Atualmente, há um grande incentivo governamental para a produção de biodiesel a partir de óleo de mamona. O aumento na fabricação desse óleo irá aumentar a produção de torta de mamona, que tem grande potencial de emprego na tecnologia de material biodegradável e utilização na alimentação animal, se destoxificada. Os objetivos desta tese foram a caracterização química da torta de mamona, a extração de suas proteínas para desenvolvimento de material biodegradável, e a caracterização do resíduo sólido do processo de extração, visando seu uso na alimentação animal. A extração das proteínas da torta de mamona foi feita por solubilização em meio alcalino. Inicialmente, diversos parâmetros (velocidade de agitação, concentração da torta na solução extratora e temperatura de extração) foram testados no intuito de aumentar o rendimento de extração das proteínas, utilizando-se NaOH (pH = 9). Em seguida, diversos experimentos foram realizados para se avaliar os efeitos do pH (8-12) e/ou do tipo de agente alcalino (NaOH, KOH e Ca(OH)2) na extração das proteínas da torta de mamona, sempre à temperatura de 50°C, velocidade de agitação de 400rpm, e concentração da torta na solução extratora de 20%. Os extratos proteicos obtidos foram liofilizados (EPL), e os resíduos foram desidratados em estufa (40°C/24 horas). Análises para determinação da composição bromatológica, minerais, amido, compostos fenólicos totais, ácidos graxos, aminoácidos, fibra dietética, da microestrutura, atividade alergênica, eletroforese dimensional e identificação de ricina foram feitas na matéria prima, nas proteínas extraídas e nos resíduos. A composição de aminoácidos foi analisada no extrato proteico liofilizado (EPL) e nos resíduos somente nas amostras preparadas com NaOH. A composição bromatológica das matérias primas (semente, torta e farelo) mostrou elevados teores de óleo (43,6%) e fibras (29,5%) para as sementes; bem como altos teores de proteína (36-40%) e fibras (29-30%) para a torta e o farelo de mamona. A semente de mamona apresentou valores de minerais considerados razoáveis para oleaginosas, e a torta de mamona apresentou alta proporção de K; Ca e P, importantes para alimentação animal. Verificou-se alto teor de ácido ricinoléico (79-90%) nas matérias primas avaliadas. A torta de mamona apresentou altos teores de ácido glutâmico (15%), arginina (11%) e triptofano (9%), sendo que os teores dos outros aminoácidos variaram entre 2 e 7%. Os resultados da caracterização química dos EPL e dos resíduos foram dependentes principalmente do pH de extração das proteínas. Observou-se aumento da concentração de proteína (64-68%) e de cinzas (13-19%), nos EPL, em função do aumento do pH (10 - 12), independente do tipo de agente alcalinizante. Os resíduos obtidos da extração dos EPL apresentaram teores de proteína, cinzas e fibras variando entre 20 e 34%, 12 e 17% e 39 e 42%, respectivamente. Em relação ao perfil de minerais dos EPL, verificou-se aumento nas concentrações de Na e Mn com o aumento do pH para ambos agentes alcalinizantes. O perfil de minerais dos resíduos mostrou aumento significativo nos teores de Na e K com aumento do pH de extração, ajustado com NaOH e KOH, respectivamente. O teor de ácido ricinoléico foi menor no resíduo, e maior no EPL, obtidos em pH 12 ajustado com NaOH. A composição de aminoácidos dos resíduos sofreu efeito do pH apenas para o pH 12. Os aminoácidos presentes em maior concentração nos EPL e nos resíduos foram ácido glutâmico, triptofano e arginina. Os EPL e os resíduos obtidos da extração das proteínas apresentaram menor poder alergênico avaliado pelo teste de desgranulação de mastócitos obtidos do lavado peritoneal de ratos. O perfil eletroforético dos EPL e dos resíduos mostrou maior proporção de proteínas com massa molecular entre 29-36kDa, seguidos por proteínas de massa molecular ao redor de 20kDa e menor proporção de proteínas de massa molecular entre 45-66kDa. Não foi observada a presença da ricina nos resíduos obtidos em pH 12. Os resultados de todos os experimentos de extração das proteínas da torta de mamona permitiram escolher os melhores parâmetros de extração: temperatura = 50°C, velocidade de agitação = 400rpm, concentração da torta na solução extratora = 20%, pH = 12 e agente alcalinizante = NaOH. Em conclusão, o processo de extração de proteínas por solubilização alcalina permitiu a produção de concentrado proteico com características interessantes para a produção de material biodegradável (agricultura e biofilmes), e de resíduos ainda com alto teor de proteínas e de fibras, isento