MSc., Faculty of Science, University of the Witwatersrand, 2011 / The 2-isopropenyl-2,3-dihydrobenzofuran moiety is found in many naturally occurring compounds including rotenone, a complex pentacyclic molecule isolated from several leguminous plants of the Derris and Lonchocarpus species. Interest in rotenone stems from the fact that it possesses significant pesticidal and piscicidal properties which have been employed for centuries. Furthermore, as it has three stereogenic centres, rotenone poses an interesting and challenging synthetic target for organic chemists. Although various syntheses of this natural compound have been reported, none of these were stereoselective. The first stereoselective total synthesis of rotenone is described in this dissertation. Initially, a model study was conducted in which the simplest of the natural rotenoids, munduserone, was synthesised. The key step in this transformation involves the use of a platinum catalysed 6-endo-hydroarylation reaction of an alkynone intermediate, thus affording munduserone in 6 steps and an overall yield of 23%. We then attended to the synthesis of the more complex rotenoid, rotenone. Rotenone was synthesised by the initial assembly of a chiral (-)-(R)-2-isopropenyl-2,3-dihydrobenzofuran-4-ol moiety, asymmetrically accessible using a stereoselective Pd π-allyl mediated cyclisation of (E)-4-(2,6-dihydroxyphenyl)-2-methylbut-2-enyl methyl carbonate. Having constructed the dihydrobenzofuran in an enantiomeric excess of 94.8%, the chromene part of rotenone could then be synthesised. To this end, the LDA mediated coupling reaction of the formylated dihydrobenzofuran and 1,2-dimethoxy-4-(prop-2-ynyloxy)benzene, gave a secondary alcohol which was subsequently oxidised to the corresponding alkynone, (-)-(R)-(6,7-dimethoxy-2H-chromen-4-yl)(4-methoxy-2-isopropenyl-2,3-dihydrobenzofuran-5-yl)methanone. A 6-endo-hydroarylation reaction was employed as a mild strategy to construct the chromene moiety, (-)-(R)-(6,7-dimethoxy-2H-chromen-4-yl)(4-methoxy-2-isopropenyl-2,3-dihydrobenzofuran-5-yl)methanone. Finally, a deprotection and a base-catalysed intramolecular oxo-Michael addition concluded the first stereoselective synthesis of rotenone in 17 steps and an overall yield of 0.02%

The bumblebees (genus Bombus) are an ecologically and economically important group in decline. Their decline is driven by many factors, but parasites are believed to play a role. This thesis examines the factors that influence the diversity and distribution of multihost viruses in bumblebees using molecular and modelling techniques. In Chapter 2, I performed viral discovery to isolate new multihost viruses in bumblebees. I investigated factors that explain prevalence differences between different host species using co-phylogenetic models. I found that related hosts are infected with similar viral assemblages, related viruses infect similar host assemblages and related hosts are on average infected with related viruses. Chapter 3 investigated the ecology of four of the novel viruses in greater detail. I applied a multivariate probit regression to investigate the abiotic factors that may drive infection. I found that precipitation may have a positive or negative effect depending on the virus. Also, we observe a strong non-random association between two of the viruses. The novel viruses have considerably more diversity than the previously known viruses. Chapter 4 investigated the effect of pesticides on viral and non-viral infection. I exposed Bombus terrestris colonies to field realistic doses of the neoticotinoid pesticide clothianidin in the laboratory, to the mimic pulsed exposure of crop blooms. I found some evidence for a positive effect of uncertain size on the infection rate of pesticide exposed colonies relative to non-pesticide exposed colonies, a potentially important result. Chapter 5 explored the evolution of avirulent multihost digital organisms across fluctuating fitness landscapes within a discrete sequence space. Consistent with theory, I found that evolution across a fluctuating discrete landscape leads to a faster rate of adaptation, greater diversity and greater specialism or generalism, depending on the correlation between the landscapes. A large range of factors are found to be important in the distribution of infection and diversity of viruses, and we find evidence for abiotic, biotic and anthropogenic factors all playing a role.

