Philosophiae Doctor - PhD (Biotechnology)

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3,3' Diindolylmethane mediated signalling and its role in Brassica napus L. responses to vanadium
(University of the Western Cape, 2016) Gokul, Arun; Keyster, Marshall
Anthropogenic activities such as mineral mining, improper watering practices, and the use of heavy metal contaminated fertilizers have caused an influx of heavy metals into arable lands. These heavy metals may have a negative impact on plant growth, as they are able to increase ROS species within plants resulting in plant metabolism deterioration and tissue damage. Heavy metals also have the ability to render important enzymes non-functional or may decrease their activity resulting in poor growth. Vanadium was used as the heavy metal of choice in this study, as South Africa is one of the top producers of this metal worldwide. In an effort to improve growth of crop plants, mechanisms have to be identified to increase growth under vanadium stress. One method to increase growth is the use of exogenously applied signalling molecules. In this study, one such compound 3,3' Diindolylmethane (DIM) was investigated to identify whether it had growth promoting properties.
Actinobacteria associated with two diverse soil environments and their multicopper oxidase diversity
(University of the Western Cape, 2024) Prins, Alaric; McCullough, Bronwyn Kirby
The Cape Floristic Region (CFR) is a biodiverse region boasting unique plant diversity with a rich concentration of endemic plants. Aspalathus linearis (Rooibos) is an indigenous plant that grows in the Clanwilliam region of the Western Cape and is cultivated for its use as an herbal tea. Emerging peatlands in the CFR have gained increasing attention over recent years through research aiming to understand the microbial diversity associated with these environments. Little is known about the actinobacterial diversity of these regions, and as such, it is necessary to investigate the diversity of the actinobacteria associated with these environments, whilst simultaneously gaining knowledge on whether the associated actinobacteria may produce enzymes of biotechnological interest. Two CFR regions (the Rooibos environment – Clanwillian, and the Springfield emerging peatland environment – Agulhas) were explored through culture-based and genomic screening. Metabarcoding analyses using actinobacterial-specific 16S rRNA gene primers showed that the major taxa contributing to the Rooibos environment were members of the families Mycobacteriaceae, Pseudonocardiaceae, Frankiaceae and Geodermatophilaceae. Members of the families Mycobacteriaceaea, Pseudonocardiaceae, Acidimicrobiaceae and Nocardioiaceae was identified as the major taxa for the Springfield environment. Through selective isolation techniques, actinobacteria from rare (underrepresented) genera were isolated, including members of the genera Dactylosporangium, Actinokineospora, Curtobacterium, Modestobacter, Leifsonia and Actinomadura. The top strains, selected based on exhibiting extracellular multicopper oxidase (MCO) activity through culture-based screening, were subjected to whole genome sequence analysis. These rare genera are also vastly underrepresented among 3 400 bacterial MCO sequences found in the Laccase and Multicopper Oxidase Engineering Database (LccED).
Analysis of the early events in the interaction between Venturia inaequalis and the susceptible Golden Delicious apple (Malus x domestica Borkh.)
(University of the Western Cape, 2014) Hüsselmann, Lizex Hollenbach Hermanus; Ndimba, Bongani K.