de ricina, podendo, portanto ser utilizado na alimentação animal. / Presently, there is large government incentive for biodiesel production from castor seed oil. The increase of this oil fabrication will raise the production of castor seed pulp, which has great potential for utilization for biodegradable material technology and for animal feed, if detoxified. The objectives of this research were the chemical characterization of the castor seed pulp, the extraction of the proteins for biodegradable material developing, and the characterization of the solid residue of the extraction process, aiming its use for animal feed. The protein extraction from the castor seed pulp was made through solubilization in alkaline medium. Initially, several parameters (stirring speed, pulp concentration in the extraction solution and extraction temperature) were tested aiming the optimization of the protein extraction yield, using NaOH (pH 9). Subsequently, several experiments were realized to evaluate the effects of pH (8-12) and/or of the type of alkaline agent (NaOH, KOH and CaOH) on the castor seed pulp protein extraction, always at 50 °C, 400rpm stirring speed, and 20 % pulp concentration in the extraction solution. The obtained protein extracts were lyophilized (EPL), and the residues were dehydrated in stove (40 °C/24 h). The raw material, the extracted proteins and the residues were analyzed for bromatologic composition, minerals, starch, total phenolic compounds, fatty acids, amino acids, dietetic fiber, microstructure, allergenic activity, dimensional electrophoresis and ricin identification. The amino acids composition was analyzed on the lyophilized extract (EPL) and in the residues of the samples prepared with NaOH. The bromatologic composition of the raw materials (seed, pulp and meal) showed high content of oil (43.6 %) and fiber (29.5 %) for the seeds; as well as high amounts of protein (36-40 %) and fiber (29-30 %) for the pulp and the castor seed meal. The castor seed had average mineral values for oil seeds, and the castor seed pulp showed high percentage of K, Ca and P, valuable for animal feed. The evaluated raw materials had high percentage of ricinoleic acid (79-90 %). The castor seed pulp showed high percentage of glutamic acid (15 %), arginine (11 %) and triptophane (9 %). The percentage of the other amino acids varied between 2 and 7 %. The results of the chemical characterization of the EPL and of the residues were mainly dependent on the pH during protein extraction. It was observed an increase of the protein concentration (64-68 %) and of the ash (13-19 %), within the EPL, due to the pH raise (10 - 12), independently of the type of alkaline agent. The residues of the EPL extraction showed protein, ash and fiber content varying between 20 and 34%, 12 and 17% e 39 and 42%, respectively. Concerning the mineral profile of the EPL, an increase of Na and of Mn was observed with the increase of pH for both alkaline agents. The mineral profile of the residues showed a significant increase of the Na and the K content with the increase of the pH of the extraction solution, adjusted with NaOH and KOH, respectively. The ricinoleic acid content was lower in the residue, and higher in the EPL, obtained at pH 12 using NaOH. The amino acid composition of the residues was affected by the pH only at pH 12. The higher content of amino acid in the EPL and in the residues was of glutamic acid, triptophane and arginine. The EPL and the residues of the protein extraction showed low allergenic capacity evaluated by the mastocyte degranulation test obtained from the peritoneal wash of rats. The electrophoresis profile of the EPL and of the residues showed higher content of proteins with molecular weight varying between 29-36 kDa, followed by proteins with molecular weight around 20 kDa and lower proportion of proteins with molecular weight between 45 and 66 kDa. Ricin was not observed in the residues obtain at pH 12. The results of all the experiments of protein extraction of the castor seed pulp allowed to select the best extraction parameters: temperature = 50 °C, stirring speed = 400rpm, castor seed pulp concentration in the extraction solution = 20 %, pH = 12 and alkaline agent = NaOH. In conclusion, through the alkaline protein extraction process it was possible to achieve a protein concentrate with interesting properties for biodegradable material production (agriculture and biofilms), and of ricinless residues which still have high content of proteins and fiber that can be used for animal feed.

