Biofumigation is an important crop protection practice that uses a plant’s natural defence mechanisms to control agricultural crop pathogens and diseases. Glucosinolates are volatile compounds found in most Brassica species and when hydrolysed, it forms a range of natural toxins including isothiocyanates that act as biofumigants. Research suggests that biofumigation is a good alternative to chemical fumigants as it is effective in controlling plant pests but with lower health and environmental risks. Several studies have confirmed the effectiveness of the breakdown products, especially isothiocyanates, as fungicidal, bactericidal and nematicidal products against a series of plant pests. However, very little information is available on the effects of glucosinolates and its breakdown products on non-target and beneficial soil organisms. Negative effects on beneficial soil organisms can have serious negative impacts on soil quality especially when essential ecosystem functions such as nutrient cycling and soil bioturbation are affected. Three biofumigants, broccoli, mustard and oilseed radish, and two chemical fumigants, metham sodium and cadusafos, were investigated for possible effects on non-target and essential soil organisms such as earthworms and the soil microbial community. Sublethal endpoints, including growth and reproductive success of the earthworms, were monitored. The genotoxicity of the biologically active compounds found in the fumigants, towards earthworms, was evaluated by means of the comet assay. The DNA damage was quantified by tail intensity parameters. Furthermore, the changes in the soil microbial community function and structure were evaluated by means of community level physiological profiling (CLPP) and phospholipid fatty acid (PLFA) analyses respectively. All exposures were done in artificial soil prepared according to the OECD standard guidelines. In the biofumigant treated soils, results varied and different effects were observed on the non-target soil organisms. Broccoli reduced cocoon production and the number of hatchlings while mustard induced more DNA strand breaks in earthworm cells compared to the control. All the biofumigants stimulated microbial growth but broccoli and oilseed radish changed the microbial functional diversity. Mustard had no lasting effect on the functional diversity but altered the microbial community structure. The chemical fumigants had a marked negative impact on the survival, growth, reproduction and the genotoxicity of the earthworms with metham sodium causing greater harm than cadusafos. The effects on the microbial community varied. Both chemicals had an inhibitory effect on the microbial growth in terms of the viable biomass determined by PLFA and the average well colour development in the Biolog™ Ecoplates. No lasting effects were observed in the community structure. Overall, cadusafos had a more pronounced effect on the microbial community functional diversity than metham sodium. Results indicated that each bioindicator species illustrates effects at their own level of organisation / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Background: Cardiovascular disease (CVD) is the leading global cause of death. CVD risk factors are considered intermediaries for the association between dietary fatty acids and CVD. Raised plasma total cholesterol, low density lipoprotein (LDL) cholesterol, raised triglycerides and decreased levels of high density lipoprotein (HDL) cholesterol, as well as reduced fibrinolytic potential (measured as increased clot lysis time) are known risk factors for CVD. Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of the fibrinolytic process and an elevated PAI-1 level is therefore considered to be a potential risk factor for CVD. The growing number of controversies around the role that fat intake (more specifically the type of dietary fat) plays in CVD risk, is making it increasingly difficult for consumers and practitioners alike to form conclusions, and make recommendations and decisions regarding fat intake. Knowledge of the intake of individual fatty acids, fatty acid status (as opposed to subgroups of fat such as polyunsaturated fatty acids) and their associations with blood lipids, PAI-1act and fibrinolytic potential is lacking in black South Africans and other populations. Therefore we aimed to investigate dietary fatty acid intake, as well as plasma phospholipid fatty acid status and their associations with blood lipids, PAI-1act and clot lysis time, as a marker for fibrinolytic potential. Methods: Cross-sectional data analysis within the Prospective Rural Urban Epidemiology (PURE) baseline study of apparently healthy black South African men and women (n=1950, 35– 70 years) from rural and urban areas in the North West Province, from whom dietary data were collected. Blood lipid analyses, as well as laboratory analyses of fibrinolysis markers such as PAI-1act and clot lysis time were also performed. Plasma phospholipid fatty acid extraction and isolation were performed on a random subsample (n = 716). Results: The intake of individual fatty acids was significantly higher in urban than rural dwellers. However, the intake of omega-3 polyunsaturated fatty acids was below recommendations in all groups (rural and urban males, and rural and urban females). Total cholesterol and LDL cholesterol were higher in females than in males, with no rural‒urban differences. Intake of alpha-linolenic acid was positively associated with total cholesterol (β=0.143) and triglycerides (β=0.256) in males. The risk of having elevated LDL cholesterol also increased with