Apple (Malus x domestica) production in the Western Cape, South Africa, is one of the major contributors to the gross domestic product (GDP) of the region. The production of apples is affected by a number of diseases. One of the economically important diseases is apple scab that is caused by the pathogenic fungus, Venturia inaequalis. Research to introduce disease resistance ranges from traditional plant breeding through to genetic manipulation. Parallel disease management regimes are also implemented to combat the disease, however, such strategies are increasingly becoming more ineffective since some fungal strains have become resistant to fungicides. The recently sequenced apple genome has opened the door to study the plant pathogen interaction at a molecular level. This study reports on proteomic and transcriptomic analyses of apple seedlings infected with Venturia inaequalis. In the proteomic analysis, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify apple leaf proteins extracted from infected and uninfected apple seedlings. Using MelanieTM 2-DE Gel Analysis Software version 7.0 (Genebio, Geneva, Switzerland), a comparative analysis of leaf proteome expression patterns between the uninfected and infected apple leaves were conducted. The results indicated proteins with similar expression profiles as well as qualitative and quantitative differences between the two leaf proteomes. Thirty proteins from the apple leaf proteome were identified as differentially expressed. These were selected for analysis using a combination of MALDI-TOF and MALDI-TOF-TOF MS, followed by database searching. Of these spots, 28 were positively identified with known functions in photosynthesis and carbon metabolism (61%), protein destination and storage (11%), as well as those involved in redox/response to stress, followed by proteins involved in protein synthesis and disease/defence (7%), nucleotide and transport (3%). RNA-Seq was used to identify differentially expressed genes in response to the fungal infection over five time points namely Day 0, 2, 4, 8 and 12. cDNA libraries were constructed, sequenced using Illumina HiScan SQTM and MiSeqTM instruments. Nucleotide reads were analysed by aligning it to the apple genome using TopHat spliceaware aligner software, followed by analysis with limma/voom and edgeR, R statistical packages for finding differentially expressed genes. These results showed that 398 genes were differentially expressed in response to fungal infection over the five time points. These mapped to 1164 transcripts in the apple transcripts database, which were submitted to BLAST2GO. Eighty-six percent of the genes obtained a BLAST hit to which 77% of the BLAST hits were assigned GO terms. These were classed into three ontology categories i.e. biological processes, molecular function and cellular components. By focussing on the host responsive genes, modulation of genes involved in signal perception, transcription, stress/detoxification, defence related proteins, transport and secondary metabolites have been observed. A comparative analysis was performed between the Day 4 proteomic and Day 4 transcriptomic data. In the infected and uninfected apple leaf proteome of Day 4, we found 9 proteins responsive to fungal infection were up-regulated. From the transcriptome data of Day 4, 162 genes were extracted, which mapped to 395 transcripts in the apple transcripts. These were submitted to BLAST2GO for functional annotation. Proteins encoded by the up-regulated transcripts were functionally categorised. Pathways affected by the up-regulated genes are carbon metabolism, protein synthesis, defence, redox/response to stress. Up-regulated genes were involved in signal perception, transcription factors, stress/detoxification, defence related proteins, disease resistance proteins, transport and secondary metabolites. We found that the same pathways including energy, disease/defence and redox/response to stress were affected for the comparative analysis. The results of this study can be used as a starting point for targeting host responsive genes in genetic manipulation of apple cultivars.
Analysis, expression profiling and characterization of hsa-miR-5698 target genes as putative dynamic network biomarkers for prostate cancer: a combined in silico and molecular approach
(University of the Western Cape, 2019) Lombe, Chipampe Patricia; Pretorius, Ashley; Meyer, Mervin
2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.
Aptamer selection against GFRa1 for its application in the prognosis of breast cancer
(University of the Western Cape, 2019) Swartz, Lauren Taryn; Meyer, Mervin
Breast cancer is the second most common cancer amongst South African women. Despite ongoing efforts to combat breast cancer, current prognostic and/or therapeutic monitoring methods are limited since very little improvement, in the rate of long term recurrence of breast cancer, has been observed. Considering this, developing novel strategies to detect breast cancer recurrence – at an early onset – is crucial for monitoring the disease and potentially preventing disease progression. Methods currently used for the detection of BC are costly and can also be very uncomfortable for the patient. These methods are also too costly to use as a routine test, following surgery or treatment to assess disease progression. Thus, developing a cost-effective detection method appears to be an appealing alternative. Serum/blood-based biomarkers are ideal targets for the development of low cost detection assays. Two candidate biomarkers, unique ligand binding protein 2 (ULBP2) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFR1) were identified using bioinformatics and proteomics, respectively. These biomarkers have demonstrated to be useful prognostic biomarkers for breast cancer. The selection of aptamers against these biomarkers can facilitate the development of cost-effective detection methods. Aptamers are short DNA or RNA oligonucleotides that have very high affinity and specificity for its targets and can potentially replace antibodies as tools for molecular recognition in detection systems, such as the enzyme-linked immunosorbent assay (ELISA), lateral flow assays and electrochemical biosensors.
Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens
(University of Western Cape, 2021) King, Maria Catharina; Kirby-McCullough, Bronwyn
Actinobacteria, a Gram-positive phylum of bacteria found in both terrestrial and aquatic environments, are well-known producers of antibiotics and other bioactive compounds. The isolation of actinobacteria from unique environments has resulted in the discovery of new antibiotic compounds that can be used by the pharmaceutical industry. In this study, the fynbos biome was identified as one of these unique habitats due to its rich plant diversity that hosts over 8500 different plant species, including many medicinal plants. In this study two medicinal plants from the fynbos biome were identified as unique environments for the discovery of bioactive actinobacteria, Aloe ferox (Cape aloe) and Sutherlandia frutescens (cancer bush).
Biological approach to improving the evaporation rates of mine wastewater desalination brine treated in evaporation ponds
(University of the Western Cape, 2021) Moyo, Anesu Conrad; Trindade, Marla
The disposal of brine effluent from inland wastewater desalination plants is a growing global problem with adverse economic and environmental implications because of the substantial cost associated with its disposal and the potential for polluting groundwater resources. Currently, the best and most economical option for brine disposal from inland desalination plants is the use of evaporation ponds, which concentrate the liquid until getting a solid waste that can be valued or directly managed by an authorized company. The effectiveness of these ponds is therefore dependent on the evaporation rate, which has previously been improved by the addition of dyes such as methylene blue. However, the addition of chemical dyes to the evaporation ponds poses a threat to the environment, wildlife, and humans.
The C-economy, nutritional benefits and symbiotic performance of dual inoculated Phaseolus vulgaris (L.) plants, under variable nutrient conditions
(University of the Western Cape, 2010) Mortimer, Peter E; Valentine, AJ
The tripartite symbiosis between Phaseolus vulgaris, arbuscular-mycorrhiza and the nodule bacteria, Rhizobia have been the focus of many studies ranging over a number of decades, however these studies have failed to answer certain questions relating the role of the symbionts in regard to host nutrition and the subsequent influence of these symbionts on the host C- economy. There is little doubt over the synergistic benefits involved in the dual inoculation of bean plants, as well as the resultant C-costs of maintaining the 2 symbionts, yet the specific contribution of the individual symbionts to the hosts overall nutrient and C-economy remain to be clarified. Thus the aim of this thesis is to help clarify these points by determining the symbiont induced photosynthetic, respiratory and nutritional changes taking place in the host. This was achieved by a series of experiments in which nodulated bean plants were split into two categories-those with and without AM colonized roots. These plants were then exposed to a range of growing conditions, including hi and low P, and a series of N treatments, ranging from zero N through to 3 mM NH/. Under these differing nutrient conditions growth, photosynthetic, respiratory, nutrient and amino acid responses were monitored, thus allowing for the determination of the symbionts influence on the host and the hosts reliance on the respective symbionts. Host reliance was noted most strongly under nutrient limiting conditions. Under low P treatment AM was the dominant symbiont as far as host C was concerned, allowing for the early establishment of the AM, thus ensuring the uptake of P for both host and nodule development. High P affected AM colonization to a greater extent than it did nodule dry weight and conversely the addition of N~ + led to a greater decrease in nodule dry weight than it did AM colonization. In spite of this decline, AM benefited the host by improving host N nutrition and relieving N-feedback inhibition of the export amino acid asparagine on BNF. These AM induced benefits did come at a cost to the host though, the dual inoculated plants had higher below ground respiratory costs and subsequently higher photosynthetic rates to compensate for the increased demand for C. The higher photosynthetic rates associated with dual inoculation were as a result of symbiont induced sink stimulation and not due to the improved nutrition of the host, as shown by the photosynthetic and nutrient response ratios. However, the respiratory costs associated with the uptake of soil nutrients were lower in AM colonized roots, thus showing an increased efficiency in nutrient gain by AM colonized roots. This improvement in host N nutrition as a result of AM colonization, coupled with the lower respiratory costs of AM nutrition led to the conclusion that under certain growing conditions nodules can become redundant and possibly parasitic.
