropy cost in Sh28GST. Stability inferences from SYPRO Orange-based thermal shift assay demonstrated Sbh26GST had increased stability in the presence of BSP and GSH. BSP proved to bind and inhibit both proteins which translate to possibly rationalized new generation anthelmintics for the treatment of schistosomiasis.
Characterization of EPNs and their bacterial symbionts
(2024) Mabena, Nokuthula Busisiwe
Many synthetic pesticides have been associated with health and environmental issues. Not only are they toxic to non-target organisms but can also pollute the soil, water and vegetation. Hence organic farming has been suggested as an alternative approach of biological pest management. Entomopathogenic nematodes (EPNs), which are obligate parasites to insects and carry pathogenic bacterial symbionts and are effective in managing and controlling populations of a variety of economically important insect pests. These insect pests pose a threat to food security as crop yield losses are primarily caused by them. Identifying natural enemies to insects is crucial as they have no harmful consequences to non-target organism and the environment and can be exploited as biological control agents. The first focus of this research was to isolate, identify and characterize a new nematode species. The nematodes were isolated from soil samples originally collected in Mahobong Leribe, Lesotho. To isolate the nematodes, the soil samples were baited with Tenebrio molitor larvae. The methods used for the identification and phylogenetic analysis of the isolated nematodes involved genomic DNA extraction, PCR amplification and Sanger sequencing of the 18S rDNA gene. The nematode that was isolated was a new, previously uncharacterized Cruznema species. The phylogenetic analysis and comparative morphometric characterization confirmed it was a new nematode. The results showed that the average body length of the female and male were 1006.45 µm and 868.31 µm, with a standard deviation of 210.95 µm and 162.75 µm. The species was also closely related to Oscheius spp., which have been reported to be entomopathogenic. Furthermore, the isolated nematode was able to cause mortality to T. molitor larvae within 3 to 6 days. Another focus of this research was to isolate and identify the bacterial symbionts of the isolated nematode. The methods that were used for the isolation involved the homogenization of sterile nematodes. The methods used for the identification and phylogenetic analysis involved genomic DNA extraction, PCR and Sanger sequencing of the 16S rDNA gene as well as culturing on selective and differential media. The isolated bacteria were identified as Alcaligenes spp., Elizabethkingia spp. and Enterobacter spp. They were also tested using lipid agar media to confirm if they were associated with the isolated nematode species. The results showed they were able to feed on and reproduce on the media. 4 The overall results of this study show that the newly identified Cruznema species has the potential of being used as an EPNs
Characterization of rhizobia and their symbiotic performance on selected important legumes under abiotic stress conditions
(2024) Khambani, Langutani Sanger
Introduction: Legumes are a source of proteins in human diet and a staple food for many cultures globally as an inexpensive meat alternative. They form a symbiotic relationship with rhizobia to form root nodules. Inside the nodules, the rhizobia fix atmospheric nitrogen (N2) by the biological nitrogen fixation (BNF) process. This provides a supply of nitrogen to plants. Lately, farmers are more open to the use of rhizobia inoculants due to the availability of effective and quality products in the market. These inoculants improve yields at a low cost when compared to chemical or artificial fertilizers. Symbiotic genes (nodA and nodC), the ACC deaminase enzyme (encoded by acdS gene) and exopolysaccharides (exoR gene) are essential for the rhizobium-legume association, particularly under abiotic stress conditions for effective nodulation. Aim: The current study aims at isolating stress tolerant SARCC (South African Rhizobium Culture Collection) isolates to be applied as inoculants under abiotic stress conditions. Materials and methods: Rhizobia spp. tolerance to various levels of temperature, acidity/alkalinity (pH5, pH7 and pH9), heavy metals (50mM, 100mM and 150mM concentrations of AlCl3.6H2O) and salinity (50mM, 100mM and 150mM concentrations of NaCl) stresses were investigated. Phylogenetic characterization of the isolates was determined using nucleotide sequence analysis of the 16S rRNA, exoR gene, acdS gene, the house keeping recA gene, as well as nodA and nodC symbiotic genes. A glasshouse nodulation efficacy test under normal and abiotic stress conditions was also conducted. Results: This study reports tolerance to abiotic stresses and the phylogenetic characterization of 40 rhizobia strains previously isolated from the root nodules of Medicago sativa, Trifolium repens, Lupinus albus, Vigna unguiculata and Phaseolus vulgaris. The isolates exhibited significant variations in their tolerance to abiotic stresses. Using molecular approaches, some of the rhizobia isolates have been detected to possess certain beneficial traits involved in the reduction of abiotic stresses such as the acdS (9 isolates) and exoR (5 isolates) genes. The symbiotic genes nodC (7 isolates) and nodA (16 isolates) were detected as well. Phylogenetic analyses based on 16S rRNA gene, housekeeping genes (recA) revealed that the isolates belongs to five genera: Sinorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium and Aminobacter. Under glasshouse nodulation efficacy test, effective isolates provided the highest plant biomass and number of nodules under normal conditions, acidity/alkalinity and salinity abiotic stresses when compared to the un-inoculated controls. Conclusion: Amid the increasing threats of the global climate change and the rising abiotic stresses, these current results provide baseline information in the selection of rhizobia for use as inoculants under abiotic stressed conditions in South Africa.
