Dissertations / Theses on the topic 'Microbial toxins'

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux.
Master of Science

L‟eutrophisation croissante des écosystèmes aquatiques favorise le développement des cyanobactéries, parmi lesquelles Microcystis est la plus représentée dans les régions tempérées. La capacité de Microcystis à produire une puissante hépatotoxine, la microcystine, est à l‟origine de diverses perturbations écologiques, et de nombreuses nuisances sanitaires. La compréhension des facteurs déterminant la toxicité des efflorescences de Microcystis constitue, de fait, un enjeu majeur des recherches actuelles. Dans ce contexte, l‟objectif premier de ce travail de thèse était d‟étudier la variabilité temporelle et l‟implication potentielle de la toxicité de Microcystis à l‟échelle de son cycle de développement annuel. Pour cela, il était nécessaire de considérer, en particulier, les parties les moins connues du cycle de développement : la phase de survie benthique, et les transitions entre les phases benthique et planctonique, via les processus de recrutement et de sédimentation. Nous avons alors étudié le potentiel toxique des populations de Microcystis grâce à des approches complémentaires menées à différentes échelles spatio-temporelles, en considérant à la fois les gènes impliqués dans la synthèse de microcystines, leur transcription et les concentrations en microcystines. Cette étude s‟est appuyée, en parallèle, sur la caractérisation de la structure génétique des populations de Microcystis dans les compartiments benthique et planctonique. La prise en compte systématique de la phase de vie benthique a tout d‟abord permis d‟améliorer nos connaissances sur cette phase du cycle de développement de Microcystis. Ainsi, Microcystis peut survivre plusieurs années en profondeur dans les sédiments, sans que les populations ne perdent leur potentiel toxique, ou que leur structure génétique soit altérée. En revanche, en surface des sédiments, le potentiel toxique et la structure génétique des populations sont variables, de manière similaire à ce qui peut être observé dans la colonne d‟eau. Enfin, ces travaux ont également mis en évidence l‟influence des phases de transition entre l‟eau et les sédiments dans la variabilité du potentiel toxique et de la structure génétique des populations de Microcystis. Les processus de recrutement benthique et de sédimentation occasionnent, en effet, une sélection génétique, qui, bien que paraissant indépendante du potentiel toxique des génotypes, peut grandement affecter le potentiel toxique des sous-populations benthiques et planctoniques de Microcystis
The increasing eutrophication of aquatic ecosystems promotes the development of cyanobacteria, among which Microcystis is the most widespread in temperate regions. The ability of this cyanobacterium to produce a potent hepatotoxin, called the microcystin, represent a serious threat for both natural life and human health. Thus, understanding the factors determining the toxicity of Microcystis blooms is a major challenge of actual research. In this context, the main goal of this work was to study the temporal variability and the potential implication of Microcystis toxicity, at the scale of its annual life cycle. For that, it was necessary to consider more particularly, the least known parts of the cycle : the benthic survival phase, and the transition between the benthic and the planktonic phases, through the benthic recruitment and the sedimentation processes. Then, we studied the toxic potential of Microcystis populations through complementary approaches conducted at different spatio-temporal scales, by considering the genes controlling the synthesis of the microcystin, their transcription and the concentrations of microcystin. In parallel, the genetic structure of Microcystis populations was characterized in both benthic and planktonic compartments. By considering systematically the benthic life stage, we were first able to improve our knowledge on this phase of Microcystis development cycle. Thus, Microcystis is able to survive several years in deep sediments, without the population‟s toxic potential or genetic structure being degraded. On the other hand, at the sediment surface, the toxic potential and the genetic structure of the populations vary, in a similar range to what observed in the water column. Furthermore, this work also shed the light on the influence of benthic-pelagic transitions in the variability of the genetic structure and the toxic potential of the populations of Microcystis. Indeed, a genetic selection occurs during the benthic recruitment and the sedimentation processes. Although such a selection does not seem to rely on the toxic potential of the genotypes, it can greatly modify the toxic potential of both benthic and planktonic sub-populations of Microcystis

