The targeting of liposomes to cells and the delivery of the liposomal contents into the cells have been investigated using either α-melanocyte stimulating hormone or Ricin-B-chain as ligands for promoting the binding of liposomes to cells. α-melanocyte stimulating hormone has been conjugated to liposomes and to Ricin-A-chain via the Lys₁₁ residue without significant loss of biological activity. The resulting conjugates were found to bind to Bl6 melanoma cells which express receptors for the hormone. Hormone targeted ricin was shown to be toxic to the cells, indicating receptor mediated internalisation of the conjugate. The hormone targeted liposomes however were unable to mediate the delivery of cytotoxic levels of methotrexate. Ricin-B-chain, a lectin which mediates membrane translocation of the toxic ricin-A-chain, was examined for its applicability for targeting of liposomes to cells. This lectin was shown to promote the binding of liposomes to cells and to mediate the delivery of cytotoxic concentrations of methotrexate. Further evidence of functional ricin-B-chain mediated intracellular delivery of the liposomal contents was shown by liposome mediated transformation of cells, and delivery of nuclease into the cell resultin in digestion of genomic DNA. The study demonstrates that α-melanocyte stimulating hormone is unsuitable as a ligand by which to achieve delivery of large quantities of material into cells, although cell-specific targeting can be achieved. Ricin-B-chain is however ideally suited for this task, though is less cell-specific. This finding may be of use in studies in which investigators wish to achieve intracellular delivery of compounds.

Haliotis midae is an economically important South African abalone species which has been cultured since the 1990s. H. midae farming constitutes approximately 93% of the country's total marine aquaculture industry. However, the slow growth rate of this animal and the potential for disease outbreaks remain a concern for farmers. Therefore, government and private institutions have joined efforts to investigate ways of enhancing abalone production, to increase abalone growth rates and improve disease resistance. Several studies have shown that probiotic microorganisms can significantly increase the growth rate and disease resistance of H. midae. To date, no comprehensive studies have been conducted to characterise these physiological improvements at the molecular level. Thus, the aim of this study was to evaluate the effect of a probiotic-supplemented diet on the haemocyte proteome in H. midae. Two probiotic-strains, Vibrio midae SY9 and Debaryomuces hansenii AY1, were introduced into H. midae via a kelp-based feed. Changes in the haemocyte proteome were analysed using isobaric tag for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS. A total of 128 haemocyte proteins were identified. Proteins that were found to vary significantly in their expression levels in haemocytes sampled from abalone fed the probiotic-supplemented diet were identified as COP9 signalosome subunit 4, phosphorylase, T-complex protein 1 subunit gamma, V-type proton ATPase subunit B, Rab 1 and Ra-related protein Rab 1A. Differential expression of COP9 signalosome subunit 4 (up-regulated) and Ras-related protein Rab 1A (down-regulated) was confirmed by western blot analysis. Bioinformatics analysis revealed proteins with immune class GO terms that functioned in metabolism, apoptosis, cell adhesion, immune response, stress response, and response to endogenous and external stimulus. Hierarchical clustering analyses showed that proteins with similar expression patterns mostly belonged to the same immune classes. Analysis of protein interaction networks indicated that all the differentially expressed proteins may indirectly interact with each other. It was also found that the neurotrophic tyrosine kinase receptor was the central molecule within the interaction network, suggesting that this protein may play a crucial role within the protein interaction network that contains all the differentially expressed proteins. Biochemical pathway analysis indicated that phagosomal maturation was the most significant canonical pathway identified, in which V-type proton ATPase and Ras-related protein Rab have fundamental importance. Changes in Ras-related protein Rab 1A expression were further investigated in the cytosolic and membrane fractions of haemocyte cells using western blot analysis and cellular immunochemistry. The expression of this protein was found to be down-regulated both in cytosolic and membrane fractions from haemocytes sampled from H. midae fed a probioticsupplemented diet. Although an association between Ras-related protein Rab 1A and F-actin (cell cytoskeleton) was not detected, confocal microscopy confirmed Ras-related protein Rab 1A down-regulation. Thus, results from this study suggest that Ras-related protein Rab 1A may play a key role in H. midae immune response, when this species of abalone is fed with a probiotic-supplemented diet. This is the first time that a large-scale proteomics approach has been used to investigate proteome changes in haemocytes sampled from H. midae fed a probiotic-supplemented diet. The findings of this study, regarding the protein profile, interaction networks, molecular pathways and a putative molecular indicator of H. midae immune response, provide a foundation from which future studies can be conducted in order to increase our understanding of how probiotics affect the abalone immune system.

