Philosophiae Doctor - PhD (Bioinformatics)

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Discovering cancer subtypes by tracking cancer progression with transcriptomic data through the multi-stage process of cancer development.
(University of the Western Cape, 2023) Livesey, Michelle Chantel; Bendou, Hocine
Background: The development of cancer is driven by genomic alterations, which become more heterogeneous as the disease progresses throughout the stages. Consequently, cancer patients have differential levels of sensitivity to treatment. Tumor heterogeneity thus contributes to therapeutic failure, which ultimately leads to the generally poor prognosis and poor overall survival outcome associated with cancer. Introduction: Transcriptomic profiles can be used to track cancer progression based on gene expression changes that occur throughout the multi-stage process of cancer development. The accumulated genetic changes can be detected when gene expression levels in advanced-stage are less variable but show high variability in early-stage. Normalizing advanced-stage expression samples with early-stage and clustering of the normalized expression samples can reveal cancers with unique gene expression patterns based on cancer progression. Aims: A computational method was employed to investigate cancer progression through RNASeq expression profiles across the multi-stage process of cancer development. The method was assessed in a subtype of the heterogeneous kidney cancer and enabled the discovery of in-depth cancer subtypes based on the differences in gene expression profiles. Methods: A preliminary study was performed by downloading RNA-sequenced gene expression and associated phenotypic and survival profiles of Diffuse Large B-cell Lymphoma, Lung cancer, Liver cancer, Cervical cancer, and Testicular cancer from the UCSC Xena database. Similarly, Kidney renal clear cell carcinoma (KIRC) was downloaded as a validation dataset. Advanced-stage samples were normalized with early-stage to consider heterogeneity differences in the multi-stage cancer progression. The normalized gene expression of the preliminary cancer datasets was subjected to weighted gene co-expression network analysis. Gene modules were linked to cancer-related proteins and pathways using enrichment analyses.
Investigating genetic diversity and microRNA of Hermetia illucens (the black soldier fly) to breed for mass production of a novel sustainable protein
(University of the Western Cape, 2023) De Raedt, Sarah Joanne; Christoffels, Alan
Introduction: A new sustainable source of protein is needed to meet the demands of the growing global population. Insect meal is a suitable replacement, and the black soldier fly (BSF) is the most used insect in industrial rearing. The black soldier fly larvae (BSFL) are not only high in sought-after nutrients (protein, fat, and chitin/source of fiber), but they also reduce organic waste that would go into landfills by consuming the waste and leaving behind a beneficial residue, which is used in fertilizers. However, little has been published on the genetics of BSF which are crucial to optimizing mass breeding programs necessary to meet the population demands. The aim of this study was to further the base of knowledge beneficial to mass rearing protocols by describing the genetic diversity of 3 populations, under differing scales of rearing, and the microRNA expression profile across 5 life stages, along with the first report of the novel microRNA of BSF.
Genomic epidemiology of Rift Valley fever in East Africa
(University of the Western Cape, 2023) Juma, John; Christoffels, Alan
Rift Valley fever (RVF) is a climate-driven zoonotic disease of significant importance to public and livestock health given its epidemic potential. The disease was first identified in 1930 in the great rift valley region of Kenya following massive abortion among pregnant ewes in a sheep farm. RVF is caused by the Rift Valley fever virus (RVFV) belonging to the genus Phlebovirus, family Phenuiviridae and order Bunyavirales. RVF primarily affects domestic ruminants, wild animals and humans with varying degrees of fatalities. In this PhD research, tools and methods that will be of immense importance in conducting genomic surveillance of RVFV were developed. Furthermore, we have applied phylodynamic and phylogeographic approaches to understand the transmission dynamics of the virus in East Africa. In the second chapter, we utilized publicly available genetic sequence data of the virus to build a tool that rapidly detects circulating lineages. A general observation made in this work is the paucity of RVFV genetic data globally. Only a handful of sequence data is available from countries that have previously experienced RVF outbreaks such as South Africa, Kenya and Madagascar. The genetic information in the sequence data is crucial in identifying lineage defining single nucleotide polymorphisms (SNPs) or mutations. Using these mutations, we developed a command line tool that rapidly characterizes RVFV isolates using the 15 alphabet (A to O) nomenclature.
