High risk human papillomaviruses (HPVs) are the causative agents of cervical cancer. The three approved prophylactic vaccines do not benefit already infected individuals; therefore, there is still an urgent need for therapeutic vaccines. The HPV oncoproteins E6 and E7 are ideal targets for the development of such vaccines, as they are expressed throughout the viral life cycle and in tumours. They could be used to elicit strong cytotoxic lymphocyte (CTL) responses which would aid in viral clearance, and could also be effective against tumours. Granadillo et al. (2011) developed an Escherichia coli-produced therapeutic vaccine candidate, consisting of the HPV-16 E7 protein and a cell membrane- penetrating and immunomodulatory peptide (LALF), whose fusion to HPV-16 E7 aided in the immunogenicity and antigen presentation of the oncoprotein. However, such vaccines need not only to be effective, but also to have a low cost. Plant expression systems represent an attractive alternative to conventional expression systems based on bacterial, yeast, mammalian and other cell cultures, and are potentially far more cost- effective. The aim of the present project was to produce LALF-E7 in Nicotiana benthamiana leaves via Agrobacterium-mediated transient expression, and to optimize its expression, extraction and purification. This was done by expressing LALF-E7 using different expression vectors, testing different subcellular localizations, and testing the effect of gene silencing suppressors. By using our group's replicating expression vector and targeting LALF-E7 to the chloroplasts, the expression of the candidate vaccine in N. benthamiana leaves was increased 26.8 fold compared to non-replicating vectors or cytoplasmic localization. Furthermore, silencing suppressors did not significantly increase the expression of LALF-E7 when expressed by the replicating vector and targeted to the chloroplasts. I showed, by fluorescence microscopy, that LALE-E7 was indeed being targeted to the plants' chloroplasts and that it possibly forms proteins bodies (PBs) that are closely associated to the chloroplast envelope. I further hypothesized a mechanism by which the PBs-like structures form. Once the expression of LALF-E7 was optimized in plant leaves, a purification strategy was developed by testing different extraction methods and using metal ion affinity chromatography. The extraction protocol developed pre-purified LALF-E7 by removing the majority of soluble proteins from the final extract. However, LALF-E7 was not fully purified by affinity chromatography, suggesting that other purification strategies should be used. Finally, I tested the partially purified plant-produced LALF-E7 candidate, and compared it to the E. coli-produced counterpart, in tumour regression experiments using mice as animal models. Due to low antigen doses and a large number tumourigenic cells used to inoculate the mice animal models, the effect of the plant-produced LALF-E7 as a therapeutic vaccine was inconclusive. However, it was suggested that it could potentially be comparable to the E. coli-produced counterpart. In summary, I report for the first time the entire chain of research involving the expression of LALF-E7 in plants, its extraction, purification and the testing of its immunogenicity in a mouse model. This research also suggests new avenues for the use of the LALF peptide as a PB-inducer which could be useful in increasing the expression of other recombinant proteins.

The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane.