Abstract Symbiotic relationships between arthropods and microorganisms are widespread in nature. In the last years these interactions are received considerable attention, as many microorganisms may play relevant roles in the biology and lifecycle of insects (Dale and Moran, 2006; Moran et al., 2008). In this perspective, researchers are directing many efforts to depict the interactions that shape the symbiosis. Furthermore, considering the importance that microorganisms play for their hosts, the modification of the microbiome structure in the insect body could support the development of sustainable strategies, alternative to chemical pesticides. To achieve the development of these methods, the knowledge and the identification of the symbionts associated to the pest of interest, is a mandatory requirement. Recent studies documented the evidence of stable associations between acetic acid bacteria (AAB) and insects characterized by a sugar-based diet. These include Diptera, Hymenoptera and Hemiptera orders (Crotti et al., 2010). It was reported that AAB are essential in the modulation of the immune homeostasis as well as metabolism and larval development. These capacities have been demonstrated in Drosophila (Ryu et al., 2008; Shin et al., 2011), but have been recently confirmed in other models, like Anopheles (Chouaia et al. 2012, Hughes et al., 2014). Along with bacteria, drosophilid flies establish a mutualistic relationship with yeasts, in particular with those belonging to the Saccharomycetaceae family: these microorganisms represent the main nutritional source for the flies, as they provide proteins, vitamins and other nutrients. Yeasts are vectored by Drosophila, from which they are dispersed, favoring the colonization of new habitats (Christiaens et al., 2014). Moreover, they can affect the fly development and fitness in terms of susceptibility to parasitism (Anagnostou et al. 2010). Yeasts share the same environments with AAB, supporting the hypothesis of possible microbe-microbe interactions. The aim of my PhD project was the characterization of the microbiome associated to the spotted wing fly Drosophila suzukii Matsumura (Diptera: Drosophilidae), an economically damaging pest of healthy soft summer fruits, rapidly spreading in many countries from South-East Asia (Lee et al., 2011). In particular, targets of the research were AAB and yeasts symbionts. Results revealed that AAB were a major component of D. suzukii bacterial community. Members of Gluconobacter, Gluconacetobacter and Acetobacter genera were the main representatives, as shown by culture-dependent (isolation by using specific media, dereplication with ITS-PCR and isolate identification through partial 16S rRNA gene sequencing) and -independent analyses (16S rRNA barcoding and Denaturing Gradient Gel Electrophoresis-PCR). The investigation was performed on specimens of different developmental stages (larvae, pupae and adults), reared on two feeding substrates (fruit or an artificial diet). The plasmid pHM2(Gfp) was introduced by electroporation in three selected AAB isolates, Gluconobacter oxydans DSF1C.9A, Acetobacter tropicalis BYea.1.23 and Acetobacter indonesiensis BTa1.1.44 to label them with Green fluorescent protein (Gfp). After oral administration to the insects, Gfp-tagged strains were visualized in the host by fluorescence microscopy. The symbionts were able to successfully reach and colonize the epithelium of the insect crop, proventriculus and midgut. Tests performed on bacterial cultures grown in liquid media showed that several AAB isolates are able to produce an extracellular matrix in which the cells are entrapped and that presumably is implicated in the bacterial adhesion to the insect epithelia and maintenance in the digestive system. By using probes specific for AAB (Texas red-labelled probe AAB455) and Eubacteria (Texas red-labelled universal Eubacterial probe Eub338), fluorescent in situ hybridization (FISH) experiments on the host dissected tissues from D. suzukii, detected AAB within the peritrophic membrane of the midgut and proventriculus. Probes specific for Gluconobacter (Cy5-labelled probes Go615 and Go618) were also designed, and used to confirm the presence of this species within the intestinal tract of the fly. Due to the abundance and intimate connection of these symbionts with D. suzukii tissues and organs, I predict that AAB have important roles in the lifecycle of the host. The capacity of D. suzukii-selected AAB isolates to emit microbial volatile compounds for flies’ specific attraction was subsequently analysed. Microbial volatiles are known to attract or repel insects, inhibit or stimulate the plant growth. For example, acetic acid was described to be an attractant molecule for Drosophila flies (Cha et al., 2014). With the aim to evaluate the different attraction capabilities of some selected AAB on D. sukuzii, a two-choice olfactometer assay was developed and attraction experiments were carried out. After the first evaluation of the bacterial growth to set up the experimental conditions, flies were exposed to volatile organic compounds (VOCs) produced by AAB isolates in comparison to a control, represented by the growth medium without bacteria. Higher attractiveness for flies than the other bacteria was obtained with Gluconobacter oxydans DSF1C.9A, Gluconobacter kanchanaburiensis L2.1.A.16 and Gluconacetobacter saccharivorans DSM1A.65A strains. Since currently traps for flies are composed by vinegar and baker’s yeast, the analyses of the best attractive molecules released by specific microorganisms might provide novel tools for D. suzukii biocontrol and the assembly of baits specifically targeted for this pest. Flies at different developmental stages (larvae, pupae and adults) reared on different food sources (fruit or artificial diet) were analyzed through cultivation-independent (DGGE-PCR, 16S rRNA 454-pyrosequencing) and -dependent (isolation trials, and isolate identification) techniques to investigate the yeast community. Most of the analyzed sequences obtained from the excised DGGE bands and pyrosequncng data had close similarity with sequences assigned to Saccharomycetales, in particular Candida, Geotrichum and Pichia genera. These yeasts comprise specialist colonizers of rotten and fermenting fruits, and the skin of intact fruits eaten by Drosophila. A collection of 237 yeast isolates were obtained from the isolation trials, with the purpose to explore the community diversity in individuals of different life stages, and reared on the two-abovementioned food sources. Identification of the yeast species was carried out using RFLP (Restriction Fragment Length Polymorphism) analysis of the ITS1-ITS2 region of the fungal rRNA gene, of all the isolates. Restriction patterns were obtained through the use of HaeIII, HinfI and TaqI endonucleases. After the analysis of the generated digestion patterns, the representative isolates of each RFLP profile were submitted to sequencing analyses of both the D1-D2 region of the large subunit (LSU) of the fungal rRNA gene and the ITS1-ITS2 region of the fungal rRNA gene. Phylogenetic trees completed the analysis. The results strengthened and enlarged the molecular data, and showed that the most abundant species recorded, i.e. Pichia occidentalis, Saccharomycopsis craetegensis and Arthroascus schoenii, were the only species isolated from all of the tree life stages (larvae, pupae, and adults). Insects reared on fruits were characterized by a higher diversity in terms of yeast species. In particular, it was also recorded the presence of Hanseniaspora uvarum, which was described in previous works as a dominant yeast genus associated to different Drosophila species, including D. suzukii (Chandler et al., 2012, Hamby et al., 2012). Data obtained from the yeast community characterization, as well as from the bacterial community one, were exploited for a screening of the possible interactions among symbionts. In particular, some yeast strains are able to compete for their own ecological niche and nutrients by producing an array of compounds named “killer toxins” (Woods and Bevan, 1968). Since the production of killer toxins could be highly affected by the culture conditions, in terms of pH, temperature and carbon source content, then the optimal ones were developed for the growth of selected yeast isolates, and subsequently antagonistic activity tests were performed. The results highlighted the capacity of a specific yeast isolate, Candida stellimalicola AF4.1.P.268, to limit the growth of several yeast and AAB isolates, by creating inhibition haloes. This feature might have a role in a pest management perspective. In conclusion, this project indicates that AAB and yeast communities establish an intimate association with D. suzukii, as they were found stably and abundantly in individuals of different stages and fed on different diets. Recolonization trials performed with AAB strains suggest, in particular, their importance for the biology of this pest. Gathered information might be a basis to develop alternative strategies for a more effective and sustainable biocontrol management of this emerging pest, for whom a successful strategy has not been found yet. References - Anagnostou C., Dorsch M. and Rohlfs M. (2010). Influence of dietary yeasts on Drosophila melanogaster life-history traits. Entomol. Exp. Appl. 136: 1–11. - Cha D. H., Adams T., Werle C. T., Sampson B. J., Adamczyk J. J., Rogg H. and Landolt P. J. (2014). A four-component synthetic attractant for Drosophila suzukii (Diptera: Drosophilidae) isolated from fermented bait headspace. Pest. Manag. Sci., 70: 324–331. - Chandler J. A., Eisen J. A. and Kopp A. (2012). Yeast communities of diverse Drosophila species: comparison of two symbiont groups within the same hosts. Appl. Environ. Microbiol. 78: 7327-7336 - Chouaia B., Rossi P., Epis S., Mosca M., Ricci I., Damiani C., Ulissi U., Crotti E., Daffonchio D., Bandi C. and Favia G. (2012). Delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts. BMC Microbiol. 12: S2. - Christiaens J. F, Franco L. M., Cools T. L., De Meester L., Michiels J., Wnseleers T., Hassan B.A., Yaksi E. and Verstrepen K. J. (2014), Cell Reports 9, 425–432 - Crotti E., Rizzi A., Chouaia B., Ricci I., Favia G., Alma A., Sacchi L., Bourtzis K., Mandrioli M. and Cherif A. (2010). Acetic acid bacteria, newly emerging symbionts of insects Appl. Environ. Microbiol. 76: 6963-6970 - Dale C. and Moran N. A. (2006). Molecular interactions between bacterial symbionts and their hosts. Cell. 126(3): 453-65. - Hamby K. A., Hernández A., Boundy-Mills K. and Zalom F. G. (2012). Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries. Appl. Environ. Microbiol. 78: 4869-4873. - Hughes G. L. , Dodson B. L. , Johnson R. M., Murdock C. C., Tsuijimoto H., Suzuki Y., Patt A. A., Cui L., Nossa C. W., Barry R. M., Sakamoto J. M., Hornett E. A. and Rasgon J. L. (2014). Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes. PNAS USA. 111: 12498-12503. v- Lee J. C., Bruck D. J., Dreves A. J., Ioratti C., Vogt H. and Baufeld P. (2011). In Focus: spotted wing drosophila, Drosophila suzukii, across perspectives. Pest Manag. Sci. 67(11):1349-1351. - Moran N.A., McCutcheon J.P., Nakabachi A. (2008).Genomics and evolution of heritable bacterial symbionts . Annu Rev Genet.42: 165-190. - Ridley E. V., Wong A. C. N., Westmiller S. and Douglas A. E. (2012.) Impact of the Resident microbiota on the nutritional phenotype of Drosophila melanogaster. .PlosONE 7: e36765. - Ryu J. H., Kim S. H., Lee H. Y., Ba J. Y., Nam Y. D.,Bae J. W., Lee D. G., Shin S. C., Ha E. M. and Lee W. J. (2008). Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science. 319: 777–782. - Shin S. C., Kim S. H., You H., Kim B., Kim A. C., Lee K.A., Yoon J.H., Ryu J.H. and Lee W.J. (2011). Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science 334: 670-674. - Woods D. R. and Bevan E. A. (1968). Studies on the nature of the killer factor factor produced by Saccharomyces cerevisiae. J. Gen. Microbiol. 51: 115-126.