Novel genomic biomarkers for pediatric and adult acute myeloid leukemia
(University of the Western Cape, 2023) Bendou, Hocine; Eshibona, Nasr O M
Acute myeloid leukemia (AML) is a heterogeneous type of blood cancer that affects individuals of all ages. AML patients are categorized into favorable, intermediate, and adverse risks based on patients' genomic features and chromosomal abnormalities. Despite this risk stratification, the progression and outcome of the disease remain highly variable in pediatric and adult patients, which emphasizes the importance of finding more accurate genomic biomarkers studying the gene expression profiling of pediatric and adult AML patients to facilitate and improve the risk stratification of the patients. Consequently, two research aims were proposed to study the prognostic heterogeneity for pediatric and adult AML. In pediatric AML, the research project was set to identify a genetic signature related to patients with FLT3-ITD mutation and poor survival. While for adult AML, this study focused on establishing a genetic signature predictive of prognosis with the ability to accurately reclassify the risk of AML intermediate group.
Computational analyses on transcriptional regulation in mammals
(University of the Western Cape, 2009) Schmeier, Sebastian; Bajic, Vladimir
The genomes of various organisms have been sequenced and their transcriptome elucidated. With the information about genes and gene products readily available it has become of the utmost importance to decipher the underlying biological mechanisms that are involved in the transcriptional control of these genes. Transcription initiation is a fundamental process in living cells. It involves the interaction of transcription factors with DNA to regulate the transcription of a gene. Despite significant research during the last few decades into transcription factors and their role in gene regulation we are still far from understanding the complete transcriptional machinery that acts within biological systems. In this dissertation two computational approaches are presented to contribute to a better understanding of the transcriptional control of genes in mammals. The first addresses the transcriptional regulation of microRNA genes and its influence on the microRNA gene expression during monocytic differentiation. This is the first large-scale approach to decipher how microRNA genes are regulated by transcription factors during monocytic differentiation. The second approach relates to combinatorial gene regulation and the physical interaction of transcription factors. Here, a computational approach is used together with a novel form of numerical representation of transcription factors to predict their interactions. In this setup, the information necessary to predict the transcription factor interactions is kept at the lowest level to minimize the data acquisition overhead that often occurs in computational prediction tasks. Both approaches enhance our insights into transcriptional control and have an impact on the further study of gene regulation.
"Development and implementation of ontology-based systems for mammalian gene expression profiling"
(University of the Western Cape, 2009) Kruger, Adele; Hide, Winston
The use of ontologies in the mapping of gene expression events provides an effective and comparable method to determine the expression profile of an entire genome across a large collection of experiments derived from different expression sources. In this dissertation I describe the development of the developmental human and mouse e VOC ontologies and demonstrate the ontologies by identifying genes showing a bias for developmental brain expression in human and mouse, identifying transcription factor complexes, and exploring the mouse orthologs of human cancer/testis genes. Model organisms represents fundamental aspects of mammal biology phenomena between model organism is complex and it is to be the meaningful, a simplified representation can be a powerful means for comparison illustrated here in two ways. Firstly, the ontologies have been used to illustrate methods to determine clusters of genes showing tissue-restricted expression in humans. The identification of tissue-restricted genes within an organism serves as an indication of the finetuning in the regulation of gene expression in a given tissue. Secondly, due to the differences in human and mouse gene expression on a temporal and spatial level, the ontologies were used to identify mouse orthologs of human cancer/testis genes showing cancer/testis characteristics. With the use of model systems such as mouse in the development of gene-targeted drugs in the treatment of disease, it is important to establish that the expression characteristics and profiles of a drug target in the model system is representative of the characteristics of the target in the system for which it is intended.
