Doctoral Degrees (Biochemistry)

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    To target or not to target: strategies for the aroma analysis of alcoholic beverages
    (Stellenbosch : Stellenbosch University, 2023-12) Williams, Cody; Buica, Astrid; Stander, Marietjie A.; Medvedovici, Andrei V.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Classically, targeted analysis has dominated analytical chemistry research, whereby analytical methods are required for separation, identification and quantitation of specific analytes of interest. The complex wine, gin and beer matrices contain several hundreds of compounds, each with varying concentration levels and unique chemistries. Furthermore, comprehensive analysis of these alcoholic beverages are challenging, as suitable instrumentation and data handling strategies are required to effectively profile these matrices. The use of non-targeted methodologies emerge as an alternative tool used for profiling the aroma space of alcoholic beverages. Non-targeted analysis is an information rich technique which focuses on profiling a sample in its entirety. More data can equal more information, however more noise is also generated; thus, suitable strategies are highly desired to overcome this caveat. The question is now, are these strategies complementary or is the generated information redundant? The aim of this dissertation was to compare targeted and non-targeted strategies for the aroma analysis of wine, beer and gin. This dissertation is comprised of seven chapters showing the evolution of both targeted and non- targeted strategies starting with wine and culminating in the application of these strategies in craft gin and beer, together with the evaluation of the results using appropriate (and sometimes complex) statistical tools. The first part of the study is a comprehensive literature review, with specific focus on terpenoid analysis. Terpenoids are an integral component to the sensory profile in wine, beer and gin. These compounds are present in various concentrations and require advanced analytical tools to profile and quantify. The origin of terpenoids as derived from the raw materials or production practices, common concentration ranges, and sample preparation methods are reported. The use of non-targeted methods is also summarised in this review for wine, beer and gin, with particular focus on the use of hyphenated instrumentation, software processing tools, and multivariate statistical analysis. The first objective (chapter 3) focused on targeted strategies in wine. Terpenoids were selected due to their importance to the aroma profile and inherent challenges associated with the analysis of these compounds. The complexity associated with the analyses stimulated interest and prompted further investigation in the quantitation of terpenoids in wine. Two sample preparation strategies, namely offline-SPE and online-HS-SPME were explored for the quantitation of 20 terpenoids in red and white wine. This study documented which sample preparation was best for the quantitation of terpenoids in wine and reported on various method performance parameters. The second objective (chapter 4) expanded on the quantitation of terpenoids in wine, whereby the method was expanded from 20 to 53 terpenoids in a single method. This was accomplished through method development, optimisation and method performance characterisation. In addition, terpenoid stability was evaluated over a period of forty days. As this extended method includes compounds not found in wine, it was applied to craft gin (n=21) and craft beer (n=34) samples, and it documents for the first time the most terpenoids quantified in a single method using authentic standards. It also constitutes the first report on terpenoids quantified in South African craft gin and beer. In the non-targeted space, the third objective (chapter 5) highlights the use of various sample prepration strategies (LLE, SPE and HS-SPME) applied to wine of different cultivar (Chenin Blanc, Sauvignon Blanc and Chardonnay) and winemaking style (wooded and unwooded). The data was acquired using a non-targeted profiling method and processed using cloud metabolomics software, XCMS online, followed by multivariate analysis. Importantly, the pipeline methodology for this approach was established to characterise both qualitative and quantitative information on the wine samples. This strategy enabled the identification of the most sensitive and best profiling sample preparation method. In addition, this application conceptualises a pipeline methodology approach, whereby sample preparation, instrument analysis, cloud data processing and multivariate analysis is applied in order to obtain meaningful relationships in complex data sets. As an extension of the targeted and non-targeted strategies reported, the fourth and final objective (chapter 6) showcases an application of this pipeline methodology to craft beer (n=91) and gin (n=67). Craft beer and gin samples were analysed using the targeted terpenoid and non-targeted profiling strategies. Subsequent data processing and curation allowed for application to multivariate statistical analysis. The configurational similarity was compared to reveal differences between the targeted and non-targeted strategies. Further insight was made into identifying characteristic biomarkers for distinguishing between lager-style and ale-style craft beers.