Clostridium perfringens type E iota toxin is implicated in some cases of fatal diarrhea in calves, lambs, and guinea pigs. A crossreacting "iota-like" toxin, produced by Clostridium spiroforme, is responsible for antibiotic-associated and weaning related enterotoxemias of rabbits. Antisera developed against culture supernatant of either organism neutralized the biological activity of iota or iota-like toxin. By using C. spiroforme antiserum and crossed immunoelectrophoresis (crossed IEP), we found two cross-reacting antigens in C. perfringens type E supernatants. C. perfringens types A, B, C, and D, which do not produce iota toxin, did not cross-react with C. spiroforme antiserum. To determine if either antigen had iota toxin activity, we separated the cross-reacting antigens of C. perfringens by preparative isoelectric focusing (IEF) and tested all IEF fractions for biological activity in guinea pigs and mice. The fraction containing the faster-migrating antigen seen in crossed IEP, designated iota b (i_b), had some guinea pig dermonecrotic and mouse lethal activity. Other fractions, including the one containing the slower migrating iota a (i_a) antigen, had little to no biological activity. When fractions containing i_a and i_b were mixed, there was an 8 and 25 fold increase in mouse lethal and dermonecrotic titers, respectively. Activity was neutralized by C. perfringens type E or C. spiroforme antisera and other fractions, when mixed with i_a or i_b, did not have a synergistic effect. Both components of C. perfringens iota toxin were purified using ammonium sulfate precipitation, DEAE anion exchange chromatography, preparative IEF, Sephadex G-100 gel filtration, and flatbed electrophoresis to yield a 12 and 5% final recovery of i_a and i_b, respectively. Each protein was homogeneous by SDS PAGE, gradient PAGE, and crossed IEP using homologous antiserum. There was at least an 8 fold increase in mouse lethal titer and 64 fold increase in dermonecrotic titer when equimolar amounts of i_a and i_b were mixed. Monospecific antisera against purified i_a and i_b neutralizd the iota or iota-like activity of crude supernatants. A sensitive and specific ELISA was developed using monospecific and C. spiroforme antisera. The i_a and i_b proteins have a pI of 5.2 and 4.2 and molecular weights of 48,000 and 71,000 (SDS PAGE), respectively. The i_a protein is heat stable (85° C/15 min) while i_b lost its activity at 55°C. Amino terminus sequencing revealed that both proteins were blocked by an unknown functional group(s). Purified i_a, but not i_b, has ADP-ribosylating activity specific poly-L-arginine in vitro. Recent evidence suggests that nonmuscle actin, involved in the cytoskeletal structure of eucaryotic cells, may act as the in situ acceptor.
Ph. D.