Characterisation of AtPNP-A - a novel arabidopsis thaliana gene with role in water and salt homeostasis
(University of the Western Cape, 2009) Bastian, René; Gehring, Chris A.; NULL; Faculty of Science
Plant natriuretic peptides (PNPs) are a novel class of extracellular, systemically mobile molecules that elicit a number of plant responses important in homeostasis and growth. Natriuretic peptides were first identified in vertebrates where they play a role in the regulation of salt and water balance. Subsequent experimental investigations have identified the presence of a natriuretic peptide hormone system in plants. While PNPs have been implicated in various physiological responses such as stomatal guard cell movements and regulation of net water uptake, its biological role has remained elusive. Here we have used co-expression and promoter content analysis tools to understand the biological role of the Arabidopsis thaliana PNP (AtPNP-A). The analysis of AtPNP-A and its co-expressed genes revealed that genes annotated as part of the systemic acquired resistance (SAR) pathway were over-represented, thus suggesting that AtPNP-A may function as a component of plant defense responses and specifically, SAR. The results further show that AtPNP-A shares many characteristics with pathogenesis related (PR) proteins in that its transcription is strongly induced in response to pathogen challenges, thus implying a newly described role for AtPNP-A in pathogen attack. Additional tissue expression analysis also indicated distinct localization of PNP activity in sepals and transcriptional meta-analysis showed that AtPNP-A may play a role in starch breakdown. Therefore, together with the finding that AtPNP-A plays a role in regulating phloem transport, we also hypothesize that AtPNP-A may play a role in phloem unloading in sepals to assist processes such as seed formation in plants. In plants, the second messenger, guanosine 3’,5’-cyclic monophosphate (cGMP) mediates a whole range of important processes including salinity tolerance, disease resistance, drought tolerance and responses to light. Since PNPs regulate water and salt homeostasis via a cGMP-dependent signaling pathways, it is thus important to analyse the transcriptome induced by the second messenger (cGMP) in Arabidopsis thaliana to give a better understanding of its mechanism of action. This study was also supplemented by the analysis of the gibberellic acid (GA) dependent transcriptome, since cGMP also plays a role its transcription pathway. This data analysis, together with promoter content investigation, revealed that genes upregulated after cGMP treatment and down-regulated in the GA insensitive mutant (ga1-3) were enriched with a GA response element (GARE), while no GARE enrichment were observed in genes up-regulated in the ga1-3 mutant. These findings suggest that GARE is indicative of GA-induced and cGMP-dependent transcriptional up-regulation. Gene ontology analysis confirmed previous reports that cGMP is involved in ion homeostasis and indicated that the transcriptional cGMP response is bi-polar in the sense that both genes up- and down-regulated in response to cGMP is involved in cation transport. Additionally, ab initio analysis of genes transcriptionally dependent on cGMP identified CHX8 as a hub gene and promoter content of CHX8 co-expressed genes show enrichment of the GARE motif. The fact that CHX8 has its highest expression levels during male gametogenesis and pollen tube growth, together with our findings, suggest that GA-induced and cGMP- dependent genes may play a key role in ion and water homeostasis in the male gametophyte. Finally, we propose that the type of analysis undertaken here can yield new insights into gene regulation networks and inform experimental strategies to unravel complex transcription regulatory systems under different developmental and stimulus specific conditions.