increased intake of alpha-linolenic acid (OR 1.49, 95% CI 1.04, 2.14). In females, dietary arachidonic acid and eicosapentaenoic acid (EPA) were positively associated with total cholesterol and LDL cholesterol, whereas docosahexaenoic acid (DHA) was negatively associated with total cholesterol and LDL cholesterol. Dietary alpha-linolenic acid was positively correlated with plasma EPA (males r = 0.19, p = 0.002, females r = 0.25, p < 0.001) and DHA (males r = 0.33, p < 0.001, females r = 0.30, p < 0.001). Plasma DHA was positively associated with triglycerides in males (β = 0.410, p< 0.001) and in females (β = 0.379, p< 0.001). PAI-1act was positively associated with clot lysis time, and plasma myristic acid and DHA were positively associated with PAI-1act in females. However, these fatty acids were not associated with clot lysis time. Different types of plasma fatty acids were associated with PAI-1act than with clot lysis time. Plasma alpha-linolenic acid (β = 0.123, P = 0.037), mead acid (β = 0.176, P = 0.019), arachidonic acid (β = 0.253, 0.025) and omega-3 docosapentaenoic acid (omega-3 DPA) (β = 0.224, P = 0.002) were positively associated with clot lysis time, while both myristic acid (β = - 0.130, P = 0.016) and EPA (β = -0.131, P = 0.021) were negatively associated with clot lysis time in male subjects. Plasma oleic acid (C18:1n9) (β = -0.411, P = 0.001) and omega-6 DPA (C22:5n6) (β = -0.285, P = 0.001) were negatively associated with clot lysis time, while dihomogamma- liolenic acid (DGLA) (C20:3n6) were positively associated (β = 0.178, P = 0.001) with clot lysis time in females. Conclusions: These results suggest that specific individual dietary fatty acids might be associated with blood lipids in males differently than in females, irrespective of rural or urban dwelling. It is not known however, if associations would still be present under conditions of greater intake of alpha-linolenic acid. Our results further suggest that a higher percentage of alpha-linolenic acid might be converted to DHA in this population with low intake of essential and long-chain polyunsaturated fatty acids compared to populations with a high intake of these fatty acids. These results suggest that plasma phospholipid fatty acids should not be used in isolation as biomarkers for intake of fat, without taking dietary intake data into consideration also. Associations between fatty acids and clot lysis time might be independent from PAI-1act. The association between mead acid and clot lysis time indicates that clot lysis time might increase with an essential fatty acid deficiency. This may be of particular concern in this population with a documented lower fat intake. Because the study design of this study is crosssectional, it is not able to determine cause-and-effect, and results should therefore be verified with a randomised controlled trial. / PhD (Nutrition), North-West University, Potchefstroom Campus, 2015

The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007

Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.

The physical and biophysical properties of a biological membrane model, phosphatidylcholine bilayers, were investigated using novel spiropyran/merocyanine molecular probes. The femtosecond to second dynamics of this system's photochemistry enabled bilayer viscosity and free volume to be studied over a broad time scale. Spiropyrans/merocyanines with different polarity were synthesized by changing the substitution of the indole moiety enabling determination of the trans-membrane properties of the bilayer. In addition, transient grating spectroscopy was used to study thermal energy transfer in phospholipid bilayers on a picosecond time scale.Femtosecond transient absorption spectroscopy was used to study the photo-induced spiropyran ring-opening and isomerization reactions that produce the highly polar merocyanine species. The hindered rotation of the merocyanine bridge results in several metastable merocyanine isomers. The merocyanine ground state was determined to be populated predominantly by two isomers (TTC and TTT). Selective photoexcitation of these isomers results in excited state isomerization producing a third isomer (τ = 60 ps). Merocyanine thermal ring-closing was observed on a seconds time scale. Reaction kinetics, and solvatochromic and photochromic properties of merocyanines and spiropyrans were used to determine the bilayer physical properties. Bilayer viscosity was determined from merocyanine isomerization kinetics. Phospholipid bilayer free volume (the unoccupied volume enclosed in the bilayer) was determined from a modified Kramers' analysis. The greatest free volume was found in the extreme interior of the bilayer, while the head-group region exhibited the least free volume in qualitative agreement with molecular dynamics simulations of these bilayer systems. Free volumes determined via ps experiments were lower than those determined on a seconds time scale due to reduced acyl chain dynamics on the ps time scale.Femtosecond transient grating spectroscopy was used to study the rate of thermal energy transfer from photo-excited porphyrin molecules to the surrounding solvent. Thermal energy transfer was observed as photo-acoustic waves propelled through the system upon relaxation of photo-excited porphyrin molecules in aqueous solution and embedded in bilayers. For liposome solutions, a bimodal energy transfer model was developed. The determined rate constants suggest that energy transfer occurs predominantly via thermal diffusion and vibrational energy transfer, while lipid dynamics (isomerizations) are not involved.