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O glufosinate é derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos Streptomyces. Atua inibindo a atividade da enzima glutamina sintetase, que é necessária para a produção do aminoácido glutamina e para a desintoxicação da amônia pela planta. Objetivou-se neste trabalho avaliar a intoxicação e alterações fisiológicas e bioquímicas causadas pelo glufosinate em plantas de soja. O experimento foi conduzido em casa-de-vegetação, no Núcleo de Pesquisas Avançadas em Matologia (Nupam), pertencente à Faculdade de Ciências Agronômicas – FCA/UNESP. Foi utilizado o cultivar de soja Nidera NS 7100 (RR) plantada em vasos de cinco de litros de solo. Foram realizados dois experimentos, sendo o primeiro experimento instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio a 2,5 L p.c. ha-1 e glufosinate de potássio 2,5 L p.c. ha-1), três períodos de coleta de folhas dois, quatro e seis dias após aplicação e um tratamento testemunha sem aplicação. O segundo experimento foi instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio e glufosinate de potássio), três doses de cada produto (0,625 L p.c. ha-1; 1,25 L p.c. ha-1; 2,5 L p.c. ha-1) e um tratamento testemunha sem aplicação. As variáveis analisadas em ambos os experimentos foram, amônia, compostos pertencentes à rota metabólica de ação do glufosinate (glutamato, glutamina, serina, ácido aminolevulênico, glufosinate), análise visual de fitotoxicicidade e ETR (taxa de transporte de elétrons). Para determinação de amônia foram desenvolvidas metodologias de extração para posterior quantificação em espectrofotômetro. Para determinação dos compostos, foram realizados...
The glufosinate is derived from phosphinothricin, a natural microbial toxin isolated from Streptomyces of two species of fungus. Acts inhibits the activity of glutamine synthetase, which is necessary for the production of the amino acid glutamine and for the detoxification of ammonia by the plant. The objective of this study was to evaluate the toxicity and physiological and biochemical changes caused by glufosinate in soybean plants. The experiment was carried out in green-house, in Faculty of Agronomic Sciences at São Paulo State University. We used soybean the cultivar NS 7100 Nidera planted in vases containing 5 liters of soil. Two experiments were carried out, the first experiment set with the following treatments: two formulations (glufosinate ammonium 2.5 L c.p. ha-1 and glufosinate potassium 4 2.5 L c.p. ha-1), three periods of leaf collection two, for and six days after application and a control without application. The second experiment was carried out with the following treatments: two formulations (glufosinate ammonium and glufosinate potassium), three doses of each product (0.625 L c.p. ha-1, 1.25 c.p. ha-1 L, 2.5 L c.p. ha -1) and a control without application. The variables analyzed in both experiments were ammonia, compounds belonging to the metabolic pathway of action of glufosinate (glutamate, glutamine, serine, aminolevulenic acid, glufosinate), visual analysis of plant injury and ETR (electron transport rate). To determine the ammonia were developed extraction methodologies for a quantification in spectrophotometric and for determination of compounds, extraction tests were performed to choose the most appropriate methodology, and development of analytical methods in LC-MS/MS (liquid chromatography and mass spectrometry). Plant injury evaluations were based on visual criteria and ETR measurements are given... (Complete abstract click electronic access below)

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Resumo: O glufosinate é derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos Streptomyces. Atua inibindo a atividade da enzima glutamina sintetase, que é necessária para a produção do aminoácido glutamina e para a desintoxicação da amônia pela planta. Objetivou-se neste trabalho avaliar a intoxicação e alterações fisiológicas e bioquímicas causadas pelo glufosinate em plantas de soja. O experimento foi conduzido em casa-de-vegetação, no Núcleo de Pesquisas Avançadas em Matologia (Nupam), pertencente à Faculdade de Ciências Agronômicas - FCA/UNESP. Foi utilizado o cultivar de soja Nidera NS 7100 (RR) plantada em vasos de cinco de litros de solo. Foram realizados dois experimentos, sendo o primeiro experimento instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio a 2,5 L p.c. ha-1 e glufosinate de potássio 2,5 L p.c. ha-1), três períodos de coleta de folhas dois, quatro e seis dias após aplicação e um tratamento testemunha sem aplicação. O segundo experimento foi instalado com os seguintes tratamentos: duas formulações (glufosinate de amônio e glufosinate de potássio), três doses de cada produto (0,625 L p.c. ha-1; 1,25 L p.c. ha-1; 2,5 L p.c. ha-1) e um tratamento testemunha sem aplicação. As variáveis analisadas em ambos os experimentos foram, amônia, compostos pertencentes à rota metabólica de ação do glufosinate (glutamato, glutamina, serina, ácido aminolevulênico, glufosinate), análise visual de fitotoxicicidade e ETR (taxa de transporte de elétrons). Para determinação de amônia foram desenvolvidas metodologias de extração para posterior quantificação em espectrofotômetro. Para determinação dos compostos, foram realizados... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The glufosinate is derived from phosphinothricin, a natural microbial toxin isolated from Streptomyces of two species of fungus. Acts inhibits the activity of glutamine synthetase, which is necessary for the production of the amino acid glutamine and for the detoxification of ammonia by the plant. The objective of this study was to evaluate the toxicity and physiological and biochemical changes caused by glufosinate in soybean plants. The experiment was carried out in green-house, in Faculty of Agronomic Sciences at São Paulo State University. We used soybean the cultivar NS 7100 Nidera planted in vases containing 5 liters of soil. Two experiments were carried out, the first experiment set with the following treatments: two formulations (glufosinate ammonium 2.5 L c.p. ha-1 and glufosinate potassium 4 2.5 L c.p. ha-1), three periods of leaf collection two, for and six days after application and a control without application. The second experiment was carried out with the following treatments: two formulations (glufosinate ammonium and glufosinate potassium), three doses of each product (0.625 L c.p. ha-1, 1.25 c.p. ha-1 L, 2.5 L c.p. ha -1) and a control without application. The variables analyzed in both experiments were ammonia, compounds belonging to the metabolic pathway of action of glufosinate (glutamate, glutamine, serine, aminolevulenic acid, glufosinate), visual analysis of plant injury and ETR (electron transport rate). To determine the ammonia were developed extraction methodologies for a quantification in spectrophotometric and for determination of compounds, extraction tests were performed to choose the most appropriate methodology, and development of analytical methods in LC-MS/MS (liquid chromatography and mass spectrometry). Plant injury evaluations were based on visual criteria and ETR measurements are given... (Complete abstract click electronic access below)
Doutor

