List of Titles

Towards development of a malaria diagnostic: Generation, screening and validation of novel aptamers recognising Plasmodium falciparum lactate dehydrogenase

- Frith, Kelly-Anne

Authors: Frith, Kelly-Anne
Date: 2020
Subjects: Plasmodium falciparum , Malaria -- Chemotherapy , Oligonucleotides , Lactate dehydrogenase , Biochemical markers , Systematic evolution of ligands through exponential enrichment (SELEX)
Language: English
Type: Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/142247 , vital:38062
Description: Malaria, caused by infection with the Plasmodium parasite, is one of the leading causes of death in under-developed countries. Early detection is crucial for the effective treatment of malaria, particularly in cases where infection is due to Plasmodium falciparum. There is, therefore, an enduring need for portable, sensitive, reliable, accurate, durable, self-validating and cost-effective techniques for the rapid detection of malaria. Moreover, there is a demand to distinguish between various infectious species causing malaria. Research in the area of malarial biomarkers has identified a unique, species-specific, epitope of P. falciparum lactate dehydrogenase (PfLDH), enhancing prospects for the development of diagnostics capable of identifying the species causing malarial infection. In recent years, improvements have been made towards the development of rapid diagnostic tests for detecting malarial biomarkers. Owing to their low cost, ease of labeling, and high thermal stability (relative to antibodies), the development and synthesis of aptamers that target the malarial lactate dehydrogenase represents one of the key innovations in the field of rapid diagnostics for malaria. This study explored the generation of aptamers that specifically target P. falciparum. Two sets of aptamers with diagnostically-supportive functions were generated independently, through parallel SELEX of recombinantly-expressed, full-length Plasmodium falciparum lactate dehydrogenase (rPfLDH), and an oligopeptide comprising the P. falciparum-specific epitope on lactate dehydrogenase (LDHp). The latter offers a promising solution for generating aptamers capable of binding with high specificity to P. falciparum. In this work, an rLDH class of aptamers was generated when SELEX was performed using the full-length rPfLDH protein as the target and the LDHp class of aptamers was generated when SELEX was performed using the oligopeptide LDHp as a target. Aptamers were successfully generated through the process of SELEX (systematic evolution of ligands through exponential enrichment) following the study and application of several optimisation steps, particularly during the amplification stage of SELEX. Optimisation steps included the study of improvements in PCR conditions; role of surfactants (Triton-X), modifying the PCR clean-up protocol; and agarose gel excision. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers both reported here and previously published, confirming their importance in recognition of the target. Novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Clades of consensus sequences were identified in both the rLDH and LDHp groups of aptamers, where sequences in the rLDH clade did not show preferential binding to rPfLDH while those in the LDHp clade (particularly LDHp 3 and 18) were able to recognise and bind only LDHp. Of the 19 sequences returned from the parallel SELEX procedures for rPfLDH (11 sequences) and LDHp (8 sequences), six rPfLDH and all eight LDHp sequences underwent preliminary screening and those with low responses eliminated. Of the eight LDHp-targeting aptamer sequences, five were preliminarily shown to bind to LDHp, whereas only two rPfLDH-targeting sequences were shown to bind to the target (rLDH 4 and 7). To this small selection of rPfLDH oligonucleotide sequences, two more (rLDH 1 and 15) were chosen for further study based on their sequences, secondary and predicted tertiary conformations. Sequences chosen for further study were therefore: rLDH 1, 4, 7 and 15 in the rLDH class, and LDHp 1, 3, 11, 14 and 18 in the LDHp class. Binding properties of the aptamers towards their targets were investigated using enzyme-linked oligonucleotide assays (ELONA), fluorophore-linked oligonucleotide assays (FLONA), electromobility shift assays (EMSA), surface plasmon resonance (SPR), and GelRed dissociation assays, while applications towards aptasensors were explored using electrochemical