Of the 800,000 suicides recorded globally every year, over a third are due to pesticide ingestion, the majority of which occur in rural Asia with organophosphorus (OP) compounds. These anticholinesterase pesticides cause an acute cholinergic syndrome characterised by decreased consciousness, excessive airway secretions and respiratory failure. A combination of these clinical features is the most common cause of death. Up to 30% of OP pesticide poisoned patients are admitted to the Intensive Care Unit (ICU) for tracheal intubation and lung ventilation, but up to half die. It is not understood why the case fatality for intubated poisoned patients is so high, but one hypothesis is that the patients, when unconscious, aspirate their stomach contents (including the OP and the solvent present in its agricultural formulation) causing a toxic lung injury which contributes to the observed high mortality. In this PhD, I aimed to characterise the lung injury caused by OP pesticide self-poisoning through both indirect (ingestion) and direct (aspiration) means. To achieve this, I analysed data from previous toxicological minipig work and designed and conducted a specific minipig pulmonary aspiration study which was complemented by an experimental OP poisoning ex vivo lung perfusion model and human data from pesticide poisoned patients in Sri Lanka. I first investigated the pulmonary pathophysiology resulting from orogastric administration of OP pesticide without aspiration. Analysis of my group’s Gottingen minipig in vivo work demonstrated that orogastric placement of agricultural OP (dimethoate EC40) produced lung injury via exposure to blood-borne pesticide. Pathological lung changes consisted of alveolar and interstitial oedema, pulmonary haemorrhage and modest neutrophilia with increased concentrations of protein, IL-6 and IL-8 when compared with controls, but with low concentrations of TNF-α and IL-10 in bronchoalveolar lavage fluid (BALF). In a second study, OP poisoned minipigs had increased concentrations of BALF protein, neutrophils, IL-8 and CRP six hours after orogastric poisoning when compared with their baseline values. Electron microscopy images of both studies demonstrated damage to the alveolar capillary membrane secondary to systemic OP poisoning. Prior to conducting the main pulmonary aspiration study in minipigs, there was considerable refinement of the processes involved through use of: (i) pilot aspiration and dose ranging studies; (ii) the development of a specific pulmonary histopathological scoring system; and (iii) employment of modern human anaesthetic equipment and intensive care patient management protocols. After this period of model development, an in vivo 48 hour study using Gottingen minipigs (n=26) was conducted to investigate the pulmonary pathophysiology in animals given either sham bronchoscopy (sham control) or 0.5 mL/kg of: saline (saline control), porcine gastric juice [GJ], OP (dimethoate EC40) + GJ [OP+GJ], or solvent (cyclohexanone) + GJ [Solv+GJ] into the right lung under bronchoscopic guidance. The results showed that in a minipig model OP and GJ placed into one lung created a direct (right) and indirect (left) lung injury significantly different to controls, and in some respects worse than GJ alone 48 hours after poisoning. The direct lung injury caused by OP+GJ was characterised by significantly worse pathology (p=0.0003) in terms of: pulmonary neutrophilia, alveolar haemorrhage, necrosis, oedema and fibrin deposition, when compared with sham controls at 48 hours. Lungs injured directly with OP+GJ also had significantly higher concentrations of BALF neutrophils (p≤0.01), protein (p≤0.05), IL-6 (p≤0.01), IL-8 (p≤0.01) and CRP (p≤0.05) at 24 hours, and BALF protein (p≤ 0.01), and CRP (p≤ 0.05) when compared with sham controls at 48 hours. The BALF from OP+GJ minipigs at 48 hours also had higher numbers of aerobic bacteria than other groups, suggesting the development of pneumonia could be a source of additional lung injury. Lung damage might also have resulted from a reduction in the surfactant component responsible for the lowering of alveolar surface tension. Direct lung injury with OP+GJ caused a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine (PC) species 16:0/16:0 [29(±4) % vs. 38(±4) %] when compared with sham controls at 48 hours. Unlike the other groups, OP+GJ (direct and indirectly-injured) lungs had type 2 alveolar cell ultrastructural morphological differences in the lamellar bodies that stored the surfactant. The lamellar bodies were more numerous and more dense in the OP+GJ lungs compared with other groups and could signify a failure of surfactant release or some other pathology pertinent to OP aspiration lung injury. Computed tomography analysis showed that direct lung injury with OP+GJ caused significantly more lung tissue to be poorly or non-aerated [77 (±13) % ; p≤0.0001 when compared with sham] as opposed to 62 (±27) % in GJ, 53(±13)% in sham and 47(±0.2)% in saline control animals by 47.5 hours and was mainly due to pulmonary haemorrhage and oedema fluid. The key differences between aspiration of OP+GJ versus GJ alone was that the majority of inflammatory markers (e.g. BALF protein, IL-6 and CRP) appeared to increase from 24-48 hours in OP+GJ treated animals, but decreased in GJ pigs, possibly signifying resolution. Treatment with GJ alone produced less severe histopathological damage, bacterial BALF numbers and percentage of poorly and non-aerated lung tissue. Importantly, there was less evidence of indirect lung injury within the GJ pigs when compared with animals treated with OP+GJ. Solvent placed into the lung seemed to offer some form of protection from the effects of GJ aspiration. This was dramatically demonstrated by the histopathology scores, proportional percentage of beneficial phosphatidylcholine (PC) species 16:0/16:0 and the percentage of poorly and non-aerated lung tissue all approaching control animal levels by 48 hours in minipigs that had Solv+GJ placed in the directly-injured (right) lung. Further evidence of benefit was provided by statistically significant reductions (p≤ 0.05) in BALF concentrations of IL-8, IL-6 and CRP in minipigs which had aspirated Solv+GJ when compared with OP+GJ and/or GJ minipig groups at 24 hours. The pathophysiology of aspirated OP+GJ was also investigated in a pilot ovine ex vivo lung perfusion (EVLP) model (n=4). Lungs directly-injured with OP+GJ had higher concentrations of total protein (4300 mg/L vs. 350 mg/L) with a proportional reduction of beneficial pulmonary surfactant phosphatidylcholine species 16:0/16:0 (27% vs.34%) when compared with control lungs. Analysis of toll-like receptor (TLR) lung tissue expression in the OP+GJ directly and indirectly-injured lungs indicated that inflammatory mechanisms might also involve upregulation of TLR 3 and 5, unlike other lung injuries e.g. those induced with lipopolysaccharide, which typically upregulates TLR 2 and 4. To compare OP-induced lung injury in humans and the minipigs, a small feasibility study was conducted in the ICUs of the University of Peradeniya hospital, Sri Lanka. Unfortunately, ethics review and recruitment proved more difficult than expected and we failed to recruit to target. We did however find raised BALF concentrations of IL-6, IL-8 and CRP and low concentrations of TNF, IL-1β, IL-10 in intubated OP poisoned patients at 24 hours when compared with controls. We also found that two plasma micro-RNA biomarkers thought to be involved in inflammation and lung injury, MiR-21 and MiR-146a, had significantly reduced expression in OP-poisoned patients with aspiration compared to non-intubated control patients from the UK (p=0.008 and p=0.0083 respectively). The work from this thesis has allowed the characterisation of both indirect and direct lung injuries caused by OP pesticide ingestion and aspiration. The minipig model showed that at 48 hours the lung injury created by aspiration of OP+GJ appeared more severe than GJ alone, but the addition of the solvent cyclohexanone seemed protective and even beneficial in the context of GJ aspiration. The cytokine expression profiles from both the human and minipig work, combined with the preliminary TLR lung tissue analysis from the EVLP model, suggest that OP+GJ aspiration is unlike normal GJ aspiration and classic ARDS. / Increased concentrations of aerobic bacteria in the minipig OP+GJ lungs at 48 hours and evidence of suppression of plasma miR-21 and miR-146a in OP poisoned patients could be linked, and may involve cholinergic immune system modulation. These molecular mechanisms need to be investigated further in both in vitro and in vivo models. These discoveries indicate the complex nature of the pulmonary injury that occurs after OP pesticide poisoning, and suggests that damage is not caused by gastric contents alone. Preliminary findings indicate that aspiration of OP+GJ could create favourable conditions for the development of aspiration or ventilator-associated pneumonia but this would need confirmation in larger clinical studies. The potential roles of micro RNA as a biomarker of OP poisoning and lung injury, and solvent as a therapy for aspiration should be explored in further pre-clinical studies.