Development of an operon detection algorithm to analyze gene regulation in drug resistant Mycobacterium tuberculosis
(University of the Western Cape, 2022) Calvert-Joshua, Tracey; Christoffels, Alan
In prokaryotes, operon structures often form to allow microorganisms to respond rapidly and efficiently to changing environmental conditions. Operons are sets of neighbouring genes which are co-regulated and co-transcribed. Studies have shown evidence of operons changing their lengths and/or maintaining their lengths while up- or downregulating their expression levels when exposed to various stresses. Since several operons have also been associated with drug resistance, having access to the operon map of Mycobacterium tuberculosis (Mtb), may give us insight into the existing mechanisms employed by Mtb to circumvent drug stress, and more importantly, it may allow us to target larger sections of a genome when designing antitubercular drugs.
Next generation sequencing approaches for novel gene discovery in South African Parkinson’s disease families
(University of the Western Cape, 2022) Pillay, Nikita Simone; Christoffels, Alan
In the last decade, next-generation sequencing (NGS) approaches have revolutionised the study of human genomics, particularly aiding the understanding of genetic diseases. Parkinson’s disease (PD) is a complex neurodegenerative disorder with a heterogenous genetic disposition. This disorder is clinically characterised by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Subsequently, this results in a severe decrease of available dopamine that manifests as a myriad of both motor and non-motor symptoms. Several genes, including α-synuclein (SNCA), parkin (PRKN), leucine-rich repeat kinase 2 (LRRK2), PTEN induced putative kinase 1 (PINK1), and protein deglycase (DJ-1), are confirmed as disease-causing in autosomal recessive (AR), autosomal dominant (AD), early-onset (EO), and late-onset (LO) forms of the disorder.
Concept Based Knowledge Discovery From Biomedical Literature
(University of the Western Cape, 2009) Radovanovic, Aleksandar; Bajic, Vladimir
Advancement in biomedical research and continuous growth of scientific literature available in electronic form, calls for innovative methods and tools for information management, knowledge discovery, and data integration. Many biomedical fields such as genomics, proteomics, metabolomics, genetics, and emerging disciplines like systems biology and conceptual biology require synergy between experimental, computational, data mining and text mining technologies. A large amount of biomedical information available in various repositories, such as the US National Library of Medicine Bibliographic Database, emerge as a potential source of textual data for knowledge discovery. Text mining and its application of natural language processing and machine learning technologies to problems of knowledge discovery, is one of the most challenging fields in bioinformatics. This thesis describes and introduces novel methods for knowledge discovery and presents a software system that is able to extract information from biomedical literature, review interesting connections between various biomedical concepts and in so doing, generates new hypotheses. The experimental results obtained by using methods described in this thesis, are compared to currently published results obtained by other methods and a number of case studies are described. This thesis shows how the technology presented can be integrated with the researchers' own knowledge, experimentation and observations for optimal progression of scientific research.
Computational analysis of multi-omic data for the elucidation of molecular mechanisms of neuroblastoma
(University of Western Cape, 2021) Giwa, Abdulazeez; Bendou, Hocine
Neuroblastoma is the most common extracranial solid tumor in childhood. The survival rates of patients with neuroblastoma, especially those in the high-risk category, are still low despite varied therapies. The detailed understanding of the molecular mechanisms underlying the pathogenesis of neuroblastoma is essential to develop better therapeutics and improve the poor survival rates. This study provides a multi-omic analysis of neuroblastoma datasets from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) neuroblastoma project and the Gene Expression Omnibus (GEO) data portals to better understand the molecular mechanisms of neuroblastoma.
Irradiation induced effects on 6h-SIC
(University of the Western Cape, 2012) Sibuyi, Praise; Maaza, M.