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    Discovery of novel anti-Candida albicans compounds from environmental bacteria
    (Stellenbosch : Stellenbosch University, 2023-12) Barnard-Jenkins, Bernice; Rautenbach, Marina; Snoep, Jacky; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The opportunistic fungal pathogen, Candida albicans (C. albicans), has the ability to colonize different epithelial tissues (e.g. vaginal, intestinal and oral tissues) causing moderate to severe and fatal infections. The fatal infections are due to fungal cells that enter the bloodstream and circulate after being dispersed from their resistant biofilms. Organisms that have adapted to form biofilms are associated with improved tolerance to drugs, which was also found for some drugs in this study. A medium-throughput multiplex assay system (MPAS) has been developed for the in-parallel study of several fungal life-cycle stages of different strains of C. albicans. MPAS was designed to use conventional 96-well plates and 96-pin lids in four different assay layouts. In MPAS, the metabolic activity measurements were used to determine the effect of antimicrobial compounds in assay targeting planktonic cell susceptibility, biofilm prevention, biofilm eradication, as well as susceptibility of cells released from biofilms. MPAS detection limits were further optimized by testing the activity of antimicrobial culture extracts from known bacterial producers on the different C. albicans life stages. The MPAS data showed which producer extracts were more active against certain strains of C. albicans, as well as highlighted which fungal forms are more sensitive to certain culture extracts. This led to the successful application of MPAS to identify active culture extracts of unknown soil microbes against planktonic and biofilm forms of C. albicans for further characterization. Ultra-performance liquid chromatography linked to high resolution tandem mass spectrometry (HR UPLC-MSᵉ) was used to identify and characterize known antifungal lipopeptides (such as surfactins, iturins and fengycins), rhamnolipids, as well as active small quorum sensing compounds in the most active culture extracts. The overall success of the MPAS together with HR UPLC-MSᵉ was confirmed by the successful identification of known and unknown compounds, from soil sample bacteria, with activity against C. albicans. The study showed the potential of the medium throughput MPAS together with HR UPLC-MSᵉ to discover and characterize potential antibiofilm and antifungal compounds for further development.
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    Phylogenetic analyses in the african fish genus nothobranchius in relation to landscape evolution
    (Stellenbosch : Stellenbosch University, 2023-03) Van der Merwe, Pieter De Wet; Bellstedt, Dirk U.; Cotterill, Fenton P. D. ; Schliewen, Ulrich Kurt; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Nothobranchius is a genus of short-lived annual to semi-annual African freshwater fishes that are found in smaller seasonal ephemeral wetlands or pools and not in larger open lakes or non-seasonal river systems. The waterbodies in which they are found are situated on vertisol-type soils that have a large capacity to absorb and retain water which is slowly released upon drying. Eggs are fertilized and are deposited into the vertisol-type substrate of the waterbody. As the soil dries during the dry season cracks are formed and the eggs, which up to that point have been in an anoxic environment, are exposed to oxygen which stimulates them to develop. Several complicated pathways exist through which the eggs develop, which can lead to full development, or to several types of delayed development called diapauses. Some will remain dormant for subsequent rainy seasons ensuring that the species can survive extended droughts. Nothobranchius hatch quickly after the rainy season starts and subsequently grow very rapidly to reach maturity to repeat the life cycle. Sequencing of two mitochondrial and three nuclear genes of the majority of the species in Nothobranchius and appropriate outgroups was used to generate suitable sequence matrices which were analysed extensively using maximum likelihood and Bayesian Inference to generate robust phylogenies and dated phylogenies of the genus. A dated phylogeny was used in a biogeographic analysis to determine dispersal routes and establish that the ancestral area of Nothobranchius is in the Nilo-Sudan. Using the recently developed technique, double digest RAD sequencing, an even greater sequence data matrix of the species in Nothobranchius and appropriate outgroups was generated and analysed using the same techniques as described above. This resulted in an even more robust phylogenetic hypothesis for the genus. From this dated phylogeny landscape evolution in a tectonically active area on south central Africa could be inferred. Four key landscape evolutionary events in this area could be inferred and dated: (i) landscape warping, faulting and rifting in the Luapula river drainage, (ii), formation of the Upemba rift in the Lufira-Lualaba river drainage system, and (iii) formation of the Congo-Zambezi Watershed between the Kafue and Luapula drainage systems disrupting the ancient Palaeo-Chambeshi river drainage system, (iv) drainage of Palaeo Lake Mweru, from a previous maximum level of 1200 masl to the present-day level of 917 masl. Lastly, this research resulted in the identification of 15 new Nothobranchius species which were described in the course of this study. .