This thesis provides an overview of case studies investigating the behaviour of freshwater crayfish, including social and reproductive interactions, decision making, and channels for recognition or processing of environmental stimuli. We described relationships between the environment and the organism and relationships among conspecifics and heterospecifics. These aspects are important, given that every decision and interaction has consequences for crayfish survival and reproduction, i.e. individual and even species success. For ethological studies in aquatic ecosystems, crustaceans, and especially crayfish, represent practical model organisms. Crayfish are advantageous over other invertebrates given their high level of social interaction in both the field and the laboratory. They exhibit unique eco- ethological behaviour in variety of feeding habits, activity cycles, habitat selection, and resource utilization at the level of species, sex, and age. Similar to vertebrates, crayfish demonstrate unique behaviours throughout their life. Subsequently, pollution impacts on native crayfish stock can result in ecosystem instability. The different reproductive strategies of females and males demonstrate that females rely on some familiarity of sexual partners as a reliable guarantee for successful mating. Crayfish females are choosier than males because of their higher energy costs for reproduction (oogenesis, incubation, parental care). While male reproduction prioritises securing their genetic information by searching for as many mates as possible. Crayfish mate preferences was analysed by facing the choosing crayfish with two equal sized mature counterparts from the same or different origin. Our conclusions could be applied in conservation research, captive breeding, and recovery programs as well as in aquaculture. The success of biological invaders has been described as the outcome from intra- and interspecific interactions. The marbled crayfish, known as parthenogenetic species with high adaptability, fast growth, early maturation, and high fecundity, established dominant status in more cases by interspecific combat victories. Fight intensity and aggressiveness decreased after dominance was established in particular pairs. Marbled crayfish seem to have an ability to conserve energy by increasing activity only at times of danger. Based on known species-specific abilities, the marbled crayfish is capable of outcompeting aggressive species, such as the red swamp crayfish. This parthenogenetic species poses a substantial threat to native ecosystems and biodiversity of aquatic environments, since it can establish not only in crayfish free habitats, but is also capable of outcompeting resident species. Recently, dilute concentrations of contaminants such as pharmaceuticals, daily-care products and pesticides have been reported to indirectly affect aquatic organisms. We found that selected pharmaceuticals can alter the behavioural patterns of crayfish, event at the very low concentration. The risks associated with surface water pollutants stresses the importance of research investigating their ecological impacts and their different/specific mechanisms of action. Prolonged exposure to the low concentrations of two pesticides exhibited changes in crayfish behavioural patterns. Various alterations in locomotion were observed after treatment in pesticide-free water. In the wild, decreased movement and activity can be a challenge for crayfish survival because of predation, even foraging to compensate for energy loss could be very difficult for less active individuals. This study suggests that crayfish behaviour assessment is a practical approach not only for investigating basic biology, or intra- and inter-specific interactions, but also for assessing the risks associated with micro-pollutants.