The framework agreement in the year 2000 by the international community to launch Generation IV program with 10 nations, to develop safe and reliable nuclear reactors gave rise to the increased interest in the studies of SiC and the effect of different irradiations on solids. Silicon carbide is a preferred candidate used in harsh environments due to its excellent properties such as high chemical stability and strong mechanical strength. The PBMR technology promises to be the safest of all nuclear technology that have been developed before. SiC has been considered one candidate material being used in the fabrication of pebble bed fuel cell. Its outstanding physical and chemical properties even at high temperatures render it a material of choice for the future nuclear industry as whole and PBMR in particular. Due to the hostile environment created during the normal reactor operation, some of these excellent properties are compromised. In order to use this material in such conditions, it should have at least a near perfect crystal lattice to prevent defects that could compromise its strength and performance. A proper knowledge of the behavior of radiation-induced defects in SiC is vital. During irradiation, a disordered crystal lattice occurs, resulting in the production of defects in the lattice. These defects lead to the degradation of these excellent properties of a particular material. This thesis investigates the effects of various radiation effects to 6H-SiC. We have investigated the effects of radiation induced damages to SiC, with a description of the beds and the importance of the stability of the SiC-C interface upon the effects of radiations (y-rays, hot neutrons). The irradiated samples of 6H-SiC have been studied with various spectroscopic and structural characterization methods. The surface sensitive techniques such as Raman spectroscopy, UV-Vis, Photoluminescence and Atomic Force Microscopy will be employed in several complimentary ways to probe the effect of irradiation on SiC. The obtained results are discussed in details.
Management and analysis of HIV -1 ultra-deep sequence data
(University of the Western Cape, 2014) Shrestha, Ram Krishna; Travers, Simon A
The continued success of antiretroviral programmes in the treatment of HIV is dependent on access to a cost-effective HIV drug resistance test (HIV-DRT). HIVDRT involves sequencing a fragment of the HIV genome and characterising the presence/absence of mutations that confer resistance to one or more drugs. HIV-DRT using conventional DNA sequencing is prohibitively expensive (~US$150 per patient) for routine use in resource-limited settings such as many African countries. While the advent of ultra deep pyrosequencing (UDPS) approaches have considerably reduced (3-5 fold reduction) the cost of generating the sequence data, there has been an even more significant increase in the volume of data generated and the complexity involved in its analysis. In order to address this issue we have developed Seq2Res, a computational pipeline for HIV drug resistance test from UDPS genotypic data. We have developed QTrim, software that undertakes high throughput quality trimming of UDPS sequencing data to ensure that subsequently analyzed data is of high quality. The comparison of QTrim to other widely used tools showed that it is equivalent to the next best method at trimming good quality data but outperforms all methods at trimming poor quality data. Further, we have developed, and evaluated, a computational approach for the analysis of UDPS sequence data generated using the novel Primer ID that enables the generation of a consensus sequence from all sequence reads originating from the same viral template, thus reducing the presence of PCR and sequencing induced errors in the dataset as well as reducing. We see that while the Primer ID approach does undoubtedly reduce the prevalence of PCR and sequencing induced errors, it artificially reduces the diversity of the subsequently analysed data due to the large volume of data that is discarded as a result of there being an insufficient number of sequences for consensus sequence generation. We validated the sensitivity of the Seq2Res pipeline using two real biological datasets from the Stanford HIV Database and five simulated datasets The Seq2Res results correlated fully with that of the Stanford database as well as identifying a drug resistance mutations (DRM) that had been incorrectly interpreted by the Stanford approach. Further, the analysis of the simulated datasets showed that Seq2Res is capable of accurately identifying DRMs at all prevalence levels down to at least 1% of the sequence data generated from a viral population. Finally, we applied Seq2Res to UDPS resistance data generated from as many as 641 individuals as part of the CIPRA-SA study to evaluate the effectiveness of UDPS HIV drug resistance genotyping in resource limited settings with a high burden of HIV infections. We find that, despite the FLX coverage being almost three times as much as that of the Junior platform, resistance genotyping results are directly comparable between both of the approaches at a range of prevalence levels to as low as 1%. Further, we find no significant difference between UDPS sequencing and the "gold standard" Sanger based approach, thus indicating that pooling as many as 48 patient's data and sequencing using the Roche/454 Junior platform is a viable approach for HIV drug resistance genotyping. Further, we explored the presence of resistant minor variants in individual's viral populations and find that the identification of minor resistant variants in individuals exposed to nevirapine through PMTCT correlates with the time since exposure. We conclude that HIV resistance genotyping is now a viable prospect for resource limited setting with a high burden of HIV infections and that UDPS approaches are at least as sensitive as the currently used Sanger-based sequencing approaches. Further, the development of Seq2Res has provided a sensitive, easy to use and scalable technology that facilitates the routine use of UDPS for HIV drug resistance genotyping.
