Theses and Dissertations (Biochemistry)
http://hdl.handle.net/2263/32446
2024-03-29T06:55:00ZChemically synthetic mycolic acids as vaccine adjuvants
http://hdl.handle.net/2263/79305
Chemically synthetic mycolic acids as vaccine adjuvants
No abstract
Dissertation (MSc)--University of Pretoria, 2013.
2014-01-01T00:00:00ZDevelopment and evaluation of a bi-enzymatic nitric oxide reduction system
http://hdl.handle.net/2263/79235
Development and evaluation of a bi-enzymatic nitric oxide reduction system
Nitric oxide is a small diatomic molecule and is part of the nitrogen radical species. As a gas, it diffuses easily across cell membranes and is involved in numerous physiological processes and inflammation. This peculiar molecule has a dual role in inflammation. NO is one of the first signals to commence an innate immune response and it is involved in the resolution of inflammation. In the control of inflammation it is crucial to resolve NO bursts to promote tissue healing. The failure thereof results in the progression of inflammation with potentially catastrophic consequences for the host.
This study aimed to develop a nitric oxide reduction system as a research tool which could facilitate the understanding of the intricate role of NO in inflammation. Numerous chemical tools have been used to study NO biology, but were found to interfere in other metabolic pathways; thereby masking the role of NO. The nitric oxide reduction system entails the reduction of NO by nitric oxide reductase (NOR) with the concomitant oxidation of glucose by glucose dehydrogenase (GDH). The latter enzyme recycles the cofactor NADH in such a way that NO is continuously reduced. This bi-enzymatic cofactor recycling system presents the advantage of NO removal without any interference of metabolic pathways. Here, we propose that the continuous reduction of NO by the NOR system could be used to elucidate the role of NO in an innate immune response.
The construction of the NOR system commenced with development of fast and reliable spectrophotometric NADH-enzyme activity assay. This assay was essential for the quantification of enzyme activity and was used throughout the study for the purification of NOR, characterisation of NOR as well as the determination of enzyme activity maintenance after enzyme immobilisation. Both enzymes were immobilised onto five carriers with two different functional group chemistries and three functional group densities. The carboxyl functionalised carrier with the lowest functional group density was the most suitable immobilisation carrier by maintaining the highest enzyme activity for NOR and GDH. Upon co-immobilisation of both enzymes, an average of 0.088 μmoles NADH.min-1 for NOR and 0.077 μmoles NAD.min-1 for GDH cofactor oxidation rate was achieved. Furthermore, the cofactor was recycled six times with the concomitant consumption of the enzymes’ substrates. Subsequently, the NOR system was evaluated for its potential as a research tool in an in vitro inflammation model.
The continuous reduction of NO was established which highlights the NOR system suitability as a research tool. However, its evaluation as a potential anti-inflammatory reagent indicated that the chosen carrier has immunogenic properties of its own. The inflammation response elicited by this carrier alone was in part abrogated by the immobilisation of enzymes in the eventual NOR system assembly, thereby providing a scope for future work and further optimisation of this anti-inflammatory reagent.
Thesis (PhD)--University of Pretoria, 2013.
2013-01-01T00:00:00ZTranscriptional regulation underlying the quantitative genetic response of maize to grey leaf spot disease
http://hdl.handle.net/2263/79215
Transcriptional regulation underlying the quantitative genetic response of maize to grey leaf spot disease
Cercospora zeina causes grey leaf spot (GLS), a yield-limiting disease on maize. The main objective of this study was to exploit maize gene expression data to dissect the quantitative disease response to C. zeina infection. The project addresses the hypothesis that there is an underlying DNA polymorphism that gives rise to a change in gene expression, which in turn affects GLS disease severity. Genomic and functional annotation of the reporters on an Agilent 44K maize microarray was carried out. This microarray was used for global gene expression profiling of earleaf samples collected from 100 recombinant inbred sub-tropical maize lines exposed in the field to C. zeina. Gene expression profiles together with GLS severity scores were used in a weighted gene co-expression network analysis to identify co-expression modules associated with disease severity. Quantitative trait locus (QTL) mapping for GLS severity was combined with expression QTL (eQTL) analyses to investigate the molecular basis of the quantitative response to GLS. An Eqtl data analysis pipeline was developed in Galaxy. The overlap of phenotypic QTLs with cis- and trans-eQTLs revealed putative causal candidate genes and potential mechanisms responsible for the QTLs, respectively. Regulatory network models were constructed for trans-eQTL hotspots coinciding with phenotypic QTLs. A genetic basis for coordinated expression responses to GLS disease was identified. For the susceptible response, the results lead to the hypothesis that a calmodulin-related protein with a cis-eQTL acts as a global regulator of various pathogenesis-related proteins that are activated too late after infection started. For the resistant response, it is hypothesised that a serine threonineprotein kinase with a cis-eQTL acts as a post-translational global regulator regulating phosphatases and kinases involved in activation of defense gene expression. The outcomes of this study were: i) the development of a systems genetics strategy and ii) several hypotheses of maize transcriptional responses to C. zeina which need to be validated with further studies. These results extend the current knowledge of GLS resistance and could aid in the improvement of maize varieties.
