There is widespread use of ionization sources (ambient and non-ambient) for a variety of applications. More recently, charged microdroplets generated by electrospray ionization and paper spray have been used to conduct chemistry at faster rates compared to bulk volumes. Uncharged droplets such as those generated by the Leidenfrost technique have also been used to explore chemistry and study the degradation of drugs in an accelerated manner. These microdroplets serve as reaction vessels in which in which some reactions are known to occur at accelerated rates. Such chemistry can be particularly useful in pharmaceutical settings to rapidly synthesize small amounts of materials in relatively short amount of time. Additionally, microdroplets may also be used to perform high throughput screening analysis. While several parameters influencing the rate of reaction in microdroplets have been explored (such as spray distance and reagent concentration), the mechanism of reaction acceleration has not been probed to a significant extent. A major portion of my dissertation describes the use of charged and uncharged microdroplets to perform quick chemistry, guide microfluidic synthesis of drugs such as diazepam, perform scale up of copper catalyzed C-O and C-N coupling reactio<a></a>ns and screen reaction conditions for pharmaceutically relevant reactions such as the Suzuki cross-coupling reaction. Additionally, work discussed here also describes development and use of existing techniques such as structured illumination microscopy to measure droplet sizes, explore the role of distances on droplet size, and study the effect of surfactants on the rate of reactions in microdroplets generated by nano-electrospray ionization. A mathematical model to understand the mechanism of increased reaction rates in microdroplets has also been presented. Additionally, this dissertation also describes ways to manipulate ions in air using various designs of 3D-printed electrodes that operate with DC potentials only and which can be easily coupled with nano-electrospray ionization sources to transmit ions over long distances

The main objective of this study was the application and comparison of different assays in assessing toxicity of cyanobacterial samples, and also characterizing toxicity of the field samples. Therefore, toxicity of purified microcystin-LR (MC-LR) and cyanobacterial samples collected from the Hartbeespoort (HBP) Dam (winter and summer seasons of 2005/2006) and Kruger National Park (KNP) were investigated and compared using the ELISA, mouse bioassay, catfish primary hepatocytes (in vitro assay) and protein phosphatase inhibition (PPi) assays. During sampling in the summer season at the HBP Dam, the dam surface was covered with a thick-green layer of cyanobacterial scum and a foul smell coming from the water surface was always present. Only blue-green streaks of cyanobacteria covered the dam surface during the winter season. All HBP Dam samples (winter and summer samples) and KNP samples (Nhlanhanzwani Dam, Mpanama Dam and Sunset Dam) were dominated by Microcystis aeruginosa with the exception of Makhohlola Dam samples which were found to have no cyanobacteria. The World Health Organization (WHO) has proposed a guideline value for human use of 1.0 µg/L (0.001 mg/L) for MC-LR, the most common microcystin (MC) variant, in drinking water (WHO 1998), whereas 2 000 Microcystis cells/mL have been recommended as the limit of cyanobacteria in drinking water for animals (DWAF 1996). Cyanotoxin concentrations exceeding the prescribed guideline value were detected in all HBP Dam samples (ELISA results ranging between 3.67 to 86.08 mg/L; PPi results ranging between 2.99 to 54.90 mg/L) and KNP samples (ELISA results ranging between 0.1 to 49.41 mg/L; PPi results ranging between 0.006 to 10.95 mg/L) using both the ELISA and PPi assays. In the current study, a dose of about 175 µg/kg of purified MC-LR was demonstrated to be lethal in male CD-1 SPF mice. The HBP Dam summer samples and Nhlanganzwani Dam samples were the only cyanobacterial samples that resulted in death (acute toxicity) of mice. In order to be able to investigate further the in vivo effects of cyanotoxins, transmission electron microscopy (TEM) was used to complement results obtained from the in vivo assay. Ultrastructural changes of varying degree were observed in livers of mice exposed to both the HBP Dam winter and summer samples. Early stages of hepatocyte to hepatocyte disassociation, slight vesiculation of endoplasmic reticulum (ER) and swollen mitochondria were the most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam winter samples. The most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam summer samples were massive hepatic haemorrhage indicated by the appearance of erythrocytes between hepatocytes and the extensive vesiculation of ER. This is the first time that the African sharptooth catfish primary hepatocyte model has been used to assess the hepatotoxicity of purified MC-LR and cyanotoxin-containing water samples. In this study, the toxicity of cyanobacterial samples and purified MC-LR to cause hepatotoxicity in mice was confirmed in vitro using the catfish primary cell line. A comparison among the cyanobacterial samples using EC50 showed the following hepatotoxicity trend in the catfish primary cell line: HBP Dam summer samples > Nhlanganzwani Dam samples > HBP Dam winter samples > Mpanama Dam samples > Sunset Dam samples > Makhohlola Dam samples. The HBP Dam samples were the most hepatotoxic and Makhohlola Dam samples were the least hepatotoxic. The EC50 for purified MC-LR using the catfish primary hepatocytes was about 91 nM. A statistical comparison of the assays used in this study (i.e. ELISA, PPi, mouse test and cytotoxicity [catfish primary hepatocyte] assays) was performed based on the Kappa coefficient (K). An almost perfect agreement (K > 0.80) was observed between the mouse test and cytotoxicity assay; mouse test and ELISA; cytotoxicity assay and ELISA; and ELISA and PPi assay. In conclusion, field samples collected during the summer season were found to have very high levels of toxins and a higher degree of toxicity when compared to the winter samples. The cytotoxicity assay using African sharptooth catfish (Clarias gariepinus) primary hepatocytes has been shown for the first time to produce results similar to those observed when using the mouse bioassay in assessing cyanobacterial toxicity. Therefore, this primary cell line may be used as a potential alternative to the mouse assay in toxicity testing of cyanotoxins. Three KNP dams (Nhlanganzwani Dam, Mpanama Dam and Sunset Dam) investigated in this study were found to contain Microcystis aeruginosa. All four KNP dams (Nhlanganzwani Dam, Mpanama Dam, Makhohlola Dam and Sunset Dam) had cyanotoxin levels above the prescribed guideline value, which is of concern and warrants further investigations to the effects on wildlife in the park. Future studies will include use of High Performance Liquid Chromatography (HPLC) to investigate the toxin profile of the field samples in order to fully describe the different classes/or types of toxins present in the samples. More validation studies that could give a more comprehensive understanding about the sensitivity of the catfish primary cell line for microcystins will also be undertaken. / Dissertation (MSc (Paraclinical Studies))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