Coding of tsetse repellents by olfactory sensory neurons: towards the improvement and the development of novel tsetse repellents
(University of the Western Cape, 2020) Souleymane, Diallo; Christoffels, Alan
Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.
Coding of tsetse repellents by olfactory sensory neurons: towards the improvement and the development of novel
(University of the Western Cape, 2020) Souleymane, Diallo; Christoffels, Alan
Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication. In the sensilla shaft the dendrite of OSNs are housed, which are protected by called the sensillum lymph produced by support cells and contains a variety of olfactory proteins, including the odorant binding protein (OBP) and chemosensory proteins (CSP). While on the dendrite of OSNs are expressed olfactory receptors. In my PhD, studies I tried to decipher the sense of smell in tsetse fly. In the second chapter, I demonstrated that G. f. fuscipes is equipped with diverse olfactory sensilla, that various from basiconic, trichoid and coeloconic. I also demonstrated, there is shape, length, number difference between sensilla types and sexual dimorphism. There is a major difference between male and female, while male has the unique basiconic sensilla, club shaped found in the pits, which is absent from female pits. In my third chapter, I investigated the odorant receptors which are expressed on the dendrite of the olfactory sensory neurons (OSNs). G. f. fuscipes has 42 ORs, which were not functionally characterised. I used behaviourally well studied odorants, tsetse repellents, composed of four components blend. I demonstrated that tsetse repellent is also a strong antifeedant for both G. pallidipes and G. f. fuscipes using feeding bioassays as compared to the attractant odour, adding the value of tsetse repellent. However, the attractant odour enhanced the feeding index. Using DREAM (deorphanization of receptors based on expression alterations of mRNA levels). I found that in G. f. fuscipes, following a short in vivo exposure to the individual tsetse repellent component as well as an attractant volatile chemical, OSNs that respond to these compounds altered their mRNA expression in two opposite direction, significant downregulation and upregulation in their number of transcripts corresponding to the OR that they expressed and interacted with odorant. Also, I found that the odorants with opposite valence already segregate distinctly at the cellular and molecular target at the periphery, which is the reception of odorants by OSNs, which is the basis of sophisticated olfactory behaviour. Deorphanization of ORs in none model insect is a challenge, here by combining DREAM with molecular dynamics, as docking score, physiology and homology modelling with Drosophila a well-studied model insects, I was able to predict putative receptors of the tsetse repellent components and an attractant odour. However, many ORs were neutral, showing they were not activated by the odorants, demonstrating the selectivity of the technique as well as the receptors. In my fourth chapter, I investigated the OBPs structures and their interaction with odorants molecules. I demonstrated that OBPs are expressed both in the antenna, as well as in other tissues, such as legs. I also demonstrated that there are variations in the expression of OBPs between tissues as well as sexes. I also demonstrated that odorants induced a fast alteration in OBP mRNA expression, some odorants induced a decrease in the transcription of genes corresponding to the activated OBP and others increased the expression by many fold in OBPs in live insect, others were neutral after 5 hours of exposure. Moreover, with subsequent behavioural data showed that the behavioural response of G. f. fuscipes toward 1-octen-3-ol decreased significantly when 1-octen-3-ol putative OBPs were silenced with feeding of double-stranded RNA (dsRNA). In summary, our finding whereby odorant exposure affects the OBPs mRNA, their physiochemical properties and the silencing of these OBPs affected the behavioural response demonstrate that the OBPs are involved in odour detection that affect the percept of the given odorant. The expression of OBPs in olfactory tissues, antenna and their interaction with odorant and their effect on behavioural response when silenced shows their direct involvement in odour detection and reception. Furthermore, their expression in other tissues such as legs indicates they might also have role in other physiological functions, such as taste.
