Anthracnose, incited by Colletotrichum cereale, is a destructive disease of physiologically stressed creeping bentgrass putting greens in Mississippi and Alabama. Anthracnose severity and frequency of occurrence have increased over the past 15 years, and fungicide resistance may have had a role in the increase. In vitro bioassays were performed to evaluate thiophanate methyl and azoxystrobin against C. cereale isolates exposed to the fungicides and baseline isolates that had not been exposed to either fungicide. All isolates collected from creeping bentgrass were uninhibited by both fungicides at discriminatory doses. Partial nucleotide sequences of the â-tubulin 2 (thiophanate methyl) or cytochrome b (azoxystrobin) gene was compared to confirm fungicide resistance. Thiophanate methyl resistance was conferred by either a point mutation from glutamic acid to alanine at position 198, or phenylalanine to tyrosine at position 200. Azoxystrobin resistance was conferred by an amino acid point mutation from glycine to alanine at position 143.

No description available.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

It is well known that ultraviolet (UV) B (280-315 nm) and blue light (400-500 nm) radiation can produce phototoxic lesions in the neural retina and the retinal pigment epithelium (RPE). In the first section of this thesis, bovine lens cells (epithelium and superficial cortical fibre cell) and human retinal pigment epithelial (ARPE-19) cells were used to characterize in vitro changes following oxidative stress with UVB radiation in ocular lens optics and cellular function in terms of mitochondrial dynamics. In the second part, human retinal pigment epithelial (ARPE-19) cells and in vitro bioassays were used together to develop an in vitro approach for UV radiation-induced retinal toxicology research. In the third chapter, the in vitro approach developed above was used with intraocular lens (IOL) materials to evaluate the UV radiation blocking efficiency of commercially available IOL’s. Lastly, narrowband blue light irradiation and in vitro assays were used to determine more precisely the wavelengths of blue light responsible for photochemical lesions of the retina as an effort to contribute to future IOL designs. The results from mitochondrial dynamics of lens cells and RPE cells show significant decreases in mitochondrial movement after UVB irradiation in a dose dependent manner. Results obtained from four in vitro assays (Alamar blue assay, confocal microscopy for mitochondrial distribution and nucleic acids damage, phagocytotic activity assay) for evaluating the UVB-induced damage in ARPE-19 show significant decreases in cell viability as well as phagocytotic activity of RPE cells after UVB radiation. In addition, the results show that UV radiation can also induce the degradation of DNA/RNA and mitochondria of RPE cells in a dose dependent manner. The results of the UV blocking efficiency test of commercially available IOL materials show very effective UV blocking ability, allowing no cellular damage at all, in comparison to an IOL uncovered control cell. The results of three different wavelengths of blue light exposure show that only 400 nm blue light radiation can cause significant damage to RPE cells, while 420 and 435.8 nm blue light radiation cause no cellular damage at all. In conclusion, UVB and blue light radiation can cause phototoxic damage to the retinal pigment epithelium as a result of oxidative stress, and in vitro bioassays used for this research may offer a sensitive, and meaningful biomarker approach, not only for evaluating RPE function after oxidative and chemical stress, but also for evaluating IOL effectiveness.