Les analogues de choline constituent une nouvelle classe d'antipaludiques qui inhibent la biosynthèse de la phosphatidylcholine (PC) de Plasmodium, parasite responsable du paludisme. Les études conduites ont mis en relief des particularités uniques de ces composés. Nous avons élucidé le mécanisme d'action biochimique de l'albitiazolium, actuel candidat clinique, caractérisant chacune des 5 étapes conduisant à la biosynthèse de PC. L'albitiazolium affecte en premier lieu l'entrée de choline dans le parasite intraerythrocytaire, choline et albitiazolium utilisant le même transporteur et affecte de façon différentielle les autres étapes de synthèse. L'activité antipaludique est fortement antagonisée par la choline indiquant que le mécanisme d'action primaire est bien l'inhibition de la synthèse de PC. L'accumulation des analogues de choline dans le parasite intracellulaire leur permet de restreindre leur toxicité aux seuls érythrocytes infectés. Des études comparatives réalisées chez Plasmodium et Babesia montrent une double compartimentation de l'albitiazolium uniquement chez Plasmodium, l'une d'elles correspondant à la vacuole digestive. L'accumulation chez Plasmodium est glucose-dépendante et exige aussi le maintien des gradients ioniques dans la cellule. Bien que les analogues de choline exercent leur effet antiparasitaire dès les premières heures de contact, l'effet dit « cheval de Troie » exige des conditions particulières pour les mesures d'activités pharmacologiques, nous amenant à comparer différents tests d'activité. Seuls les tests isotopiques basés sur l'incorporation d'hypoxanthine ou d'éthanolamine après un cycle parasitaire entier et le test fluorescent au SYBR green appliqué après 72h obtiennent des résultats fiables quel que soit le mécanisme d'action des antipaludiques. Enfin, des études de pharmacocinétique / pharmacodynamie montrent une exposition plasmatique supérieure chez les souris infectées par Plasmodium, due au recyclage de l'albitiazolium après son accumulation dans l'érythrocyte infecté. / Choline analogues form a new class of antimalarial drugs that inhibit the biosynthesis of phosphatidylcholine (PC) in Plasmodium, the malaria-causing parasite. The studies presented here highlighted the unique features of these compounds. We elucidated the biochemical mechanism of action of albitiazolium, the current clinical candidate, characterizing each of the 5 steps leading to the biosynthesis of PC. Albitiazolium primarily affects the entry of choline into the intraerythrocytic parasite and choline and albitiazolium use the same carrier. The other steps of synthesis are differentially affected. Antimalarial activity is strongly antagonized by choline indicating that the primary mechanism of action is the inhibition of PC synthesis Accumulation of choline analogs in the intracellular parasite allows them to restrict their toxicity to infected erythrocytes. Comparative studies in Plasmodium and Babesia show a double compartmentalization of albitiazolium only in Plasmodium, one of them corresponding to the food vacuole. Accumulation in Plasmodium is glucose-dependent and requires maintaining ionic gradients in the cell.Although choline analogues exert their antiparasitic effect in the first hours of contact, the “Trojan horse effect” requires specific conditions for the determination of pharmacological activity, leading us to evaluate various tests of activity. Only the isotopic tests based on hypoxanthine or ethanolamine incorporation after one parasite cycle and the fluorescent SYBR green assay applied after 72 hours give reliable results regardless of the mode of action of the tested antimalarials. Finally, pharmacokinetics/pharmacodynamics studies in Plasmodium-infected mice revealed that albitiazolium is recycled after its accumulation in the infected erythrocyte leading to increased plasma levels.