Monoclonal antibodies (mAbs) are an important tool for both therapeutic and nontherapeutic applications. Their increased demand is due to their ability to recognize and bind specifically to a wide range of antigens. In addition to full-size antibodies, one can also utilise smaller antibody fragments, single chain variable region fragments (scFvs), which like full-size mAbs, are also capable of specific antigen-binding. The constant and rapidly expanding use of antibodies and their derivatives presents a need for a fast and effective method of production. Traditionally, antibodies have been produced using hybridoma technology. They have also been successfully produced in other expression hosts such as bacteria, yeasts, insect cells and mammalian cell lines. However, these expression systems come with a few disadvantages, some of which include high maintenance costs as well as lengthy and laborious production protocols. This dissertation describes the use of phage display technology to screen for and identify scFvs that bind to three different test antigens. Phage display library technology involving the expression and presentation of antibody or antibody derivatives on the coat surfaces of phage particles. It is considered to be a preferable alternative to hybridoma technology because it eliminates the requirement for immunization of animals, making it a more rapid and animal-friendly method for the production of antibodies compared to that of hybridoma technology. A naïve mouse scFv phage display library was screened with appropriate antigens to isolate scFvs which bind to rabbit IgG, human IgG and the Shuni virus (SHUV) N protein. Isolated scFvs were sequenced, cloned and tested for binding to their cognate antigens using phage ELISA, phage dot blots and phage western blots. ScFvs displaying the highest affinities for their respective antigens were selected for cloning and expression in plants, as this expression system is scalable, cheaper, safe and facilitates posttranslational modifications to recombinant proteins such as glycosylation. Rabbit IgG and human IgG scFvs were isolated successfully from the mouse scFv phage library, however, successful binding of the scFvs to the respective antigens by western blotting and ELISAs was not demonstrated. On further investigation, it appeared that the protocols were flawed, as the secondary anti-mouse AP conjugate, iv used in the western blots and ELISAs was found to cross-react with both rabbit and human IgG. Since we were not able to pinpoint scFvs with high binding affinity, the mouse phage display library was screened for scFvs that bound to SHUV N protein instead. This was more successful in that several scFvs with high binding affinity were isolated. Three scFvs with the highest binding affinity for the SHUV N protein were selected and their nucleotide sequences determined. Due to time constraints only 2 of the identified scFvs were selected for further cloning and expression in plants. Both scFvs were cloned into the pTRA-HRPB2SEKDEL plant expression vector that contains the gene sequence for a his6x tag to assist with downstream purification as well as a horse radish peroxidase (HRP) gene. Cloning scFvs into this vector allows their fusion to HRP, resulting in the production of potential reagents for use as secondary antibodies in western blots and ELISAs. The cloned scFvs were expressed transiently in tobacco plants using Agrobacterium-mediated infiltration. Plant expression of the HRP-fused scFvs was optimized; both were optimally expressed at 5 days post infiltration (dpi) when co-expressed with a silencing suppressor (pBIN-NSs). Extraction of the scFvs from the plants was most effective when a bicine buffer with a pH of 8.4 was used. Partial purification of the scFvs was achieved by isoelectric and ammonium sulphate precipitation. Preliminary tests were done to test functionality of the partially purified scFvs, in which the ability of the scFvs to recognize and bind to the SHUV N protein in a dot blot was tested. However, both were found to be non-functional in this regard. Further investigation into the reason for the demonstration of non-functionality showed that the HRP was being spontaneously cleaved from the scFv. This study demonstrates that it is possible to isolate antigen-specific scFvs from a phage display library. However, their binding capacity needs to be analysed fully prior to incorporating them into fusion proteins which can be used as potential diagnostic reagents.

Includes bibliographical references. / Most human beings experience the pull of three different daily timers, the solar clock, their endogenous circadian clock and the societal clock. Solar time is generated by the Earth’s revolution on its axis, resulting in its surface being alternately exposed to and shielded from the sun every 24 hours. The endogenous clock, or circadian oscillator, is driven by a network of transcriptional translational feedback loops, and has a period of close to 24 hours. The circadian oscillator is synchronised to the 24 hour light-dark cycle of the solar clock. The third timer is the standardised societal clock that organises and schedules work, school, transport, appointments and free time in a 24 hour period. The way an individual’s endogenous clock synchronises to the solar clock, through advances or delays relative to sunrise and sunset, results in a phenomenon known as diurnal preference or chronotype. A person may have a morning-chronotype, where they enjoy rising and being active early in the day, an evening-chronotype where they prefer to be active later in the day into the late night, retiring in the early morning hours, or have no strong preference for early or late rising. This renders it easy for some to cope with the demands of the societal clock and others to struggle. Chronotype has both genetic and environmental influences. As society’s schedule is governed by the standardised clock, it was hypothesised that chronotype may be influenced by one’s longitudinal location within a time zone. South Africa presents an interesting case because although it uses just one time zone, in the most Easterly regions of the country, the sun rises and sets up to an hour earlier than in the most Westerly regions throughout the year. Sunrise times have an impact on the way the endogenous clock synchronises to the solar clock. It was hypothesised firstly, that South Africans living in the East of the country may have a greater preference for mornings (more morningchronotypes) than those living in the West; and secondly, that this difference would not be due to genetic differences in the populations, particularly two gene polymorphisms previously shown to influence chronotype. Therefore the aims of this study were to describe and compare the distribution of chronotype in Eastern (n=222) and Western (n=205) sample populations with the use of a validated tool, the Horne–Östberg Morningness, Eveningness Questionnaire. Secondly to describe the genotype and allelic frequency distributions of the PER2 single nucleotide polymorphism (SNP) G3853A (rs934945) in the Eastern (n= 184) and Western (n=186) populations, and the PER3 variable number tandem repeat (VNTR) polymorphism in the Eastern (n=143) and Western (n=176) populations from buccal cell samples. There was a significantly higher proportion of morning-types in the Eastern population (60.6%) than in the Western population (40.5%) (p<0.001). Whereas there were higher proportions of neither-types and evening-types in the Western population (50.8% and 8.7% respectively) than in the Eastern population (35.1% and 4.3% respectively) (p<0.001). There were no significant differences in distribution of the PER2 genotype (p=0.121) and allele frequencies (p=0.051) between the Eastern and Western populations nor in the PER3 genotype (p=0.879) and allele (p=0.075) frequencies. Although previous studies have shown associations between chronotype and PER2 G3853A and PER3 VNTR genotypes, no significant associations were observed in either the Eastern (PER2 p=0.769; PER3 p=0.221) or the Western (PER2 p=0.584; PER3 p=0.733) populations. These findings indicate that, in South African populations, longitude influences chronotype independently of genotype. Factors that may contribute to this may be the difference in the rising times of the sun, which is exacerbated to some extent by the study areas being at dissimilar latitudes and thus experiencing slight differences in climate. The impact of the differences in chronotype but the maintenance of the same societal temporal organisation in the Eastern and Western regions were not assessed. However, they may be revealed by investigating certain general health indicators in such as quality of sleep and prevalence of depressive symptoms which are affected when there is incongruence between societal time and endogenous time.