impedance spectroscopy (EIS) and fluorescent microscopy. Some inconsistencies were seen for specific aptamer to target binding interactions using specific techniques; however, generally, binding to the targets was observed across the techniques assessed. These varied responses demonstrate the need to screen and validate aptamers using a variety of techniques and platforms not necessarily specific for the proposed application. From the aptamer binding screening studies using ELONA, the most promising aptamers generated were identified as LDHp 11, rLDH 4, rLDH 7 and rLDH 15. Aptamer rLDH 4, which was generated against rPfLDH, exhibited preferential and specific binding to the lactate dehydrogenase from P. falciparum, over the recombinantly-expressed lactate dehydrogenase from Plasmodium vivax (rPvLDH), albeit with lowered responses compared to LDHp 11 in ELONA and EMSA studies. However, in kinetic ELONA studies rLDH 4 showed binding to both rPfLDH and rPvLDH. Aptamer rLDH 7 showed high affinity for rPfLDH and rPvLDH in kinetic studies using ELONA. However, screening studies with ELONA indicates that aptamer rLDH 7 may not be suitable for diagnostic tests in serum samples given its non-specific binding to human serum albumin (HSA). Aptamer rLDH 15 exhibited species specificity for rPfLDH in screening studies using ELONA but showed affinity towards rPvLDH (albeit lower relative to its affinity for rPfLDH) in kinetic studies using ELONA. LDHp 11, generated against the PfLDH peptide, showed a clear preference for rPfLDH when compared to rPvLDH and other control proteins, in both sets of ELONA studies conducted, as well as EMSA, thus possessing a strong ability to identify the presence of Plasmodium falciparum owing to its generation against the species-specific epitope. While LDHp 1 demonstrated binding to plasmodial LDH in a flow-through system (SPR), so reiterating ELONA responses, it did not perform well in the remaining methodologies. Aptamers rLDH 1 and 15 and LDHp 3, 14 and 18 exhibited a mixed set of results throughout the target protein screening analyses and were, thus, not considered for selective binding in P. falciparum parasite bodies. In studies aimed at exploring biosensor assemblies utilising the developed aptamers, both rLDH 4 and LDHp 11, along with rLDH 7, LDHp 1 and pL1, demonstrated in situ binding to the native PfLDH in fluorescent microscopy. LDHp 11 exhibited FITC-based fluorescence equivalent to the anti-rPfLDHp IgY antibody in confocal fluorescent microscopy indicating superior binding to the native PfLDH compared to the remaining aptamers. An examination of electrochemical impedance as a platform for a biosensor assembly did not, in these studies, exhibit the required sensitivity using physiologically relevant concentrations of analyte expected for pLDH following infection with Plasmodium spp. Malstat/LDH activity was explored for application in a colorimetric aptasensor. A decrease in both rPfLDH and rPvLDH activity was observed following incubation with the tested aptamers, but rLDH 1, rLDH 7 and LDHp 14 did not exhibit similar decreases in rPvLDH activity. Aptamers rLDH 1, 4 and 7 and LDHp 11 and 14 were, therefore, not selected as candidates for LDH capture in LDH activity-based diagnostic devices for P. falciparum. The decreases in pLDH activity in the presence of aptamers could hold promise as direct or antagonistic malaria therapeutic agents. Preliminary studies on the application of aptamers as malaria therapeutic agents, while of interest, should be viewed with due caution given the challenges of aptamers reaching the intracellular native plasmodial LDH hosted within the red blood cells. In conclusion, this work has shown the ability of the LDHp 11 aptamer, generated in these studies, to selectively bind rPfLDH over rPvLDH, and to bind to the native PfLDH in fluorescent microscopy, indicating that this aptamer holds promise as a biorecognition element in malaria biosensors and other diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax. The use of a species-specific epitope of P. falciparum as a target in aptamer generation paves the way for similar such studies aimed at generating aptamers with species selectivity for other Plasmodium species.
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Exploration of challenges in bringing traditional medicine into SA’s healthcare system, using medicinal plants for treatment of waterborne diarrhoeal diseases as a case study