Characterisation of microbial communities associated with hypolithic environments in Antarctic Dry Valley soils
(University of the Western Cape, 2008) Khan, Nuraan; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
The Eastern Antarctic Dry Valley region is a polar desert, where conditions of extreme aridity, high temperature fluctuations and high irradiation levels make it one of the most extreme environments on earth. Despite the harsh environment, the soils in this region yield a wide range of bacterial and eukaryotic phylotypes in greater abundance than previously believed. In the Dry Valleys, highly localized niche communities colonise the underside of translucent quartz rocks and present macroscopic growth.
Characterization and engineering of Bacillus megaterium AS-35, for use in biodegradation of processed olive wastewater
(University of Western Cape, 2005) Van Schalkwyk, Antoinette; Cowan, Don A
The popularization and health benefits associated with the "Mediterranean diet" saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds.
Characterization of the role of Zea mays burp domain-containing genes in maize drought responses
(University of the Western Cape, 2016) Phillips, Kyle; Ludidi, Ndiko
Global climate change has resulted in altered rainfall patterns, causing annual losses in maize crop yield due to water deficit stress. Therefore, it is important to produce maize cultivars which are more drought-tolerant. This not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of water deficit stress. The RD22-like protein subfamily is expressed in response to water deficit stress. Members of the RD22-like subfamily include AtRD22, GmRd22 and BnBDC1 which have been identified in Arabidopsis thaliana, Glycine max and Brassica napus respectively. This study aims at characterising two putative maize RD22-like proteins (designated ZmRd22A and ZmRd22B) by identifying sequence/domain features shared with characterised RD22-like proteins. Semi-quantitative and quantitative PCR techniques were used to examine the spatial and temporal expression patterns of the two putative maize Rd22-like proteins in response to, water deficit stress and exogenously applied abscisic acid in the roots and 2nd youngest leaves of maize seedlings. Using an in silico approach, sequence homology of the two putative maize Rd22- like proteins with AtRD22, GmRD22 and BnBDC1 has been analysed. Online bioinformatic tools were used to compare the characteristics of these Rd22-like proteins with those of the two maize proteins. It was shown that the putative maize RD22-like proteins share domain organisation with the characterised proteins, these common features include a N-terminal hydrophobic signal peptide, followed by a region with a conserved amino acid sequence, a region containing several TxV (x is any amino acid) repeat units and a C-terminal BURP domain-containing the conserved X₅-CH-X₁₀-CH-X₂₃-₂₇-CH-X₂₃-₂₆-CH-X₈-W motif. The putative maize Rd22-like protein appears to be localized in the apoplast, similarly to AtRD22, GmRD22 and BnBDC1. Analysis of the gene's promotor regions reveals cis-acting elements suggestive of induction of gene expression by water deficit stress and abscisic acid (ABA). Semi-quantitative and quantitative real time PCR analysis of the putative maize RD22-like gene revealed that the genes are not expressed in the roots. Exposure to water deficit stress resulted in an increase of ZmRD22A transcript accumulation in the 2nd youngest leaves of maize seedlings. ZmRD22A was shown to be non-responsive to exogenous ABA application. ZmRD22B was highly responsive to exogenous ABA application and responded to water deficit stress to a lesser degree. Transcript accumulation studies in three regions of the 2nd youngest leaves in response to water deficit stress showed that ZmRd22A transcripts accumulate mainly at the base and tips of the leaves. A restricted increase in ZmRD22A transcript accumulation in the middle of the leaves was observed. ZmRD22B showed a similar, but weaker transcript accumulation pattern in response to water deficit stress. However, ZmRD22B showed increased transcript accumulation in the middle region of the leaves. In response to exogenous ABA application, ZmRd22B exhibited high transcript accumulation at the base of the 2nd youngest leaves, with the middle showing higher transcript accumulation than the tip of the leaves. It was concluded that ZmRD22A and ZmRD22B share the domain organisation of characterised RD22-like proteins as well as being responsive to water deficit stress, although only ZmRD22B was shown to be responsive to exogenous ABA application.