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O glufosinate é um herbicida derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos do gênero Streptomyces. O mecanismo de ação é a inibição direta da enzima glutamina sintetase, que resulta no aumento da concentração de amônio, sendo tóxico para as células. A pesquisa teve como objetivo avaliar a velocidade de absorção do glufosinate e seus efeitos em plantas daninhas e na cultura do algodão. O experimento foi conduzido em casa-de-vegetação com o cultivar de algodão FiberMax 910 e as plantas daninhas Brachiaria decumbens e Ipomoea grandifolia plantadas em vasos. O experimento foi instalado com duas formulações do herbicida, glufosinate de amônio (2,0 L p.c. ha-1) e glufosinate de potássio (2,0 L ha-1), cinco períodos sem a ocorrência de chuvas (1; 3; 6; 24 e 48 horas após aplicação) e uma testemunha sem aplicação com quatro repetições por tratamento. As plantas para as análises laboratoriais foram coletadas com dois dias após aplicação, quando começaram a aparecer os primeiros sintomas de intoxicação visual. As variáveis analisadas foram teor de amônia, glutamato, glutamina e glufosinate; sintomas de intoxicação visual e taxa de transporte de elétrons (ETR). Foi observado...
Glufosinate is derived of phosphinothricin, a natural microbial toxin isolated from two species of the Streptomyces fungus. The mechanism of action is through direct inhibition of the glutamine synthetase enzyme, which results in the increase of ammonium concentration, becoming toxic to the cells. The objective of this study was to evaluate the rate of absorption of glufosinate and its effects on weeds and cotton culture. The experiment was conducted in a green house, the cultivar of cotton FiberMax 910, Brachiaria decumbens and Ipomoea grandifolia planted in 5 liter pots. The experiment was conducted with two formulations of the herbicide, glufosinate ammonium (2.0 pc L ha-1) and glufosinate potassium (2.0 L ha-1), five periods without rainfall (1, 3, 6, 24 and 48 hours after application) and an untreated control with all treatments were replicated four times. The plants for laboratory analysis were collected within 2 days after application, when the first visual symptoms of intoxication began to appear. The analyzed variables were the ammonia content, the content of compounds that belongs to the glufosinate metabolic route (glutamate and glutamine), glufosinate content, visual symptoms of intoxication and electron transport rate (ETR). It was observed that the absorption of glufosinate occurs within 48 hours of application for both formulations and evaluated species. The amount of ammonia in the commercial formulation did not interfere with ammonia levels observed in intoxicated plants. The highest levels of ammonia for cotton occurred at 5 hours without rain, and for B. decumbens and I. grandifolia with 6 hours without rain. We have observed a significant reduction of glutamate and glutamine in plants treated with the two formulations of glufosinate, with the lowest levels found in cotton and B. decumbens carried out in... (Complete abstract click electronic access below)

Orientador: Caio Antonio Carbonari
Coorientador: Edivaldo Domingues Velini
Banca: Fernando Tadeu de Carvalho
Banca: Ana Catarina Cataneo
Resumo: O glufosinate é um herbicida derivado do fosfinotricina, uma toxina microbiana natural isolada a partir de duas espécies de fungos do gênero Streptomyces. O mecanismo de ação é a inibição direta da enzima glutamina sintetase, que resulta no aumento da concentração de amônio, sendo tóxico para as células. A pesquisa teve como objetivo avaliar a velocidade de absorção do glufosinate e seus efeitos em plantas daninhas e na cultura do algodão. O experimento foi conduzido em casa-de-vegetação com o cultivar de algodão FiberMax 910 e as plantas daninhas Brachiaria decumbens e Ipomoea grandifolia plantadas em vasos. O experimento foi instalado com duas formulações do herbicida, glufosinate de amônio (2,0 L p.c. ha-1) e glufosinate de potássio (2,0 L ha-1), cinco períodos sem a ocorrência de chuvas (1; 3; 6; 24 e 48 horas após aplicação) e uma testemunha sem aplicação com quatro repetições por tratamento. As plantas para as análises laboratoriais foram coletadas com dois dias após aplicação, quando começaram a aparecer os primeiros sintomas de intoxicação visual. As variáveis analisadas foram teor de amônia, glutamato, glutamina e glufosinate; sintomas de intoxicação visual e taxa de transporte de elétrons (ETR). Foi observado... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Glufosinate is derived of phosphinothricin, a natural microbial toxin isolated from two species of the Streptomyces fungus. The mechanism of action is through direct inhibition of the glutamine synthetase enzyme, which results in the increase of ammonium concentration, becoming toxic to the cells. The objective of this study was to evaluate the rate of absorption of glufosinate and its effects on weeds and cotton culture. The experiment was conducted in a green house, the cultivar of cotton FiberMax 910, Brachiaria decumbens and Ipomoea grandifolia planted in 5 liter pots. The experiment was conducted with two formulations of the herbicide, glufosinate ammonium (2.0 pc L ha-1) and glufosinate potassium (2.0 L ha-1), five periods without rainfall (1, 3, 6, 24 and 48 hours after application) and an untreated control with all treatments were replicated four times. The plants for laboratory analysis were collected within 2 days after application, when the first visual symptoms of intoxication began to appear. The analyzed variables were the ammonia content, the content of compounds that belongs to the glufosinate metabolic route (glutamate and glutamine), glufosinate content, visual symptoms of intoxication and electron transport rate (ETR). It was observed that the absorption of glufosinate occurs within 48 hours of application for both formulations and evaluated species. The amount of ammonia in the commercial formulation did not interfere with ammonia levels observed in intoxicated plants. The highest levels of ammonia for cotton occurred at 5 hours without rain, and for B. decumbens and I. grandifolia with 6 hours without rain. We have observed a significant reduction of glutamate and glutamine in plants treated with the two formulations of glufosinate, with the lowest levels found in cotton and B. decumbens carried out in... (Complete abstract click electronic access below)
Mestre

Thesis (M. Tech. Environmental health) -- Central University of technology, Free State, 2011
In nature microorganisms do not exist alone, but in association with one another. These kinds of associations can also be found in food industries, where cells of the same or different species can attach to pipes (biofilm formation) and a variety of surfaces in food processing environments and in food product such as yoghurt which can contain both yeast and bacteria originating from the starter culture as well as fruit. To control food spoilage organisms and food-borne pathogens preventative measures such as good manufacturing processes, the use of sanitizers and preservatives as well as hazard analysis critical control points (HACCP) are crucial in food industries. Sanitation of the working surface, floors, pipes, containers and equipment is a stepwise application of a detergent, acid or alkali rinse, a disinfectant treatment followed by final rinsing. If rinsing of the sanitizer is not done properly it may end up in the product in sub-lethal doses. In this study the influence of Liquid Hypochlorite (LH) and Liquid Iodophore (LI) sanitizers on organism growth and toxicity was evaluated. The organisms investigated included Escherichia coli 0113, Escherichia coli 026 and Zygosaccharomyces bailii Y-1535 in yeast malt broth, which was supplemented with LH and LI at sub-lethal concentrations 0.05% LH, 0.2% LH and 0.075% LI. Subsequently, bacterial and yeast growth responses as pure cultures and in combination (E. coli + Z. bailii) were measured as colony forming units and optical density values. Incorporation of the sanitizers in the growth media resulted in different levels of growth inhibition. Z. bailii proved more robust and the growth rate was not influence significantly by the addition of sanitizers or communal growth with either E. coli strains. The growth rate of both E. coli strains decreased where grown in combination with Z. bailii as well as in the presence of sanitizers, with the most influence exerted by LH. Changes in endotoxicity following the growth of the test samples (stressed cells) and the control (unstressed) were measured by the limulus amoebocyte lysate (LAL) and porcine IL-6 ELISA methods. Where E. coli strains were cultured together with Z. bailii the toxicity of tire mixture showed a decrease over time when measured with the limulus amoebocyte assay method. Interestingly the communal growth of the E. coli strains and Z bailii produced different toxicity profiles when the IL-6 porcine method was used, hi both cases, where E. coli strains were cultured together with Z. bailii the toxicity of the mixture showed an increase over tune when measured by this assay. Other than a similar toxicity profile for E. coli 0113 grown in pure culture, the comparison between results obtained using the LAL or porcine IL-6 methods yielded no correlation in determined toxicity. It was established that LH and LI sanitizers as well as communal growth had an influence in the toxicity of LPS/EPS and the method used to determine such toxicity should be carefully considered.