Aluminium toxicity is the main limiting factor in crop production on acid soils. The main symptom of aluminium toxicity is a rapid inhibition of root growth, but the mechanism of root growth cessation remains unclear. In this diploma thesis we deal with the question of whether phospholipases PLDα1 and PLDδ may play a role in the mechanism of aluminium toxicity. We compared the responses of plants lacking PLDα and PLDδ with WT plants. Growth analysis of roots was performed in hydroponic conditions. The most sensitive part of roots was transient zone in which cells were dying earlier. It was further found that pldα1 plants were less sensitive on aluminium toxicity because their roots showed less growth inhibition than WT. Pldδ plants did not differ from WT plants in their response to aluminum. During further analysis of the pldα1 reactions, it was found that the root cells were capable of cell expansion during aluminum toxicity, and the cellular malformations were formed on the roots. This phenomenon was associated with faster reorientation and even depolymerization of cortical microtubules in response to toxic aluminium in pldα plants compared to WT plants. The results indicated that PLDα1 molecule affects the stability of cortical microtubules. Microtubules were less stable and they depolymerized...

Doctor of Philosophy / Division of Biology / Sherry D. Fleming / Ischemia/reperfusion (IR) events result in severe tissue damage and often death. The complex network of molecular and cellular mechanisms that contributes to intestinal IR-induced pathology has hindered a comprehensive understanding of IR-induced injury and limited the success of medical intervention. Although several of the mechanisms contributing to intestinal IR-induced injury have been identified, the initiating event(s) remains unclear. Mouse models have been instrumental in the unraveling of the many components and interactions that ultimately result in tissue damage. It is clear that leukocyte infiltration, complement activation, eicosanoid and pro-inflammatory cytokine production are involved. Toll-like receptors and antibodies also play critical roles. Based on the literature, and especially data demonstrating a significant role for anti-phospholipid antibodies, we hypothesized that ischemia induces phospholipid alterations that result in the exposure of a neoantigen which is recognized by anti-phospholipid antibodies. Furthermore, we hypothesized that endothelial cells are the primary cell type involved in the initial molecular events that result in intestinal IR-induced pathology. A mouse model of intestinal IR as well as an in vitro cell culture system was used to explore these hypotheses. Mass spectrometry-based lipidomics was utilized to assess lipid responses to IR and hypoxia/re-oxygenation (HR). No inherent differences in intestinal phospholipid composition were found between wildtype and several strains of knock-out mice. It was determined that the lack of antibody production by Rag-1[superscript]-[superscript]/[superscript]- mice is responsible for protection against intestinal IR-induced injury, as antibody is needed to induce prostaglandin E[subscript]2 production, through up-regulation of cyclooxygenase 2 transcription. Unexpectedly, the presence or absence of toll-like receptor 9 was found to be inconsequential for tissue damage caused by intestinal IR. The results of several analyses point to endothelial cells as being directly involved in IR-induced pathology. Importantly, the activation of phospholipid scramblase 1 has been identified as a potential molecular mechanism by which subsequent molecular and cellular responses are elicited as a consequence of IR.

Water-soluble deep-cavity cavitands provide a rare opportunity to study self-assembly driven by the hydrophobic effect. These molecular hosts dimerize in the presence of certain guest molecules to form water-soluble molecular capsules. These systems have given rise to numerous novel chemical phenomena and have potential use in drug delivery. The host octaacid (OA) has been particularly well-characterized, but studies are limited to basic pH because of limited host solubility. Herein we report an improved synthesis of OA and the syntheses of three new water-soluble deep-cavity cavitands. The new hosts are soluble at neutral pH, increasing relevance for biological studies. The new syntheses are versatile enough to apply to the synthesis of additional water- soluble cavitands in the future. We also describe preliminary characterization of the molecular recognition properties of the new hosts. Binding of organic guest molecules to form 1:1 host:guest complexes and 2:1 host:guest capsules was qualitatively similar to that of OA. However, binding of anions spanning the Hofmeister series revealed interesting new behavior. The new hosts bound a wider range of anions inside the hydrophobic pocket with much higher association constants. Moreover, external binding of several anions to the cavitand pendant feet was observed. Looking towards biological applications, we desired to learn how these molecules interact with phospholipid membranes. Six water-soluble cavitands were tested for their ability to permeabilize liposomal POPC membranes. One host showed very high potency in permeabilizing membranes, while three other hosts showed moderate activity. Host binding of POPC was found to be at least one factor in host-induced permeabilization. A requenching assay to determine leakage mechanism strongly supported all-or-none leakage, whereby some vesicles lose all contents while others lose none. These results suggest that these cavitands induce partial transient leakage of vesicles by the formation of transient membrane pores. These findings show potential for the use of these hosts as drug delivery carriers, antimicrobial compounds, and tools in membrane alteration studies.

