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Digitaria sanguinalis, a widespread African grass, was micropropagated from immature inflorescences. Embryogenic calli generated was transformed with XvPerl using the Biolistic gene gun. XvPer 1 shares high similarity to a recently discovered group of antioxidants, the 1-Cys PRX's. A homologue was shown to be absent in D. sanguinalis by northern, Southern and weotstern Bls.

Epithelial sodium channels (ENaC) located in the apical membranes of transporting epithelia, such as cells of the renal distal nephron, constitute the rate limiting step in the transcellular transport of sodium. The regulation of sodium ion reabsorption in the kidney, which is essential for salt and water homeostasis, is therefore governed by the cellular control of ENaC activity. It is well established that the neurohypophysial hormone vasopressin (antidiuretic hormone) is responsible for both water and sodium regulation and, in renal cells, increases sodium permeability by regulating ENaCs at their apical membranes. The mechanisms involved in acute ENaC regulation have however not been demonstrated unequivocally. Evidence exists to suggest that sodium ion transport rates could be increased either by direct activation of membrane-resident ENaCs, or recruitment of channels from cytoplasmic storage pools. In order to address the issue, this study aimed to investigate the hypothesis that vasopressin regulation of ENaC occurred by targeted exocytic delivery of channels to the apical membrane from sub-apical pools. All investigations were carried out on the sodium transporting A6 cultured cell line derived from Xenopus laevis kidney. The secondary messenger pathway of vasopressin stimulation had not been extensively characterised in these cells and this was the first study undertaken. Once it was established that an adenosine-3',5'-cyclic monophosphate (cAMP) secondary messenger pathway was in operation, attention was shifted to ENaC regulation in response to a raised intracellular cAMP level. The electrophysiological technique of current-fluctuation analysis was employed to characterise the single channel response to cAMP production. These studies demonstrated that the main factor responsible for increased sodium transport was a dramatic increase in the number of channels at the apical surface despite declines in both single channel conductance and channel open probability. Investigating intracellular membrane traffic by marker-uptake and confocal microscopic techniques demonstrated that cAMP pre-stimulation significantly altered the rates of membrane endocytosis from the apical surface implicating regulated recruitment and retrieval mechanisms for ENaC regulation. These methods provided indirect evidence for the shuttling hypothesis and established that the vesicle trafficking machinery was responsive to cAMP, but did not associate ENaC directly with the process. In order to demonstrate the movement of ENaC in conjunction with membrane trafficking mechanisms, a green fluorescent protein (GFP) tagged-ENaC was employed. By using live cell confocal microscopic techniques, the dynamic movement of GFP-ENaC in response to cAMP was demonstrated. Shuttling of ENaC to the apical surface was observed on cAMP stimulation with endocytic retrieval on removal of stimulus. For the first time in live cells these data directly demonstrate the dynamic regulation of ENaC by shuttling mechanisms.

