Photoimmunotheranostic targeting of CSPG4-positive melanoma cells using SNAP-tag technology

Melanoma is one of the most aggressive and inherently resistant cancers and the most dangerous skin cancer. While it accounts for fewer than 5% of skin cancer cases, 80% of skin cancer related deaths are attributed to melanoma. While resection remains the gold standard for melanoma treatment, surgery is only effective in providing local control of the disease if the cancer is detected in the early stages. Once melanoma enters the later stages, and particularly in the metastatic phase, recurrence is probable, and no adequate treatment exists. Previous work in this group has shown that photodynamic therapy (PDT) presents an opportunity to induce cell death in melanoma cells through the production of ROS and singlet oxygen at doses high enough to overwhelm the resistance mechanisms of the cancer. In this study, we investigated the use of recombinant SNAP-tag-based antibody fusion proteins as a means of delivering phototoxic molecules directly to cancer cells expressing the CSPG4 and PD-L1 cell surface receptors. SNAP-tag is an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase. It reacts autocatalytically and in a strictly 1:1 coupling chemistry with substrates that have been modified with benzylguanine (BG). Through genetic fusion of this self-labelling protein with a tumour targeting antibody, we developed a recombinant immunoconjugate able to carry BG-modified photosensitizers to selectively target and eliminate malignant melanoma cells. Conjugation of the SNAP-tag fusion protein with the fluorescent dye Alex Fluor 488 showed that anti-CSPG4-SNAP binds specifically to melanoma cells expressing the CSPG4 surface antigen. Binding was tested across a range of cell lines presenting melanoma in its radial and vertical growth phases, in the metastatic growth phase, in its chemoresistant form, and in both its pigmented and unpigmented forms. This binding data thus confirms CSPG4 as a suitable targeted for this treatment strategy. Conjugation of the fusion protein with the BGmodified photosensitizer IRDye 700DX (IR700) has produced no phototoxicity as of yet. In light of the convincing binding analysis, it is concluded that inefficient solubilization of the lyophilized product resulted in inadequate conjugation of BG-IR700 with SNAP-tag. Nonetheless, steps have been planned to resolve the problem in future ongoing work on this project, and we remain confident in the applicability of this technology. The results for the PD-L1 fusion protein were inconclusive. In summary, SNAP-tag technology offers a simple and efficient method for immunofluorescent detection of cancerous cells. These fusions proteins are versatile as they 1) can contain any antibody targeting a tumour-associated or tumour-specific antigen of choice and 2) can be endowed with a wide variety of substrates, as long as the latter contains the BG moiety

The impact of quantitative easing on capital flows to the BRICS economies

Doctor EducationisA possible effect of quantitative easing (QE) undertaken by the United States of America (USA) Federal Reserve Bank (Fed) may have been an increase in capital flowing into emerging market economies (EMEs). The 2008 global financial crisis created an environment in which traditional monetary policies – cutting policy rates – became ineffective in stimulating growth. Faced with this policy environment, several high-income countries including the USA resorted to unconventional monetary policies notably QE, to grow their economies. While QE was effective in lowering interest rates in high-income countries, some argued that investors switched to higher yielding assets, mostly EME assets. Therefore, QE is perceived to have increased capital flows into EMEs. Using a dynamic panel data model with fixed effects this mini-thesis investigates empirically whether QE worked through unobservable channels to increase gross private capital inflows to Brazil, Russia, India, China and South Africa (BRICS) in the period 2000-2015. The study finds evidence in support of the view that QE increased capital inflows to EMEs. The results reveal that gross private capital inflows to the BRICS increased during the QE intervention period and that the increase was higher in the first period of QE than in subsequent QE periods. The empirical results also reveal differences in the way types of capital flows responded to QE; portfolio flows, and in particular equity flows were the most responsive to QE

Continuity or rupture? : the shaping of the rural political order through contestations of land, community, and mining in the Bapo ba Mogale traditional authority area

A research project submitted at the University of the Witwatersrand, Department of Political Studies, School of Social Sciences, in fulfilment of the Master of Arts (Research) Degree.South Africa’s countryside’s are rich in ‘new’ high-demand metal and energy minerals, like platinum and uranium, as well as vast, untapped reserves of industrial staples, above all coal. Yet, these are also characterised by deep rural poverty and legally insecure systems of ‘customary’ tenure, under the local administrative control of traditional authorities. Here, new mining activity is setting in motion significant processes dispossession and Immiseration that are at once tracing, reconfiguring and widening the class, gender and other social divisions that define these rural settings. Communal land is frequently alienated with little or no compensation, local residents forcibly removed to make way for surface infrastructure, and scarce water and other natural resources polluted and depleted. At the same time political tensions are arising from the assumption that local chiefs are ‘custodians’ of the mineral-rich land under their jurisdiction. Questions of land, livelihood and rural democracy are thus intimately bound together on the new frontiers of the regional extractives boom in ways that are having profound implications for growing numbers of the rural poor. Using a case study of the Bapo ba Mogale traditional Authority in the North West Province, South Africa, this thesis seeks to explore how these new mining activities are shaping and reconfiguring the heightened political contestations over the institution of traditional leadership in the area, the definitions of community and belonging/exclusion, and the struggles over land ownership and how mining capital is shaping these struggles and is connected with these strugglesEM201