[Not OCR'd]

Bibliography: pages 304-309. / The field of metal determination as a part of pollution studies, has been critically examined and metal pollution may be defined in one simple statement: The presence of metal binding proteins confirms toxic metal pollution. It has been shown that current methods of metal determination in biological systems are of little use. This has been illustrated by both a review of metal concentration in Southern African coastal water, sediments and biotopes, and by a comparative baseline study of organisms from Gough Island and Mar ion Island. These showed that extrapolation of results from one geographical area to another are invalid and that this interpretation is made difficult by factors such as age, sex, size life stage of the organisms. Furthermore, it was shown that many reports on metal pollution do not even mention fundamental information such as the size or the sex of the animals. Metal pollution could be linked to metal binding protein through an independent pollution er i ter ia, for example, the out of season moulting of crayfish. The new definition of metal pollution has then been tested by application to five different organisms (crayfish, Jasus lalandii; hermit crab, Diogenes brevirostris; shrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis and limpet, Patella granularis) kept under identical conditions and it was shown that a much more meaningful interpretation of the results could be made. The new definition was al so tested with two naturally occurring metal accumulating organisms (whelk, Bullia digitalis and "kikuyu" grass) and it was shown that dramatic increases in metal may not necessarily be toxic. It was concluded that less effort and time should be spent on metal analysis in determination of metal pollution and attention should rather be directed to the presence or absence of metal-binding proteins.

Very little is known about the molecular and genetic mechanisms controlling pigmentation within the bird kingdom. The aim therefore, of this study was to contribute towards the understanding of the genetic regulation of avian pigmentation by the cloning and characterisation of the chicken Tyrosinase-related protein (TRP) gene family. To accomplish this goal, neural crest cells from 500 black chick embryos were cultured under conditions supportive of melanocyte differentiation and proliferation. Using RNA extracted from these pigmented melanocyte cultures, a novel embryonic chick cDNA library was constructed. Screening of this library for chicken equivalents of the mammalian TRP gene family yielded more than 200 cDNA clones. After sequencing, three of these clones, 88.3, pcTRP- 1.6 and pcTRP -2. 10, were found to encode chicken Tyrosinase (Tyr), Tyrosinase-related protein-1 (Tyrp1) and Tyrosinase-related protein-2 (Tyrp2), respectively. In addition, a chicken Microphthalmia (Mi) cDNA clone (M156) was isolated using a mouse Mi cDNA probe. Comparative analyses revealed that chicken Tyr, Tyrp1 and Tyrp2 share approximately 68%, 72% and 70% amino acid sequence identity with their vertebrate orthologues. Northern blot hybridisation analysis demonstrated that the chicken TRPs are expressed in RNA from cultured retinal pigment epithelial (RPE) cells as well as in whole eye RNA. The major transcript sizes for the chicken Tyr, Tyrp1 and Tyrp2 genes are 2.5 kb, 2.3 kb and 3.5 kb, respectively. In situ hybridisation studies confirmed that both chicken Tyr and Tyrp2 genes are expressed in a pigment cell-specific fashion with signals detected in both the skin and RPE of chick embryos. Genomic Southern blot hybridisation analyses strongly suggested that all three chicken TRP genes contain several introns that are likely to be conserved within the vertebrate TRP gene family. Furthermore, the chick Tyr, Tyrp1 and Tyrp2 genes were found to span approximately 5-19 kb, 5-11 kb and 15-30 kb, respectively of the chicken genome. Comparisons between a black and white chick breed at the Tyr and Tyrp1 loci revealed no gross rearrangements at either of these loci. However, 1-2 kb alterations were observed between the same breeds at the Tyrp2 locus. The nature and significance of this alteration is not known. The cloning of the chicken Tyr, Tyrp1 and Tyrp2 cDNAs constitutes the first molecular cloning and characterisation of any avian TRP gene family. Taken together therefore, this study contributes towards the further understanding of the molecular mechanisms regulating pigmentation as well as the evolution of gene families.