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-02Bitstream added on 2014-06-13T20:21:44Z : No. of bitstreams: 1 celli_mg_dr_sjrp.pdf: 756707 bytes, checksum: 4e5cc119fa93aadaf2ac40ff2235e038 (MD5) / A patulina, toxina com potencial carcinogênico, mutagênico e teratogênico produzida por Penicillium spp., Aspergillus spp. e Byssoclamys spp., pode ser encontrada em maçãs, sucos comerciais e outros produtos não fermentados constituindo-se num problema neste setor agroindustrial. Em bebidas fermentadas, porém, não é detectável mesmo que a matéria-prima esteja visivelmente contaminada. O presente trabalho visou quantificar a patulina tanto no tecido deteriorado de maçãs in natura quanto na parte sadia ao redor da lesão, monitorar a degradação no processo fermentativo típico utilizando Saccharomyces cerevisiae e degradar a toxina presente em produtos derivados de maçã pela ação do composto bioativo produzido pela levedura. A micotoxina foi quantificada por CLAE em sistema isocrático de fase reversa e detector UV a 275 nm. O grau de recuperação alcançado foi de 86,24% e os limites de detecção e de quantificação foram 4,3 µg/L e 8,6 µg/L, respectivamente. Foram analisados 35 frutos de maçã in natura cultivar Fuji, tendo sido constatada a presença de patulina em 32 amostras, em concentrações que variaram de 1,01 a 120,4 mg/Kg de tecido na porção deteriorada e de 0,02 a 5,02 mg/Kg de tecido, na sadia. Para avaliação da cinética de degradação de patulina, suco de maçã contendo 5,0 µg de patulina/mL foi inoculado com 0,25g de células de levedura seca ativa/L, sendo observada uma maior redução no teor de toxina de 81,6% nas primeiras 48 horas de fermentação. Este tempo foi utilizado como parâmetro para obtenção do extrato bruto contendo o composto bioativo capaz de degradar a toxina. Na cinética de degradação de patulina pelo extrato bruto estéril inoculado com 3,0 µg de patulina/mL, foi obtida a maior taxa de degradação (50%) da toxina nas primeiras 48 horas, seguido de uma degradação mais lenta de apenas 5,7% nas 48 horas... / Patulin, a toxin with potential carcinogenic, mutagenic and teratogenic effects produced by Penicillium sp., Aspergillus sp. and Byssoclamys sp., can be found in apples, juices and other commercial non-fermented products making up a problem in the agribusiness sector. However, patulin is not detectable in fermented beverages even if the raw material is visibly contaminated. This paper aimed to quantify patulin in both the tissue deteriorated fresh apples and at the sound around the lesion, to monitor the degradation in typical fermentation process using Saccharomyces cerevisiae and degrade the toxin present in products derived from apple by the action of the bioactive compound produced by yeast. The mycotoxin was quantified by HPLC in isocratic system of reversed phase and UV detection at 275 nm. The degree of recovery achieved was 86.24% and the limits of detection and quantification were 4.3 µg/L and 8.6 µg/L, respectively. Thirty five fresh apple fruits Fuji were analyzed and it was, found the presence of patulin in 32 samples in concentrations that ranged from 1.01 to 120,4 mg/Kg of tissue in the damaged portion and 0.02 to 5, 02 mg/Kg of tissue in sound. To evaluate the kinetics of degradation of patulina, apple juice with 5.0 µg patulin/mL was inoculated with 0.25 g of cells active dry yeast/L, being observed a greater reduction in the toxin content of 81.6% in the first 48 hours of fermentation. This time was used as a parameter to obtain the crude extracts containing bioactive compound capable of degrading the toxin. The kinetics of patulin degradation by the crude extract inoculated with 3.0 µg patulin/mL showed the highest rate of the toxin degradation (50%) in the first 48 hours, followed by only 5.7% degradation after the remaining 48 hours of analysis. The kinetics of degradation of patulin by the extract dialysate inoculated with 3.0 µg patulin/mL showed... (Complete abstract click electronic access below)