Comparing changes in gene expression across three myeloid cell lines during monocyte-to-macrophage differentiation
(2022) Cosser, Duncan Alexander
Monocytes and macrophages exhibit wide heterogeneity in phenotype and function. Despite this variety, cells from model cell lines are often used in experiments without accounting for these differences. We therefore sought to characterize the gene expression profiles of three model myeloid cell lines as they differentiate from a monocyte-like state to a macrophage-like state in order to identify core pathways across cell lines central to the differentiation process, as well as pathways uniquely involved in differentiation in specific cell lines. Using an RNA-seq analysis pipeline we developed, we examined gene expression in three myeloid cell lines – HL60, U937, and THP-1 – during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation. Using gene expression data from three experiments which induced macrophage differentiation in cells from these cell lines using PMA, we constructed gene expression profiles for the cells before and after PMA treatment. We performed differential gene expression analysis to find the genes that are differentially expressed in differentiation, followed by pathway overrepresentation analysis to identify key pathways involved in this process. We identified 1789 genes which were differentially expressed in all cell lines, as well as 2131 genes marked as differentially expressed only in HL60 cells, 1711 in U937 cells, and 2159 genes uniquely differentially expressed in THP-1 cells. Through subsequent investigation of specific gene clusters, we found that there were conserved pathways involved in the monocyte-to-macrophage transition in all cell lines, particularly those involved in phagocytosis and adherence. All cell lines also downregulated maintenance pathways involved in DNA replication and cell cycle control, a behaviour associated with differentiation. Additionally, we found that there were differences in differentiation between the cell lines: the ribosome and oxidative phosphorylation pathways were strongly down-regulated during the transition in HL60 cells, and multiple different components in the cytokine-cytokine receptor interaction pathway were up- and down-regulated in THP-1 cells. A core gene expression profile is associated with PMA-induced differentiation of myeloid cell lines to a macrophage state. However, differentiation of HL60 and THP-1 cells also induces distinct changes in metabolism and cellular signalling. Our research recommends discretion in cross-comparison between experimental results incorporating gene expression studies of these myeloid cells.
De novo production of Taxol intermediates by Saccharomyces Cerevisiae
(2022) Rabie, Renée
Taxol is an invaluable anticancer molecule produced by Yew trees and their endophytic fungi. Harvesting taxol is difficult and often has low yields. For these reasons, a method to produce taxol heterologously in a fast-growing, well-studied, safe microbe is desirable. In this study, artificial genes were designed for the expression of two taxol biosynthesis pathway enzymes, as well as an assisting NADPH-cytochrome P450 reductase. The genes were designed to be compatible with the pCut transformation technique, which allows genomic integration into Saccharomyces cerevisiae strain BY4742. The genes were then divided into seven fragments. Two additional DNA fragments were amplified directly from the yeast genome because their complexity made them difficult and expensive to synthesise. These nine DNA fragments were designed to be assembled into three linear fragments of equal length for transformation of S. cerevisiae. Attempts at assembling these nine fragments into three inserts failed for various reasons, which largely came down to the complexity and integrity of the DNA, as well as the size of the fragments.
DNA damage associated lncRNAs, PANDA, ANRIL and DDSR1, are constitutively active in HT29 cells under hypertonic stress
(2022) Patel, Aalia
Our genomes undergo DNA damage regularly with the most lethal being DNA double-strand breaks (DSBs). Long non-coding RNAs (lncRNAs) have distinct biological functions and play roles in DNA damage response (DDR). We hypothesized that lncRNA expression would be altered in response to NaCl-induced DNA damage and that these effects may be different in cancer and non-cancer cell lines. Exposure of cells to high NaCl concentrations promotes DSBs in DNA regions near the nuclear periphery. These are rapidly repaired after NaCl withdrawal. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed on HEK293 and HT29 cell lines to determine the EC50 for NaCl. For confocal microscopy, cells were treated with NaCl for 6 hrs or 24 hrs followed by 5 min NaCl withdrawal, and Mre11 was detected. Results indicated that meiotic recombination 11 homolog 1 (Mre11) was localized in the cytoplasm in the presence of NaCl and translocated to the nucleus during 5 min NaCl withdrawal in HEK293 cells. Under high NaCl, Mre11 was present in both the nucleus and cytoplasm in HT29 cells. DSB assessment by western blot analysis for gH2AX revealed no significant change in expression in HEK293 and HT29 cells, but slightly lower levels in HT29 suggest DNA repair may occur in the presence of NaCl. PCR and qPCR were performed for lncRNAs p21 associated ncRNA DNA damage activated (PANDA), Antisense non-coding RNA in the INK4 locus (ANRIL), and DNA damage sensitive RNA1 (DDSR1). qPCR data shows that PANDA, ANRIL, and DDSR1 expression is reduced in HEK293 during NaCl treatment, while in HT29s PANDA was upregulated in response to NaCl treatment and NaCl withdrawal, ANRIL was only upregulated after 4 hrs NaCl and in the 6 hr 5 min NaCl withdrawal treatment and DDSR1 expression was significantly higher in the 6 hr treatment only. Whilst hyperosmotic stress was used to induce DNA damage, we have observed that it results in reduced expression of lncRNAs that play roles in DNA repair and cell cycle regulation in a non-cancer cell line, and enhanced expression in a cancer cell line. This suggests that in hypertonic environments the HT29 cells may still be undergoing cell cycle arrest, DNA repair, and proliferation.