Bluetongue is a disease of domestic and wild ruminants caused by Bluetongue virus (BTV). It has caused several serious outbreaks, the most recent occurring in Northern Europe in 2006 during which high mortality rates of livestock were reported. The only vaccines currently approved and commercially available for use are live-attenuated or inactivated virus strains and although these are effective, there is the risk of reversion in the case of live-attenuated strains to more virulent forms by recombination. Another drawback associated with the use of live-attenuated virus vaccines is that they are not DIVA (differentiate infected from vaccinated animals) compliant, this means that naturally infected animals cannot be distinguished from vaccinated animals. Recombinantly produced vaccines would be preferable to minimize the risks associated with live-attenuated virus vaccines and also enable the development of candidate vaccines that are DIVA-compliant. A number of recombinant vaccine candidates have been developed against BTV, with the most promising vaccine consisting of BTV virus-like particles (VLPs). BTV VLPs were successfully produced in insect cells by the co-expression of the four BTV capsid proteins (VP2, VP3, VP5 and VP7). Sheep vaccinated with insect cell-produced BTV VLPs were shown to be protected against challenge with wild type virus. However, the high costs associated with the production and scale-up of BTV VLPs in insect cells has possibly limited their widespread application. Plants – such as N. benthamiana – provides a safe, efficient and cost effective system for the production of recombinant proteins. In this study the best plant expression vector with which to co-express the four BTV serotype 8 (BTV-8) VPs – which direct formation of BTV-8 VLPs – was identified. Expression and purification of the BTV-8 VLPs was optimised with the aim of producing a VLP-based vaccine for BTV-8. It was further undertaken to develop two novel second generation plant-produced protein body (PB) vaccines that are DIVA compliant. Mice were immunised with the plantproduced VLP and PB vaccines in order to analyse their ability to elicit humoral immune responses.

As a consequence of an immobile lifestyle, plants have had to evolve appropriate perception mechanisms and responses to diverse environmental stresses. Stress can be the result of both biotic and abiotic agents and the ORTHOLOG OF SUGAR BEET HS1 PRO-1 (HSPRO 1) and HSPRO2 genes were previously shown to be induced in response to several stresses including infection with Pseudomonas syringae and drought stress in Arabidopsis thaliana. The aim of this study was to characterise the biological role(s) played by these proteins in Arabidopsis. Several bioinformatics approaches provided evidence that supported function of both genes in response to both biotic and abiotic stresses and identified potential regulatory elements that may drive HSPRO gene expression during stress responses. Accordingly, analysis of null hspro mutants revealed antagonistic functions of the two proteins in PAMP-triggered immunity to P. syringae infections of shoot tissues and osmotic stress tolerance in plant roots. HSPRO proteins have been shown to interact with a central integrator of stress and energy signalling, SUCROSE NON-FERMENTING-1-RELATED KINASE1 (SnRK1) and microarray analysis of the null mutants suggested potential roles in carbohydrate signalling. An array of energy responsive genes including a subset of SnRK1 targets were misregulated in hspro mutants under standard growth conditions supporting involvement of HSPRO in energy signalling. Mutant phenotype and gene expression analysis revealed that HSPRO2 may be of importance in energy perception as hspro2 seeds were hypersensitive to exogenous glucose during germination, and that perception and/or signalling of low energy status may require HSPRO2. Although HSPRO2 expression may be driven via perception of environmental stress cues, promoter-luciferase assays revealed a diurnal expression pattern of the gene that was driven by the circadian clock. However, phenotypic analysis did not reveal a requirement of HSPRO2 for normal clock modulation. Since stress perception typically causes fluctuations in energy levels, it is proposed that HSPRO genes are important for the integration of energy and stress signalling in an effort to maintain a homeostatic balance between coping with environmental stress and normal growth and development.