Coding of tsetse repellents by olfactory sensory neurons: towards the improvement and the development of novel tsetse repellents
(University of Western Cape, 2021) Souleymane, Diallo; Christoffels, Alan
Tsetse flies are the biological vectors of human and animal trypanosomiasis and hence representant medical and veterinary importance. The sense of smell plays a significant role in tsetse and its ecological interaction, such as finding blood meal source, resting, and larvicidal sites and for mating. Tsetse olfactory behaviour can be exploited for their management; however, olfactory studies in tsetse flies are still fragmentary. Here in my PhD thesis, using scanning electron microscopy, electrophysiology, behaviour, bioinformatics and molecular biology techniques, I have investigated tsetse flies (Glossina fuscipes fuscipes) olfaction using behaviourally well studied odorants, tsetse repellent by comparing with attractant odour. Insect olfaction is mediated by olfactory sensory neurons (OSNs), located in olfactory sensilla, which are cuticular structures exposed to the environment through pore and create a platform for chemical communication.
Optimisation of proteomics techniques for archival tumour blocks of a South African cohort of colorectal cancer
(University of Western Cape, 2020) Rossouw, Sophia Catherine; Christoffels, Alan; Rigby, Jonathan
Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin.
Observations on the ecology and life-history of Chrysaora fulgida (Reynaud 1830) (Scyphozoa: Semaeostomeae) and other pelagic cnidarians in the inshore waters off central Namibia
(University of the Western Cape, 2019) Skrypzeck, Heidi; Gibbons, Mark
Although jellyfish are recognised recently as key components that can influence ecosystem functioning and trophic flows in the northern Benguela upwelling ecosystem, the number of published studies on their abundance, seasonality, life history and ecological roles off Namibia is strictly limited. Chrysaora fulgida is one of the most common and conspicuous medusae in the plankton off Namibia, and has flourished in the region, following the decline of the pilchard fishery at the end of the 1960s. It is said that their biomass (together with Aequorea forskalea) exceed that of the commercially important fish stocks off Namibia. In addition, this species is also capable of forming large swarms in northern Benguela where they are a nuisance to fisheries operations. The objective of this study is to try and fill gaps regarding our knowledge of the biology and ecology of Chrysaora fulgida off Namibia, with a view to improve our understanding of its success in the northern Benguela ecosystem. In the Chapter 1, a general overview on the current knowledge and population dynamics of jellyish blooms and their ecology is compiled. Other key topics of the thesis such as jellyfish life cycles and their reproduction are also introduced. Chapter 2 investigates the temporal changes in the jellyfish community in Walvis Bay over a 23-month period from biweekly plankton samples. All twelve of the recovered taxa were characteristically neritic, and included meroplanktonic Hydrozoa and Scyphozoa, as well as cydippid ctenophores and shallow water siphonophores. Whilst, ephyrae of Chrysaora fulgida were dominant overall, and peaked in abundance during mid-spring (Year 2012: 168 933 ind. 100 m-3) and late winter (Year 2013: 23 389 ind. 100 m-3), they were not present all year round, being replaced (in part) by Obelia in summer and autumn, Bougainvillia in spring and summer, and Muggiaea atlantica in summer. Seasonal changes in the composition and structure of the community were driven primarily by bottom water temperature and day length (explaining 24% of the variability in community structure), with wind speed and moon illumination playing a secondary role. The recruitment of ephyrae of C. fulgida to the plankton off Walvis Bay is confirmed not to be continuous throughout the year. Chapter 3 present the first detailed investigation on the identification, morphological development and growth of wild caught ―ephyrae‖ of the scyphozoan Chrysaora fulgida and Chrysaora africana in Walvis Bay, off Namibia. Concrete morphological dissimilarities are documented to distinguish C. africana from C. fulgida, despite the limited sample size of C. africana: coloration differences and the presence/absence of branched