Bibliography: pages 148-171. / Bacteria are constantly faced with harsh environmental conditions to which they have to adapt. These adaptive mechanism generally involve the use of two-component sensory systems, comprising of sensor proteins interacting with their cognate response regulator proteins. To survive fluctuating environments such as osmotic conditions, certain bacterial species employ the ompR-envZ (ompB) two-component system to monitor and respond to the osmotic cue. The EnvZ protein functions as the sensor and relays information regarding changes to the external environment, to the response regulator, OmpR. OmpR, in turn, regulates the porins, OmpF and OmpC in a reciprocal manner, so that one porin predominates over the other, depending on osmotic conditions. Erwinia chrysanthemi, which causes "soft rot" in a wide range of economically important crops, has been demonstrated to contain porin-like proteins similar to OmpF and OmpC. The expression of these porins was regulated in a similar manner to OmpF and OmpC with respect to medium osmolarity. Furthermore, preliminary studies have shown that changes in osmolarity affect the expression of pathogenecity genes. Evidence for an osmoregulatory system analagous to the ompB system of Escherichia coli was, therefore, sought. Primers specific for conserved regions in ompR were designed and used to PCR amplify a 631 bp fragment from E. coli. This fragment was cloned into the vector, pBluescriptSk, and end-sequenced to confirm its authenticity. The same strategy was followed, using envZ-specific primers to generate an E. coli envZ clone. Southern hybridisation analyses, using an ompR probe, confirmed the presence of an ompR homologue in E. chrysanthemi. An E. chrysanthemi genomic library was thus constructed and screened and a clone homologous to the ompR probe was isolated. The resulting plasmid, pRZ69, was partially characterised and determined to have both envZ and ompR homologues resident. Southern hybridisation analyses were employed to localise the ompR and envZ genes on the plasmid. A 1200 bp EcoRV-Pst1 fragment containing the ompR homologue and a 2000 bp EcoRV-EcoRV fragment containing the envZ homologue, were subcloned into pBluescriptSk, generating the plasmids, pRS1 and pZS2 respectively.

Studies on resurrection plants and other anhydrobiotic organisms, have shown that Late Embryogenesis Abundant (LEA) proteins are expressed upon the onset of desiccation and are therefore inferred to be associated with the desiccation tolerance response. To date, despite some 25 years of research on these proteins, there is still very little understanding of the physiological function(s) of the majority of LEAs. This is because they lack tertiary structure in the hydrated state, making assigning of physiological roles difficult. This MSc study was undertaken to investigate the gene expression of a set of 21 putative LEAs during dehydration and subsequent rehydration stress, in the resurrection plant Xerophyta humilis (Baker). Recombinant proteins were expressed for 3 of the LEA genes from this set in order to perform structural studies and to ascertain their LEA status. These studies were conducted with the purpose of shedding light on the role of LEAs in desiccation tolerance, to add to the ever-growing transcriptomic and proteomic data, and to the current knowledge of these enigmatic proteins. Quantitative real-time gene expression (qPCR) analysis was conducted on the set of 21 full length X. humilis cDNA clone nucleotide sequences, with similarities to late embryogenesis mRNA sequences, derived from a study conducted by Collett et al., (2004). Expression analysis was conducted in both leaves and roots, across a dehydration and rehydration profile of X. humilis. Of this total group of 21 full length cDNA clones, three LEAs; XhLEA2-3 and XhLEA2-6 (two putative Group 2 LEA genes) and XhLEA3-5 (a putative Group 3 LEA gene), were chosen for cloning and expression studies. cDNAs of these XhLEAs were cloned into a modified bacterial expression vector and recombinant protein expression was attempted in E. coli.