Les méthodes actuellement disponibles pour la surveillance de la qualité du milieu se réfèrent au suivi de composés définis par les réglementations et ne permettent pas d’appréhender l’(éco)toxicité réelle de tous les composés présents dans l’environnement (e.g. composés inconnus, produits de transformation, effets de mélange…). Dans ce contexte, une méthode d’analyse pluridisciplinaire intégrative, l’analyse dirigée par l’effet (EDA), a été développée. Cette approche bio-analytique vise à établir un lien de causalité entre une exposition aux contaminants et l’effet biologique observé. La démarche est basée sur la simplification séquentielle d’un échantillon guidée par les bio-essais afin d’individualiser puis identifier, par des techniques analytiques chimiques performantes, les composés biologiquement actifs. Les objectifs de cette thèse ont porté sur (1) le développement et la validation d’une méthodologie, allant du pré-traitement jusqu’au fractionnement, adaptée à différentes matrices environnementales ; (2) la mise en place d’une stratégie d’identification moléculaire par spectrométrie de masse haute résolution ; (3) l’application de la démarche EDA à plusieurs échantillons afin d’illustrer l’adaptabilité et l’apport d’une telle méthode à des problématiques environnementales. Ces travaux de thèse permettent de disposer aujourd’hui d’une démarche bio-analytique complète de la préparation d’échantillon à l’identification de molécules, et adaptée à tous types d’échantillons. Les différentes études environnementales réalisées au cours de ces travaux ont permis, entre autre, d’établir une liste de composés diverses non recherchés pouvant représenter un risque pour l’environnement. / To date, environmental risk assessment is based on targeted chemical analyses. These analyses allow the detection of known active compounds and this preselected set does not often explain the observed toxic effects in complex environmental samples (e.g. unknown compounds, transformation product, cocktail effect...). For an integrative contamination assessment, a multidisciplinary approach, Effect-Directed Analysis (EDA) was developed. This approach combining biological tools and chemical analyses allows determining active compounds and identitying them in such environmental complex mixtures. EDA aims at the establishment of cause-effect relationships by sequential reduction of the complexity of environmental mixtures, eventually to individual toxicants. The aims of this thesis were (1) to develop and to validate the EDA-based strategy on various environmental complex samples; (2) to set up an identification strategy for identifying non targeted or unknown molecules using high resolution mass spectrometry; (3) to prove the usefulness of EDA approach for the identification of active compounds in environnemental complex samples. This thesis work provides to a complet bio-analytical approach from the preparation step of samples to the identification of molecules. The potential of this tool was fully confirmed on various environmental studies. These investigations allowed establishing a list of non-target compounds which can represent a risk for the environment.

Coal mining and coal utilization in Mpumalanga have increased over the years due to national reliance on coal as a source of power generation. In general, this has caused significant deterioration of water quality wherever streams are impacted by acid mine drainage (AMD). The aim of this research was to assess the use of in vitro bioassays as a complement to, or potential future replacement of, waste effluent testing in whole animals from AMD impacted watersheds subjected to passive and active treatment, correlating observed changes with water chemistry analysis. To accomplish this goal, water samples were collected and in vitro bioassays carried out to investigate generation of reactive oxygen species by the water samples and cytotoxicity against Vero kidney cells, C3A liver cells and trout RTgill-W1 cells. Primary fish gill cultures were established and used as sensitive in vitro models for assessing possible contaminants in water, measuring the induction of cytochrome P450 (CYP) 1A and resultant increase in 7-ethoxyresorufin-o-deethylase activity as a potential biomarker in fish gill cells exposed to polycyclic aromatic hydrocarbons. The genotoxic potential of AMD water on commercially available cell lines was also determined. / The study site was an impacted stream located downstream of a coal mine discharge point whose effluent flowed away from the mine. Water chemistry results suggested high AMD impact evidenced by acidity, elevated sulphates, increased conductivity and presence of heavy metals. Al, Fe, Zn, Mn and Si were the major metals of potential concern in the AMD impacted stream; sulphates and major ions like Ca, K, Na and Mg were present at levels above target water quality range (TWQR) for effluents in receiving stream. The AMD impacted stream caused increased generation of reactive oxygen species (ROS) detectable in vitro in selected cell lines (Vero, C3A and RTgill-W1 cell lines), an indication of oxidative stress. In-stream, active treatment with caustic soda was efficient at reducing metal burden, with subsequent reduction in ROS generation in fish gill cell lines. For in vitro cytotoxicity tests, passive and active treated AMD water was cytotoxic to cell lines (Vero and RTgill-W1), with the fish RTgill-W1 cells exhibiting greater sensitivity compared to the mammalian Vero cells. Mitochondria played a larger role in observed loss in cellular viability (increased vacuolization, mitochondrial membrane swelling and damage), which was detected using mitochondrial specific stains, and by transmission electron microscopy (TEM). Increased dose- dependent cytotoxicity was observed in the fish gill and mammalian cell lines. Cells exposed to water samples (AMD and reference sites) revealed significant differences (p < vi 0.05) between the AMD impacted watershed and a relatively pristine site (reference site) where exposure to the same cells maintained approximately 100% viability at all concentrations for up to 72h exposure. The observed differences in effect in this study demonstrate that the effluent from the coal mine negatively impacted surface water quality, resulting in toxicity to cell lines, therefore creating an environment that would not be conducive for the survival of biological aquatic communities and potentially of concern for downstream human end users. / The induction of cytochrome P450 (CYP) 1A and resultant increase in 7- ethoxyresorufin-o-deethylase activity in primary fish gill cultures exposed to polycyclic aromatic hydrocarbons B[a]P, a known AhR agonist contaminant associated with coal mining, showed that there was as increase in EROD activity which was not observed using the RTgill-W1 cell lines. Gill epithelial cells isolated from the gills of Tilapia fish (Oreochromis mossambicus) bear close similarities to fish gills in vivo and their capacity to respond to the presence of AhR indicates that they may serve as a simple, cost-effect screening tool for assessing PAHs and dioxin-like compounds in fresh water. / For genotoxicity evaluation, the Ames test performed without metabolic activation using bacterium Salmonella typhimurium TA98 and TA100 strains revealed no indication of genotoxic activity in any of the water samples. Genotoxicity assessment of all water samples using the comet assay however exposed DNA damage to Vero and RTgill-W1 cell lines. A significant reduction in DNA damage was observed following active treatment. The results suggest that neither treatment technologies employed were efficient at removing all potential genotoxicants so further improvements are required. The comet assay proved sensitive enough to detect genotoxicity in reference water samples despite no known untoward effluent inputs at the site, suggesting potential for this assay to be integrated into an environmental monitoring framework. / The results obtained support the use of in vitro bioassays for evaluating toxicity of industrial effluent through biological responses in test systems elicited following exposure, improving ability to detect AMD polluted water. This could be beneficial when assessing the degree and extent of impact of AMD in natural water sources, and the possible environmental impact resulting from hazardous elements present in effluent water. In conclusion, these results suggest that in vitro techniques involving cell lines and primary cultures from fish may serve vii as simple, rapid and cost-effective tools for assessing risk and potential toxic effects of contaminants in AMD waters. / Thesis (PhD)--University of Pretoria, 2016. / The National Research Foundation / Department of Paraclinical Sciences (University of Pretoria) / Schlumberger Stichting Fund, Netherlands / Paraclinical Sciences / PhD / Unrestricted