Bluetongue virus (BTV) infects ruminants but predominantly causes severe and often fatal haemorrhagic fever, known as Bluetongue (BT) disease, in sheep. Increasing global temperatures have contributed to the global dissemination of BTV. This is a result of the Culicoides insect vectors which function optimally in warm and wet climates. In South Africa, the eradication and control of BTV is made difficult due to the circulation of 21 of the 28 known serotypes and their limited serological cross-reactivity. Current vaccine strategies include live attenuated and inactivated vaccines. Although they have been successful in protecting animals, there are many limitations and risks associated with these vaccine strategies such as reversion to virulence, re-assortment and short-lasting immunity. There is thus a need for vaccines that are safe, scalable, economically viable and effective against multiple serotypes. A variety of recombinant BTV vaccine strategies have been developed which address and improve upon some of the limitations of the commercially available vaccine strategies. One of the most promising strategies is that of the virus-like particle (VLP). BTV VLPs comprise four structural proteins, namely VP2, VP3, VP5 and VP7. VP2 is highly immunogenic and is responsible for eliciting neutralising antibodies against the virus. Although a number of different BTV VLPs have been produced in traditional protein expression systems, such as insect cells, these production methods are considered too expensive to compete with those of the commercial vaccines. This has led to the consideration of plants as an alternative vaccine expression system. Plants are easily scaled up, upstream processes are cost effective, they are easy to work with, and do not require sterile environments and expensive infrastructure to maintain. A number of studies have shown that when the four major BTV structural proteins are transiently co-expressed in Nicotiana benthamiana, they self-assemble into VLPs which can be used as vaccines to protect animals against homologous serotypes. The aim of this study was to develop and compare two different particulate BTV candidate vaccines made in plants and determine their ability to elicit specific immunity in guinea pigs. The first vaccine approach was to develop a chimeric BTV VLP vaccine. This was achieved by substituting the immunogenic tip domain of the VP2 gene of BTV serotype 8 (BTV8) with that of the corresponding domain of BTV serotype 1 (BTV1) generating a chimeric VP2 which, when co-expressed in plants with the remaining BTV8 VP3, VP5 and VP7, resulted in chimeric BTV1/8 VLPs. The second approach involved the display of the immunogenic BTV1 VP2 tip domain on a capsid protein particle. Here, the domain was displayed on the surface of the bacteriophage AP205 particle through the application of the SpyTag (ST)/SpyCatcher (SC) bioconjugation method. It was anticipated that these vaccine candidates would be safe to use and allow for rapid production and scalability. Moreover, only a small fragment of the BTV8 VP2 gene would need to be modified to allow for a VLP to be made against a new serotype. Chimeric BTV1/8 VLP protein expression in plants, VLP extraction, and purification protocols were optimised using previously made homologous BTV8 VLPs for comparison. Plants were vacuum coinfiltrated with the chimeric BTV1/8 VP2 protein and the BTV8 VP3, VP5 and VP7 proteins. Protein was extracted from the plants and VLPs were subsequently purified by density gradient ultracentrifugation. The VLP proteins were detected on Western blots and Coomassie-stained gels. These methods were optimised to maximise protein yields by increasing the salt concentration of the extraction and purification buffer, maintaining an alkaline pH throughout the extraction and maturation process and harvesting at five days post-infiltration. Transmission electron microscopy (TEM) confirmed the presence of a mixture of core-like particles (CLPs), assembly intermediates and fully formed VLPs. These optimised methods were sufficient to produce high enough yields of BTV8 and BTV1/8 VLPs with protein yields of 35mg/kg fresh leaf weight (FLW) and 34mg/kg FLW, respectively, to be used in immunogenicity trials in guinea pigs. The alternative vaccine strategy involved the display of the BTV1 VP2 tip domain on phage AP205 particles. We utilised the ST/SC antigen display technology for the display of the BTV1 antigenic tip domain on the surface of the AP205 capsid. ST was fused to the N-terminal of the AP205 protein (STAP205) while SC was fused to the C-terminal of the BTV1 VP2 tip domain used in the chimeric VLPs (BTV1Tip-SC). Both components were expressed in plants and extracted and purified separately before combining for in vitro coupling. The ST-AP205 particles were purified by density gradient ultracentrifugation while the BTV1Tip-SC proteins were purified by nickel affinity chromatography. The purified components were coupled in vitro in a molar ratio of 1:3 (ST-AP205:BTV1Tip-SC). The 60kDa coupled complex was detected on Western blots and Coomassie gels with an estimated protein concentration of approximately 0.03ug/uL and a coupling efficiency of 44%. Finally, since there was insufficient coupled protein product for immunisation doses, only the immunogenicity of the plant produced chimeric BTV1/8 VLPs compared with the BTV8 VLPs were tested. Five guinea pigs per vaccine group (BTV8 VLPs and chimeric BTV1/8 VLPs) were immunised with 15ug of the appropriate vaccine and boosted 13 days later. Serum was collected 41 days post immunisation and used to determine whether there was an immunogenic response to the vaccines by Western blotting and indirect enzyme-linked immunosorbent assays (ELISAs). This preliminary immunogenicity trial found that both VLP candidate vaccines induced an immune response in guinea pigs. While the BTV1 VP2 tip display vaccine strategy still requires further optimisation to generate more dose-appropriate yields, the VLP vaccine strategy tested here shows great potential for further development into a BTV vaccine candidate that is safe, scalable and has potential for multivalency.

