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A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Doctor of Philosophy. Gauteng, Johannesburg, 2016. / Overexpression of RBBP6 in cancers of the colon, lung and oesophagus makes it a potential target in anticancer therapy. This is especially important because it associates with the tumour suppressor gene p53, inactivation of which has been linked to over 50% of all cancer types. Cancer is an enormous burden of a disease globally. Today, more people die from cancer than HIV/AIDS, tuberculosis and malaria combined. And in females, breast and cervical malignancies remain the most common types. Currently, cervical cancer is the most diagnosed gynaecological cancer type, whose mortality rate is the highest in developing countries due to the asymptomatic nature of the disease coupled with inadequate cancer control tools and facilities. Breast cancer incidence rate has increased beyond that of lung cancer, making it the most common malignancy among women. / GR2016

A Dissertation submitted to the Faculty of Science, University of Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. 2015. / Prdm1/Blimp1 is a transcription factor whose mechanism of action is mainly repression; however it has been identified as an activator in some cases. As a transcriptional repressor, it plays multiple roles during embryonic development, including neural crest specification. Prdm1 acts by repressing large sets of genes via sequence specific recruitment of co-repressors, many of which are epigenetic modifiers. Neural crest is a transient, migrating cell population that gives rise to a number of diverse cell lineages that form important structures in the vertebrate embryo. Examples of these include peripheral nervous system, melanocytes and cranial cartilage. Prdm1 is expressed during neural crest specification in Xenopus, zebrafish and lamprey. The expression of Prdm1 had not yet been investigated in the neural crest during chick embryonic development. The mechanism of Prdm1 action or the nature of possible binding partners that mediate its effects in the neural crest had not yet been addressed. Prdm1 binding partners are known to play important roles during embryonic development, yet in many cases no spatiotemporal expression analysis during early vertebrate development has been performed. Single and double in situ hybridization for Prdm1 and all the binding partners was performed to determine localization of mRNA during early stages of chick embryonic development. We report the expression patterns of Prdm1 and seven of its known or putative binding partners (Hdac1, Hdac2, Tle1, Tle3, G9a, Prmt5 and Lsd1) during early stages (HH4-HH10) of chicken embryogenesis. Prdm1 expression was observed in the neural plate border and pre-migratory neural crest during chick development. Six Prdm1 binding partners (except Tle1) are co- expressed with Prdm1 in the prospective neural plate border at HH4-HH6, and all seven show strong and specific expression in the neural plate border at HH7-HH8, suggesting all of them co-operate with Prdm1 during neural crest development in chick embryos. Future work will focus on protein interaction studies in order to directly demonstrate the association between Prdm1 and the binding partners it co-localizes with.