Evaluating the activity of novel natural and synthetic antifungal agents against select pathogenic yeast
(2024) Paul, Alyssa
Fungal infections are believed to affect over a billion people worldwide, with more than 150 million people estimated to be suffering with serious or fatal fungal diseases. Many of these infections are caused by opportunistic fungal pathogens within the Candida and Cryptococcus genera. Current treatment options are limited, costly and can come with significant toxic side effects. This has contributed towards the emergence of novel, multidrug resistant pathogens. With the rise of microbial resistance, it is imperative that efforts are made to provide alternative antifungal treatments that are not only effective, but also non-toxic. To this end, plant based extracts and silver compounds serve as promising treatment opportunities. This study screened a variety of novel silver (I)-phosphine compounds and native plant extracts for their antifungal and antibiofilm activity against pathogens in the Candida and Cryptococcus genera. Furthermore, the toxicity of select compounds was assessed using Caenorhabditis elegans as a model organism. Lastly, the efficacy of select compounds was assessed in vivo through the use of the C. elegans infection model. In Chapter 1, important fungal pathogens within the Candida and Cryptococcus genera are examined. The difficulties associated with the treatment of systemic infections including resistance mechanisms, such as biofilm formation, employed by fungal pathogens is reviewed. The importance of the need for new antifungal treatment opportunities is stressed with silverbased molecules and plant extracts serving as promising antifungal treatment opportunities. Lastly, the use of C. elegans as a model organism to assess toxicity and efficacy of novel antifungal drugs is assessed. In Chapter 2, seven novel silver (I)-phosphine complexes (SPC) as well as eleven native plant extracts are screened for their antifungal activity against a variety of clinically important pathogens within the Candida and Cryptococcus genera. This screening showed SPC 7 as wells as the ethanolic extracts of Hypoxis hemerocallidea, Kigelia africana and Pelargonium zonale possess promising antifungal activity. As such the minimum inhibitory and fungicidal concentrations for those compounds were determined. Chapter 3 examines the antibiofilm properties of the K. africana extract and SPC 7 against pathogens within the Candida and Cryptococcus genera. The results demonstrated that neither of the compounds were able to prevent or disrupt biofilm formation, however when the biofilms were exposed to SPC 7, reductions in the metabolic activity of the biofilms was observed. In Chapter 4, the toxicity of SPC 7 and the ethanolic extracts of H. hemerocallidea, K. africana and P. zonale is assessed using C. elegans as a model organism. The K. africana extract was found to be the least toxic of the plant extracts with SPC 7 also showing low levels of toxicity against the nematode. As such, the efficacy of those compounds was assessed in vivo against Candida auris and Cryptococcus neoformans infected nematodes. The infected nematodes showed significant reductions in mortality when treated with SPC 7. As such, SPC 7 was identified as a promising antifungal treatment showing potent fungistatic, fungicidal, and antibiofilm properties against Candida and Cryptococcus pathogens. Furthermore, SPC 7 shows to exhibit low toxicity towards nematodes while also improving the lifespan of C. auris and C. neoformans infected nematodes. Thus, future work should be conducted to elucidate the mechanisms of action to further assess its suitability as a novel antifungal treatment.
Genomic characterisation of the thermoadaptation strategies in the penguin genus Eudyptes
(2022) Jackson, Debora
Penguins are some of the most well adapted vertebrates with regards to cold. They have managed to achieve this through maintaining thermal balance by reducing the amount of heat lost to the environment and by increasing heat production within the body through the use of various behavioural, physiological and molecular mechanisms. The genus Eudyptes is the most specious group within the family Spheniscidae and these species inhabit a range of different thermal environments ideal for studying the molecular mechanisms that drive coldadaptation. This study aimed to investigate the molecular determinants underlying thermoregulatory adaptation and their evolution among Eudyptes penguin species from different thermal environments. A phylogenetic assessment was undertaken using two approaches. Orthologous and paralogous proteins were identified, annotated and classified to their function and gene ontology using OrthoFinder and EggNOG-mapper. Amino acid analysis and selection analysis were then undertaken on proteins that were found to have a putative role in thermoregulation using BaCoCa and the Selection Server, respectively. The penguins that inhabited sub-Antarctic environments were shown to form a clade in both core genome ML phylogeny and a house-keeping gene ML phylogeny that consisted of the macaroni and royal penguins. A clade was also formed with the penguins that occupy temperate environments i.e. the temperate climate penguin clade, composed of the southern rockhopper, eastern rockhopper, northern rockhopper, Fiordland, snares and erect-crested penguins. This temperate climate clade was further divided into two sub-clades. When the Eudyptes pan-genome was compared with the yellow-eyed penguin (outgroup), 16,511 orthogroups were identified and of these approximately 60% were shared among the nine compared taxa (core proteins). The unique proteins in the cold climate penguin clade only made up 1.28% of the total pan-genome complement compared to the 10.9% that were found in the temperate climate penguin clade. Thirty-five of these unique proteins found in the cold climate Eudyptes penguins play potential roles in various thermoadaptation and thermoregulation strategies. Paralogous proteins were then investigated among the temperate climate and cold climate penguin clades to discover whether they play a potential role in cold-adaptation as gene duplication may serve as a mechanism for tolerating cold stress, especially in penguins. Approximately 19.4% of the total pan-genome protein complement comprised paralogous copies, of which 90 were found to be over-represented in the cold clade penguins by > two-fold relative to the temperate climate penguins. Of these, thirteen were linked to a potential role in thermoregulation with TUBA1C having the most copies present in the macaroni penguin compared to the temperate climate penguins. 