canals on the periphery of velar and rhopalial canal tips. In the case of C. fulgida the morphological development from an ephyra (Stage 0) to a juvenile medusa could be described successfully in six stages, whilst missing stages were noted for C. africana. In general, the development of ephyrae described here agrees with patterns described for other species in the genus from elsewhere. The ephyrae stages of C. fulgida illustrated a low overall growth rate (4.33 and 3.45% d-1, respectively) and longer ontogenic development (~164 days), respectively, than most other jellyfish species. Through the histological examination of medusa gonads, Chapter 4 investigates the sexual reproduction and maturation of both Chrysaora species, collected off Walvis Bay, Namibia. Both species were non-brooding, gonochoristic, displayed a 1:1 sex ratio and exhibited no clear sexual dimorphism features. Gametogenesis in both species was similar to that displayed by other Discomedusae, whilst some differences in gonad maturity were evident between them – Chrysaora fulgida displayed aseasonal, reproductive heterogeneity (maturing at ~300 mm diameter) and individuals were semelparous, whilst C. africana appeared strongly seasonal but iteroparous. Through stable isotope analysis (𝛿13C, 𝛿15N and C:N ratios), Chapter 5 examines the presence of tissue, ontogenetic, seasonal, spatial and interspecific variability in medusae of Chrysaora fulgida and Chrysaora africana off Walvis Bay, in the northern Benguela, Namibia. This study did not only illustrate size-associated shifts in trophic ecology, but also revealed spatial, inter-species and some tissue differences in the northern Benguela upwelling system. Size would appear to be the over-riding factor that influences the isotope signatures of Chrysaora fulgida; size being linked in turn to space. A clear negative relationship is illustratred between 𝛿15N and individual size for two scyphozoans (C. fulgida and C. africana) off central Namibia, indicating that larger jellyfish feed lower down the food chain than smaller ones in both species. This is explained by the need and ability of ephyrae and small medusae to access the microbial food web which consists of many trophic steps and hence numerous opportunities for enrichment of nitrogen isotopes, resulting in higher 𝛿15N values of smaller individuals. Chapter 6 provides a synthesis of the main findings of the thesis, and makes recommendations on ways that the research can be carried forward.
Baobab LIMS: An open source biobank laboratory information management system for resource-limited settings
(University of the Western Cape, 2019) Bendou, Hocine; Christoffels, Alan
A laboratory information management system (LIMS) is central to the informatics infrastructure that underlies biobanking activities. To date, a wide range of commercial and open source LIMS are available. The decision to opt for one LIMS over another is often influenced by the needs of the biobank clients and researchers, as well as available financial resources. However, to find a LIMS that incorporates all possible requirements of a biobank may often be a complicated endeavour. The need to implement biobank standard operation procedures as well as stimulate the use of standards for biobank data representation motivated the development of Baobab LIMS, an open source LIMS for Biobanking. Baobab LIMS comprises modules for biospecimen kit assembly, shipping of biospecimen kits, storage management, analysis requests, reporting, and invoicing. Baobab LIMS is based on the Plone web-content management framework, a server-client-based system, whereby the end user is able to access the system securely through the internet on a standard web browser, thereby eliminating the need for standalone installations on all machines. The Baobab LIMS components were tested and evaluated in three human biobanks. The testing of the LIMS modules aided in the mapping of the biobanks requirements to the LIMS functionalities, and furthermore, it helped to reveal new user suggestions, such as the enhancement of the online documentation. The user suggestions are demonstrated to be important for both LIMS strengthen and biobank sustainability. Ultimately, the practical LIMS evaluations showed the ability of Boabab LIMS to be used in the management of human biobanks operations of relatively different biobanking workflows.
