Masters Degrees (Molecular Biology and Human Genetics)
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- ItemEffect of oxidative stress on MEP pathway dependent enzyme and cytokinin production in Mycobacteria(2023-11) Sinalo, Mdolo; Gabriel Tshwahla, Mashabela; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB) remains a significant global public health concern and is exacerbated by the emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of the disease. The survival strategy employed by the pathogen in the face of oxidative stress and antibiotic onslaught is tolerance. Therefore, gaining insights into such tolerance mechanisms is critical to understanding M. tb infection and towards development of antitubercular agents. This study focused on investigating contribution of tRNA delta(2)-isopentenylpyrophosphate transferase (MiaA) to the tolerance mechanism employed by the bacteria. MiaA mediates (i6A37) tRNA isopentenylation in which isoprenyl group is transferred to tRNA molecule at N-6 of adenine in position 37. The loss of (i6A37) tRNA isopentenylation increased translational infidelity, which is associated with tolerance of oxidative and osmotic stress in some prokaryotes. To unravel the potential role of nitric oxide in translational infidelity-induced drug tolerance through the low (i6A37) tRNA isopentenylation mechanism, we employed targeted metabolomics and CRISPRi technology targeting the Mycobacterium smegmatis miaA (MSMEG_2734) gene. Subsequently, various phenotypic characterizations were performed to investigate MiaA potential physiological roles. Using LC-MS/MS method, we also investigated the impact of hydrogen peroxide and nitric oxide on intracellular concentration of cytokinin, the degradation end-product of (i6A37) isopentenylated tRNA. Oxidant tolerance was assessed by exposing the miaA CRISPRi hypomorph to varying concentrations of potassium nitrite and hydrogen peroxide, while drug tolerance was assessed using isoniazid and ethambutol. Additionally, we evaluated the ability of the miaA CRISPRi hypomorph to withstand acidic pH conditions, while the viability of stimulated THP-1 cells was assessed using commercial cytokinins. The miaA M. smegmatis CRISPRi strain was successfully generated and confirmed by plasmid sequencing, and exposure of the miaA mutant to 200 ng/ml ATc for 24 hours depleted 80% of miaA, and slowed mutant growth by 5%. The miaA hypomorph displayed 4-fold susceptibility to superoxide radical generating agents, cadmium and paraquat, while susceptibility to the first-line TB antibiotics, isoniazid and ethambutol was unchanged. Interestingly, long miaA depletion induced minor but significant tolerance to hydrogen peroxide and potassium nitrite. Furthermore, miaA depletion was found to increase cytokinin 2-fold, which was further increased 2- and 4-fold by potassium nitrite and hydrogen peroxide treatment, respectively. Further evidence was presented to support degradation of (i6A37) isopentenylated tRNA as the only route for cytokinin biosynthesis in mycobacteria. Finally, cytokinin was found to inhibit THP-1 growth in a concentration dependent manner. Overall, using miaA hypomoprph, we showed that deficient (i6A37) tRNA isopentenylation increased hydrogen peroxide and potassium nitrite tolerance and that cytokinin production occurs via degradation of isopentanylated (i6A37) tRNA in mycobacteria.
- ItemA comparative analysis of fungal agents with anti-glioblastoma properties(Stellenbosch : Stellenbosch University, 2023-12) Lai, Daanyaal; Baatjies, Lucinda; Mavumengwana, Vuyo; Mabasa, Lawrence; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Cancer can be defined as a disease where cells within the body grow uncontrollably and may spread throughout the body. Globally cancers have been found to amass a high mortality rate, with central nervous system (CNS) tumors increasing substantially over the past 30 years more specifically within the elderly. Primary treatment regimens for CNS tumors include but are not limited to chemotherapy, radiation therapy and surgical resection. However, these treatments carry a lot of toxic side effects, thus highlighting the need to develop alternative treatment strategies. In the current study, fungal crude extracts were evaluated for their anti-glioblastoma (GBM) potential. The fungal crude extracts were derived from different fungal species which were obtained from crustose lichens, plants, and marine vertebrates such as ascidians. A total of 45 different fungi were grown with 15 fungi from each source undergoing sequencing to identify their genus and species. The fungi which were successfully identified via ITS and cultured for metabolite extraction. The metabolites were extracted by soaking the fungal mat in methanol and consequently concentrating using a rotary evaporator. Fungal endophytes Mucor janssenii (P13) and Diaporthe rudis (R3.10) which came from a lichen and a plant respectfully, showed the greatest cell growth inhibitory activity when screened against the GBM cancer cell line. Fungal crude extracts of Mucor janssenii (P13) showed inhibition of 55% at 250 µg/mL and Diaporthe rudis (R3.10) showed a 50% inhibition at 83 µg/mL against the U251 GBM cell line. These extracts showed no cytotoxicity against the Vero cell line, which was used as the normal control cell line. Nanoparticles (NPs) were synthesized, functionalized with crude extracts Mucor janssenii (P13) and Diaporthe rudis (R3.10) and their inhibitory effects against the GBM cell line assessed. U251 GBM cell line was treated with the functionalized NPs at concentration range of 100 µg/mL to 12.5 µg/mL. The functionalized NPs displayed inhibition only at 100 µg/mL with both Mucor janssenii (P13) and Diaporthe rudis (R3.10) functionalized NPs displaying inhibition of 60% and 65% respectfully. Untargeted metabolite profiling was conducted with both crude extracts P13 and R3.10 along with their “spent” fractions from NP functionalization. The present study has concluded and highlighted the importance of fungi as a reservoir of natural products.