- Keche, Priscilla

Authors: Keche, Priscilla
Date: 2019
Subjects: Traditional medicine -- South Africa , Waterborne infection -- South Africa , Diarrhea -- South Africa , Healers -- South Africa , Medical care -- Quality control , Medicinal plants -- South Africa , Diarrhea in children -- South Africa , World Health Organization
Language: English
Type: thesis , text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/118035 , vital:34588
Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biotechnology Innovation Centre (RUBIC), 2019.
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An investigation of mitochondrial dynamics and networks observed within human undifferentiated and differentiated cell lines

- Houseman, Pascalené Shannon

Authors: Houseman, Pascalené Shannon
Date: 2018
Subjects: Mitochondria , Mitochondrial pathology , Degeneration (Pathology) , Mesenchymal stem cells , Neural stem cells , Cell lines , Reactive oxygen species (ROS)
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/60687 , vital:27816
Description: Mitochondrial dynamics refers to a series of constant division and fusion cycles that form interconnecting networks within healthy cells. Reactive oxygen species (ROS) are the byproducts of cellular redox reactions, and, when in excess, have been linked to degenerative diseases and aging. Mesenchymal stem cells (MSCs) require a niche that presents with low levels of ROS; this enables the stem cell to maintain its “sternness”, the stem cell population, as well as the ability to adhere, migrate, and proliferate. If ROS levels increase within the MSC niche, inhibition of cellular adhesion and migration occurs. In contrast, neural stem cells require a niche that presents with a high level of ROS, aiding in their proliferative, self- renewing capacities. Investigations into what constitutes a healthy mitochondrial network versus the disease state of the network are required in order to determine what promotes degeneration and aging within stem cells. It was hypothesized that increased levels of ROS would stunt the ability of MSCs to attach and migrate, and hinder their abilities of proliferation and differentiation. In contrast, neuronal differentiation would present with an increased proliferation. This led to the investigation into the effects of ROS and oxidative stress, and the resulting mitochondrial dynamics, have on undifferentiated and differentiated mesenchymal stem and SH-SY5Y cells. Upon the addition of non-lethal S3I-201 (STAT3 has been linked to a reduction in ROS) to MSCs, an increase in ROS was observed. Higher concentrations of STAT3 inhibitor resulted in a decrease in MSC attachment and proliferation. When exposed to similar conditions, the SH-SY5Y cells underwent an increased proliferation; due to multiple restrictions, they were not used any further within the study. Mitochondrial dynamics were observed using a fusion promoter (M1) and a fission inhibitor (Mdivi-1); the MSCs were dosed with varying concentrations in order to determine the effects that mitochondrial dysfunction may have on the established networks, and cell survival. The mitochondria within MSCs migrated to the extensions of the cell, and displayed an alteration in morphology, or were clustered around the nucleus and/or the lipid deposits. These high density clusters correlated with a high intensity of fluorescence using 2’,7’- dichlorofluorescein diacetate. In conclusion, varying concentrations of ROS have different effects on MSCs in terms of overall maintenance and function; mitochondrial dynamics play an important role in cell survivability and the fate of stem cell differentiation. Further investigation into the mitochondrial dynamics and networks of these cell lines and their differentiated progeny is required.
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Development of a low-cost bioprinting system for the fabrication of cell-laden sodium alginate hydrogels