Research was conducted 2014-2016 to determine how tobacco thrips (Frankliniella fusca) (Hinds) resistance to neonicotinoid insecticides impact thrips resistance to neonicotinoid insecticides in cotton, Gossypium hirsutum (L.).Studies included bioassays to determine severity and mechanism of resistance and evaluation of host plant characteristics in multiple cotton varieties. Another aspect of research focused on the neonicotinoid insecticide clothianidin and its leaching ability as a seed treatment on corn by evaluating soil type, water regime, and amount found in tissue.

The fungus Macrophomina phaseolina (Tassi) Goidanich (Mp) is the causal agent of charcoal rot in soybean and infects over 500 plant species worldwide. Mp produces various mycotoxins and is suspected of utilizing a toxin-mediated process to penetrate host tissue. Identification and evaluation of secondary metabolites produced by Mp will further elucidate the pathogenesis mechanisms used by the fungus. Mp cultures isolated from soybean were evaluated for phytotoxicity in a hydroponic soybean bioassay and chemically analyzed by LC-MS/MS. All Mp cultures at two dilutions induced phytotoxicity symptoms including chlorosis, necrosis, wilting, stunting, and death. Analysis identified 13 unreported secondary metabolites including mellein, a compound with various biological activities. The phytotoxicity of mellein was evaluated against soybean seedlings in hydroponic culture, and symptoms of wilting and stunting were observed at levels above 40 MUg/L. Observations suggest that mellein does not directly contribute to the phytotoxic effects of Mp cultures.

Ultraviolet filters are active ingredients in sunscreen that protect us from harmful UV radiation. However, organic UV filters are thought to have adverse effects on the environment and humans. In recent years, fear of harmful impacts of sunscreen has caused a surge of coral reef safe sunscreens to hit the market. These sunscreens, which contain inorganic metal oxides as UV filters, have been accepted as safe for humans and the environment until recently. Metal oxides in reef safe sunscreens may form intermediates in the water that can harm marine life and can absorb through the skin and into the blood, possibly disrupting normal bodily function. In this study, a 48-hour bioassay was run with Artemia salina and various UV filters at different concentrations to determine at what levels of exposure and to which UV filters the organism is sensitive. Three trials were run with one organism in each of the 200 bioassay wells and 20 replicates per treatment. At each data collection time, organism survival outcomes were recorded. Results showed significant difference between trials but not between treatments. This project serves to research the impact sunscreen has on A. salina and potential environmental and human health impacts.

No description available.

No description available.

