Doctor Educationis / Maize (Zea mays L.) is grown globally as an important grain crop in South Africa, United States, China and Brazil and plays a major role in the worldwide economy. In South Africa, the grain is utilised for food consumption, livestock feed, for malting purposes and bioethanol production. Maize contains approximately 72% starch, 10% protein, 4% fat and supplying an energy density of 365 Kcal/100 g. The production of grain crops in South Africa is restricted by various factors such as abiotic and biotic stresses. The fungal genus Aspergillus is one of the most important biotic stresses affecting maize in the country. Aspergillus flavus can contaminate a wide range of agricultural commodities either in storage or field. Hydrogen sulfide appears to have a potential in the mechanism of resistance against pathogen attack by Aspergillus flavus. / 2023

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<p> Exercise intensity is one of the major factors determining the utilization of carbohydrates (CHO) and lipids in mammalian skeletal muscle. Using indirect calorimetry, we determined maximal oxygen uptake (VO2max) and whole-body rates of CHO and lipid oxidation in rats selectively bred for high and low running capacity (HCR's and LCR's) during exercise at 50, 60, 70 and 80%VO2max. Previous studies have revealed a pattern of selection where mammals with different aerobic capacities use the same proportions of lipids and CHO when exercising at the same relative exercise intensity and as intensity increases, CHO use increases and lipid use decreases. The present results showed that the HCR's had a VO2max and distance run to exhaustion that was 1.3 and 4.0 times greater than the LCR's respectively. Also, both groups of rats followed the pattern of fuel selection seen in previous studies where the same proportions (in%) of lipids and CHO are used at the same relative exercise intensity. On an absolute scale, the HCR's used more lipids and CHO than the LCR's at all exercise intensities but the results were not always statistically significant. We also determined the exercise intensity that elicited the greatest lipid use to be 60% VO2max in both groups.</p> <p> In order to explain these patterns of fuel selection, metabolic indicators, metabolites and enzymes, in skeletal muscle were measured at rest and post exercise for one hour at 60%VO2max. Specifically, ATP and phosphocreatine (PCr) metabolite concentrations were determined in the medial and lateral gastrocnemius, extensor digitorum longus (EDL), tibialis anterior (TA), and soleus muscle. The medial gastrocnemius and soleus were analyzed (pre and post exercise samples were combined) for their oxidative and glycolytic enzyme activity by measuring citrate synthase (CS), cytochrome oxidase (COX), β-hydroxyacyl CoA dehydrogenase (HOAD), and lactate dehydrogenase (LDH) . PCr and ATP concentrations did not change pre and post exercise and between the HCR's and LCR's except for the EDL where there was a significant decrease (P<0.05) in both metabolites after exercise in both groups of rats. For the enzyme measurements, CS and COX activities were higher (P<0.05) in the HCR's for the soleus and HOAD activities were also higher in the HCR's medial gastrocnemius compared to the LCR's. We concluded that the HCR's have a greater oxidative capacity as shown by their greater aerobic and endurance capacity (VO2max and distance to exhaustion), their ability to oxidize a greater absolute amount of lipids and CHO's at the same relative exercise intensity, and their higher activities of oxidative enzymes in the soleus (CS and COX) and medial gastrocnemius (HOAD). Future research into the mechanisms involved in explaining these patterns of fuel selection may include examining fatty acid transport proteins, fatty acid and CHO availability, fiber types, and catecholamines.</p> / Thesis / Master of Science (MSc)

Recent work in microbial ecology has focused on elucidating controls over biogeographic patterns and connecting microbial community composition to ecosystem function. My objective was to investigate the relative influences of landscape legacies and contemporary environmental factors on the distribution of soil microbial communities and their contribution to ecosystem processes across a glacial till sequence in Taylor Valley, Antarctica. Within each till unit, I sampled from dry areas and areas with visible evidence of recent surface water movement generated by seasonal melting of ephemeral snow packs and hillslope ground ice. Using T-RFLP 16S rRNA gene profiles of microbial communities, I analyzed the contribution of till and environmental factors to community similarity, and assessed the functional potential of the microbial community using extracellular enzyme activity assays. Microbial communities were influenced by geochemical differences among both tills and local environments, but especially organized by variables associated with water availability as the first axis of an NMDS ordination was strongly related to shifts in soil moisture content. CCA revealed that tills explained only 3.4% of the variability in community similarity among sites, while geochemical variables explained 18.5%. Extracellular enzyme activity was correlated with relevant geochemical variables reflecting the influence of nutrient limitation on microbial activity. In addition, enzyme activity was related to changes in community similarity, particularly in wet environments with a partial Mantel correlation of 0.32. These results demonstrate how landscape history and environmental conditions can shape the functional potential of a microbial community mediated through shifts in microbial community composition. / Master of Science