Cadherins are a family of cell-surface proteins mediating adhesion that are important in development and maintenance of tissues. The family is defined by the repeating cadherin domain (EC) in their extracellular region, but they are diverse in terms of protein size, architecture and cellular function. The best-understood subfamily is the type I classical cadherins, which are found in vertebrates and have five EC domains. Among the five different type I classical cadherins, the binding interactions are highly specific in their homo- and heterophilic binding affinities, though their sequences are very similar. As previously shown, E- and N-cadherins, two prototypic members of the subfamily, differ in their homophilic K_D by about an order of magnitude, while their heterophilic affinity is intermediate. To examine the source of the binding affinity differences among type I cadherins, we used crystal structures, analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and electron paramagnetic resonance (EPR) studies. Phylogenetic analysis and binding affinity behavior show that the type I cadherins can be further divided into two subgroups, with E- and N-cadherin representing each. In addition to the affinity differences in their wild-type binding through the strand-swapped interface, a second interface also shows an affinity difference between E- and N-cadherin. This X-dimer interface, which is a weakly binding kinetic intermediate in E-cadherin, has a much stronger affinity in N-cadherin: nearly as strong as N-cadherin wild-type binding. In the swapped and X-dimer interactions of E- and N-cadherin, differences in hydrophobic surface area can mostly account for the affinity difference. However, several mutants of N-cadherin have a K_D an order of magnitude stronger even than the wild-type N-cadherin. In these mutants, the source of the strong affinity seems to be entropic stabilization through an equilibrium between multiple conformations with similar energies. We thus have a molecular-level understanding of vertebrate classical cadherins, with a detailed understanding of their adhesive mechanism and their binding affinity determinants. However, the adhesive mechanisms of cadherins from invertebrates, which are structurally divergent yet function in similar roles, remain unknown. We present crystal structures of the predicted N-terminal region of Drosophila N-cadherin (DN-cadherin). Of the 16 total predicted EC domains, we have crystallized the EC1-3 and EC1-4 segments. While the linker regions for the EC1-EC2 and EC3-EC4 pairs display binding of three Ca^2+ ions similar to that in vertebrate cadherins, domains EC2 and EC3 are joined in a bent orientation by a novel, previously uncharacterized Ca^2+-free linker. Based on sequence analysis of the further ECs of DN-cadherin, we predict another such Ca^2+-free linker between EC7 and EC8. Biophysical analysis demonstrates that a construct containing the first nine predicted EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins. Intriguingly, this segment contains both the crystallized and predicted Ca^2+-free linkers, suggesting a complex binding interface. Sequence analysis of the cadherin family reveals that similar Ca^2+-free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins. In cases of long cadherins, there are frequently multiple Ca^2+-free linkers in a single protein chain. It thus appears that a combination of calcium-binding and calcium-free linkers can allow cadherins to form three-dimensional arrangements that are more complex than the extended, calcium-rigidified structures in classical cadherins. Discovery of the Ca^2+-free linker, together with the differing numbers and arrangements of ECs and other domain types, implies that the cadherin superfamily is more structurally diverse than previously thought. Because little is known about the function and even less about the structure of the majority of the superfamily, studying the linear architecture (i.e. the precise sequence of ECs and the characteristics of the interdomain linkers) at the scale of the superfamily would give significant new insights on the structure and function of less-understood cadherins. With this motivation, we have constructed a cadherin database with relevant information on two different scales: the protein and the domain. On the whole protein level, we represent the architecture of each cadherin by recording the arrangement of ECs, different linker types, and other (non-EC) domain types in the protein. On the individual EC level, based on the sequence, we record the domain characteristics that give rise to the different structural features at the protein level. We have annotated over 9,600 proteins from 560 organisms, containing over 69,000 ECs; and built an online interface to search and access this information. Our aim is to provide a tool for understanding the protein architecture, function, and relationships among cadherins, a structurally diverse protein family. Together, these studies examine the relationships between sequence, structure and function of cadherins at different scales. In the classical cadherin study, small changes of one or two residues can dramatically alter the dimer conformations and thus lead to large differences in binding affinity between highly related cadherins, or between wild-type and mutant proteins. These seemingly small mutations can result in even higher binding affinity with the effect of entropic stabilization by multiple conformations. In DN-cadherin, the absence of certain calcium-binding motifs in adjacent ECs leads to a new linker type and a new interdomain orientation. This, in turn, has great implications in the global shape, and possibly the binding mechanism of the protein. The cadherin database aims to provide information at different structural levels in order to allow users to draw connections between primary sequence, domain structure and protein architecture, to ultimately learn about protein function.

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and the mechanistic pathway of covalent binding to nucleophilic thiols. The BQ binding by Cys34 on human serum albumin was studied, and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudo-first order rate constants (k) of methyl-, t-butyl-, and chlorine-substituted BQD reactions with NBT, whereas electron pair resonance (EPR) studies were performed to investigate the possible free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine substituted (activated) BQD to be more reactive toward NBT, than the methyl and t-butyl-substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA of NBT onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQD stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to allergenic potency. Hammett and Taft (HT) constants were then used to estimate the influence of these substituents on chemical reactivity. HT values demonstrated chlorine substituted BQD to be more reactive than methyl substituted BQD. BQ and BQD dermal allergenicity, as evaluated in the murine local lymph node assay, (LLNA) was consistent with that predicted by reactivity and HT parameters. These results demonstrate the effect of substituents on BQ reactivity and dermal allergic sensitization, and suggest the potential utility of chemical reactivity data and HT values for electrophilic allergen identification and potency ranking.

Copy of author's previously published article in pocket on back end-paper. Includes bibliography (leaves 135-185). Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 60-71).

Graduation date: 1991