90 Single copy orthologous proteins conserved in both cold and temperate clade penguins were subjected to microevolutionary scale analysis at the individual protein and gene level. SCOs contributed nearly 47% of the Eudyptes pan-genome complement. Through amino acid bias analysis, biases in the SCO aligned aa sequence data were identified using various statistical tests (RCFV, chi-square and p-value). Fourteen SCOs met at least one of the statistical cut-off criteria and were found to be linked to thermal regulation. Furthermore, two were discovered to be under overall positive selection (DLL4 and FADS1). Amino acid composition of the fourteen candidate thermoregulation proteins (as is supported in the literature) revealed that cold-adapted proteins prefer conformational flexibility over conformational stability. This is then achieved through an increase in the number of small, neutral amino acids such as serine as well as amino acids that increase the solubility of a protein i.e. aspartic acid. These observed changes are modest but may have a large potential impact on protein function in cold conditions regardless. All these analyses have shown that various molecular mechanisms can be linked to main thermoregulatory mechanisms such as glucose metabolism, lipid metabolism, adipocyte differentiation, vascular development, feather development and skeletal muscle functioning. There are a few limitations to this study. Without looking at other factors such as morphology, integumentary data and behavioural data we cannot definitively say that all eight Eudyptes species are separate species, but our molecular data does suggest this is the most likely case. Another limitation is that many proteins still need to be fully functionally evaluated and annotated and many more proteins may play a role in thermoregulation and thermoadaptation. As a result, they may provide more information on mechanisms that help in cold adaptation. Future studies should incorporate all extant penguin species, especially the species localised in Antarctica i.e. the emperor and king penguins, and by the equator (the Galápagos penguin) to get an even more in-depth account of cold adaptation in penguins. Studies could also look at molecular mechanisms in their temperate counterparts in order to see how climate change may affect the different taxa. In conclusion, this study provides primary information in order to start uncovering the molecular mechanisms that allow penguins to inhabit various environments as well as to uncover the molecular mechanisms that drive cold adaptation in penguins.
Identifying the core microbiome of two South African lichens
(2024) Naude, Pascale Josine
Lichens are a symbiotic relationship formed between fungi (the mycobiont) and algae and/or cyanobacteria (the photobiont). The microbiome (i.e. fungi and bacteria) associated South African lichens have not been investigated. Given the unique South African climate, and the many different lichen taxa found in South Africa, lichens could represent a rich source of novel microorganisms that produce biotechnologically relevant molecules and enzymes and could serve as a tool for environmental and climate change monitoring. The aim of this study was to investigate the microbiomes of two lichens, Parmotrema and Flavopunctelia in three locations in Gauteng, across both the winter and summer seasons, to determine the influence of biotic and abiotic factors on the microbial community structure of these lichens. The total genomic DNA was extracted from the lichens and subjected to 16S ribosomal RNA (16S rRNA) / Internal Transcribed Spacer (ITS) based tag-encoded FLX amplicon pyrosequencing (bTEFAP) at MR DNA (Texas, USA) to identify the bacterial and fungal components of the lichen microbiome, respectively. The core microbiome was identified, and the alpha and beta diversities were calculated to determine the composition of the microbiome and the influence of the lichen mycobiont, lichen sampling location, and seasonal changes on its structure. The core bacteriome was found to be mainly denominated by the phyla Proteobacteria, Acidobacteria and Bacteroidetes. The core mycobiome was largely made up of black meristematic fungi which provide lichens with nutrient sources and protection in nutrient poor environments and was dominated by the phylum Ascomycota. The lichen mycobiont was observed to have little to no influence on the composition and diversity of the lichen microbiome. This finding may be attributed to that fact that both sampled lichens are members of the same family. In contrast, significant differences in the microbial composition and diversity were observed between the sample locations and the seasons, which are characterised by differences in temperature and rainfall. Additionally, the microbial diversity was found to increase with an increase in rainfall. All of this shows that the lichen microbiome may vary with different environmental conditions. This study provides the first look at the South African lichen microbiome and paves the way for more studies to investigate a broader range of lichen taxa over a wider geographical area and over a longer study period.
Identifying the core microbiome of two South African lichens
(2024) Naude, Pascale Josine
Lichens are a symbiotic relationship formed between fungi (the mycobiont) and algae and/or cyanobacteria (the photobiont). The microbiome (i.e. fungi and bacteria) associated South African lichens have not been investigated. Given the unique South African climate, and the many different lichen taxa found in South Africa, lichens could represent a rich source of novel microorganisms that produce biotechnologically relevant molecules and enzymes and could serve as a tool for environmental and climate change monitoring. The aim of this study was to investigate the microbiomes of two lichens, Parmotrema and Flavopunctelia in three locations in Gauteng, across both the winter and summer seasons, to determine the influence of biotic and abiotic factors on the microbial community structure of these lichens. The total genomic DNA was extracted from the lichens and subjected to 16S ribosomal RNA (16S rRNA) / Internal Transcribed Spacer (ITS) based tag-encoded FLX amplicon pyrosequencing (bTEFAP) at MR DNA (Texas, USA) to identify the bacterial and fungal components of the lichen microbiome, respectively. The core microbiome was identified, and the alpha and beta diversities were calculated to determine the composition of the microbiome and the influence of the lichen mycobiont, lichen sampling location, and seasonal changes on its structure. The core bacteriome was found to be mainly denominated by the phyla Proteobacteria, Acidobacteria and Bacteroidetes. The core mycobiome was largely made up of black meristematic fungi which provide lichens with nutrient sources and protection in nutrient poor environments and was dominated by the phylum Ascomycota. The lichen mycobiont was observed to have little to no influence on the composition and diversity of the lichen microbiome. This finding may be attributed to that fact that both sampled lichens are members of the same family. In contrast, significant differences in the microbial composition and diversity were observed between the sample locations and the seasons, which are characterised by differences in temperature and rainfall. Additionally, the microbial diversity was found to increase with an increase in rainfall. All of this shows that the lichen microbiome may vary with different environmental conditions. This study provides the first look at the South African lichen microbiome and paves the way for more studies to investigate a broader range of lichen taxa over a wider geographical area and over a longer study period.