Characterization of two Arabidopsis thaliana genes with roles in plant homeostasis
(University of the Western Cape, 2004) Ludidi, Ndomelele Ndiko; Gehring, C. A.
Plants are continuously exposed to varying conditions in their environment, to which they have to adapt by manipulating various cellular processes. Environmental (abiotic) and pathogen (biotic) stress are challenges against which plants have to defend themselves. Many plant responses to stress stimuli are a result of cellular processes that can be divided into three sequential steps; namely signal perception, signal transduction m1d execution of a response. Stress signal perception is, in most of these cases, facilitated by cell surface or intracellular receptors that act to recognize molecules presented to the cell. In several cases, hormones are synthesized in response to stress signals and in turn these hormones are perceived by cellular receptors that trigger signal transduction cascades. Propagation of signal transduction cascades is a complex process that results from activation of various signaling molecules within the cell. Second messengers like calcium (Ca2+) and guanosine 3', 5'-cyclic monophosphate (cGMP) play a vital role in mediating many signal transduction processes. The result of these signal transduction cascades is, in most instances, expression of genes that contribute to the plant's ability to cope with the challenges presented to it. Plant natriuretic peptides (PNPs) are novel plant hormones that regulate water and salt homeostasis via cGMP-dependent signaling pathways that involve deployment of Ca2+. The aim of this study is to partially characterize a PNP and a guanylyl cyclase, both from Arabidopsis thaliana. Guanylyl cyclases synthesize cGMP from the hydrolysis of guanosine 5' -triphosphate (GTP) in the cell. The study also aims to investigate the effect of drought and salinity on cGMP levels in plants, using sorbitol to mimic the osmolarity/dehydration effect of drought and NaCl as a source of salinity stress and thus link NaCl and sorbitol responses to both AtPNP-A and cGMP up-regulation.
Cloning, expression and characterization of Novel Lipase and Esterases from Burkholderia multivorans UWC10
(2005) Rashamuse, Konanani J; Cowan, Don A
An esterase and lipase producing Burkholderia multivorans strain was isolated by culture enrichment strategies. A shotgun library of Burkholderia multivorans genomic DNA (prepared in E. coli/pUC18) was screened for lipase and esterase activities. Three positive recombinant clones, pTEND5, pHOLA6 and pRASHI4, conferring esterolytic and lipolytic phenotypes respectively, were identified. Full-length sequencing of DNA inserts was performed using subeloning and "primer-walking" strategies. Nucleotide sequence analysis revealed that the pRASH14 plasmid DNA consisted of two open reading frames (ORPI and ORP2) encoding 356 and 350 amino acids, respectively. Database searches revealed that ORPI and ORP2 were homologous to lipases and chaperones from subfamily I.2. In the pTEND5 sequence, an open reading frame consisting of 978 bp, encoding 326 amino acids, was identified. Database searches revealed that this open reading frame was homologous to family Vesterases. Nucleotide sequence analysis revealed that pHOLA6, plasmid DNA consisted of 1194 bp encoding 398 amino acids and showed homology to family VIII esterases. The primary structures of LipA, EstEFH5 and EstBL from pRASHI4, pTEND5 and pHOLA6, respectively, showed a classical GxSxG motif, which is conserved in many serine hydrolases. In addition, EstBL also showed a consensus SxxK motif, the serine of which acts as a catalytic nucleophile in class C B-lactames and some peptidases.