Thesis (M. Tech. Environmental health) -- Central University of technology, Free State, 2011
The threat to the world food supply and the concern for public health as a result of food-borne diseases has been established as a constant global problem. The safety of food, in particular, is of significance to consumers and producers alike. Regarding the diseases related to food-borne pathogens, the disease syndromes affecting the entire human body has become inestimable. The focus of the study was to establish the effect of sanitisers, detergents and household storage temperatures on the growth profiles and toxicity of typical food related organisms. The endotoxin, LPS of these Gram-negative organisms in communal growth as compared to pure culture was the focus of the investigation. Pure and communal samples were grown in the presence of the extrinsic stresses including storage temperature. The change in toxicity was measured using the Limulus amoebocyte lysate test and the possible change in the immune response was determined using the porcine-IL-6 test. The first obvious finding was that the overall sensitivity of organisms was similar for the same sanitiser and the same detergent. The sensitivity of the community varied slightly but in principle followed the same pattern as the individual organisms. The LD50 for all growth samples were as follows: 32 X 104 PPM for sanitiser 1 and sanitiser 2, and 16X 104 PPM for detergent 1 and detergent 2. Growth in community was found not to be the arithmetic sum of the individual growth patterns. The detergents had a marked effect on the growth of all samples throughout the growth cycle. The sub-optimum household storage temperatures inhibited the growth throughout the cycle but growth did not cease entirely. This finding may have revealed that the acceptable refrigeration temperatures still allows for pathogen growth and thus for biofilm formation. Furthermore, the response of the community to the extrinsic stresses appears to be entirely different to the pure culture and therefore needs further exploration to address the problem. Regarding the quantification by LAL, it was found that the enumeration of the food-borne pathogens isolated from households might not be indicative of acclimatisation obtained over short periods of time and the causal stress turning these organisms into more or less toxic pathogens. The sanitisers and detergents induced competition in colonial fashion and the growth varied between feast and famine. The extrinsic stresses had a more observable effect on the older biofilm as this was shown by a decrease in toxicity. The toxicity as quantified by porcine-IL-6 yielded a mixture of stimulation levels for the cytokine. The toxicity change indicated by the test showed a variation between lowering and noticeable elevation for pure cultures. A marked elevation in toxicity was detected in community at storage temperature 4°C. The study would suggest that porcine IL-6 is not an accurate biomarker for pyrogenicity since its sensitivity is questionable and its inability to indicate toxicity if there is a possible change in the LPS structure. It should be said that further elucidation is needed to support this finding. Having said all that, it is no surprise that the validation for the two tests favours the LAL procedure. The large room for pre-test stimulation in pigs’ blood also tends to cast a shadow on the IL-6 findings. The findings of the study contribute to the body of knowledge covering the effects and quantitative analysis of toxins in food. This should add to safety assurance by sensitizing the industry regarding the most suitable analytical methodologies to apply.