The relationship between progestin-only injectable contraception and risk of HIV-1 acquisition is controversial. Most clinical data suggests that the injectable contraceptive medroxyprogesterone acetate (MPA), unlike norethisterone enanthate (NET-EN), increases susceptibility to infections such as HIV-1. The first part of this thesis investigated the differential effects, molecular mechanisms of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 and HeLa cell line models for the endocervical epithelium, a key point of entry for pathogens in the lower female genital tract (FGT). Quantitative real-time PCR analysis showed that MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IκBα genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the cervical cell lines and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR siRNA experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone (DEX). This is at least in part consistent with direct effects on transcription, without a requirement for new protein vi synthesis. This is the first study to show direct proof for a GR-mediated mechanism of action in anti-inflammatory effects of MPA. Dose response analysis shows that MPA has a potency of ~24 nM for transactivation of the anti-inflammatory GILZ gene and ~4 - 20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that MPA effects on genital mucosal immune function and susceptibility to infections are likely to be very different to those of NET and P4, when mediated by the GR The second part of this thesis investigated the effects of the virion associated HIV-1 protein, Vpr, on GR-regulated inflammatory genes in the presence of the ligands.

Psittacine beak and feather disease (PBFD), the most prevalent viral disease affecting psittacines, is caused by beak and feather disease virus (BFDV). An outbreak of the disease has been reported in wild endangered Cape parrots (Poicephalus robustus), which is endemic to South Africa. No treatment or vaccine is commercially available. In this study, an investigation into the outbreak was undertaken. BFDV diversity was assessed and viral load and clinical signs correlated. A plant-produced BFDV subunit vaccine was produced in parallel with a corresponding challenge model. Cape parrots were assessed and 53 blood samples collected. Viral load was determined using quantitative real-time PCR (qPCR), and 22 BFDV full-length genome sequences acquired to infer phylogenetic relatedness. The capsid gene (cp) was optimised for transient Agrobacterium-mediated expression in whole-plant Nicotiana benthamiana (N. benthamiana). Virus-like particles (VLPs) were purified and analysed using transmission electron microscopy. Virions from a Palm cockatoo (Probosciger aterrimus) were purified and a BFDV dsDNA molecular clone was synthesised and replication assessed in 293TT mammalian cells and N. benthamiana using rolling circle replication and qPCR. Two distinct BFDV phylogenetic clusters were reported for Cape parrots, and a direct correlation was seen between viral load in the blood and clinical signs in PBFD-afflicted birds. The CP was successfully expressed in N. benthamiana, and increased through optimisation of Agrobacterium infiltration density and the inclusion of the NSs silencing suppressor. The CP formed VLPs, which were shown to be morphologically similar to infectious virions. The dsDNA molecular clone was shown to replicate autonomously in mammalian 293TT cells, and in plants with the assistance of the Bean yellow dwarf virus replication associated protein (Rep). BFDV genetic diversity in Cape parrots highlights the importance of ensuring new strains are not inadvertently introduced into the wild. This is the first systematic investigation of virus diversity in Cape parrots and assessment of BFDV viral load in a wild psittacine population. The CP was successfully produced in planta and presence of VLPs suggests the possibility of developing pseudovirions. This is the first reported replication of BFDV in tissue culture, and will greatly expand the scope of available research.

Includes bibliographical references / Reciprocal modulation between the glucocorticoid receptor (GR) and gonadotropin-releasing hormone (GnRH) signalling pathways is a potential mechanism for integrating cellular responses to stress with reproductive function. This study investigated if membrane rafts play a role in GR and GnRH receptor (GnRHR) crosstalk and explored the mechanism involved in the mouse pituitary gonadotrope LβT2 cell line by dexamethasone (Dex), GnRH and both together.