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Computer and Applied Sciences) Vaal University of Technology / This study seeks to design and test specific short hairpin RNA (pshRNA) for their efficiency in knocking down the GALT gene RNA products thereby limiting the resultant enzyme activity. The following objectives were followed in designing the current study: 1. Designing a shorthairpin RNA (pshRNA) to target different regions of the coding sequence of the target GALT gene. 2. Propagating the pshRNAs in Escherichia coli (E.coli) and subsequently isolation of the respective plasmids for transfection. 3. Transfection of HeLa cells to test the efficiency of relevant pshRNAs in knocking down the GALT gene expression. 4. Transfection was followed by extraction of total mRNA, purification and quantification of total mRNA. 5. The GALT gene expression was qualitatively quantified against a house-keeping gene, glyceraldehyde phosphate dehydrogenase (GAPDH) to evaluate efficiency of knockdown using real time PCR. The three newly designed pshRNA (pshRNA2, pshRNA3 and pshRNA4) targeting the GALT gene expression showed a knockdown efficiency of 171 %, 48 % and 200 %, respectively. The results of this study will be useful for future evaluation of the possible long term glycosylation patterns under proper UDP glucose/UDP galactose levels compared with variable defective GALT gene levels.

Cette thèse traite de la conception de sondes fluorogènes incorporant des bras espaceurs auto-effondrables répondant à l’activité enzymatique.Ces travaux commencent par la synthèse d’une sonde modèle à trois composantes pour détecter l’activité de la Leucine AminoPeptidase (LAP). Le cœur de cette sonde est un espaceur cyclisant efficace (t1/2 cyclisation≈7sec) qui unit un substrat enzymatique à un fluorophore précipitant avec une stabilité exemplaire (pas de dégradation sur 15h d’incubation). Appliquée sur des cellules vivantes, cette sonde produit des précipités fluorescents qui marquent durablement les cellules. L’élaboration de sondes pour d’autres enzymes a cependant soulevé l’importance d’abaisser le seuil de solubilité du fluorophore.Cette thèse se penche également sur deux nouvelles conceptions de sondes à la spécificité améliorée. Alors que la première tente de réutiliser les efforts déployés pour obtenir des inhibiteurs hautement sélectifs, la seconde est basée sur deux transformations successives par deux enzymes indépendantes. Si la première solution a échoué jusqu’alors, la seconde nous a effectivement permis de différencier des populations cellulaires.Enfin, ce manuscrit détaille le développement d’une nouvelle génération d’espaceur permettant l’affinement de certaines propriétés des sondes. Focalisés sur l’amélioration de l’hydrosolubilité, les premiers exemples sont très prometteurs. Particulièrement, une sonde pour Péncilline G Amidase possède une hydrosolubilité 3500 fois supérieure à celle de son analogue commercial. Véritables multiprises chimiques, ces espaceurs devraient permettent de relever certains des grands défis de la chimie médicinale moderne. / This thesis concerns the design and evaluation of fluorogenic molecular probes that respond to enzyme activity via the help of self-immolative spacers.This work starts with the synthesis of a model three-component probe that detects the activity of Leucine AminoPeptidase (LAP). The heart of this probe is an efficient cyclizing spacer (t1/2 cyclization≈7sec) that links a specific enzyme substrate to a precipitating fluorophore with an exemplary stability (no false positive signal over 15h incubation). When incubated with live cells, this construct is processed by active LAP to yield fluorescent precipitates which lead to long-term cell-tagging. However, probes susceptible to other enzyme activity have indicated the interest in further reduction of the solubility threshold of ELF®97.This manuscript also describes two new strategies to improve the specificity of the probes. While the first tries to take advantage of the efforts made to develop highly selective inhibitors, the second is based on two consecutive transformations by two independent enzymes. The first strategy has not yet been successfully applied in our hands, but the second has led to a first prototype that allowed discriminating between different cell lines.Lastly, this thesis relates the design and synthesis of a new generation of cyclizing spacer which opens up a great number of possibilities to optimize probes’ properties. For example, a probe targeting Pencilline G Amidase and containing such a spacer possesses a hydrosolubility 3500 times higher than its commercial analogue. As true “molecular hubs”, these spacers may turn out to address the big challenges of modern imaging agent and prodrug development.