Entrepreneurial orientation and practice: three case examples of historically disadvantaged primary schools

Historically disadvantaged schools are mostly in a position where they cannot easily practise entrepreneurial customs like innovation, proactiveness and risktaking. However, some of these schools perform well under similar circumstances and show strong entrepreneurial inclinations. In fact, in research conducted in 2006, Lebusa and Xaba found that there were very strong prospects of fostering entrepreneurial customs at historically disadvantaged schools. It was found that schools were already practising innovativeness, proactiveness and risk-taking, albeit unintentionally. We report on the practice of innovativeness, proactiveness and risk-taking at historically disadvantaged schools. Case examples of three successful entrepreneurial historically disadvantaged primary schools are presented. Results indicate novel and innovative ventures undertaken at these schools and clearly indicate that some historically disadvantaged schools are indeed entrepreneurially oriented

Multi-Grade Teaching and Inclusion: Selected Cases in the Free State Province of South Africa

Multi-grade teaching is a commonly used pedagogic strategy in rural schools. However, this form of teaching continues to present challenges to both the teacher and the learner in particular since the advent of inclusive education in South Africa. The policy on inclusive education is silent on how it could be enhanced in a multi-grade class. This paper therefore attempts to analyse and deconstruct the tensions, contradictions and anomalies within the practice of inclusion as a pedagogic discourse in a multi-grade teaching system. The qualitative study on which this article is based employed a critical emancipatory lens and critical realist analysis as instruments with which to analyse narratives from selected cases at multi-grade schools in the Free State province of South Africa. The findings of the study indicated that, while the knowledge and skills teachers need to enhance inclusion within a multi-graded system were limited, well-designed teaching practices in a multi-grade class may enhance an inclusive pedagogy and promote inclusive learning

The impact of the Construction Regulations 2014 on a water utility’s projects’ health and safety (H&S) performance in South Africa

The construction industry plays a major role in South Africa’s economic development. Despite its importance, the poor health and safety (H&S) performance and associated costs to the industry are a concern. The literature review indicates that improved H&S performance can be achieved on a project where there is a collaborative approach among all stakeholders during the six project stages. This study focuses on the impact of the Construction Regulations 2014 on a water utility’s projects’ H&S performance. It determines the H&S involvement of client representatives and internal project stakeholders during the six project stages. It also addresses their commitment towards H&S, measures taken by them during planning and design, in contracts to improve H&S, and to select conscious contractors, and the extent of their H&S participation in construction H&S. Mixed methods research (a questionnaire survey and four case study projects) was used to collect the data required for the study. A total of 67 responses were obtained from the five stakeholder groups from the water utility, namely client representatives, designers, quantity surveyors (QSs), project managers (PMs), and construction H&S (CHS) professionals to obtain information regarding their H&S involvement during the six project stages. Four case study projects (three pre- Construction Regulations 2014 and one post- Construction Regulations 2014) were obtained, using a stratified random sampling technique and a checklist to obtain information regarding the stakeholder H&S involvement during the six project stages and the overall project performance. The study found that the Construction Regulations 2014 are perceived to have had an impact on the water utility’s H&S performance. There is a direct relationship between stakeholder H&S involvement and project H&S performance. The integration of H&S in the initial project phases; early involvement of CHS professionals, and H&S training for stakeholders are among the recommendations arising from this study.&nbsp

Multi-Grade Teaching and Inclusion: Selected Cases in the Free State Province of South Africa

Multi-grade teaching is a commonly used pedagogic strategy in rural schools. However, this form of teaching continues to present challenges to both the teacher and the learner in particular since the advent of inclusive education in South Africa. The policy on inclusive education is silent on how it could be enhanced in a multi-grade class. This paper therefore attempts to analyse and deconstruct the tensions, contradictions and anomalies within the practice of inclusion as a pedagogic discourse in a multi-grade teaching system. The qualitative study on which this article is based employed a critical emancipatory lens and critical realist analysis as instruments with which to analyse narratives from selected cases at multi-grade schools in the Free State province of South Africa. The findings of the study indicated that, while the knowledge and skills teachers need to enhance inclusion within a multi-graded system were limited, well-designed teaching practices in a multi-grade class may enhance an inclusive pedagogy and promote inclusive learning