The number of drones and genetic diversity among drones are essential components to a well mated queen. Varroa destructor preferentially parasitizes drone brood, and is thought to be responsible for the loss of feral populations that once provided additional drones for honey bee mating areas. It is necessary to use miticides (e.g. coumaphos) in managed colonies to control V. destructor. Little is known about the sublethal effects of these compounds, which are directly introduced into the hive. In response to growing concerns about the successful mating of honey bee queens, drone honey bees were exposed to coumaphos, during drone development. Sperm and seminal vesicles were sampled among drones that were exposed to coumaphos and drones that were not exposed to coumaphos, but were parasitized by Varroa destructor. There were no significant differences found between the two treatments in terms of seminal vesicle size and sperm numbers. These results indicate that drones parasitized by V. destructor have similar sperm quantities as drones exposed to coumaphos. / Graduation date: 2004

Volatile loss rates of pesticides from turfgrass were measured using the Backward-Time Lagrangian Stochastic Dispersion model (Flesch et al., 1995). Solar radiation, ambient temperature, surface temperature, relative humidity, wind direction, and wind speed were monitored continuously. Growth regulator was applied to the turf plot several days before pesticide application to maintain a constant grass height and aerodynamic roughness length during the experiment. No irrigation occurred following application. Pesticides were applied as mixtures to allow direct comparison of evaporative loss. Mixtures studied were chlorpyrifos + triadimefon + ethofumesate and triclopyr (acetic acid) + propiconazole + cyfluthurin. Airborne flux estimates correlated with temperature, solar radiation, wind speed, time, and vapor pressure of the active ingredient. A log vapor pressure vs. 1/Temperature (K) relationship was observed between flux and surface temperature over a single day for most pesticides. An exponential attenuation of flux was observed over a period of several days and correlated with attenuation of dislodgeable surface residues for two of the pesticides. A fugacity-based model for predicting initial evaporative loss rates from turf grass is presented. Input parameters include pesticide vapor pressure, molecular diffusion coefficient, surface temperature, wind speed profile, atmospheric stability, surface roughness, and average upwind fetch. The GC retention method (Jensen, 1966) was used to estimate pesticide vapor pressures over an environmentally relevant temperature range. The model predicts fluxes that are an order of magnitude greater than measured values. This bias may be due, in part, to deviation from the assumption of pesticide saturated vapor density at the foliar surface. In addition, sensitivity analysis suggests improved estimates of leaf surface temperature and pesticide vapor pressures have the greatest potential to improve model performance. / Graduation date: 2003