Impact of the hinge region L38↑H↑L double insertion mutation on the structure and function of the HIV-1 South African subtype C protease
(2022) Setshedi, Mpho
The development of mutations in the human immunodeficiency virus (HIV) genome has become a crucial factor in limiting antiretroviral therapy for HIV/AIDS treatment. By the end of June 2021, approximately 37 million people globally were infected with HIV. South Africa, in particular, bears the brunt of the HIV-1 subtype C epidemic with 7.7 million infections. The HIV protease is a homodimeric protein that naturally contains 99 amino acids per monomeric subunit. The protease is vital in the HIV life cycle because it cleaves Gag and Gag-Pol precursor polyproteins into proteins necessary for viral assembly, maturation, and infection. Herein, we performed a comparative study between the HIV-1 subtype C protease (wild-type) and a protease containing a double amino acid insertion (histidine and leucine at codon 38), L38↑H↑L. Each recombinant protease was overexpressed and isolated from E. coli BL21 (DE3) pLysS cells, and purified using ion-exchange chromatography. Far-UV circular dichroism spectra of the WT-CSA and L38↑H↑L protease displayed a minimum at 218 nm which was indicative of a predominantly beta-sheeted protein. Size exclusion chromatography results indicated that the L38↑H↑L protease has a homodimeric molecular weight of approximately 22 kDa which is consistent with characteristics pertaining to the WTCSA protease. Steady-state enzyme kinetic assays were performed by using a fluorogenic substrate that mimics a cleavage site on the Gag polyprotein. The L38↑H↑L protease displayed a 15-fold reduction in KM and a 1.25-fold reduction in the catalytic turnover number, kcat relavite to the WT-CSA protease. The L38↑H↑L protease was found to be 11 times more efficient in catalyzing the hydrolysis of the fluorogenic substrate relative to the WT-CSA protease. The L38↑H↑L protease displayed a 2.25-fold and 0.71-fold reduction in VMax and specific activity relative to the WT-CSA protease. The data suggested that amino acids in the hinge region of HIV-1 proteases indirectly affect enzyme catalysis and substrate specificity but has no effect on the structural stability of the L38↑H↑L protease. Enzyme inhibition studies in the presence of Saquinavir, Darunavir and Atazanavir showed that the L38↑H↑L binds the inhibitors more tightly relative to the WT-CSA protease. This was confirmed by the lower IC50 concentrations of the respective protease inhibitors relative to the WT-CSA protease. Thermodynamics data obtained from performing displacement isothermal titration calorimetry showed that the L38↑H↑L protease displayed a 2-fold increase (-79.02 ± 6.81 kJ/mol) in ∆H when bound to Atazanavir and a 1-fold reduction in ∆H when bound to ii Saquinavir (-9.35 ±3.1 kJ/mol) and Darunavir (-27.61 ± 1.76 kJ/mol) relative to the WT-CSA protease. The binding of Saquinavir and Darunavir to the L38↑H↑L protease was found to be entropically unfavourable (where -T∆S was -32.81 ± 6.40 kJ/mol and -22.74 ± 3.70 kJ/mol, respectively), whereas the binding of the protease to Atazanavir was entropically unfavourable (-T∆S was 37 ± 4.83 kJ/mol). Upon analysis of the Kd, it was noted that L38↑H↑L protease displayed a 2-fold increased drug susceptibility towards Saquinavir (79 ± 7.50 nM), and 2-fold reduced drug susceptibility towards Atazanavir (83.63 ± 7.10 nM), while the drug susceptibility for Darunavir (3.3 ± 1.20 nM) remained unchanged relative to the WT-CSA protease. Due to the important role of the hinge and flap region in accommodating substrates and inhibitors into the active site of the protease, the mutations affected the kinetics of substrate hydrolysis and the thermodynamics of drug binding
In silico identification of transcription based biomarkers for early diagnosis of inflammatory bowel disease and predicting progression to colorectal cancer
(2022) Khan, Farhat
Inflammatory Bowel Disease (IBD) is a complex intestinal inflammation disorder that negatively impacts the quality life of patients. Diagnosis is confirmed through colonoscopy and in paediatrics this procedure is traumatic which highlights the need of less-invasive screening methods for clinical use. Circumstances is additionally entangled by the way that IBD patients have 20-fold increased risk of developing colorectal cancer (CRC). Due to advancements in computer technologies and development of new methods, bioinformatics has become an essential component of biology research. We hypothesized that utilizing bioinformatics methods to identify transcription-based biomarkers can be used for early diagnosis and screening of IBD, and for predicting the danger of its development to CRC in these patients. Transcription factors (TFs) are regulatory proteins that bind primarily to the promoter region of the target genes and control some of the key fundamental processes in a cell. We aim to identify key transcriptional regulators of IBD, and CRC associated genes by generating and understanding the transcription regulatory networks through our proposed methodology. To our knowledge there is currently no IBD genes database created so far. We first aimed at developing manually curated database of 289 IBD genes that are experimentally validated. Furthermore, to achieve other aims, we used various computational tools. To identify TFs and mapping of transcription factor binding sites (TFBSs) to mammalian matrices were achieved by OPOSSUM and JASPAR respectively. Using Cytoscape, we constructed largest transcriptional regulatory networks of top ranked TFs that were mapped to CRC genes. The pathway analysis was done by KEGG, and gene ontology was conducted using DAVID. Majority of the pathways identified were involved in the processes, known to play potential role in IBD. The top upregulated pathways in IBD identified were cytokine, immune response, autophagy, WNT signalling, transmembrane signalling. Further testing of expression of these key regulators in using publicly available published data were done using ONCOMINE database. We identified serological biomarkers of CRC and combined them with transcription-based biomarkers of IBD (CEA + TIMP1 + CA724 + RUNX1) to predict early diagnosis of IBD progressing to CRC. This project will form the basis to develop a kit in future for use in pathology laboratories and clinical settings.