Comparative analysis of sugar-biosynthesis proteins of sorghum stems and the investigation of their role in hyperosmotic stress tolerance
(University of the Western Cape, 2015) Njokweni, Anathi Perseverence; Ndimba, Bongani
Sorghum bicolor (L.) Moench is an important cereal crop currently explored as a potential bio-energy crop due to its stress tolerance and ability to ferment soluble sugars. Physiological studies on sorghum varieties have demonstrated that part of drought tolerance is attributed to sugar accumulation in the sorghum stems. Despite the agronomic advantages of sorghum as a bio-energy crop, more research efforts towards the molecular elucidation of sorghum traits that confer drought tolerance are necessary. Particular focus on traits, which could potentially contribute to an efficient bio-energy production under environmental constraints, would be an added advantage. This study examined the role of sugar biosynthesis proteins in conferring tolerance to drought-induced hyperosmotic stress, and ultimately osmotic adjustment in sorghum varieties. Sorghum bicolor (L.) Moench varieties (ICSB338, ICSB73, ICSV213 and S35) with different levels of drought tolerance, were grown under watered conditions until early anthesis after which, a 10-day water deficit period was introduced
Comparison of actinobacterial diversity in Marion Island terrestrial habitats
(University of the Western Cape, 2008) Sanyika, Walter Tendai; Cowan, Donald A.; Faculty of Arts
The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats.
A computational framework for transcriptome assembly and annotation in non-model organisms: the case of venturia inaequalis
(University of the Western Cape, 2014) Kimbung, Stanley Mbandi; Christoffels, Alan; Jasper, D; Rees, G
In this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date
Deciphering the determinants of molecular physiological responses to drought and heat stress in sorghum lines contrasting in stress tolerance via a proteomics approach
(University of the Western Cape, 2022) Ali, Ali Elnaeim Elbasheir; Ludidi, Ndiko
Sorghum is an important crop in Sub-Saharan Africa and South Asia. The predicted rise in global temperatures will increase the probability of exposing sorghum to heat waves in combination with drought. Thus, production and availability of sorghum and its products will be negatively affected. Although much progress has been made in identifying molecular processes involved in some crop responses to drought or heat stress, knowledge on such responses in sorghum is limited and in fact does not exist for combined drought and heat stress in sorghum lines that respond differently to these stresses.
Defining the African green monkey (Chlorocebus Aethiops): expression behaviour of selected lipid metabolism genes in response to niacin
(University of Western Cape, 2012) Chauke, Chesa Gift; Arieff, Zainunisha; Seier, Jürgen; Kaur, Mandeep
In this century most major medical advances have resulted in part from research on animals and non-human primates such as the African green monkey and therefore often serve as a critical link between basic research and human clinical application. Due to its close evolutionary relationship to humans, the African green monkey is known to be an excellent and most sought after models for studies of human cardiovascular disease (CVD). While the human genome project and some others related to model organisms are very well advanced or even complete, little sequence information has been acquired for the African green monkey. Given the importance of this species in biomedical research generally and CVD specifically, and the fundamental significance of sequence data, it is critical that this paucity of genome information concerning this specific animal model be addressed in order to better define the molecular basis and to further understand the mechanism of cholesterol metabolism in this species which will also contribute immensely to primatology. There is a growing interest in the role of genetic polymorphisms in predicting susceptibility to disease and responsiveness to drug interventions. Since plasma lipid abnormalities are risk factors for coronary atherosclerosis, determination of these plasma lipid concentrations, especially for genes involved in lipid transport and metabolism may be influenced by genetic variations. In this study, the African green monkey was used as a model to evaluate the effect of niacin on plasma lipids and reverse cholesterol transport by examine gene expression and the influence of several polymorphisms found in genes that are involved in cholesterol metabolism in humans. A survey of genetic variation spanning ten prioritised “candidate” genes was conducted, all of which are known to produce proteins that play key roles in the reverse cholesterol pathway (RCT), and in the homeostatic regulation of blood lipid