Background HER2-positive breast cancer is a poor prognostic subgroup, even if treated with anti-HER2 directed therapy. Trastuzumab is an important HER2-targeting antibody but only limited patients respond to this drug, and acquired resistance is a common problem. HER3 has been shown to be a key candidate in mediating resistance to trastuzumab and other ErbB inhibitors. The aims of the project are to investigate the resistance mechanisms and the relevant biomarkers in relation to trastuzumab treatment and resistance in HER2-positive breast cancer, in particular, HER3 subcellular localisation and HER3 phosphorylation. Methods Effects of trastuzumab on HER3 subcellular localisation and HER3 phosphorylation in relation to MET receptor were studied using western blots, nuclear fractionation, confocal microscopy, and immunoprecipitation in a panel of HER2-positive cell lines, including SKBr3 and BT474 breast cancer cells in which trastuzumab resistance was induced by long-term drug exposure. Effects of drug and knockdown experiments were tested by cell viability and proliferation assays. HER3 and MET expression was assessed by immunohistochemistry in xenograft tumours and human tissue samples, and clinical impact was assessed in different cohorts of HER2-positive breast cancer patients. Results Acquired trastuzumab resistant SKBr3 cells showed an increase of nuclear HER3_100kD, which was derived from C-terminus of HER3. Nuclear HER3_100kD could be due to the proteolytic cleavage of HER3 since it was reduced by ADAM17 or gamma-secretase inhibitor. In a panel of HER2-positive cell lines and xenograft samples, nuclear HER3 was observed only in the resistant cells. In addition, nuclear HER3 was associated with poor progression-free and overall survivals in HER2-positive breast cancer patients. It was also found that HER3 phosphorylation was maintained in acquired trastuzumab resistant cells, which was contributed by the ligand independent interaction of MET and HER3. Higher MET expression was associated with better overall survival in HER2-positive, breast cancer patients who were not treated with trastuzumab. Conclusions Nuclear HER3 was found in trastuzumab resistant cells and appeared to result from HER3 proteolytic cleavage mediated by ADAM17 and gamma-secretase. Further studies are required to investigate its mechanism and to identify the HER3 cleavage sites. MET was a key factor in maintaining HER3 phosphorylation during trastuzumab resistance. Lastly, nuclear HER3 and MET could be two potential biomarkers in HER2-positive breast cancer.

Dans cette étude, nous avons démontré par des essais de type Pull-Down la capacité de la queue C-terminale du CysLT1 d'interagir in vitro avec les importines [alpha]1, [alpha]4 et [alpha]5. Cette interaction est comparable à celle du NLS de l'antigène grand T du SV40 et du NLS de la nucléoplasmine de Xenopus laevis avec les mêmes importines. Nos études démontrent aussi que les déterminants structuraux de l'interaction ne sont pas limités seulment au NLS: des mutations dirigées contre les résidus-clé qui forment le NLS n'ont pas empêché l'interaction entre la queue C-terminale et les importines. Nos résultats suggèrent que d'autres résidus que ceux qui forment le NLS potentiel sont impliqués dans la liaison avec les importines. Nous avons démontré pour la première fois la phosphorylation in vitro de la queue C-terminale du CysLT1 par la PKA, et que cette phosphorylation peut moduler la capacité d'interaction avec l'importine [alpha]4. Nos travaux visaient aussi l'étude de l'expression cellulaire du récepteur CysLT1 et de son comportement suite à une stimulation au LTD[indice inférieur 4] .--Résumé abrégé par UMI.

Through this study, a strategy for the localisation and development of the South African nuclear industry was developed. As background, the Korean localisation experience was investigated, along with international recommendations regarding nuclear localisation, and South African governmental policies. This research was used as foundation for the formulation of a localisation strategy. The possibility of using localisation and nuclear industry development as a means to address governmental socio-economic development goals was investigated. From the literature investigation localisation principles were identified. The focus areas of the localisation strategy were subsequently based on these principles. The principles are:  Aggressive human resource development  Governmental leadership and support  International co-operation The localisation strategy addresses general localisation recommendations, needed human resource development, structure of the Nuclear Energy Project Implementation Organization (NEPIO), roles of the participants of the NEPIO, and finally the supply-chain development and technology transfer guidelines. It was assumed that three nuclear power plants, consisting of two reactors each would be constructed. For localisation to be successful, a fleet approach must be followed to ensure economy of scale, and local participation must be incrementally increased with each power plant. The localisation strategy was circulated to industry for validation, and changes were made, based on industry feedback. The needed human resource development amounts to the training of 4 012 labourers per year (see Table 1). The local participation for each consecutive power plant is 30%, 50%-55% and 75%-80%, respectively. It was found that 100% localisation is not feasible. The planned nuclear power programme is too small to justify the development of globally leading components such as ultra-heavy forgings. The structure of the NEPIO is shown in Figure 1. It was found that the localisation and nuclear industry development would serve as a vehicle to help achieve governmental socio-economic development programmes. It was finally concluded that South Africa has the potential for localisation, but obstacles such as a lack of governmental commitment, negative public perception, and lack of industry confidence will be detrimental to the localisation efforts. If these, and other obstacles are not urgently addressed, South Africa will miss out on a much needed development opportunity. / MIng (Development and Management Engineering), North-West University, Potchefstroom Campus, 2013