Au Sénégal, dans les zones maraîchère (zone des « Niayes ») et cotonnière, le traitement des cultures se fait respectivement avec des pulvérisateurs à dos et avec des cannes centrifuges. Des enquêtes ont été réalisées sur le terrain et un ensemble de paramètres comme le temps dapplication et de rinçage, les surfaces traitées, les volumes appliqués, la température et la vitesse du vent, ont été mesurés pour analyser les facteurs de risque encourus par les opérateurs. Lanalyse statistique des résultats montre que les risques de contamination des opérateurs sont importants. En effet, en se référant à la classification établie par lOMS, plusieurs produits sont classés hautement dangereux. Le faible niveau de formation des opérateurs ne leur permet pas de prendre connaissance des informations concernant lutilisation correcte des produits. Les techniques dapplication sont insuffisamment maîtrisées, lapplication nest pas réalisée au moment où les conditions climatiques sont les plus favorables et les opérateurs travaillent sans aucune protection. Des mesures dexposition simulant les conditions de travail rencontrées en régions maraîchère et cotonnière ont été effectuées dans un tunnel aérodynamique. Quil sagisse du pulvérisateur à dos ou de la canne centrifuge, la position qui expose le moins lopérateur est la position latérale par rapport au vent. Avec le pulvérisateur à dos, la contamination se produit essentiellement au niveau des tibias, tandis quavec la canne centrifuge, la contamination est généralisée à lensemble des parties corporelles. La présence de végétation augmente lexposition par rapport à la pulvérisation sur sol nu. Ces mesures permettent dune part de proposer un équipement de protection adapté en fonction du type de pulvérisation réalisée et dautre part destimer lexposition spécifique dermique de lopérateur pendant lapplication des pesticides. Synthétisant les mesures de terrain et celles réalisées en tunnel aérodynamique, un modèle estimant lexposition du corps par voie dermique pendant la phase dapplication est proposé pour le Sénégal. Il se base sur les principes généraux de calcul dexposition adoptés dans les modèles déterministes européens. Partant de ce modèle, les doses moyennes absorbées sont estimées à 1,40 et 19,64 mg de substance active par personne et par jour, respectivement avec un pulvérisateur à dos et une canne centrifuge. Avec un équipement de protection individuelle, lestimation conduit à des valeurs dix fois plus faibles.

A recent development in drift-control for agricultural ground sprayers is the installation of a hood (or shroud) over the boom. Hoods are designed to provide a protected zone in which droplets can be sprayed and deposited on the target with limited interference from the wind. This study compared chemical drift using hooded and open-boom pesticide sprayers in various configurations. The hooded sprayer included an air-foil mounted on top of the hood, designed to re-direct the airflow and further decrease drift. The principal objective was to test the performance of the hood, the air-foil, and nozzle size in reducing drift. A fluorescent dye and water solution was sprayed adjacent to a series of parallel string collectors. The amount of drifting material was determined by rinsing the string and testing the fluorescence of the rinsewater. Wind speed and direction, temperature, and relative humidity were monitored during each field test so that the influence of weather conditions on drift could be assessed. Statistical and graphical comparisons were based on the development of a regression model to describe downwind drift for each sprayer configuration under a set of variable weather conditions. Only parameters found to be important to drift were included in the final model. These parameters included sprayer configuration, wind speed, temperature, and the interactions of configuration with wind speed and temperature. Results of the comparisons indicated that for a larger droplet spectrum, the hood reduced drift significantly, while for smaller droplets the hood was ineffective. The air-foil was found to have no influence on drift. / Graduation date: 1991

Reproduction de : Thèse de doctorat : Qualité et Sécurité des Aliments : Toulouse, INPT : 2007. / Titre provenant de l'écran-titre. Bibliogr. 158 réf.

Thesis (MTech (Chemical Engineering))--Peninsula Technikon, 2003. / Word processed copy. Summary in English. Includes bibliographical references (leaves 93-96). Also available online.