Isolation, identification, and characterisation of fungi from a platinum mine
(2022) Haripersad, Kiara
Mycohydrometallurgy is the application of fungi and their metabolites such as organic acids(OAs) for the extraction and recovery of metals through mechanisms such as bioleaching. The purpose of this study was to identify and characterise fungal isolates from a platinum mine to determine potential fungal candidates for bioleaching of a platinum concentrate. Fungi were isolated from platinum mining samples using traditional microbiological techniques and sixty-six isolates were identified based on their internal transcribed spacer (ITS) regions using Sanger sequencing, as members of either Ascomycota, Basidiomycota or Mucoromycota. Qualitative OA screening was conducted by inoculating isolates into Potato Dextrose Agar containing a pH indicator to select OA producing isolates. These isolates were quantitatively screened using high-performance liquid chromatography and showed the production of acetic, butyric, formic, lactic, propionic, and indole-3-acetic acid. Based on their OA production, Penicillium sp., Rhizopus microsporus, and Aspergillus terreus underwent tolerance tests to copper, chromium, and nickel. Their overall tolerance was low, suggesting that they are promising candidates for two-step direct bioleaching as it involves the growth of fungi prior to their exposure to the metal-containing solid material.
Novel hinge insertions in South African HIV-1 subtype C protease: implications for biochemical and biophysical properties
(2022) Ismail, Zaahida Sheik
Although treatable, human-immunodeficiency virus infections remain distressingly high in sub-Saharan Africa, particularly due to the prevalence of the subtype C variant in this location. While advances have been made in tackling drug resistance globally, subtype C infections are majorly understudied specifically with regards to the effect of mutations in this subtype on drug resistance to currently available inhibitors. The HIV protease is responsible for the production of new infectious viral progenies. Cleavage of the Gag and Gag-Pol polyprotein precursors by the protease produces mature viral particles that can infect new host cells. The indispensable role of the HIV protease in the lifecycle of the virus makes it a major drug target for combatting this disease. Mutations in the protease accumulate both within and distal to the active site, often contributing to drug resistance. More recently, insertions within the hinge region of the protease have been identified. The effect of these hinge region insertions on the HIV protease is not fully understood. The study was based on a clinical isolate containing two insertions, Asn and Leu, at position 38 in the hinge region as well as four mutations: K20R, E35D, R57K and V82I (L38↑N↑L +4 ). The aim of this study was to determine the direct effect of these hinge region insertions on the characteristics of the protease. As such, a variant subtype C protease containing only the double insertion of Asn and Leu was created (L38↑N↑L -4 ). The variant L38↑N↑L -4 protease was successfully overexpressed and purified using a newly adapted ion-exchange chromatography purification protocol. Circular dichroism and size exclusion-high performance liquid chromatography experiments showed that the secondary and oligomeric structures of the variant protease was similar to the wild-type (WT). Active site titrations indicated that the purification of L38↑N↑L -4 resulted in a decrease of actively prepared sample in comparison to the WT. The specific activity and turnover number of L38↑N↑L -4 was significantly reduced in comparison to the WT while the catalytic efficiency was increased. Induced-fit docking studies showed that the WT, L38↑N↑L -4 and L38↑N↑L +4 do bind the protease inhibitors atazanavir (ATV), darunavir (DRV), ritonavir (RTV) and saquinavir (SQV) albeit with different affinities. In vitro displacement titration experiments confirmed that L38↑N↑L -4 does bind these inhibitors. The binding affinity of L38↑N↑L -4 to the protease inhibitors ATV, RTV and SQV was significantly reduced with changes to the overall binding energetics of these vi reactions. Darunavir did not display any change in binding affinity to L38↑N↑L -4 ; however, the overall energetics of this reaction was altered in comparison to the WT. In addition, the stability of these L38↑N↑L -4 complexes were significantly diminished implicating these insertions in drug binding interactions. Differential scanning calorimetry results showed that the transition temperature of the apo L38↑N↑L -4 protease was 5 °C higher than the apo WT protease indicating the role of these insertions in the stability of the protease. Molecular dynamic simulations confirmed the increased structural stability of the apo mutated proteases which exhibited more flexible hinge regions than the WT. Additionally, both hinge region mutants, L38↑N↑L -4 and L38↑N↑L +4 , exhibited a mainly closed conformation thus explaining the decreased catalytic properties of the protease. Furthermore, the presence of inhibitors altered the dynamics, conformations, and flexibility of the three proteases. The inhibitor bound mutated proteases, L38↑N↑L -4 and L38↑N↑L +4 , displayed more flexible flap and hinge regions in comparison to the WT. Except for SQV, the presence of inhibitors shifted the flap conformers of the mutated proteases from closed to semiopen as well. Further analysis indicated changes in the number of hydrophobic interactions, hydrogen bonding and water bridge formations between the proteases and the inhibitors. Altogether, these results implicate the hinge region insertions in drug binding affinities and interactions with protease inhibitors as well as structural stability, dynamics, and flexibility of the protease both in the absence and presence of protease inhibitors.