Through this study, a strategy for the localisation and development of the South African nuclear industry was developed. As background, the Korean localisation experience was investigated, along with international recommendations regarding nuclear localisation, and South African governmental policies. This research was used as foundation for the formulation of a localisation strategy. The possibility of using localisation and nuclear industry development as a means to address governmental socio-economic development goals was investigated. From the literature investigation localisation principles were identified. The focus areas of the localisation strategy were subsequently based on these principles. The principles are:  Aggressive human resource development  Governmental leadership and support  International co-operation The localisation strategy addresses general localisation recommendations, needed human resource development, structure of the Nuclear Energy Project Implementation Organization (NEPIO), roles of the participants of the NEPIO, and finally the supply-chain development and technology transfer guidelines. It was assumed that three nuclear power plants, consisting of two reactors each would be constructed. For localisation to be successful, a fleet approach must be followed to ensure economy of scale, and local participation must be incrementally increased with each power plant. The localisation strategy was circulated to industry for validation, and changes were made, based on industry feedback. The needed human resource development amounts to the training of 4 012 labourers per year (see Table 1). The local participation for each consecutive power plant is 30%, 50%-55% and 75%-80%, respectively. It was found that 100% localisation is not feasible. The planned nuclear power programme is too small to justify the development of globally leading components such as ultra-heavy forgings. The structure of the NEPIO is shown in Figure 1. It was found that the localisation and nuclear industry development would serve as a vehicle to help achieve governmental socio-economic development programmes. It was finally concluded that South Africa has the potential for localisation, but obstacles such as a lack of governmental commitment, negative public perception, and lack of industry confidence will be detrimental to the localisation efforts. If these, and other obstacles are not urgently addressed, South Africa will miss out on a much needed development opportunity. / MIng (Development and Management Engineering), North-West University, Potchefstroom Campus, 2013

No description available.

No description available.

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

Class I PI3Ks are a family (α, β, δ and γ) of ubiquitous lipid kinases that can be activated by cell surface receptors to 3-phosphorylate PI(4,5)P2 (phosphatidylinositol(4,5)-bisphosphate) and generate the signalling lipid PI(3,4,5)P3. The PI(3,4,5)P3 signal then activates a diverse collection of effector proteins involved in regulation of cell migration, metabolism and growth. The importance of this network is evidenced by the relatively high frequency with which cancers acquire gain-of-function mutations in this pathway and huge efforts to make PI3K inhibitors to treat cancer. The canonical model describing these events suggests class I PI3Ks are activated at the plasma membrane and generate PI(3,4,5)P3 in the inner leaflet of the plasma membrane where its effectors are activated. The PI(3,4,5)P3 signal can be terminated directly, by the tumour-suppressor and PI(3,4,5)P3-3-phosphatase PTEN, or modified to a distinct PI(3,4)P2 signal, by SHIP-family 5-phosphatases. The PI(3,4)P2 is removed by INPP4-family 4-phosphatases. Published work has shown that PI(3,4,5)P3 signalling can also occur in endosomes and nuclei, however, there is very little data defining the intracellular distribution of endogenous class I PI3Ks that supports these ideas; this is as a result of technical problems such as; their very low abundance, poor antibody-based tools and artefacts generated by overexpression of PI3Ks. Past work has indicated that, in PTEN-null mouse models of prostate tumour progression, either PI3Kβ or PI3Ks α and β, have important roles. Furthermore, the cell types and mechanism involved remained unclear. Recent published work in the host laboratory had indicated that there is an unexpectedly large accumulation of PI(3,4)P2 in PTEN-null cells that might be an important part of its status as a major tumour suppressor. The explanation and prevalence of this observation was unclear but potentially a result of PTEN also acting as a PI(3,4)P2 3-phosphatase in vivo. MEFs were derived from genetically-modified mice expressing endogenous, AviTagged class I PI3K subunits and used in experiments to define the subcellular localisation of class I PI3Ks. We found that following stimulation with PDGF, class IA PI3K subunits were unexpectedly depleted from the adherent basal membrane, in contrast, p85α and p110α, but not p85β and p110β, accumulated transiently in the nucleus. Interestingly, p110β, but none of the other subunits, was constitutively localised in the nucleus. These results support the idea that class I PI3K and PI(3,4,5)P3 signalling occurs in the nucleus. In organoids derived from WT, PI3Kγ-null or PTEN-null mouse prostate, application of PI3K-selective inhibitors revealed that PI3Kα had a dominant role in generating PI(3,4,5)P3 in prostate epithelial cells. The levels of PI(3,4)P2 were also elevated substantially in PTEN-null, compared to WT prostate organoids, use of PI3K-selective inhibitors suggested that it was also generated by PI3Kα. These data were consistent with the idea that PTEN can act as a PI(3,4)P2 3-phosphatase. Surprisingly, raising the pH of the organoids medium dramatically increased accumulation of PI(3,4,5)P3 and PI(3,4)P2, although the cause of this effect was unclear, we hypothesised the pH of the local environment may influence signalling via class I PI3Ks.