De nos jours, l’évaluation de la qualité d’un milieu naturel repose traditionnellement sur des analyses ciblant des molécules déjà connues pour être biologiquement actives. Cependant ces molécules ne reflètent que partiellement les effets biologiques observés et la complexité des matrices environnementales. Par ailleurs, les contaminants dans l’environnement subissent des phénomènes de dégradation (hydrolyse, biodégradation, photodégradation). Ces processus peuvent conduire à la génération de produits de transformation qui sont encore trop peu pris en compte dans l’évaluation du risque, alors qu’ils sont susceptibles d’occasionner des effets toxiques plus importants que les molécules mères. L’impact écotoxicologique de ces mélanges nécessite préalablement l’obtention d’une vision globale quant à la présence et au devenir de ces molécules.Dans ce contexte, ces travaux appliquent une méthodologie pluridisciplinaire afin d’identifier des molécules préoccupantes et pertinentes à surveiller. Pour cela, ce travail s’appuie sur des analyses ciblées ultra-traces, des outils bioanalytiques, et des analyses non ciblées, qui ont été appliqués sur des échantillons environnementaux ou issus d’études en laboratoire mimant l’évolution des matrices environnementales. Au final, cette étude a permis : (1) d’identifier des molécules préoccupantes en estuaire de Seine sur la base de leur occurrence et de leur persistance, (2) d’identifier de nouveaux produits de transformation et (3) d’identifier les molécules responsables d’activités biologiques dans des matrices très complexes. / To date, environmental risk assessment is based on a restricted number of molecules assessed by targeted chemical analyses. However, this approach give a partial picture of co-occurrence of known and unknown compounds. Moreover, in the aquatic environment, chemicals are not completely mineralized and are subject to abiotic and biotic processes. Transformation products (TPs) can be more toxic and more persistent than the parent compound. However, TPs are not typically included in classical monitoring and risk assessment. Because of complexity and variability of these matrices and the restricted number of molecules focused in targeted chemical analyses, selection of relevant molecules for environmental monitoring is often laborious.In this context, an integrative approach was used in order to identify chemicals of concern for a classical monitoring. This work has been realized on environmental complex samples and on laboratory-generated samples. A combination of targeted, non-targeted analyses and in vitro bioassays was performed and allowed to: (1) identify polar chemical of concern in the Seine estuary on the basis of occurrence and persistence, (2) identify new transformation products and (3) identify compounds responsible of biological activity observed in complexes matrices.

No description available.