Bacterial plant pathogens have evolved a wide range of mechanisms to suppress the immune response that they trigger in their hosts, including the production of effectors and phytotoxins. The tri-carboxylic acid citrate, which is secreted into the apoplast by both bacterial pathogens and plant hosts has previously been shown to increase the virulence of the gram negative pathogen Pseudomonas syringae DC3000 (Pst DC3000), by acting both as a chemoattractant and as an inducer of genes associated with the type III secretion system (T3SS) and phytotoxin production. The effect of citrate on the host is less clear, though microarray analysis of Arabidopsis thaliana has demonstrated that application of exogenous citrate leads to the differential expression of 1876 genes suggesting that it might act as a metabolic signal for transcriptional reprogramming. In this study, functional enrichment analysis revealed statistically significant enrichment for gene ontology terms associated with defence in both citrate up-regulated and down-regulated gene sets. Furthermore this project demonstrated that exogenous citrate can increase the success of virulent Pst DC3000 infection in Arabidopsis; bacterial titres in plants pre-treated with citrate 24 hours prior to infection were significantly higher than those in control plants. This phenomenon was also observed in plants pre-treated with a non-metabolisable citrate analogue but not in plants pre-treated with another TCA cycle intermediate, malate, suggesting that it is citrate specific. However, it remains unclear whether the increased apoplastic citrate concentrations lead to increased bacterial titres through a suppressive effect on the host immune response, an enhanced induction of the T3SS system in Pst DC3000, or a combination of both.

In response to abiotic stresses, organisms throughout the plant kingdom, as well as microorganisms and micro-animals such as nematodes or tardigrades, have been observed to express Late Embryogenesis Abundant (LEA) proteins as protective mechanisms. However, despite two decades of research, little is understood about their physiological functions and this has led to extensive nomenclature, with a large amount of redundancy. The primary reason for this lack of insight into LEA protein functions is their highly hydrophilic and intrinsically disordered nature. Intrinsically disordered proteins (IDPs) cannot be studied using conventional methods of structural analyses such as X-ray crystallography and, therefore, alternative techniques are required. A combination of transgenic and in vitro studies have also shown that LEA proteins are most likely to behave as molecular chaperones by binding water and ions, preventing macromolecular aggregation and protecting enzymatic activity during dehydration. This study characterized two dehydrins that were expressed during dehydration in the desiccation tolerant plant, Xerophyta humilis. From a transcriptome analyses on X. humilis, cDNA for the two dehydrins were obtained. These sequences were first analysed using various in silico tools in order to identify putative dehydrin-specific characteristics. Subsequently, these two dehydrins were cloned and expressed for production of recombinant dehydrin protein. These proteins were then analysed in terms of structural and functional characteristics. Structurally, through the use of circular dichroism in an in vitro system, both dehydrins demonstrated the shift towards being increasingly alpha-helical when placed in environments of decreasing water content. The role of these two dehydrins in stabilizing enzymes during dehydration was subsequently investigated using citrate synthase (CS) and lactate dehydrogenase (LDH). The preservation of enzyme activity was observed in both CS and LDH. This preservation of enzyme activity was further maintained by the presence of trehalose. Anti-aggregation roles were also investigated, however, neither dehydrin demonstrated significant ability to minimize the aggregation of LDH. This study hopes to establish a pipeline for characterizing LEA proteins using structural and functional assays in order to provide alternative means of LEA protein classification.

Includes bibliographical references (leaves 100-108). / Climate change is predicted to have a negative impact on world food security in the next 40 years. Resurrection plants can withstand highly variable and harsh climatic conditions. This makes them ideal candidates to elucidate possible mechanisms which can be used to adapt crop plants to tolerate variable climatic conditions associated with climate change.

Summary in English. / Bibliography: pages 105-125. / A survey was conducted by researchers at ARC-PPRI on dry beans (Phaseolus vulgaris) during 1993. All the viruses known to occur on dry beans in South Africa were found, as well as a few unidentified viruses. Of these, samples 93/1 and 93/65 form the basis of this thesis. Electron microscopy (EM) indicated that these viruses could be potyviruses, as they were flexuous particles of approximately 700 to 800 nm. Observation of pinwheels in ultrathin sections of Nicotiana benthamiana infected with isolate 93/1 and Phaseolus vulgaris infected with isolate 93/65, confirmed that the viruses probably belonged to the Potyvirus genus, family Potyviridae. Further serological tests indicated that the viruses were related but not homologous to strains of clover yellow vein (CIYW) and blackeye cowpea mosaic (BICMV) viruses respectively. None of these viruses have previously been described as occurring in South Africa. As we were unable to positively identify the viruses with serological methods, we needed to characterise these viruses on a molecular level. Potyvirus specific oligonucleotide primers were used for PCR amplification of viral eDNA The primers amplify an approximately 700 bp fragment of the virus genome, spanning the 3' noncoding region as well as a part of the coat protein gene: one primer is complementary to the poly(A) tail, and the other to a sequence coding for a conserved block of amino acid sequences (also known as the WCIEN block) in the mid-region of the coat protein. The nucleic acid sequences of the PCR products were compared to that of other potyviruses to positively identify these isolates.