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    Quantifying the insulin response in mouse C2C12 skeletal muscle: a minimal modelling approach
    (Stellenbosch : Stellenbosch University, 2021-12) Kuhn, Stefan; Snoep, Jacky L.; Westerhoff, Hans V.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The insulin signalling cascade is one of the most important regulatory and signalling pathways in humans. Dysregulation or dysfunction of the insulin signalling path- ways often underlies the molecular ætiology of diseases such as diabetes, obesity, and Alzheimer’s. In turn, these diseases are the harbingers of various co-morbidities such as cardio-vascular disease, chronic inflammation, and dementia. The healthcare, eco- nomic, personal, and mortality burden of these diseases cannot be overstated. Mathematical modelling of insulin signalling is indispensable in the effort to un- derstand the dynamics of the insulin signalling cascade and how malfunctions therein lead to disease. However, despite the availability and complexity of existing models, few have explicitly connected the signalling cascade, glucose transporter activity, and metabolism with one another. In order to study these interactions, a ‘three-module’ approach was adopted that defined the signalling cascade, glucose transporter activ- ity, and metabolism as core, ‘input-output’ modules. The present work is limited to the signalling cascade and glucose transporter activity modules whereas work by Dr. Cobus van Dyk is concerned with the metabolic module. With this in mind, this thesis sets forth three aims. Firstly, to establish standard- ised culturing conditions which can be used to determine the basal state of insulin signalling and glucose transporter activity. Secondly, to develop a core, mathemati- cal model based on Western blotting and radio-labelled glucose -assay data which is able to describe the concentration- and time-dependence of the signalling cascade and glucose transporter activity in response to insulin. Thirdly, to determine the clustering behaviour of GFP-tagged GLUT4 molecules in response to insulin. The first goal was to standardise culturing conditions. Herein, the ability of high (25mM), medium (15mM), and low (5mM) glucose culturing conditions were evalu- ated with regards to their ability to sensitise or desensitise the insulin signalling cascade as well as the degree to which they are able to induce the differentiation of C2C12 my- oblasts into myocytes. The glucose and lactate concentrations in the external media were used to determine the glucose-lactate flux of the C2C12 cells. This served as a proxy for the induction of insulin-dependent glucose transport and metabolism. A modified Ladd staining protocol was used to assess the degree to which C2C12 cells could differentiate under the culturing protocols. The second goal was to construct a core, mathematical model of insulin signalling and glucose transporter activity. The time-dependent phosphorylation and dephos- phorylation of the insulin receptor and the serine 473 and threonine 308 sites of Akt in response to varying insulin concentrations was investigated using Western blotting techniques. The glucose transporter (GLUT4) activity was assayed using radio-carbon glucose. The data were used to optimise parameters for a core, ODE-based model of the signalling and glucose transporter modules. The third goal, to investigate the clustering behaviour of GLUT4 in response to insulin, was investigated by using confocal microscopy to image GFP-tagged GLUT4 molecules before and after being stimulated with insulin. A hierarchical clustering algorithm as well as further geometric and statistical analyses were used to determine the number, size, density, and distribution of GLUT4 clusters pre and post insulin exposure. Of the remaining chapters, Chapter 1 discusses the background, context, scope, and aims of this study as well as further elaborating on the ‘three module’ approach. The literature review in Chapter 2 provides an overview of the relevant literature as delineated by the scope and aims of this study. The materials and methods are provided in Chapter 3, with specific alterations or methodologies being further discussed in the relevant experimental chapters. The final chapter, Chapter 7, provides the reader with general discussions, limitations, and final thoughts concerning this work.