Overexpression, characterisation, and encapsulation of LRP/LR for treatment of neurodegenerative disorders
(2024) Madhav, Chandni
The neurodegenerative diseases of Parkinson's disease (PD) and Alzheimer’s disease (AD) are debilitating conditions affecting millions of people worldwide. Both diseases pose a significant economic burden and require therapeutic strategies. AD is identified by intracellular neurofibrillary tangles and the accumulation of amyloid beta (Aβ) plaques, while PD is characterised by the loss of dopamine-producing neurons and mutations in PINK1, parkin, and α-synuclein proteins. The 37 kDa/67 kDa laminin receptor (LRP/LR) is a multifunctional receptor found to impede the progression of AD and PD. In-vitro studies have revealed that the LRP::FLAG overexpression increases hTERT expression and telomerase activity while rescuing cells from Aβ-mediated cytotoxicity. Cuttler et al. 2019 also revealed that LRP::FLAG overexpression decreased phosphorylated tau levels and tauopathy-related proteins. Within PD, in vitro studies demonstrated that LRP::FLAG overexpression plays a protective role, through the degradation of α-synuclein and rescuing cells from MPP+ cytotoxicity. Thus, the current study comprised of three parts. The first part aimed at investigating the effect of overexpressing LRP::FLAG in HEK293 cells by assessing LRP/LR and PINK1 protein levels, as well as cell viability after treatment with MPP+, TBHP, and Aβ!", in vitro AD and PD models. The results demonstrated that the overexpression of LRP::FLAG rescued cells from MPP+, TBHP, and Aβ!" induced cytotoxicity, and increased LRP/LR and PINK1 protein levels. The second part of the study focussed on overexpressing, purifying, and structurally characterizing the 37 kDa LRP protein. The protein was successfully overexpressed and purified through Co2+-IMAC while structural characterisation indicated that protein was correctly folded and predominantly αhelical, as expected. The purified LRP protein was then encapsulated in PLGA nanoparticles to develop an efficient in vivo delivery system. Thus, the third part of the study investigated the effect of empty and LRP-encapsulated PLGA nanoparticles on cell viability, LRP levels, and telomerase activity in SH-SY5Y and HEK 293 cells. The results demonstrated that LRPencapsulated PLGA nanoparticles successfully delivered the therapeutic protein and increased exogenous LRP protein levels. Additionally, SH-SY5Y cells treated with LRPencapsulated PLGA nanoparticles exhibited increased cellular viability and telomerase activity. Therefore, the LRP-encapsulated PLGA nanoparticles could be a possible therapeutic for conditions such as ageing and age-related diseases including AD and PD.
Regulation of PXDN in eye development and PXDN gene variant screening within a South African cohort of patients presenting with anterior segment dysgenesis
(2024) Marutha, Tebogo Rector
Peroxidasin (PXDN) is an extracellular matrix-associated haem-peroxidase predominantly expressed in the vasculature and eye. PXDN crosslinks collagen IV through sulfilimine bond formation in the basement membrane. Aberrant PXDN expression is associated with fibrosis, heart failure and cancer, and various pathologies of the eye, where PXDN likely provides structural support via basement membrane synthesis in the cornea and lens during eye development, as well as protect the lens, trabecular meshwork and cornea against oxidative damage. Furthermore, PXDN pathogenic variants have been associated with anterior segment dysgenesis (ASD), congenital cataracts and corneal opacity. To further understand the role of PXDN in the eye, first we aimed to identify PXDN as a novel target of key transcriptional regulators of eye development, namely PAX6, FOXC1 and PITX2, and second, to screen a cohort of South African patients with ASD to look for pathogenic variants in PXDN and other ASD genes. Protein expression of PAX6, FOXC1, PITX2 and PXDN, in response to Fibroblast Growth Factor-2 (FGF-2) were quantified by western blotting and localisation visualised using immunofluorescence confocal microscopy. Chromatin immunoprecipitation-PCR and luciferase assays were employed to detect transcription factor-PXDN promoter interactions. Expression data established that PXDN, PAX6, FOXC1 and PITX2 were induced by FGF2 at varying time points. Putative binding sites for all three transcription factors were identified in the PXDN promoter and ChIP-PCR confirmed that PAX6, FOXC1 and PITX2 interact with various regions of the promoter. Luciferase reporter assays are currently underway. Next Generation Sequencing of genomic DNA from South African patients exhibiting ASD disorders. identified disease causing variants in PAX6 and GJA8. Variants of uncertain significance were identified in PXDN, BCOR, EPHA2 and LTBP2 genes and are being investigated further. In conclusion, we identified PXDN as a novel target of PAX6, FOXC1 and PITX2 that further supports for the integral role of PXDN in eye development.