In the past few years the Utah Fish and Game Department, as well as the fish and game departments of other states, has been spending fisheries money for fish eradication on both lakes and streams. The Utah Fish and Game Department recently suggested to the University that research be initiated on the subject of toxaphene as a fish poison. Consequently, a better insight into conditions that exist for a given water type could be developed. To date there is a very small amount of material written on toxaphene. This is due primarily to the fact that toxaphene was not developed until the 19hO's. Toxaphene is used mainly as an insecticide. Such crops as tomatoes, beans, alfalfa, clover and cotton are protected by the use of toxaphene (Hudd and Genelly, 1956).

Zinc, copper and cadmium accumulation was measured in two herbivorous gastropod molluscs Austrocochlea constricta and Bembicium auratum from Lake Macquarie NSW an area with a history of trace metal pollution. The investigation consisted of three main parts. The first part examined the influence of organism mass and location within the Lake on whole body tissue metal concentrations. This part of the study also compared the distributions of tissue metal concentrations of populations from Lake Macquarie, a known polluted environment, with those of populations from Jervis Bay NSW, an unpolluted environment, to establish whether either species is a net accumulator of zinc copper or cadmium. The second part of the investigation examined a range of factors which may influence whole body metal concentrations. One location in Lake Macquarie was sampled monthly from August 1995 to July 1996. The factors examined were temporal variation, gender, breeding cycle, and tissue distribution. The final part of the investigation examined the detoxification and storage of excess metals in the gastropods from Lake Macquarie. The mechanisms studied were metallothioneins and granules. The tissue metal concentrations of both species were found to be independent of mass. Location within Lake Macquarie did not significantly influence tissue metal concentrations. Variation between individuals was the most significant contribution to overall variation, resulting in a positive skewing of sample trace metal distributions. B. auratum populations from Lake Macquarie had significantly higher copper and cadmium tissue concentrations and A. constricta populations had significantly higher zinc, copper and cadmium tissue concentrations than the populations from Jervis Bay. This suggests that regulation of these metals is not occurring. A. constricta may therefore be considered a net accumulator of zinc, copper and cadmium and B. auratum of copper and cadmium. Tissue metal concentrations did not vary significantly over time. It is suggested that the organisms are in equilibrium with their environment. B. auratum has higher natural equilibrium concentrations than A. constricta particularly for copper and cadmium, suggesting different routes of exposure, uptake or accumulation for the two species. Gender and breeding cycle did not significantly influence tissue metal concentrations. Most of the variability in total copper and cadmium concentrations of both species was explained by variability in gonad tissue metal concentration, while variability in the gonad and somatic tissues zinc concentration explained about an equal amount of the variability in total zinc concentration. A. constricta and B. auratum were both found to induce a cadmium binding protein which has some features in common with metallothionein. A protein of around 10 000 Da which binds approximately 60% of the soluble cadmium was isolated using gel filtration. This protein was further separated into two isoforms using anion exchange. The first isoform eluted at the same time as MT I and the second at the same time as MT II rabbit liver standard. Large cells containing granular material which stained positive for calcium were observed interspersed among the connective tissue immediately behind the columnar epithelial cells lining the gut wall in both species under a light microscope. Calcium positive granular particles were also observed within the columnar epithelial cells of B. auratum. These species have been shown to be net accumulators of the trace metals investigated, with the exception of zinc in B. auratum. It has also been established that organism mass, gender and reproductive state, the partitioning of metals between tissues, and temporal effects are not confounding factors for the purposes of comparing trace metal concentrations between populations. They should therefore be effective biomonitors of the trace metals investigated, with the exception of zinc in B. auratum.

Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.

Le maintien de l'homéostasie rédox des thiols, dépendant du pouvoir réducteur du<br />NADPH, est un processus vital pour la cellule faisant intervenir deux voies supposées<br />distinctes : 1) thiorédoxine réductase/thiorédoxines et 2) glutathion<br />réductase/glutathion/glutarédoxines. En dépit de leur importance, on connaît mal la spécificité<br />des glutarédoxines et des thiorédoxines [1]. C'est particulièrement vrai chez les organismes<br />photosynthétiques, dont le métabolisme rédox, dépendant de la photosynthèse, est essentiel à<br />la biosphère (production d'oxygène, assimilation du carbone et de l'azote inorganiques<br />nécessaires à la production de biomasse pour la chaîne alimentaire). La photosynthèse,<br />comme la respiration, produit des molécules oxydantes (les espèces activées de l'oxygène) qui<br />perturbent, entre autres, l'homéostasie rédox des thiols.<br />Dans le cas des glutarédoxines, il a été montré, chez les organismes hétérotrophes, que ces<br />enzymes utilisent le pouvoir réducteur du glutathion (re-réduit par le NADPH via la<br />glutathion réductase) pour contrôler l'état rédox des thiols des résidus cystéines (Cys) des<br />protéines (réduire les ponts disulfure inter- ou intra-moléculaires). Les glutarédoxines se<br />répartissent en deux grandes familles, selon la composition de leur centre rédox actif : les<br />glutarédoxines à dithiol (possédant un site actif de type CysXXCys) et les glutarédoxines à<br />monothiol (avec un site actif de type CysXXSer).<br />Au cours de ma thèse, j'ai analysé, in vitro et in vivo, les glutarédoxines avec un organisme<br />photosynthétique modèle: la cyanobactérie unicellulaire Synechocystis PCC6803<br />(Synechocystis), qui possède un petit génome (3,57 Mb) entièrement séquencé et<br />génétiquement manipulable avec les vecteurs plasmidiques développés au laboratoire.<br />Synechocystis possède 3 glutarédoxines (Grx): deux à dithiol (Grx1 et Grx2) et une à<br />monothiol (Grx3). Dans un premier temps, j'ai inactivé les 3 gènes correspondants,<br />indépendamment ou non. Tous les mutants correspondants (simples, doubles et triple) sont<br />parfaitement viables, dans les conditions standard de croissance. Par contre, leur tolérance aux<br />stress diffère de celle de la souche sauvage. Le mutant Dgrx1 est sensible au mercure (HgCl2)<br />et à l'uranium ((CH3COO)2UO2). Le mutant Dgrx2 est sensible au peroxyde d'hydrogène<br />(H2O2), au cadmium (CdSO4) et au sélénate (NaSeO4). Le mutant Dgrx3 est sensible au bleu<br />de méthylène. Le triple mutant Dgrx1Dgrx2Dgrx3 est sensible à chacun des toxiques<br />précédemment cités, et aussi, de façon spécifique par rapport aux autres mutants, à un excès<br />(x25) de zinc (ZnCl2). Ces résultats indiquent que les Grx possèdent une certaine sélectivité,<br />qui s'exerce vraisemblablement au travers d'interactions spécifiques.<br />11<br />Pour rechercher des partenaires protéiques des Grx, j'ai utilisé un système bactérien de<br />"double hybride", basé sur 2 plasmides dans lesquels on peut cloner, indépendamment, les<br />gènes codant pour les protéines "appâts" (chacun des trois gènes grx) et les protéines "proies"<br />(les partenaires possibles des Grxs). Pour identifier ces dernières, nous avons cloné,<br />indépendamment, une cinquantaine des gènes impliqués dans métabolisme rédox, sans se<br />limiter au contrôle de l'homéostasie rédox des thiols. Les 1000 tests d'interaction réalisés<br />nous ont permis d'identifier 10 interactions totalement nouvelles impliquant des Grx. Ensuite,<br />j'ai analysé les interactions (1) Grx1-MerA (MerA est la réductase du mercure) et (2)<br />thiorédoxine réductase-Grx1-Grx2, par une approche multidisciplinaire: (i) mutagenèses<br />dirigées, tests double hybride et études in vivo; (ii) approche biochimique (GST Pulldown); et<br />(iii) tests d'activité in vitro.