Mestrado em Microbiologia
As estirpes eméticas de Bacillus cereus produzem uma toxina altamente resistente, denominada cereulida. Esta toxina é o agente responsável por uma intoxicação alimentar caracterizada por emese. A cereulida é um dodecadepsipéptido cíclico com uma estrutura semelhante à da valinomicina, um composto antimicrobiano produzido por Streptomyces spp. Ambos os compostos são ionóforos de potássio, facilitando o movimento the K+ através das membranas celulares com concomitante dissipação do potencial de membrana. Devido às suas propriedades ionofóricas, a valinomicina e a cereulida são ambas tóxicas para as mitocôndrias. As similaridades com a valinomicina sugerem que a cereulida possa ser um composto antimicrobiano. A actividade antimicrobiana da cereulida e da valinomicina foi testada avaliando o seu efeito no crescimento das bactérias seleccionadas sob diferentes condições de crescimento. A valinomicina e a cereulida exibiram actividade antimicrobiana contra as bactérias Gram positvo testadas. Por outro lado, as bactérias Gram negativo revelaram-se insensíveis a estes ionóforos de potássio sob as condições testadas. À excepção de Enterococcus faecalis, o crescimento bacteriano foi fortemente inibido pela valinomicina e pela cereulida a pH 8.5. Enquanto que a pH 6.5 estes ionóforos foram ineficazes. O oxigénio revelou-se um importante factor para a eficiência dos ionóforos. As células aeróbicas foram mais sensíveis à acção da valinomicina e da cereulida do que as células anaeróbicas. As estirpes eméticas revelaram uma menor suscepibilidade aos ionóforos testados. Curiosamente, a estirpe não emética Bacillus cereus ATCC 14579 mostrou, ao contrário das outras estirpes não eméticas, alguma resistência à valinomicina.
Emetic Bacillus cereus produce an highly resistant toxin, named cereulide. This toxin is the causative agent of a food-borne disease characterized by emesis. Cereulide is a cyclic dodecadepsipeptide with a structure similar to the one of valinomycin, an antimicrobial compound produced by Streptomyces spp. Both of these compounds are potassium ionophores, facilitating the movement of K+ across cell membranes with concomitant dissipation of the membrane potential. Due to their ionophoric properties, valinomycin and cereulide are both toxic to mitochondria. The similarities with valinomycin suggest that cereulide may be an antimicrobial compound. The antimicrobial activity of cereulide and valinomycin was evaluated in relation to growth of selected bacteria under different growth conditions. Valinomycin and cereulide were found to exhibit antimicrobial activity effective against the Gram positive bacteria tested. Gram negative were insensitive to these potassium ionophores in the conditions used. With the exception of Enterococcus faecalis, bacterial growth was strongly inhibited by valinomycin and cereulide at pH 8.5. While at pH 6.5 these potassium ionophores were inefective. Oxygen was found to be an important effector of ionophore efficiency. Aerobic cells were more sensitive to valinomycin and cereulide than anaerobic cells. The emetic Bacillus strains were found to be less susceptible to the tested ionophores. Interestingly, the non-emetic Bacillus cereus ATCC 14579 showed, unlike the other tested non-emetic strains, some resistance to valinomycin.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O presente estudo avaliou a qualidade microbiológica, as características físicoquímicas e a presença de resíduos de antimicrobianos em leite de mulheres, vacas e cabras, bem como investigou a multirresistência bacteriana à antimicrobianos e a detecção de genes e produção de toxinas em linhagens de Staphylococcus spp. Foram colhidas 240 amostras de leite de mulheres encaminhadas ao Banco de Leite Humano (BLH) de Botucatu, SP, 200 amostras de leite de vacas com mastite e 200 de vacas sem mastite. Iguais quantidades de amostras de leite foram colhidas de cabras com e sem mastite. Dentre os tetos amostrados de vacas e cabras com mastite, 85,50% e 97,50% respectivamente acusaram mastite subclínica no CMT. A mastite clínica foi observada em 14,50% dos tetos de vacas e 2,50% dos tetos de cabras amostrados. A presença de micro-organismos em leite de vacas e cabras sem mastite foi verificada em cerca de 25,00% das amostras de leite testadas, alertando para a presença de animais portadores de patógenos no rebanho. A acidez dornic do leite de mulheres revelou que 95,42% encontraram-se dentro dos limites aceitáveis pela Rede Nacional de BLH. O aporte calórico do leite humano (LH) apresentou ampla variação nos teores de creme, gordura e valor energético. A acidez dornic do leite de vacas e cabras com e sem mastite apresentaram ampla variação indicando que outros fatores, além da contaminação microbiana do leite, podem interferir nos índices de acidez titulável. Staphylococcus spp. e Enterobacter spp. foram os isolados mais frequentes em amostras de LH. Streptococcus spp., Staphylococcus spp. e Corynebacterium bovis foram os micro-organismos mais comumente...
The present study evaluated the microbiological quality, the characteristics physicist-chemistries and the presence of antimicrobials residues in milk of women, cows and goats, as well as investigated the bacterial multidrug resistance and the detection of genes and/or toxin production in Staphylococcus spp. strains. Was collected 240 milk samples from women referred to the Human Milk Bank (HMB) in Botucatu, 200 milk samples from cows with mastitis and 200 cows without mastitis. Equal amounts of milk samples were collected from goats with and without mastitis. Among the sampled cows and goats teats with mastitis, 85.50% and 97.50% respectively accused subclinical mastitis on CMT. Clinical mastitis was observed in 14.50% of cows teats and 2.50% of the sampled goats teats. The presence of microorganisms in milk from cows and goats without mastitis was found in about 25.00% of the milk samples tested, warning of the presence of pathogens from animals in the herd. Dornic acidity of woman’s milk revealed that 95.42% were within acceptable limits by the National Network of HMB. Calorie intake of human milk (HM) showed wide variation in the amounts of cream, fat and energy value. Dornic acidity of cows and goats milk with and without mastitis showed wide variation indicating that other factors in addition to microbial contamination of milk can interfere with acidity indices. Staphylococcus spp. and Enterobacter spp. were the most frequently isolated in samples of human milk. Streptococcus spp., Staphylococcus spp. and Corynebacterium bovis were the microorganisms most commonly isolated in milk cows with and without mastitis. In milk samples from goats with and without... (Complete abstract click electronic access below)

Orientador: Márcio Garcia Ribeiro
Coorientador: José Paes de Almeida Nogueira Pinto
Banca: Antonio Carlos Paes
Banca: Hélio Langoni
Banca: Maria de Lourdes Ribeiro de Souza da Cunha
Banca: Nilson Roberti Benites
Resumo: O presente estudo avaliou a qualidade microbiológica, as características físicoquímicas e a presença de resíduos de antimicrobianos em leite de mulheres, vacas e cabras, bem como investigou a multirresistência bacteriana à antimicrobianos e a detecção de genes e produção de toxinas em linhagens de Staphylococcus spp. Foram colhidas 240 amostras de leite de mulheres encaminhadas ao Banco de Leite Humano (BLH) de Botucatu, SP, 200 amostras de leite de vacas com mastite e 200 de vacas sem mastite. Iguais quantidades de amostras de leite foram colhidas de cabras com e sem mastite. Dentre os tetos amostrados de vacas e cabras com mastite, 85,50% e 97,50% respectivamente acusaram mastite subclínica no CMT. A mastite clínica foi observada em 14,50% dos tetos de vacas e 2,50% dos tetos de cabras amostrados. A presença de micro-organismos em leite de vacas e cabras sem mastite foi verificada em cerca de 25,00% das amostras de leite testadas, alertando para a presença de animais portadores de patógenos no rebanho. A acidez dornic do leite de mulheres revelou que 95,42% encontraram-se dentro dos limites aceitáveis pela Rede Nacional de BLH. O aporte calórico do leite humano (LH) apresentou ampla variação nos teores de creme, gordura e valor energético. A acidez dornic do leite de vacas e cabras com e sem mastite apresentaram ampla variação indicando que outros fatores, além da contaminação microbiana do leite, podem interferir nos índices de acidez titulável. Staphylococcus spp. e Enterobacter spp. foram os isolados mais frequentes em amostras de LH. Streptococcus spp., Staphylococcus spp. e Corynebacterium bovis foram os micro-organismos mais comumente... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The present study evaluated the microbiological quality, the characteristics physicist-chemistries and the presence of antimicrobials residues in milk of women, cows and goats, as well as investigated the bacterial multidrug resistance and the detection of genes and/or toxin production in Staphylococcus spp. strains. Was collected 240 milk samples from women referred to the Human Milk Bank (HMB) in Botucatu, 200 milk samples from cows with mastitis and 200 cows without mastitis. Equal amounts of milk samples were collected from goats with and without mastitis. Among the sampled cows and goats teats with mastitis, 85.50% and 97.50% respectively accused subclinical mastitis on CMT. Clinical mastitis was observed in 14.50% of cows teats and 2.50% of the sampled goats teats. The presence of microorganisms in milk from cows and goats without mastitis was found in about 25.00% of the milk samples tested, warning of the presence of pathogens from animals in the herd. Dornic acidity of woman's milk revealed that 95.42% were within acceptable limits by the National Network of HMB. Calorie intake of human milk (HM) showed wide variation in the amounts of cream, fat and energy value. Dornic acidity of cows and goats milk with and without mastitis showed wide variation indicating that other factors in addition to microbial contamination of milk can interfere with acidity indices. Staphylococcus spp. and Enterobacter spp. were the most frequently isolated in samples of human milk. Streptococcus spp., Staphylococcus spp. and Corynebacterium bovis were the microorganisms most commonly isolated in milk cows with and without mastitis. In milk samples from goats with and without... (Complete abstract click electronic access below)
Doutor

The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria.
Ph. D.