- ItemTargeting of myeloid-derived suppressor cells by all-trans retinoic acid as host-directed therapy for human tuberculosis(Elsevier Inc., 2021-06) Leukes, Vinzeigh N.; Dorhoi, Anca; Malherbe, Stephanus T.; Maasdorp, Elizna; Khoury, Justine; McAnda, Shirley; Walzl, Gerhard; Du Plessis, NelitaConventional anti-tuberculosis (TB) therapies comprise lengthy antibiotic treatment regimens, exacerbated by multi-drug resistant and extensively drug resistant mycobacterial strains. We assessed the ability of all-trans retinoic acid (ATRA), as repurposed compound serving as host-directed therapy (HDT), to counteract the suppressive effects of myeloid-derived suppressor cells (MDSCs) obtained from active TB cases (untreated or during week one of treatment) on T-cell responsiveness. We show for the first time that MDSCs suppress non-specific T-cell activation and production of interleukin (IL)-2, IL-4, IL-13 and GM-CSF via contact-dependent mechanisms. ATRA treatment decreases MDSC frequency, but fails to mature MDSCs to non-suppressive, terminally differentiated myeloid cells and does not restore T-cell function or cytokine production in the presence of MDSCs. The impact of ATRA treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.
- ItemImplementing a pipeline for analysing single-cell RNA sequencing data(Stellenbosch : Stellenbosch University, 2023-03) Ahiavi, Kwame; Tromp, Gerard; van der Spuy, Gian; Maasdorp, Elizna; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Single-cell RNA sequencing (scRNA-seq) has permitted the dissection of gene expression at single-cell resolution and provides novel insights into the composition of apparently homogeneous cell types and transitions between cell states — thereby deepening our understanding of the cell as a functional unit. The data generated by scRNA-seq are characterised by sparsity, heterogeneity, and high-dimensionality as well as large scale. As a result of biological and technical limitations, scRNA-seq data are “noisier” and more complex than their bulk RNA-seq counterparts. Thus, analysing scRNA-seq data demands new statistical and computational methods. Analytical algorithms employed in scRNA-seq pipelines are prone to producing different results depending on the state at the start of the analysis and the number of iterations of computation, complicating reproducibility. I developed a highly robust, scalable, and reproducible analysis pipeline for scRNA-seq data, implemented in Nextflow — a workflow management system that complies with current best practices in bioinformatics. The pipeline implements pre-processing and comprehensive downstream analyses for scRNA-seq data. With the publicly available datasets used for testing, the pipeline identified cell types and differentially expressed genes that enabled the identification of cell subtypes. Trajectory inference also showed the differentiation trajectory of cells, identifying subclusters within cells. In addition, the pipeline documents all steps and transformations, records software packages and versions, and incorporates ontological metadata annotation. Containerisation of pipeline processes ensures that software dependencies are satisfied — contributing to consistent, robust, and reproducible science.
- ItemPipeline and tools for the analysis of multiplexed ELISA data(Stellenbosch : Stellenbosch University, 2023-03) Asimeng, Jesse; Tromp, Gerard; Maasdorp, Elizna; Gian, van der Spuy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: A cornerstone of scientific progress is independent data verification. It is, therefore, necessary to develop robust analysis pipelines that can ensure reproducible and verifiable analyses. The pipeline should also record all steps and software that generated the results. The analysis of multiplexed ELISA data (Luminex data) can be challenging due to its complexity and variability. In particular, the data preprocessing stage has many steps and is often ad hoc, leading to inconsistency, non-standard approaches and lack of reproducibility. An existing in-house data reprocessing pipeline, the Luminex Pipeline, addresses some of the aforementioned challenges. However, there remains substantial work to extend its utility, robustness, and overall reproducibility. Thus, in this work, I improved the summary statistic reports by using Rmarkdown and implemented unit testing of pipeline components using the R Testthat package. Unit testing ensured the greater robustness of the code, which was compiled into an R package. The pipeline execution was also automated by using the Nextflow workflow management system. Finally, I deployed the pipeline in a Singularity container for execution on any platform including high-performance computing clusters.