Atualmente, a demanda por alimentos com apelo funcional tem se tornado cada vez mais recorrente dentre os consumidores devido a uma crescente busca por hábitos de vida mais saudáveis. Sendo assim, o desenvolvimento de técnicas que possibilitem uma adição mais efetiva de ingredientes funcionais em matrizes alimentícias se torna uma necessidade. Essas técnicas devem possibilitar principalmente (i) a incorporação de mecanismos de liberação sustentada na formulação; (ii) o aumento da bioacessibilidade e biodisponibilidade aos ingredientes, a partir do controle da microestrutura do alimento. Esse projeto visa contemplar essas duas premissas, ao propor a encapsulação de dois bioativos hidrofóbicos, a curcumina e a vitamina D3, conhecidos pelas suas propriedades antioxidantes e nutracêuticas, em carreadores de origem lipídica, os lipossomas, estabilizando-os com diferentes hidrocoloides - goma xantana, goma guar e inulina. Os lipossomas foram produzidos por hidratação de prolipossomas e suas propriedades físico-químicas foram caracterizadas ao longo de 42 dias de armazenagem, a partir de análises de diâmetro médio hidrodinâmico, potencial zeta, colorimetria instrumental e quantificação de bioativos encapsulados. Análises que permitiram a caracterização da microestrutura das dispersões produzidas também foram realizadas, sendo elas: calorimetria diferencial de varredura (DSC), espalhamento de raios-X a baixos ângulos (SAXS) e ensaios reológicos. As análises de SAXS mostraram que lipossomas produzidos na presença de curcumina são mais estáveis que àqueles produzidos na ausência da mesma e que não houve mudança na estrutura da bicamada lipídica das vesículas após a adição de vitamina D3, mesmo quando uma alta concentração foi incorporada ao sistema (80.000 UI). Por fim, verificou-se que a coencapsulação foi possível em lipossomas multilamelares estabilizados apenas com gomas guar e xantana, resultado que pode ser comprovado pelo alto teor de retenção dos bioativos ao longo do tempo de armazenagem. / Currently, the demand for food with functional appeal has become increasingly recurrent among the consumers due to a growing search for healthier living habits. Therefore, the development of techniques that allow a more effective addition of functional ingredients in food matrices becomes a necessity. These techniques should mainly enable to (i) incorporate a sustained release mechanisms into the formulation; (ii) increase the bioaccessibility and bioavailability to these ingredients, from the control of the food microstructure. This project aims to contemplate these two premises by proposing the encapsulation of two hydrophobic bioactives, curcumin and vitamin D3, known for their antioxidant and nutraceutical properties, in liposomes - lipid carriers - stabilizing them with different hydrocolloids - xanthan gum, guar gum and inulin. Liposomes were produced by proliposomes hydration and their physicochemical properties were characterized during 42 days of storage, including analyzes of hydrodynamic average diameter, zeta potential, instrumental colorimetry and quantification of encapsulated bioactives. Analyzes that allowed the microstructure characterization of the produced dispersions were also performed, including: differential scanning calorimetry (DSC), small-angle X-ray scattering and rheological tests. The SAXS analysis showed that liposomes produced in the presence of curcumin were more stable when compared to the empty ones and that there was no change in the lipid bilayer of the vesicles after the addition of vitamin D3, even when a high concentration was incorporated into the system (80,000 IU). Finally, it was concluded that the coencapsulation was possible in multilamellar liposomes stabilized with guar and xanthan gums, a result that can be evidenced by the high content of bioactives retained throughout the storage time.