A watershed scale water quality evaluation system was developed for assessing spatial variation of subsurface pesticide movement. The system consists of a linked-transport model component for performing simulation and a GIS component for processing spatially-related data. The surface heterogeneity caused by agricultural activities, topographic, hydrologic, and soil type variations in a watershed was handled by partitioning the watershed into homogeneous subfields. The subsurface soil profile and aquifer heterogeneities were considered by dividing the subsurface domain into root zone, intermediate vadose zone, and saturated zone, respectively. On each of the homogeneous subfields, the physically-based models, PRZM and VADOFT, were linked to simulate pesticide transport in the root and intermediated vadose zones. Pesticide movement in groundwater underneath the watershed was simulated by linking the other two models with SUTRA. An irregular shape finite element mesh generator was developed for fitting the irregular shape watershed boundary and reducing the number of nodes of the finite element mesh. Either transient or steady state flow and transport simulation could be performed with the system. The system is able quantitatively to produce detailed spatial variation maps of pesticide concentrations at any desired depth in the unsaturated zone and in groundwater. The system requires spatially-distributed information as inputs. Management of large volumes of spatially-referenced data which represent the heterogeneous properties of the watershed were facilitated by a developed GIS component. The GIS data processing component was composed of spatial data manipulation and display, attribute database management, and model input information extraction subcomponents. The spatial data processing component consists of data format conversion, map registering, map editing, new information generation, and map display subcomponents. / Ph. D.

No / Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Erythropoietin is a hematopoietic hormone and the main physiological function is the induction of erythropoiesis. Recombinant DNA technology enabled cloning and expression of rhEPO gene to produce recombinant human erythropoietin (rhEPO). It is a sialoglycoprotein composed of 165 amino acids chain and about 40% of the molecule consists of carbohydrates, important for biological activity due to the presence of sialic acid residues at the termini of chains affecting its half-life. In the present study an alternative in vitro cell culture-based assay using TF-1 cell line was validated, to be used in conjunction with a reversed-phase liquid chromatography method with fluorescence detection (F-RP-LC) validated to determine the content of sialic acids. The values obtained for the sialic acids were higher than 126.83 ng/μg, and the biotechnology-derived products were subjected to the cell culture bioassay giving potencies 2.91% ± 0.85 lower related to the bioassay in normocythaemic mice, with significant correlation calculated by the Pearson coefficient (r = 0.9947). In parallel, it was determined the content/potency of the products by the validated reversed-phase and size-exclusion liquid chromatography methods that showed mean results 3.14% higher and 2.87% lower, respectively, compared to the in vitro bioassay. It was demonstrated that in vitro cell culture bioassay represent a valid alternative to the in vivo bioassay for the potency assessment of rhEPO, in the context of the reduction and/or replacement of animals. Likewise, correlation of the results obtained with the physicochemical methods, represents advances for the characterization of the biomolecule, which can be applied to the production steps and for the quality control, contributing to assure the batch-to-batch consistency of the bulk and the finished biological products of rhEPO. / A eritropoietina é um hormônio hematopoiético cuja principal função fisiológica é a indução da eritropoiese. Através da tecnologia do DNA recombinante foi possível a clonagem e expressão do gene para produzir a eritropoietina humana recombinante (rhEPO). É uma sialoglicoproteína composta por cadeia de 165 aminoácidos e, aproximadamente, 40% da molécula é constituída de carboidratos, importantes para a atividade biológica devido à presença de resíduos de ácidos siálicos nas extremidades das cadeias, que influenciam na meia-vida da biomolécula. No presente estudo foi validado ensaio alternativo baseado na cultura da linhagem de células TF-1 in vitro, e método por cromatografia líquida por fase reversa com detecção por fluorescência para determinação de ácidos siálicos, para avaliação em conjunto da potência de rhEPO. Determinaram-se teores de ácidos siálicos acima de 126,83 ng/μg e os produtos biotecnológicos foram submetidos ao bioensaio por cultura de células fornecendo potências 2,91% ± 0,85 menores em relação ao ensaio biológico em camundongos normocitêmicos, com correlação significativa calculada pelo coeficiente de correlação de Pearson (r = 0,9947). Paralelamente, determinou-se o teor/potência dos produtos pelas metodologias validadas por cromatografia líquida por fase reversa e por exclusão molecular, que forneceram média de resultados 3,14% maior e 2,87% menor, respectivamente, em relação ao bioensaio in vitro. Demonstrou-se que o bioensaio por cultura de células in vitro, constitui-se em alternativa ao ensaio biológico in vivo para a avaliação de potência de rhEPO, no contexto da redução e/ou substituição do uso de animais. Do mesmo modo, a correlação com os resultados fornecidos pelos métodos físico-químicos, representa avanços para caracterização da biomolécula, que podem ser aplicados nas etapas do processo de produção e para o controle de qualidade, contribuindo para garantir a consistência lote-a-lote da solução concentrada e dos produtos biológicos acabados de rhEPO.