The study of cationic amphiphilic peptides with anti-cancer selective toxicity

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014.The exposure of organisms to environmental stresses and pathogens results in rapid activation of a range of defensive pathways that act as part of the innate immune system. The most common innate immunity response is the activation of cationic amphiphilic peptides in response to microbial infection. Moreover, cationic amphiphilic peptides possess desirable attributes for the pharmaceutical development of cancer-selective drugs. They selectively and rapidly kill cancer cells without killing normal mammalian cells and have a broad spectrum of mechanisms of action. The aim of this exploratory study was to screen for cationic amphiphilic peptides with anti-proliferative activity that is induced by genotoxicity. GeneFishing® technology, 2-D gel analysis and bioassays were used to identify and analyse molecules induced in response to genotoxic stress in an embryonic cell line originating from the dung beetle Euoniticellus intermedius. Bioassay results revealed that the cell line has constitutive expression of probable cationic amphiphilic proteins that are further induced by camptothecin treatment. GeneFishing® and 2-D gel analysis showed changes in gene expression at both transcriptional and translational levels, respectively. Overall, the study failed to identify the involvement or induction of cationic amphiphilic peptides in response to genotoxic stress. However, gene expression analyses revealed changes in the expression of classes of proteins involved in stress response, oxidative phosphorylation, mitochondrial maintenance, protein translation, cytoskeletal proteins and immunophilins. The results show that the cell line constitutively expresses probable cationic amphiphilic peptides which are further induced by camptothecin

Is inflation targeting an appropriate framework for monetary policy? : experience from the inflation-targeting countries

Is inflation targeting an appropriate framework for monetary policy? Experience from the inflation-targeting countries countries are optimistic about inflation targeting as a monetary-policy framework. South Africa is also following this trend. The international literature review of the topic offers lessons to be learnt from the common experience of the countries considered. It shows that inflation targeting is not a universal remedy to modern economic ills -- there is an emerging danger of assigning monetary policy a larger role than that which it can perform; a danger of expecting monetary policy to accomplish tasks that it cannot achieve; and a danger of preventing monetary policy from making the contribution that it is capable of doing. Therefore, inflation targeting cannot address all the macroeconomic problems that face many countries, except for inflation. Nonetheless, it plays a crucial role in improving macroeconomic performance.EconomicsM.A. (Economics

Characterisation of N-terminal fragments of Retinoblastoma Binding Protein 6 for structural analysis

>Magister Scientiae - MScRetinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa RING finger-containing protein that plays a role in 3'-end poly-adenylation of mRNA transcripts as well as acting as an E3 ubiquitin ligase against a number of proteins involved in tumourigenesis, including p53. Since the human protein is too large and poorly structured for heterologous expression in bacteria, it would be advantageous to identify smaller fragments suitable for expression in bacteria. Many E3 ubiquitin ligases form homo-dimers and dimerisation is important for their activity; structural studies of the isolated RING finger of RBBP6 showed that it forms a weak homo-dimer. This poses the question of whether the complete RBBP6 protein forms homo-dimers in vivo, and, if so, whether a fragment of RBBP6 containing the RING finger could be identified which would be suitable for structural as well as functional studies. Such a construct would allow detailed investigation of the homo-dimeric state of the fragment, the relationship between dimerisation and ubiquitination activity, and the role of domains such as the DWNN domain and zinc finger in ubiquitination. A fragment consisting of the first 335 residues of RBBP6, dubbed R3 because it contained the first three domains of the protein, was expressed, along with three variants expressing mutations known to disrupt the dimerisation of the isolated RING finger. Size exclusion chromatography showed that R3 forms a strong homo-dimer that was not disrupted by the mutations, suggesting that additional parts of R3 outside of the isolated RING finger form part of the interface. To identify whether this included the DWNN domain or the zinc finger, a shorter fragment dubbed R2, excluding the N-terminal DWNN domain, was cloned and expressed. This was also found to form a strong homo-dimer, suggesting that the DWNN domain may not form an essential part of the dimer interface. Availability of the RING finger samples and monomerising mutations allowed investigation of whether the RING finger from RBBP6 was able to auto-ubiquitinate itself. Using a fully in vitro ubiquitination assay supplemented with intact proteasomes purified from human cell lysates, we found that wild type RING auto-ubiquitinates itself very efficiently, catalysing its own destruction in the proteasome. This provides an answer to the question of why RBBP6 is so difficult to detect in mammalian cells. Surprisingly, monomeric mutant RING fingers were also able to auto-ubiquitinate and catalyse their own destruction, although perhaps not as efficiently as wild type. This result would appear to rule out the hypothesis that dimerisation of RBBP6 is required for ubiquitination activity. Finally, samples of the RING finger from human MDM2 were expressed in bacteria and used to investigate whether the RING fingers of RBBP6 and MDM2 interact directly with each other. If so, this may provide a mechanism whereby RBBP6 and MDM2 cooperate in ubiquitination of p53. The results of a GST pull down assay using GST-MDM2-RING as ''bait'' and RBBP6-RING as ''prey'' provides evidence that such an interaction between the RING does exist. This work lays the foundation for future structural studies of the RING-RING hetero-dimer using protein Nuclear Magnetic Resonance Spectroscopy