<br />J'ai montré que l'activité de réduction du mercure de MerA est inhibée, de façon réversible,<br />par la glutathionylation (fixation d'une molécule de glutathion) d'une cystéine (Cys78) de son<br />site actif. Grx1 réactive MerA en catalysant la réaction de déglutathionylation du site actif de<br />MerA. Outre la tolérance de Synechocystis au mercure (HgCl2), j'ai montré que Grx1 et MerA<br />interviennent également dans la résistance à l'uranium (CH3COO)2UO2.<br />Parallèlement, j'ai analysé les interactions thiorédoxine réductase-Grx1-Grx2. J'ai montré que<br />la thiorédoxine réductase est capable de réduire Grx1, qui peut à son tour réduire Grx2. Cette<br />voie rédox originale compense l'absence de glutathion réductase chez Synechocystis. J'ai<br />également montré que cette "nouvelle" voie rédox est capable de réduire le sélénate. Ces<br />résultats in vitro sont confortés par certains de mes résultats qui suggèrent que Synechocystis<br />répond au sélénate par la formation d'un hétérodimère Grx1-Grx2.<br />Les deux "nouvelles" voies rédox caractérisées au cours de ma thèse sont particulièrement<br />intéressantes car elles permettent de relier deux processus rédox, homéostasie rédox des thiols<br />et détoxication des métaux lourds par réduction, qui n'étaient jusqu'ici pas considérés comme<br />étant étroitement imbriqués.

Background: Ambient air pollution is associated with adverse health effects, but the sources and components, which cause these effects is still incompletely understood. The aim of this thesis was to investigate the pulmonary effects of a variety of common air pollutants, including diesel exhaust, biomass smoke, and road tunnel and subway station environments. Healthy non-smoking volunteers were exposed in random order to the specific air pollutants and air/control, during intermittent exercise, followed by bronchoscopy. Methods and results: In study I, exposures were performed with diesel exhaust (DE) generated at transient engine load and air for 1 hour with bronchoscopy at 6 hours post-exposure. Immunohistochemical analyses of bronchial mucosal biopsies showed that DE exposure significantly increased the endothelial adhesion molecule expression of p-selectin and VCAM-1, together with increased bronchoalveolar lavage (BAL) eosinophils. In study II, the subjects were exposed for 1 hour to DE generated during idling with bronchoscopy at 6 hours. The bronchial mucosal biopsies showed significant increases in neutrophils, mast cells and lymphocytes together with bronchial wash neutrophils. Additionally, DE exposure significantly increased the nuclear translocation of the aryl hydrocarbon receptor (AhR) and phosphorylated c-jun in the bronchial epithelium. In contrast, the phase II enzyme NAD(P)H-quinone oxidoreductase 1 (NQO1) decreased after DE. In study III, the 2-hour exposures took place in a road tunnel with bronchoscopy 14 hours later. The road tunnel exposure significantly increased the total numbers of lymphocytes and alveolar macrophages in BAL, whereas NK cell and CD56+/T cell numbers significantly decreased. Additionally, the nuclear expression of phosphorylated c-jun in the bronchial epithelium was significantly increased after road tunnel exposure. In study IV, the subjects were exposed to metal-rich particulate aerosol for 2 hours at a subway station with bronchial biopsy and BAL sampling at 14 hours. The subway exposure significantly increased the concentration of glutathione disulphide (GSSG) in BAL, with no airway inflammatory responses. In contrast, the number of neutrophils in the bronchial mucosa and the nuclear expression of phosphorylated c-jun in the bronchial epithelium tended to decrease after the subway exposure. In study V, the exposure to biomass smoke lasted 3 hours. Bronchoscopy was conducted 24 hours post exposure. The investigated biomass combustion emissions resulted in a significant increase in total glutathione and reduced glutathione in BAL, without any evident acute airway inflammatory responses.     