Sub-Saharan Africa is severely deficient in vaccine manufacturing facilities that can keep up with the rate of emergence of viral pathogens. As seen with the Covid-19 pandemic, outbreaks of disease can be extremely detrimental to economies and put severe strain on the public health sector. Vaccination offers a solution to reduce the difficulties that accompany viral outbreaks. Lujo virus, an emerging arenavirus responsible for causing a devastating haemorrhagic fever with an 80% mortality rate, currently has no vaccines or diagnostic tools available. In this study we developed a production pipeline in HEK293T cells and N. benthamiana for the protein LUJV GP-C∆TM (Lujo virus glycoprotein precursor without the transmembrane region). The LUJV GP-C∆TM was constructed from gene sequences of the envelope glycoprotein of Lujo virus and then adapted to production in HEK293T cells using the DNA expression vector pTHpCapR. An N. benthamiana plant protein expression system was developed in parallel to compare the production utility of both systems with an emphasis being placed on the plant system. Plant expression systems are arguably cheaper and more easily automatable than traditional mammalian expression technologies. This makes them suitable for vaccine protein production in lower socio-economic countries that have an overwhelming burden of disease and poor health care systems. LUJV GP-C∆TM was successfully expressed in both HEK293T cells and N. benthamiana. It was confirmed that the LUJV GP-C∆TM undergoes the post translational modifications glycosylation and proteolytic cleavage in recombinant HEK293T cells and was produced in a conformation that allowed successful purification via a His tag sequence that was inserted into the LUJV GP-C∆TM protein gene sequence. On the other hand, N. benthamiana does not endogenously express the Site-1 protease needed for cleavage of the Lujo glycoprotein, and thus this needed to be co-expressed. Attempts were made to detect the Site-1 protease including extraction into buffers with different pHs and ammonium sulfate precipitation to concentrate the protein. However, I was not able elicit proteolytic cleavage of the LUJV GP-C∆TM nor detect the Site-1 protease in N. benthamiana. This is probably attributable to the innate responses against the hostile nature of proteases to non-native expression hosts. Proteins were expressed in N. benthamiana through the use of previously established protocols utilizing recombinant Agrobacterium tumefaciens to deliver synthesized genes to plant cells and induce their expression. It was determined that co-expression of the molecular folding chaperone calreticulin is necessary for LUJV GP-C∆TM to accumulate at detectable levels. Co-expression of an oligossacharyl transferase LmSTT3D, isolated from Leishmania major, may increase the glycan occupancy of the LUJV GP-C∆TM protein, indicated by a molecular mass shift. However, further experimental lines of evidence are needed -such as glycosylation mapping to determine if this is the case. Other parameters such as the optimal day of protein harvest and optical density of recombinant Agrobacterium tumefaciens strains were recorded. Collectively these findings serve as a prototype pipeline for the production of commercially relevant immunogens, diagnostic tools and virus-like particles. This study narrows the focus for bottlenecks in plant protein production, not just for Lujo virus proteins, but for arenaviruses generally and potentially also other haemorrhagic fever-causing viruses. Future efforts should be directed towards addressing the barriers to plant production of complex viral antigens, and to further investigate the utility of mammalian cell-produced LUJV GPC∆TM in animal studies.

The brain is arguably the most complex organ of the body: Controlling muscles, maintaining homeostasis, processing information. It has the longest developmental period of any organ, and hormones are essential during its development. Endocrine disruptive chemicals can disturb hormones in any part of their signaling pathway and can act as agonists or antagonists on their receptors. Should the balance of stem cell or progenitor cell proliferation be skewed in any direction during early brain development, it may lead to cognitive disfunctions or diseases. In this study, v-myc induced mouse-derived neural progenitor cells (NPCs) were exposed to retinoid and glucocorticoid receptor agonists and antagonists to investigate the involvement of these pathways on NPC proliferation. NPCs were exposed for 24 hours, after which they were read in a Tecan. Statistical analyses revealed that no treatment yielded statistically significant responses, however in the case for mifepristone (glucocorticoid receptor antagonist), dexamethasone (glucocorticoid receptor agonist) and Agn (retinoid receptor antagonist), a small negative response trend could be observed. Cell viability analyses also revealed that the concentrations of chemicals did not induce cytotoxicity. The unexpectedly high and variable data suggest errors were made during the seeding of the 96-well microplates. In conclusion, these findings suggest that with further development, this in vitro method could be used in conjunction with other in vitro models for the assessment of neurodevelopmental toxicity and for identification of EDCs in the future. / ENDpoiNTs

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No description available.

No description available.