Leveraging Dendrimer Macromolecules for the Encapsulation and Stabilisation of Nano-Sized Ruthenium Catalysts: Evaluation of Catalytic Reaction Kinetics in the Reduction of Pollutants Organic Dyes, Oxidation of Alcohols and Alkenes as Well as Hydrogenation Reactions

Encapsulation of nano-sized metal catalysts within the dendrimers macromolecules’ frameworks has been well documented thus far. Dendrimers are described as symmetric, monodispersed macromolecules resembling a tree-like branched structure and have been utilised as both a template and stabilising agent for the fabrication of metal (noble and non-noble) nano-catalysts. For this purpose, different types of dendrimers can be employed. The use of dendrimers for metal catalysts stabilisation or encapsulation offers several advantages in catalysis. For example, the dendrimer template allows the synthesis of catalytically active monodispersed nanoparticles and the dendrimers template itself does not passivate the metal active atoms during the catalytic process. Additionally, dendrimers have the potential to act as a “vehicle” that can be leveraged for the fabrication of heterogeneous catalysts. For example, surface groups of the dendrimers can be functionalised to chemically link the dendrimer-encapsulated nanoparticles (DENs) with solid supports such as silica. A significant number of studies on the synthesis and catalytic evaluation of dendrimer-metal nanocomposite materials (e.g. Ruthenium-based) onvarious reactions can be found in the literature. This chapter, however, will particularly focus on the recent developments on the synthesis, characterisation and catalytic applications of dendrimer-derived (colloidal and supported) Ruthenium catalysts

ВИКОРИСТАННЯ ІМПУЛЬСНОГО МЕТОДУ ВИМІРЮВАННЬ ДЛЯ ВИЗНАЧЕННЯ ПОШКОДЖЕННЯ КАБЕЛЬНИХ ЛІНІЙ

В даній роботі розглядаються особливості використання приладу ІКЛ-5 для швидкого визначення точності імпульсних вимірювань в пошкодженнях кабельних ліній і точного знаходження міста пошкодження кабелю. Для досягнення поставленої мети запропонований імпульсний метод, який визначає місце і характер пошкодження кабельних ліній. Обраний у роботі підхід дає можливість вимірювання при будьякому складному характері ушкодження. Завдяки цьому забезпечується зручність, простота і швидкість вимірювань

MphayaNER: Named Entity Recognition for Tshivenda

Named Entity Recognition (NER) plays a vital role in various Natural Language Processing tasks such as information retrieval, text classification, and question answering. However, NER can be challenging, especially in low-resource languages with limited annotated datasets and tools. This paper adds to the effort of addressing these challenges by introducing MphayaNER, the first Tshivenda NER corpus in the news domain. We establish NER baselines by \textit{fine-tuning} state-of-the-art models on MphayaNER. The study also explores zero-shot transfer between Tshivenda and other related Bantu languages, with chiShona and Kiswahili showing the best results. Augmenting MphayaNER with chiShona data was also found to improve model performance significantly. Both MphayaNER and the baseline models are made publicly available.Comment: Accepted at AfricaNLP Workshop at ICLR 202

Coordination chemistry of amide-functionalised tetraazamacrocycles: structural, relaxometric and cytotoxicity studies