The characterization and crystallization of the TBR1 T-box domain in the presence and absence of the T-box Binding Element
(2023) Mayet, Riyaadh
TBR1 is a neuron-specific transcription factor involved in numerous developmental events in the brain. It has recently emerged as a master regulator of the genes implicated in autism spectrum disorders. The protein contains an evolutionarily conserved DNA-binding domain, known as the T-box, and binds to a consensus DNA sequence known as the T-box Binding Element. The key to understanding the function of a macromolecule, such as the TBR1 T-box domain, is to determine and understand its structure at all levels. The aim of this study was to determine the DNA-binding mechanism of the protein through structural characterizations, DNA-binding studies, X-ray crystallography and computational methods. The TBR1 T-box domain was successfully overexpressed in E. coli. The protein was purified by liquid chromatography, and its purity was confirmed using SDS-PAGE and absorbance spectroscopy. The protein was confirmed to be correctly folded through intrinsic tryptophan fluorescence. The secondary structure and thermal stability were characterized by far UV circular dichroism. The protein was β-sheeted and had a Tm of 63 °C. The secondary and tertiary structures of the protein are conserved upon DNA-binding. Under reducing conditions, the protein is monomeric in solution and binds the DNA as a monomer. Furthermore, the protein binds the DNA with high affinity in the nanomolar range (KD = 179.6 nM), and the affinity is unaffected by the presence of Mg2+. After several rounds of optimization, very thin plate-like protein crystals were obtained. These crystals did not yield any significant diffraction. Protein modelling, disorder predictions and molecular docking were then used to predict the structures of the protein in the presence and absence of DNA. The structure of the protein, both in the presence and absence of DNA, was very similar to other T-box proteins. DNA-binding results in conformational changes in the side chains of residues present in the protein-DNA interface. The TBR1 T-box domain uses the same DNAbinding mechanism utilized by the TBX5 T-box domain. The protein contacts the DNA in the minor groove by insertion of helix 310C, which is an inducible recognition element that only becomes structured upon DNA-binding. The results were also used to make structural interpretations of pathogenic point mutations in the TBR1 T-box domain.
The roles of miRNA-128 and miRNA-223 in cholesterol-mediated drug resistance in breast cancer
(2024) Palma, Gabriella Bianca Henriques
Breast cancer (BC) is the most prevalent cancer in women, with 70% of BC cases being hormone responsive (estrogen receptor positive (ER+)). This ER+ BC subtype relies on estrogen for enhanced cell proliferation and survival. The main therapeutic strategy to prevent hormone responsive BC recurrence is with the use of endocrine therapy such as Tamoxifen (TAM). Despite the success in reducing mortality rates of BC patients with the use of adjuvant TAM and chemotherapy, cancer drug resistance remains a significant challenge. A major contributor to this resistance is the dysregulation of cholesterol homeostasis in these cells. BC cells have elevated intracellular cholesterol levels, which is associated with cancer progression. In our previous research, we observed that the use of a cholesterol depleting agent, Acetyl Plumbagin (AP) in combination with TAM, led to the induction of cell death via cholesterol depletion. These results therefore warranted further investigation into the molecular mechanisms in which TAM + AP are involved in. MicroRNAs (miRNAs) regulate cholesterolrelated and cancer drug resistance pathways, and the aberrant expression of these miRNAs are often associated with increased cancer proliferation and resistance. It was therefore predicted that manipulating the expression of these target miRNAs could lead to a reduction in BC related drug resistance via cholesterol depletion. Thus, we aimed at investigating the roles of miRNA128 and miRNA-223 in cholesterol-mediated TAM resistance. Three BC cell lines (MCF-7 (estrogen-dependent), MDA-MB-231 (estrogen-independent), and Long-Term Estrogen Deprived (LTED)) were treated with a combination of 1 µM TAM and 10 µM AP following transfection with a miR-128 inhibitor or a miR-223 mimic. Cell viability and cholesterol levels were assessed following treatments. In addition, gene and protein expression levels involved in cancer drug resistance and cholesterol homeostasis were also assessed. It was found that the combination treatment with altered miRNA expression led to reduced cell viability and proliferation due to a reduction in free cholesterol, cholesteryl esters, and lipid rafts in all three BC cell lines. Moreover, miR-128 inhibition lowered the expression of genes involved in cholesterol synthesis and transport (HMGCR, HMGCS1, SREBF1/2, CETP, LCAT, and LDLR), drug resistance (ABCC5 and UGCG), and cell signalling (ESR1, EGFR, and IGF1R) in MCF7 cells. Whereas overexpression of miR-223 led to decreased expression in EGFR, ESR1, ABCC5, CETP, LCAT, LDLR, HMGCR, SREBF1, and SREBF2, with increased expression in ABCG1, PTEN, and TP53 in MDA-MB-231 cells. Therefore, the current study demonstrated that miR-128 and miR-223 could be potential targets in reducing TAM resistance through the depletion of excess cholesterol.

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Browsing 3. Electronic Theses and Dissertations (ETDs) - All submissions by School "Molecular and Cell Biology"

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