profiles related to cardiovascular health and disease. everse transcription polymerase chain reaction (RT-PCR) was used to evaluate mRNA expression of those “candidate” genes. Twenty two coincident singlenucleotide polymorphisms (cSNPs), reported to play a vital role in RCT, were genotyped within these genes. This study’s findings implicate a subset of six of the twenty two genetic variants, spanning five “candidate” genes. To assess possible involvement of these prioritised “candidate” genes and their polymorphisms, biochemical analyses of known risk factors of coronary artery disease such as HDL-C and LDL-C were conducted. Eight healthy African green monkeys were entered in this study of which four were treated with niacin at an escalating dosage. Their mean lipid-lowering response following drug therapy was analysed, compared to those with the same genotype in a control group. Niacin treatment was associated with a considerable reduction in LDL-Cholesterol, up-regulation of HDL synthesis, and increase of apo A-1 levels. Gene expression had minimal effect on niacin treatment, except CYP7A1 which was down-regulated at the same time when considerable change in HDL-C, LDL-C and apoA-1 levels was observed. The presence of CYP7A1:Asn233Ser polymorphism may have played a critical role in metabolising niacin and influencing the up-regulation of HDL-C synthesis in the African green monkey. Although cholesterol lowering alone may explain the anti-atherosclerotic effect of niacin on HDL-C, in this study, gene expression data also shed some light in supporting the hypothesis that genetic variants may influence the expression of genes involved in RCT, which may also have played a role in the anti-atherosclerotic effect of the drug.
The detection of meningococcal disease through identification of antimicrobial peptides using an in silico model creation
(University of the Western Cape, 2019) Abdullah, Gadija; Pretorius, Ashley; Den Haan, Riaan
Neisseria meningitidis (the meningococcus), the causative agent of meningococcal disease (MD) was identified in 1887 and despite effective antibiotics and partially effective vaccines, Neisseria meningitidis (N. meningitidis) is the leading cause worldwide of meningitis and rapidly fatal sepsis usually in otherwise healthy individuals. Over 500 000 meningococcal cases occur every year. These numbers have made bacterial meningitis a top ten infectious cause of death worldwide. MD primarily affects children under 5 years of age, although in epidemic outbreaks there is a shift in disease to older children, adolescents and adults. MD is also associated with marked morbidity including limb loss, hearing loss, cognitive dysfunction, visual impairment, educational difficulties, developmental delays, motor nerve deficits, seizure disorders and behavioural problems. Antimicrobial peptides (AMPs) are molecules that provide protection against environmental pathogens, acting against a large number of microorganisms, including bacteria, fungi, yeast and virus. AMPs production is a major component of innate immunity against infection. The chemical properties of AMPs allow them to insert into the anionic cell wall and phospholipid membranes of microorganisms or bind to the bacteria making it easily detectable for diagnostic purposes. AMPs can be exploited for the generation of novel antibiotics, as biomarkers in the diagnosis of inflammatory conditions, for the manipulation of the inflammatory process, wound healing, autoimmunity and in the combat of tumour cells. Due to the severity of meningitis, early detection and identification of the strain of N. meningitidis is vital. Rapid and accurate diagnosis is essential for optimal management of patients and a major problem for MD is its diagnostic difficulties and experts conclude that with an early intervention the patient’ prognosis will be much improved. It is becoming increasingly difficult to confirm the diagnosis of meningococcal infection by conventional methods. Although polymerase chain reaction (PCR) has the potential advantage of providing more rapid confirmation of the presence of the bacterium than culturing, it is still time consuming as well as costly. Introduction of AMPs to bind to N. meningitidis receptors could provide a less costly and time consuming solution to the current diagnostic problems. World Health Organization (WHO) meningococcal meningitis program activities encourage laboratory strengthening to ensure prompt and accurate diagnosis to rapidly confirm the presence of MD. This study aimed to identify a list of putative AMPs showing antibacterial activity to N. meningitidis to be used as ligands against receptors uniquely expressed by the bacterium and for the identified AMPs to be used in a Lateral Flow Device (LFD) for the rapid and accurate diagnosis of MD.

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