Three different tetraazamacrocyclic ligands containing four amide substituents that feature groups (namely allyl, styryl and propargyl groups) suitable for polymerisation have been synthesised. Gadolinium(III) complexes of these three ligands have been prepared as potential monomers for the synthesis of polymeric MRI contrast agents. To assess the potential of these monomers as MRI contrast agents, their relaxation enhancement properties and cytotoxicity have been determined. A europium(III) complex of one of these ligands (with propargyl substituents) is also presented together with its PARACEST properties. In addition, to gain further insight into the coordination chemistry of the tetra-propargyl substituted ligand, the corresponding zinc(II) and cadmium(II) complexes have been prepared. The X-ray crystal structures of the tetra-propargyl ligand and its corresponding gadolinium(III), zinc(II) and cadmium(II) complexes are also presented

Exploring steric and electronic parameters of biaryl phosphacycles

Please read abstract in article.Research Centre for Synthesis and Catalysis (University of Johannesburg) and Sasol (Pty) Ltd as well as the Centre for High Performance Computing (CHPC), South Africa for providing computational resources.https://pubs.rsc.org/en/journals/journal/njhj2023Chemistr

Development of integrated processes for the coproduction of inulin, protein, and ethanol from Jerusalem artichoke tubers in a biorefinery

Thesis (PhD)--Stellenbosch University, 2021.ENGLISH ABSTRACT: Biorefining is an attractive approach to simultaneously address food supply, energy security and global warming. Furthermore, biorefining offers a sustainable strategy to utilise biomass, for energy production, thereby, reducing overreliance on fossil resources. The principal aim of the study was to evaluate the feasibility of a Jerusalem artichoke (JA) tuber-based biorefinery by integrating protein extraction to the conventional inulin extraction process, and subsequently hydrolysing the tuber residues with an enzyme cocktail of crude inulinases and commercial Cellic® CTec3 and Pectinex, for ethanol fermentation. The tuber mash was pressed for protein extraction from the juice and water extraction from the solid residues. Sequential water-extraction was used for protein and inulin, in the first and second step, respectively. The resulting tuber residues from the sequential extraction was hydrolysed and fermented into ethanol. Fed-batch culture was used to optimise the bioprocess conditions for recombinant endoinulinases production by Aspergillus niger. Comparison of sequential extraction sequences demonstrated that protein extraction in the first and second step, respectively, maximised the selectivity of the extraction and product yields. Both extraction steps utilised water as a solvent, and were optimised with respect to pH, solids loading and temperature for the selective extraction from each dedicated step from tubers. The soluble protein fraction contained a cumulative 71.8% of the protein present in tubers, while 17.1% was present in the inulin extracted in the subsequent step. The inulin yield was 67.6% of the inulin in the tubers, while 11.8% was co-extracted with the protein product. The protein extract was augmented by protein present in the press juice, obtained from tubers prior to the water extraction steps. High cell density fermentation of Aspergillus niger for recombinant endoinulinase production, was achieved through an exponential fed batch method. Endoinulinase production was growth associated at higher growth rates, achieving the highest volumetric activity (670 U/ml) and biomass concentration (33 g/L) at a growth rate (μ) of 0.07 h-1. Moreover, the significant decrease in enzyme activity (506 U/ml) and biomass substrate yield (0.043 gbiomassDW/gglucose) at low μ (0.04 h-1) was due to the high maintenance energy requirement. High biomass concentrations resulted in broth viscosity, which necessitated increased agitation for mixing and oxygen transfer. However, this led to pellet disruption and biomass growth in mycelial. Moreover, enzyme production profiles, product (Yp/s) and biomass (Yx/s) yield coefficients were not affected. High gravity simultaneous saccharification and fermentation (SSF) of the extraction residues, enriched in cellulose and inulin, was achieved with an optimised cocktail of enzymes. A combined inulin and