Melanina é um polímero constituído por uma grande heterogeneidade de monômeros tendo como característica comum a presença de grupos indóis. Por outro lado, a eumelanina produzida pela oxidação enzimática da tirosina é um polímero mais simples constituído principalmente de monômeros 5,6-dihidroxindol (DHI) e de indol-5,6-quinona (IQ). Tirosinase é a enzima chave na produção de melanina, sendo que a sua atividade cinética é medida em função da formação do intermediário dopacroma. Nanopartículas (NPs) de sílica são partículas nanométricas compostas de oxido de silício e são obtidas pelo processo sol-gel desenvolvido por Stöber de hidrólise e condensação de tetraetilortosilicato (TEOS), usando etanol como solvente em meio alcalino. As NPs foram funcionalizadas com 3-Aminopropiltrietoxissilano (ATPES) e depois com glutaraldeído. Este último permitiu a imobilização da tirosinase na superfície da sílica. Caracterizamos as NPs antes e após a reação da enzima, a atividade catalítica da enzima ligada à NP e o mecanismos de formação de melanina na superfície da sílica. As NPs foram caracterizadas por espectrofotometria de absorção e de reflectância, termogravimetria e microscopia eletrônica. A síntese da NP de sílica retornou partículas esféricas com 55nm de diâmetro e a funcionalização da partícula mostrou modificar eficientemente a sua superfície. A imobilização da tirosinase por ligação covalente foi de 99,5% contra 0,5% da adsorção física. A atividade da tirosinase foi caracterizada pela formação de dopacroma. O Km da enzima imobilizada não sofreu alteração em comparação com a tirosinase livre, mas a eficiência catalítica - que considera a eficiência recuperada - foi de apenas 1/3 para a enzima ligada covalentemente, significando que 2/3 das enzimas ligadas não estão ativas. Obtivemos NPs revestidas com melanina a partir de oxidação de tirosina solubilizada em duas preparações: NP com tirosinase ligada covalentemente na superfície e NP funcionalizada com glutaraldeido dispersa em solução de DHI e IQ. O revestimento de melanina foi na forma de um filme fino com espessura ~1,9nm, conferindo perfil de absorção luminosa equivalente ao da própria melanina. Mostramos que o mecanismo de polimerização passa pela oxidação da tirosina pela tirosinase, que gera intermediários oxidados (principalmente DHI e IQ) que vão para solução (mesmo quando a tirosinase está ligada covalentemente na sílica). Estes intermediários ligam-se ao glutaraldeido e a superfície da sílica passa a funcionar como ambiente de polimerização da melanina. / Melanin is a polymer consisting of a large heterogeneity of monomers having as a common feature the presence of indole groups. Contrarily, eumelanin produced by enzymatic oxidation of tyrosine is a simpler polymer consisting mainly of 5,6-dihidroxindol (DHI) and indole-5,6-quinone (IQ) monomers. Tyrosinase is the key enzyme in melanin production, and its kinetic activity is measured by the formation of the intermediate dopacroma. Nanoparticles (NPs) are made of silica nanoparticles of silicon oxide and are obtained by sol-gel method developed by Stöber of hydrolysis and condensation of tetraethylorthosilicate (TEOS), using ethanol as solvent in an alkaline medium. NPs were functionalized with 3-Aminopropyltriethoxysilane (ATPES) and then with glutaraldehyde. The latter allows the immobilization of tyrosinase on the silica surface. We characterized NPs before and after the reaction of the enzyme, the catalytic activity of the enzyme bound to the NP and melanin-forming mechanisms on the silica surface. NPs were characterized by absorption spectrophotometry and reflectance, electron microscopy and thermogravimetric analysis. The synthesis of silica NP returned spherical particles of 55nm diameter and particle functionalization showed efficiently modify its surface. The immobilization of tyrosinase by covalent bond was 99.5% versus 0.5% by physical adsorption. The activity of tyrosinase was characterized by the formation of dopacroma. The Km of the immobilized enzyme did not change compared to the free tyrosinase, but the catalytic efficiency - considering the recovered efficiently - was only 1/3 for the enzyme covalently bound, meaning that 2/3 of the enzymes are not connected active. We obtained melanin coated NPs from tyrosine oxidation in two preparations: NP with covalently bound tyrosinase in the NP surface and NP functionalized with glutaraldehyde dispersed in DHI and IQ solution. The melanin coating was in the form of a thin film with the thickness of ~ 1,9 nm, giving light absorption profile equivalent to that of melanin itself. We showed that the polymerization mechanism involves the oxidation of tyrosine by tyrosinase, which generates oxidized intermediates (especially DHI and lQ) that go into solution (even when tyrosinase is covalently bound to the silica). These intermediates bind the glutaraldehyde and the surface of the silica begins to function as an environment for melanin polymerization.