Thesis (PhD)--University of Pretoria, 2014.
2014-01-01T00:00:00ZBasal promoter landscape in Eucalyptus grandis : annotation of distal transcription start sites and core promoter usage
http://hdl.handle.net/2263/79196
Basal promoter landscape in Eucalyptus grandis : annotation of distal transcription start sites and core promoter usage
Transcription is a complex biological phenomenon, whereby RNA is transcribed from single
stranded template DNA by assembling targeted regulatory inputs at the promoter region.
Transcription is regulated through many hierarchically organised mechanisms, including
chromosome positioning and organisation, the binding of transcription factors, and DNA’s
secondary and tertiary structures at the region of transcription initiation. The core promoter is
the distinct functional unit of DNA overlapping the transcription start site, which possesses
linear regulator capacity and renders DNA permissive to transcription. In plants, core
promoter and enhancer studies are of particularly high impact for those traits which under
strong transcriptional control. Cellulose biosynthesis in immature xylem, the tissue which
forms wood, is one such trait, and is studied extensively in the herbaceous model plant
organism, Arabidopsis thaliana, and the economically important woody perennial,
Eucalyptus grandis. The release of the E. grandis genome sequence has provided a muchneeded
reference to study transcriptional control, not only for those traits that make it a
dominant fibre crop, but genome-wide. We aimed to use empirical transcript evidence to
perform a high-throughput genome-wide curation of the 5’ UTR annotations and empirically
infer transcription start sites (TSSs) of the nascent E. grandis genome annotation. We then
aimed to use the curated TSSs to define core promoter classes based on their sequence
Magister composition and to determine the putative expression profiles and functional associations of each.
We used deep E. grandis mRNA sequencing data across seven diverse tissues and PASA
assembled E. grandis ESTs to empirically curate 5’ UTR annotations. We improved 17,085
annotations, added 7,596 for which there was no previous annotation and retained 3,675 that
possessed only a predicted TSS without empirical evidence. These complementary data were
used to define distal transcription start sites (dTSS) by a novel, prioritising, computational
rule-based method. From these dTSS annotations, we extracted the core promoters (from
-100 to +50) and described the core promoter landscape by hexamer positional overrepresentation
analysis. We found three types of hexamer over-representation in the core
promoter, that being broad, spiked and low. Broad hexamers were classified into 5 distinct
core promoter classes, including TA, CT, GA, W and S. These were further assessed for
putative expression profiles (specificity and level) and functional associations. TA resembles
the conserved TATA-box core promoter, although displays a bimodal distribution, low
expression levels and the greatest tissue specificity. CT and GA are over-represented both up
and downstream of the dTSS and show narrow windows of greater enrichment with phasic
constraint. W and S occur in close proximity to the dTSS, with S displaying the most
constitutive and highest expression profile. Spiked hexamers occur in close proximity to the
dTSS and low hexamers are enriched for those pyrimidine-rich hexamers found in
Arabidopsis thaliana and Oryza sativa core promoters as the Y Patch. We found that E.
grandis core promoters include those such as the TATA-box class which is conserved across
kingdoms, the CT and GA classes, which are conserved in Arabidopsis, and a number of
classes which, thus far, appear unique to Eucalyptus. We postulate possible underlying
mechanisms of each core promoter class based on their sequence composition and suggest
regulation by TBP binding (TA), nucleosome positioning (W), DNA stability (S), and non-BDNA
conformation (CT and GA). This research provides a basal understanding of cistranscriptional
regulation at the core promoter in this economically important woody plant
species and provides insight into the mechanisms of permissive transcription across plant
species.
Dissertation (MSc)--University of Pretoria, 2014.
2014-01-01T00:00:00Z