Analysis of the molecular and physiological effects following treatment with BC204 in Arabidopsis thaliana and Solanum lycopersicum

Institute for Plant Biotechnolog

Genetic engineering of recombinant anti-mycolic acid antibody fragments for use in tuberculosis diagnostics

Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs.Dissertation (MSc)--University of Pretoria, 2012.Biochemistryunrestricte

The validation of nursing measures for patients with unpredictable outcomes

Ph.D. (Nursing Education)--University of the Witwatersrand, Faculty of Health Sciences, 2012The sciences of restorative nursing are unknown in South Africa, leaving patients with restorative needs with rather unpredictable outcomes. This study investigated the validity of four prospective nursing scales to be used for patients requiring nursing where the focus is to improve their functionality. Such patients are usually found in sub- and non-acute nursing units and suffering with chronic debilitating diseases, mental illness or recovering from trauma. Typically they are in need of rehabilitation, palliative care, geriatric services or long-term care to restore or maintain their functional independence. Inspired by the theories of nursing pioneers such as Florence Nightingale, the definitive nurse who was also an astute healthcare reform statistician, as well as Ida Jean Orlando, better known as the originator of the nursing process, the researcher, a general medical practitioner, has explored the intuitive knowledge of experienced nurses to document the links between their observations, interpretations and predictions of patient functioning. This information was used to develop four interrelated nursing scales to be used routinely by nurses to provide raw patient scores on patient functional changes. As nursing intuition was used to develop the measures, the working hypothesis was that the scales are considered valid. Therefore, the approach towards the study was deductive in nature, seeking the evidence to confirm this assumption. As the purpose of the study was to offer nurses useful scales to provide validated empirical evidence of human functional status, the research question was how scientific evidence can be used to conclude that these four scales have indeed the integrity to deliver a measurement function to the nurses. The researcher’s hypothesis of validating routine nursing measures is supported by two concepts: nursing utility and constructs validity. If nursing utility fail, construct validity is of no value to the nursing profession. With this in mind, the v study objectives were to first validate nursing utility using a qualitative design to collect descriptive data from nurses who have implemented the scales. Once positive findings were reported on the usefulness of the scales to the nursing profession, then construct validity was explored using the Rasch measurement model to qualitatively analyse the scale’s raw data collected in various sub- and non-acute nursing facilities. One scale was discarded, and three showed good to excellent results on both utility and construct validity. It has provided the restorative nursing sciences with a methodology to routinely collect patient-based empirical evidence for parametric analysis. In so doing, it delivered the missing link in Orlando’s nursing process theory; it also confirmed Nightingale’s theory that healthcare evidence provided routinely by nurse is the stepping stone for healthcare reform, provided it is useful, meaningful and valid. The ultimate beneficiaries of this new knowledge are patients who previously would have had unpredictable outcomes resulting in a poor prognosis

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD

Tools for strigolactone research : Towards a strigolactone-responsive promoter and a strigolactone-activity inhibitor