- Honiball, John Robert

Authors: Honiball, John Robert
Date: 2018
Subjects: Regenerative medicine , Tissue engineering , Alginates , Colloids , Three-dimensional printing
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/59204 , vital:27470
Description: Bioprinting is a rapidly expanding technology with the ability to fabricate in vitro 3D tissues in a layer-by-layer manner to ultimately produce a living tissue which physiologically resembles native in vivo tissue functionality. Unfortunately, large costs associated with commercially available bioprinters severely limit the amount of people/research groups with access to the technology. Here, we investigated the potential for modifying a commercially available RepRap Prusa iteration 3 (i3) three-dimensional (3D) printer, by replacing the traditional plastic-based print-head with various open-source syringe-housed microextrusion print-head units, such that deposition of composite bioinks consisting of cells, biopolymer scaffolds and/or biomolecules may be achieved at a relatively low cost. Using adipose-derived human mesenchymal stromal stem cells (ad-HMSC) induced for adipogenic differentiation, as well as human umbilical vein endothelial cells (HUVEC), the potential for fabricating vascularised adipose tissue was investigated. The non-toxic, inexpensive algal polysaccharide, sodium alginate, was used to test the printability of the system, as well as for investigating the functionality unmodified sodium alginate has for use as a potential bioink in adipose tissue engineering. Cell viability assays, namely WST-1 and fluorescein diacetate (FDA)/propidium iodide (PI) live/dead cell staining, revealed that ad-HMSC were viable after 7 days of culture. However, viability of HUVEC encapsulated hydrogels revealed significantly lower cell viability. Live/dead cell staining revealed that the modified printing system was able to print ad-HMSC/HUVEC co-cocultures with a large degree of cell viability after 1 day of culture. However, after 7 days of culture, the majority of cells were revealed to be dead. Furthermore, due to the lack of mechanical integrity possessed by alginate in a liquid-like state, printing sodium alginate hydrogels in air consistently resulted in deformation of printed constructs. The newly developed 3D printing technique termed freeform reversible embedding of suspended hydrogels (FRESH) was therefore investigated as a means for achieving 3D spatial control of printed hydrogels using the modified system. Printing cell-free sodium alginate hydrogels within gelatin sacrificial support baths allowed for fabricating constructs in a spatially defined manner. However, overprinting and swelling of alginate hydrogels negatively affected the overall printing accuracy. The present study aimed to pave the way for further system modifications and refinements, such that the ultimate goal of low-cost bioprinting may be achieved. Further optimisation of printing parameters, hydrogel characteristics and sterilisation techniques may allow for fabricating viable, physiologically relevant tissues using the modified system developed.
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Characterization of the Mitochondrial Plasmodium falciparum heat shock protein 70

- Nyakundi, David Onchong’a

Authors: Nyakundi, David Onchong’a
Date: 2017
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/44449 , vital:25408 , https://doi.org/10.21504/10962/44448
Description: Malaria remains a global health problem and accounts for many deaths and illnesses in subSaharan Africa. Plasmodium falciparum, the causative agent of the most fatal form of malaria, expresses a repertoire of heat shock proteins for cytoprotection, survival and pathogenesis. The parasite genome encodes six Hsp70 proteins found in various cell compartments. However, the putative parasite mitochondrial Hsp70 (PfHsp70-3) has not been investigated. The J-proteins, Pfj1 and PFF1415c, were proposed to function as co-chaperones of PfHsp70-3. The biochemical characterization of PfHsp70-3 was initially complicated by the fact that the protein was insoluble when expressed in E. coli cells. Various approaches to solubilize it resulted in inactive protein. A general characteristic of eukaryotic mitochondrial Hsp70s is their insolubility and their reliance on an Hsp70 escort protein (Hep) for solubility and ultimate functions. In this study, a putative Hep protein was identified in the genome of P. falciparum that is referred to as PfHep1. Coexpression of PfHep1 with PfHsp70-3 resulted in soluble and biochemically active PfHsp70-3. Size exclusion chromatography was employed to separate PfHsp70-3 from PfHep1 after coexpression. PfHep1 suppressed thermally induced aggregation of PfHsp70-3 but not the aggregation of malate dehydrogenase or citrate synthase, thus showing specificity for PfHsp70-3. Zinc ions were also found to be essential for maintaining the functions of PfHep1, as EDTA chelation abrogated its abilities to suppress the aggregation of PfHsp70-3. Furthermore, PfHep1 did not stimulate the basal ATPase or increase refoldase activities of PfHsp70-3 hence displaying no co-chaperone roles. The full-length putative mitochondrial type I J protein, Pfj1, could not be produced in E.coli but a truncated protein containing the J-domain was produced which stimulated both the ATPase and refoldase activities of PfHsp70-3. Further, this study demonstrated that both PfHep1 and PfHsp70-3 localized to the mitochondrion in the erythrocytic stage of P. falciparum development thus confirming in silico predictions of their localization. Besides, PfHsp70-3 was expressed during all stages of the intraerythrocytic cycle of parasite development and was heat inducible. Generally, the data obtained in this study will enhance the existing knowledge on the biology of the parasite mitochondrial chaperone functions and open the possible avenue of drug targeting considering the specificity of PfHsp70- 3 and PfHep1 partnerships.
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