Conclusion: The present thesis presents data from exposures of healthy subjects to a variety of common air pollutants, as compared with an air reference. Oxidative as well as bronchial mucosal and bronchoalveolar responses differed between these air pollutants, with the most pronounced airway effects seen after exposure to diesel exhaust. This may be due to differences in pulmonary deposition, physicochemical characteristics, toxicological pathways and potency. Additional studies will assist in addressing dose-response and time kinetic aspects of the airway responses.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

This thesis investigates two aspects of phytoalexin metabolism by the phytopathogenic fungus <i>Sclerotinia sclerotiorum</i> (Lib) de Bary: (i) determination of detoxification pathways of structurally different molecules; (ii) design and synthesis of potential inhibitors of enzyme(s) involved in detoxification steps.<p>First, the transformations of important cruciferous phytoalexins by the economically important stem rot fungus, <i>S. sclerotiorum</i>, were investigated. During these studies a number of new metabolic products were isolated, their chemical structures were determined using spectroscopic techniques, and further confirmed by synthesis. The metabolic products did not show detectable antifungal activity against <i>S. sclerotiorum </i> which indicated that these metabolic transformations were detoxification processes. Overall, the results of these transformations suggested that <i>S. sclerotiorum</i> produces various enzymes that can detoxify cruciferous phytoalexins via different pathways. While the detoxifications of strongly and moderately antifungal phytoalexins such as brassilexin, sinalexin, and 1-methoxybrassinin were fast and led to glucosylated products, the transformations of the weakly antifungal phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin were very slow and yielded non-glucosylated compounds.<p>Next, the design of potentially selective inhibitors of the brassinin detoxification enzyme, BGT, was sought. Two sets of potential inhibitors of BGT were designed: (i) a group was based on the structure of brassinin, where the indole ring of brassinin was replaced with benzofuran, thianaphthene, 7-azaindole and pyrazolo[1,5-a]pyridine and/or the position of side chain was changed from C-3 to C-2; and (ii) another group based on the structure of camalexin where the thiazole ring of camalexin was replaced with a phenyl group. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal activity, as well as screening using fungal cultures and cell-free extracts of <i>S. sclerotiorum</i>, were examined. The results of these screening indicated that 3-phenylindoles, 3-phenylbenzofuran, 5-fluorocamalexin, methyl (indol-2-yl)methyl-dithiocarbamate, methyl (benzofuran-3-yl)methyldithiocarbamate and methyl (benzo-furan-2-yl)methyldithiocarbamate could slow down the rate of detoxification of brassinin in fungal cultures and also in cell-free extracts of <i>S. sclerotiorum</i>. Among the designed compounds, 3-phenylindole appeared to be the best inhibitor both in fungal cultures and in cell-free extracts. Metabolism studies of all the designed compounds using fungal cultures of <i>S. sclerotiorum</i> indicated that they were metabolized by <i>S. sclerotiorum</i> to glucosyl derivatives, although at much slower rates.<p>It is concluded that some inhibitors that can slow down the rate of metabolism of brassinin could be good leading structures to design more active inhibitors of BGT.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.