Exploring the role of the “glycan-shield” of human immunodeficiency virus in susceptibility to, and escape from, broadly neutralising antibodies
(University of the Western Cape, 2018) Ferreira, Roux-Cil; Travers, Simon A.
The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Despite the importance of the HIV- 1 Env glycans, limited computational analyses have been employed to analyse these glycans. Here, the Env glycans of two HIV-1 wild-type subtype C isolates are examined, in detail, using computational approaches. These particular strains were used since in vitro data showed that the removal of a single glycan had a substantially different impact on the neutralisation sensitivity of the two strains. Molecular dynamics simulations, and the subsequent analyses, were carried out on the computationally determined, fully glycosylated, Env structures of these two wild-type strains and their N301A mutant counterparts. Detailed comparison of the molecular dynamics simulations demonstrated that unique glycan dynamics and conformations emerged and that, despite shared HXB2 reference sequence positions, the glycans adopted distinct conformations specific to each wild-type model. Furthermore, different changes in conformations were observed for each wild-type model compared to its N301A mutant counterpart and, interestingly, these N301A mutant model-specific glycan conformations were directly associated with the protein residues ultimately found to be exposed, which may explain the varied resistance to neutralising antibodies observed, in vitro, for the two N301A mutant strains.
Development of a data processing toolkit for the analysis of next-generation sequencing data generated using the primer ID approach
(University of the Western Cape, 2018) Labuschagne, Jan Phillipus Lourens; Travers, Simon
Sequencing an HIV quasispecies with next generation sequencing technologies yields a dataset with significant amplification bias and errors resulting from both the PCR and sequencing steps. Both the amplification bias and sequencing error can be reduced by labelling each cDNA (generated during the reverse transcription of the viral RNA to DNA prior to PCR) with a random sequence tag called a Primer ID (PID). Processing PID data requires additional computational steps, presenting a barrier to the uptake of this method. MotifBinner is an R package designed to handle PID data with a focus on resolving potential problems in the dataset. MotifBinner groups sequences into bins by their PID tags, identifies and removes false unique bins, produced from sequencing errors in the PID tags, as well as removing outlier sequences from within a bin. MotifBinner produces a consensus sequence for each bin, as well as a detailed report for the dataset, detailing the number of sequences per bin, the number of outlying sequences per bin, rates of chimerism, the number of degenerate letters in the final consensus sequences and the most divergent consensus sequences (potential contaminants). We characterized the ability of the PID approach to reduce the effect of sequencing error, to detect minority variants in viral quasispecies and to reduce the rates of PCR induced recombination. We produced reference samples with known variants at known frequencies to study the effectiveness of increasing PCR elongation time, decreasing the number of PCR cycles, and sample partitioning, by means of dPCR (droplet PCR), on PCR induced recombination. After sequencing these artificial samples with the PID approach, each consensus sequence was compared to the known variants. There are complex relationships between the sample preparation protocol and the characteristics of the resulting dataset. We produce a set of recommendations that can be used to inform sample preparation that is the most useful the particular study. The AMP trial infuses HIV-negative patients with the VRC01 antibody and monitors for HIV infections. Accurately timing the infection event and reconstructing the founder viruses of these infections are critical for relating infection risk to antibody titer and homology between the founder virus and antibody binding sites. Dr. Paul Edlefsen at the Fred Hutch Cancer Research Institute developed a pipeline that performs infection timing and founder reconstruction. Here, we document a portion of the pipeline, produce detailed tests for that portion of the pipeline and investigate the robustness of some of the tools used in the pipeline to violations of their assumptions.

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