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    The development of standardized ion mobility and mass spectrometry metabolomics methods for the characterization of plant phenolics
    (Stellenbosch : Stellenbosch University, 2021-04) Masike, Keabetswe; Stander, Marietjie; De Villiers, Andre; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Plants and plant-derived products contain a plethora of phenolic compounds, with a broad range of health benefits and useful applications such as in drug design. These phenolic compounds are often amplified by conjugation and rearrangements, thus producing isobaric and isomeric species. The standard analytical method for the identification and characterization of plant phenolics, liquid chromatography (LC) hyphenated to photo diode array (PDA) and/or high-resolution mass spectrometry (HR-MS) detectors, is not able to discriminate isomeric species in complex plant samples. The incorporation of ion mobility spectrometry (IMS) to LC-PDA-HR-MS workflows is being recognized as an additional orthogonal dimension of separation to HR-MS; where ions are separated through a drift region, filled with gas, based on their size, shape, and charge. The attractive component of IMS is the determination of the collisional cross section (CCS/Ω) values, which describes the unique rotationally averaged surface area of the ion, as it interacts and travels through the gas-filled drift region. The CCS as a feature, can be beneficial in the development of an in-house phenolics compound library in analytical laboratories, which can help expedite the characterization of phenolic compounds in varying research fields, such as in plant metabolomics and food science. The goal of the work reported in this thesis was, therefore, to develop LC-PDA-IM-HR-MS methods capable of structurally characterizing plant phenolics, found in South African indigenous herbal teas and plant species, based on a range of structural descriptors (e.g., tR, spectroscopic data, mass spectral information (including high resolution and MS/MS data), and CCS value). In the first part of the study, the phenolic profiles of Protea pure and hybrid cultivars were characterised by LC-PDA-IM-HR-MS for the first time in detail. Whereby, IMS in conjunction with other structure elucidation techniques, namely tandem MS data, UV-vis spectroscopy, nuclear magnetic resonance (NMR) were used for characterization of 67 metabolites. With the aid of NMR an undescribed hydroxycinnamic acid-polygalatol ester, caffeoyl-O-polygalatol (1,5-anhydro-[6-O-caffeoyl]-sorbitol(glucitol)) was isolated and characterised for the first time. Furthermore, positional isomers with similar MS/MS profiles were resolved by the IMS-dimension and consequently could be distinguished by their differences in CCS values. The CCS values obtained using two IMS platforms (drift-time ion mobility spectrometry (DTIMS) and travelling wave IMS (TWIMS)) were compared, and it was observed that the CCS values obtained with a TWIMS instrument were underestimated for compounds with CCS values below 200 Å2. Conversely, good agreement was obtained between both instruments for compounds with higher CCS values. Poor calibration of the TWIMS platform was attributed to the underestimation of the CCS values below 200 Å2. In the second part of the study, a detailed comparison of phytochemical profiles of a much larger set of Protea species, selections, and cultivars was reported. Using metabolomics tools and the data collected and documented (in the previous study) of phenolic compounds characterised based on their UPLC-PDA-IM-HR-MS profiles, plant metabolites associated with a post-harvesting disorder, leaf blackening, in Protea were identified/and annotated. Species, selections, and cultivars susceptible to leaf blackening contained features identified as benzenetriol- and/or hydroquinone-glycoside derivatives. On the other hand, stems not prone to blackening were linked to phenolic compounds with known protective properties against biotic and abiotic stressors. CCS values of the metabolites with protective features against leaf blackening, and those for compounds that instigate the process, were determined. Such observations serve as preliminary insights that can help accelerate plant improvement and aid in the selection of trait-specific markers in plant metabolomics. In the final portion of the study, using direct injection-IM-MS (DI-IM-MS) and descriptive chemometrics, differences between adulterated herbal teas (rooibos and honeybush) were observed. The diagnostic value of marker compounds in distinguishing the three commercialised honeybush species (Cyclopia intermedia, C. genistoides and C. subternata) for the quality control purpose were observed. To derive CCS values using the TWIMS instrument two calibrants, poly-DL-alanine and poly-L-malic acid, were compared. Poly-DL- alanine is a widely used TWIMS calibrant which results in an underestimation for compounds with CCS values below 200 Å2 (initial study), when compared to poly-L-malic acid an improvement in the underestimation of CCS values below 200 Å2 was observed. DI-IM-MS proved to be a useful tool for quality control purposes, particularly considering the analysis time is 1 minute per sample.