cellulose conversion yield of 74% was achieved during fermentation at 21% w/v solids loading. The optimised enzyme cocktail improved the saccharification and fermentation of the residues, with an ethanol concentration and yield of 38 g/L and 83%, respectively, compared to an unoptimized cocktail with the same protein dosage, with 32 g/L and 59%, respectively, both at the maximum attainable solids loading of 21% w/v. Therefore, the current data demonstrated the potential of integrating a protein extraction with conventional inulin extraction from JA tubers and fermenting the residues into ethanol with an optimised enzyme cocktail.AFRIKAANSE OPSOMMING: Bioraffinering is ’n aantreklike benadering om gelyktydig voedselverskaffing, energiesekuriteit en aardverwarming aan te spreek. Verder bied bioraffinering ’n volhoubare strategie om biomassa te gebruik, wat aanhorigheid op fossielhulpbronne verminder. Die hoofdoel van die studie was om die uitvoerbaarheid van ’n aardartisjok (JA) -knolgebaseerde bioraffinadery te evalueer deur proteïenekstraksie saam met die konvensionele inulienekstraksieproses te integreer, en vervolgens die knolresidu’s met ’n ensiemmengsel van ru-inulienase en kommersiële Cellic® CTec3 en Pectinex te hidroliseer, vir etanolfermentasie. Die knolmengsel is gepers vir proteïenekstraksie vanuit die sap-en-waterekstraksie vanuit die soliede residu’s. Sekwensiële waterekstraksie is gebruik vir proteïen en inulien, in die eerste en tweede stap onderskeidelik. Die resulterende knolresidu’s vanuit die sekwensiële ekstraksie is gehidroliseer en gefermenteer na etanol. Gevoerde-lotkultuur is gebruik om die bioproseskondisies vir rekombinante endo-inulienaseproduksie deur Aspergillus niger te optimeer. Vergelyking van sekwensiële ekstraksiereekse het gedemonstreer dat proteïenekstraksie in die eerste en tweede stap, onderskeidelik, die selektiwiteit van die ekstraksie- en produkopbrengste gemaksimeer het. Beide ekstraksiestappe het water as oplosmiddel gebruik, en is geoptimeer met betrekking tot pH, vastestoflading en temperatuur vir die selektiewe ekstraksie vanuit elke toegewyde stap van knolle. Die oplosbare proteïenfraksie het ’n kumulatiewe 71.8% van die proteïen teenwoordig in knolle, bevat, terwyl 17.1% teenwoordig was in die inulien wat geëkstraheer is in die opvolgende stap. Die inulienopbrengs was 67.6% van die inulien in die knolle, terwyl 11.8% geëkstraheer is saam met die proteïenproduk teenwoordig in die perssap, verkry uit knolle voor die waterekstraksiestappe. Hoë seldigtheid van Aspergillus niger vir rekombinante endo-inulienaseproduksie is bereik deur ’n eksponensiële voerlotmetode. Endo-inulienaseproduksie is groei geassosieer by hoër groeitempo’s, wat die hoogste volumetriese aktiwiteit (670 U/ml) en biomassakonsentrasie (33 U/ml) by ’n groeitempo (μ) van 0.07 h-1, bereik het. Verder het die hoë onderhoudenergievereiste ’n beduidende afname in ensiemaktiwiteit (506 U/ml) en biomassa-substraatopbrengs (0.043 gbiomassaDW/gglukose) by lae μ (0.04 h-1) tot gevolg gehad. Hoë biomassakonsentrasies het sopviskositeit tot gevolg gehad, wat verhoogde roering vir vermenging en suurstofoordrag genoodsaak het. Hierdie het wel tot korrelversteuring en biomassagroei in miseliaal gelei. Ensiemproduksieprofiele, produk- (Yp/s) en biomassa- (Yx/s) opbrengskoëffisiënte is verder nie geaffekteer nie. Hoë gravitasie, gelyktydige sakkarifikasie en fermentasie (SSF) van die ekstraksieresidu’s, verryk in sellulose en inulien, is bereik met ’n geoptimeerde mengsel van ensiemes. ’n Kombinasie van inulien en sellulose omsettingsopbrengs van 74% is bereik deur fermentasie by 21% w/v vastestoflading. Die geoptimaliseerde ensiemmengsel het die sakkarifikasie en fermentasie van die residu’s verbeter, met ’n etanolkonsentrasie en -opbrengs van 38 g/L en 83%, onderskeidelik, in vergelyking met ’n mengsel wat nie geoptimeer is nie met dieselfde proteïendosis, beide by die maksimum vastestoflading van verkrygbare vastestoflading van 21% w/v. Daarom het die huidige data die potensiaal gedemonstreer van integrering van ’n proteïenekstraksie met konvensionele inulienekstraksie uit JA-knolle en fermentering van die residu’s na etanol met ’n geoptimeerde ensiemmengsel.The financial assistance of the Centre for Renewable and Sustainable Energy Studies (CRSES) and National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the CRSES and NRF.Doctora