Cette thèse de doctorat traite de la conception et de l’évaluation de sondes magnétogènes et fluorogènes pour la détection in vivo d’activités enzymatiques.Des molécules capables d’acquérir un moment magnétique à partir d’un état diamagnétique et en réponse à l’action d’une enzyme seraient d’un grand intérêt pour l’imagerie moléculaire par résonance magnétique. Deux exemples de telles sondes magnétogéniques avaient été mis au point précédemment, l’un pouvant opérer en conditions physiologiques, l’autre nécessitant une acidification du milieu pour devenir paramagnétique. En préparant de nouveaux analogues du premier exemple, j’ai pu trouver une molécule dont la fragmentation a lieu trois fois plus rapidement que la molécule originale. J’ai ensuite travaillé à la conception de sondes dérivées du deuxième exemple et répondant à des activités enzymatiques ; de telles molécules permettraient de réaliser la quantification in vitro d’une activité enzymatique à des fins diagnostiques. À ce titre, j’ai participé à l’élaboration de deux preuves de concept de dispositifs dédiés à la mesure du temps de relaxation longitudinale de micro-volumes. J’ai enfin entamé le développement de nouveaux complexes s’inspirant du second exemple mais capables de fonctionner dans le milieu biologique.Le deuxième volet de mes travaux porte sur la réalisation de sondes fluorogènes précipitantes pour des activités glycosidases. Une sonde pour la leucine aminopeptidase profitant des propriétés exceptionnelles de stabilité, de luminescence et de solubilité du fluorophore ELF®-97 avaient démontré une grande efficacité pour marquer rapidement des cellules HeLa. J’ai mis au point une nouvelle architecture de sondes qui permet le ciblage de glycosidases via l’utilisation d’un tandem d’espaceurs cyclisants. Deux sondes ont été préparées, l’une pour la beta-galactosidase, l’autre pour la cellulase. La première a prouvé son bon fonctionnement pour marquer les cellules exprimant l’enzyme en bénéficiant d’une grande sensibilité. La seconde a pu être utilisée pour quantifier l’activité cellulase sécrétée par des levures, avec l’objectif d’obtenir un moyen économiquement intéressant de produire du bioéthanol à partir des déchets végétaux. / This PhD thesis deals with the design and evaluation of magnetogenic and fluorogenic probes for the in vivo detection of enzyme activities.Molecules capable of switching from a diamagnetic to a paramagnetic state in response to an enzyme stimulus would be of great interest for molecular magnetic resonance imaging. Two examples of such magnetogenic probes had been designed in a previous work : one can operate in physiological conditions, whereas the other needs an acidification of the water medium to become paramagnetic. I prepared new analogues of the first probe ; one molecule displayed fragmentation three times faster than the original compound. Then I designed and synthesized probes derived from the second example and responsive to enzyme activities ; such molecules are suitable for the in vitro quantification of enzyme biomarkers for diagnosis purposes. I participated to the conception of two proofs of concept of devices dedicated to the measurement of longitudinal relaxation times in micro-volumes. Finally, I started the development of a new family of molecules inspired by the second example but able to work at the physiological pH.I also worked on precipitating fluorogenic probes for the detection of glycosidase activities. A former probe for leucine aminopeptidase, based on the exceptional characteristics of the fluorophore ELF-97 in terms of solubility, luminescence and stability, had demonstrated great efficiency to label live HeLa cells. I designed a new architecture of probes responding to glycosidases via an original tandem of selfimmolative spacers. Two probes have been prepared, one targets beta-galactosidase and the second detects cellulase. The first probe performed a fast and sensitive labelling of beta-galactosidase-expressing cells. The second molecule was employed successfully to quantify the cellulase activity secreted by yeasts, which will be useful for the high-throughput screening of yeasts capable of producing bioethanol from vegetal waste.