Thesis (MSc)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Strigolactones are a novel group of phytohormones reported to control branching in plants. Strigolactones also plays a pivotal role in the establishment of symbiotic relationships between plants and symbiotic fungi. Furthermore, its presence in the soil is responsible for the germination of the seeds of devastating plant parasitic plants known as broomrapes and witchweeds. They have also been implicated in playing roles in root development and architecture, secondary growth, adventitious root formation and leaf senescence. These phytohormones are derived from the carotenoid synthetic pathway, with β-carotene as the precursor. Several genes and the proteins (enzymes) they code for have been identified by reverse and forward genetics. A few components of the perception and signalling mechanism have been identified, but most of the pathway remains unknown. This highlights the need for more innovative scientific tools in order to fully elucidate the role of strigolactones in higher plants. A specific inhibitor of strigolactone signalling and strigolactone-responsive reporter are two tools that could aid in the further characterization of this poorly defined pathway. In this study, the effects of furanone-derivatives, with the main focus being on trimethylbutenolide (TMB), on the growth of Arabidopsis thaliana were investigated. The aim of this study was to determine if TMB is a competitive inhibitor of strigolactone signalling, because currently there are no known inhibitors of strigolactone signalling available. Having such an inhibitor would enable researchers to study the effects of strigolactones in non-model plant species where no mutants are available. Such an inhibitor would also aid in the further elucidation of the strigolactone signalling pathway. For the second part of this study, an attempt was made to create a strigolactone-response reporter construct in A. thaliana that is activated only in the presence of exogenously applied strigolactone. Having such a reporter-construct in A. thaliana would be valuable, as strigolactones are difficult to detect and quantify in these plants due to them being bioactive at picomolar concentrations in the plant. Such a reporter-construct would also aid in the further elucidation of the strigolactone pathway, the discovery of more functions and any interactions with other phytohormones and biochemical processes in the plant. During this study, it was found that the presence of TMB induced an increase in lateral root formation in wild-type A. thaliana seedlings, suggesting that it may act as a competitive inhibitor of strigolactone signalling, at least in terms of lateral rooting. It was also found that TMB affects the expression of a small group of strigolactone-responsive genes in an opposite way than GR24, a racemic mixture of strigolactone analogues. This effect on gene expression was observed after prolonged treatment of A. thaliana seedlings with TMB. For the second part of the project, we were unable to create a strigolactone-specific reporter construct, although the results suggest that the 990 bp region immediately upstream of AtBRC1 does respond more strongly to the presence of GR24 than the full, native promoter. It was also found that the 1480 bp and 990 bp regions immediately upstream of AtBRC1 is already severely deregulated version of the native AtBRC1 promoter. From these results, it was concluded that DNA binding motif/s for SMAX-like repressor proteins probably lie upstream of the 1480 bp promoter region of AtBRC1, while possible strigolactone-responsive motif/s lie downstream of the 990bp promoter region of AtBRC1.AFRIKAANSE OPSOMMING: Strigolaktone is ‘n nuwe groep fitohormone waaroor daar berig is dat hul vertakking in plante reguleer en beheer. Strigolaktone speel ook ‘n deurslaggewende rol in die aanknoping van simbiotiese verhoudings tussen plante en arbuskulêre mikorisaie swamme. Verder is die teenwoordigheid van strigolaktone in die grond ook verantwoordellik vir die ontkieming van die saadjies van verwoestende parasitiese plante beter bekend as besemrape en kopseerblomme. Daar word geïmpliseer dat hulle rolle speel in wortelontwikkeling en argitektuur, sekondêre groei, onverwagte wortelformasie en blaarveroudering. Hierdie fitohormone word afgelei van die karotenoïed sintese padweg, met beta-karoteen as die voorloper molekule. Verskeie gene en die proteine (ensieme) waarvoor hulle kodeer is reeds geidentifiseer met behulp van keer en stuur genetika. ‘n Paar komponente van die persepsie en sein meganismes is reeds geïdentifiseer, maar meeste van die padweg bly grootliks onbekend. Dit beklemtoon die behoefte vir meer innoverende wetenskaplike gereedskap om die volledige rol van strigolactone in hoër plante te ontrafel. ‘n Strigolaktoon sein-inhibeerder en strigolaktoon-spesifieke verklikker is twee instrumente wat kan help met die verdere karakterisering van hierdie swak gedefinieërde padweg. Deur die loop van hierdie studie is die effek van furanoon-afgeleides, met trimetielbutenoliet (TMB) as die hoof fokus, op die groei van Arabidopsis thaliana ondersoek. Die doel van hierdie studie was om te bepaal of TMB ‘n kompeterende inhibeerder van strigolaktoon-seine is, want tans is daar geen bekende inhibeerder van strigolaktoon-seine beskikbaar nie. Met so ‘n inhibeerder sal wetenskaplikes in staat wees om die effek van strigolaktone in nie-model plant spesies waar geen mutante beskikbaar is nie te ondersoek. So ‘n inhibeerder sal ook help met die verdure ontrafeling van die strigolaktoon sein padweg. Vir die tweede gedeel van hierdie studie is ‘n poging aangewend om ‘n strigolaktoon-reagerende verklikkerkonstruk te skep in A. thaliana wat slegs geaktiveer word in die teenwoordigheid van eksogene toegevoegde strigolaktone. So ‘n verklikker-konstruk in A. thaliana sal waardevol wees, want strigolaktone is moeilik om te bepaal en te kwantifiseer in hierdie plante, te danke aan die feit dat hul aktief is by piko-molêre konsentrasies in die plant. So ‘n verklikker-konstruk sou ook help met die verdure ontrafeling van volledige strigolaktoon-padweg, die ontdekking van meer funksies en enige interaksies met ander fitohormone and biochemiese prosesse in die plant. Tydens hierdie studie is daar bevind dat die teenwordigheid van TMB ‘n toename in laterale wortelvorming in wilde-tipe A. thaliana saailinge veroorsaak, wat daarop dui dat dit kan dien as ‘n kompeterende inhibeerder van strigolaktoon-seine, ten minste in terme van laterale wortels. Dit is ook bevind dat TMB ‘n invloed het op ‘n klein groupie van strigolaktoon-reagerende gene in ‘n teenoorgestelde manier as GR24, ‘n rasemiese mengsel van strigolaktoon-analoë. Hierdie effek op die uitdrukking van gene was waargeneem na langdurige behandeling van A. thaliana saailinge met TMB. Vir die tweede gedeelte van hierdie projek, was ons nie in staat gewees om ‘m strigolaktoon-spesifieke verklikker-konstruk te skep nie, alhoewel die resultate daarop dui dat die 900 bp streek onmiddellik stroomop van AtBRC1 sterker reageer in die teenwordigheid van GR24 as die volle, inheemse promotor. Daar is ook bevind dat die 1480 bp en 990 bp streke onmiddellik stroomop van AtBRC1 reeds ‘n dereguleerde weergawe van die inheemse AtBRC1 promotor is. Vanaf hierdie resultate is die gevolgtrekking gemaak dat DNS-bindende motief/motiewe vir SMAX-agtige onderdrukker proteïne waarskynlik verder stroomop van die 1480 bp promotor streek van AtBRC1 lê, terwyl moontlike strigolaktoon-reagerende motief/motiewe stroomaf van die 990 bp promoter streek geleë is

Die invloed van inligtingstegnologie op bestuursbesluitvorming

Proefskrif (M. Comm.) -- Universiteit van Stellenbosch, 1973.Full text to be digitised and attached to bibliographic record

Die plek van liggaamlike opvoeding in militêre opleiding in die Suid-Afrikaanse leër

MEd, North-West University, Potchefstroom Campus"No abstract"Master

Logistieke beplanning met behulp van objek-georienteerde simulasie

Study project (M.Phil.) -- University of Stellenbosch, 1999.ENGLISH SUMMARY: This report gives an overview of the combined use of object-oriented programming and simulation, with the objective of planning and controlling the logistic business functions. A case study to explain the application and a software package named SIMPLE++ to access the calculation is included. This package contains a simulation function to emulate the process by means of defined objects and their interaction. An object approach is used in the analysis of the reception and distribution function of a courier company, in order to evaluate the process: Results from the simulation study showed an imbalance in the capacities of the workstations. The company's utilisation of most of its resources is fairly good, but it is possible to obtain a more efficient process by restructuring its activities. This model is the very first electronic emulation that can be used as a planning, tool for the company's business activities, and in that way it fills a gap in its current planning function. The complete simulation model only reaches its potential when used as a strategic operational tool, by forecasting the expected results of changes in the working methods and physical structure.AFRIKAANSE OPSOMMING: Hierdie werkstuk bied 'n uiteensetting van die gekombineerde gebruik van objekgeorienteerde prograrnmering en simulasie, met die doel om 'n ondememing se logistieke funksies te beplan en te bedryf. 'n Gevallestudie word gebruik om die toepassing daarvan te verduidelik, en die berekeninge vir die simulasie word ondersteun deur rekenaarprograrnmatuuf bekend as SIMPLE++. Die program bied 'n simulasiebenadering vir die stelselnabootsing deur middel van objekformulering en interaksie tussen objekte . 'n Objekbenadering word gebruik in die analise van 'n koeriermaatskappy se ontvangste- en verspreidingsaanleg, met die oog op evaluering van die huidige stelsel. Die simulasieresultate het aangetoon dat daar 'n wanbalans in die kapasiteitsbelading van sornmige werkstasies bestaan. Die maatskappy se benutting van die meeste van sy hulpbronne is redelik goed, maar deur 'n herstrukturering van aktiwiteite is dit moontlik om 'n meer doeltreffende stelsel te verkry. Die model is die eerste elektroniese voorstelling en beplanningshulp beskikbaar van die maatskappy se aktiwiteite, en vul 'n leemte in hulle beplanningsfunksie. Die voltooide model bereik sy potensiaal wanneer dit gebruik word as strategiese operasionele gereedskapstuk, waarmee die verwagte resultate van aanpassings in die werkswyse en fisiese veranderinge voorspel kan word