Les Escherichia coli enterohémorragiques (EHEC) sont responsables de maladies graves comme la colitehémorragique (CH), le syndrome hémolytique et urémique (SHU) ou le purpura thrombotiquethrombocytopénique (PTT). Les infections humaines sont principalement dues à la consommationd’aliments contaminés, en particulier la viande d’origine bovine, le lait ou les légumes. Le principalréservoir naturel des EHEC est le tractus gastro-intestinal (TGI) des bovins. Les fèces de bovins sont, parconséquent, responsables de la contamination de plusieurs types d’aliments mais également de ladissémination environnementale de ces bactéries pathogènes. En relation avec cette problématique, lesdeux objectifs de ce projet étaient (i) de développer une nouvelle méthode d’immuno-capture pouraméliorer l’isolement des principaux sérogroupes de EHEC impliqués dans les infections humaines; et (ii)de développer un nouveau DFM (direct fed microbial) utilisable chez les bovins pour diminuer laprévalence des EHEC chez les animaux. Cette thèse a permis le développement d’une méthoded’immuno-capture basée sur l’utilisation d’une microplaque à 96 puits, coatée avec des anticorpsspécifiquement dirigés contre E. coli O157 ou d’autres sérogroupes. Cette méthode, appelée immunomicroplatecapture (IMC) est efficace et facile d’utilisation pour l’isolement de souches d’E. coli O157 ;O26 ; O103 et O111 dans les produits alimentaires. L’IMC pourrait être une alternative à l’utilisation desbilles immuno-magnétiques qui sont classiquement utilisées pour la détection des EHEC dans lesproduits alimentaires, mais qui représentent une technique longue et fastidieuse lors de l’analysesimultanée de nombreux échantillons. La seconde partie de cette thèse a permis de sélectionner 5souches de bactéries lactiques qui présentent une activité antagoniste vis-à-vis d’E.coli O157 et d’autressérogroupes in vitro. La résistance de ces souches aux conditions du tractus gastro-intestinal (conditionsacides, présence de sels biliaires, présence de jus de rumen) a été évaluée in vitro. Leur innocuité a étévérifiée chez des souris Balb-C par l’administration de chacune des souches incorporées à la nourriture(109 cfu/g). Enfin, la lyophilisation des souches n’a pas affecté leur activité antagoniste in vitro. Lesrésultats obtenus dans les différents tests in vitro suggèrent que les 5 souches pourraientpotentiellement être utilisées comme DFM chez les bovins pour diminuer la colonisation de leur TGI parles EHEC et par conséquent diminuer le risque de contamination des aliments et le risque dedissémination environnementale
Enterohemorrhagic E. coli (EHEC) are responsible for important diseases such as hemorrhagic colitis,hemolytic and uremic syndrome or thrombotic thrombocytopenic purpura. Human infections occurprincipally by consumption of contaminated food particularly beef meat, milk or vegetables. The mainnaturally reservoir of EHEC is the gastro-intestinal tract of cattle. Cattle feces are therefore responsiblefor contamination of various types of food but also environment dissemination of the pathogenicbacteria. Related to this problematic, the two objectives of this project were (i) to develop a newimmuno-capture method to improve the isolation of the main serogroups of EHEC involved in humaninfections in food; and (ii) to develop a new direct fed microbial usable in cattle to decrease prevalenceof EHEC in animals. This thesis allowed the development of an immuno-capture method based on theuse of 96-well microplates coated with specific antibody directed against E. coli O157 and otherserogroups. This method, called immuno-microplate capture (IMC) was efficient and user-friendly forthe isolation of E. coli O157; O26; O103 and O111 in foods. This could be an alternative to the use ofimmuno-magnetic beads which are currently used for the detection of EHEC in foods, but are timeconsumingand labor intensive when large number of samples is analyzed simultaneously. The secondpart of this thesis allowed the selection of 5 lactic acid bacteria strains which presented highantagonistic activity against E. coli O157 and other serogroups in vitro. Resistance of these strains togastro-intestinal conditions (acidic conditions, presence of bile salts and rumen fluid) was evaluated invitro. The safety of the 5 strains was checked in Balb-C mice by administration of each strain mixed infeed at 109 cfu/g. Finally, freeze-drying did not affect the antagonistic activity of the 5 strains, suggesteda possible large scale use of these strains. According to the various results obtained in vitro, the 5 strainscould potentially be used as DFM in cattle to decrease colonization of their gastro-intestinal tract byEHEC and consequently decrease the risk of food and environment contaminations

The aim of the work is to evaluate the ability to keep stabile functioning of microbial community of Negev Desert clay (Israel) in the presence of typical for damaging effect toxic metals - Cu2 + and Hg2+ . The results show the stability of Negev desert microbial community to the extremely high bactericidal concentrations of toxic metals (1000 mg/l Cu2+and 10 mg/l Hg2+), despite the trace concentrations of these metals (0,23 mg/l Cu2+) in the desert ecosystem. This indicates a high resistance and ability of microbial cenosis to adapt to extreme factors. It can be supposed, according to obtained results, that Negev desert microbial community is able to interact with toxic metals and involve them in biogeochemical cycles. On the base of metal resistant microorganisms from Negev desert ecosystem development the technology of industrial heavy metal wastewater purification is available.

Cooperation presents a major challenge for evolutionary theory: how can competition favor a trait that imposes a cost on the individual expressing it while benefitting another? This challenge has been answered by theory that emphasizes the importance of assortment between individuals that tend to cooperate and those who tend to behave selfishly, or `cheat'. Microbial cooperation remains puzzling, given the generally high genetic and taxonomic diversity of most microbial communities. Many microbial populations rely on shared, beneficial extracellular products for an array of functions in nature. However, when these lineages are maintained in liquid cultures, many are invaded and outcompeted by spontaneous `cheater' mutants that forego investments in these products while benefitting from those produced by neighbors. The apparent evolutionary instability of microbial investments in extracellular products in well-mixed laboratory cultures finds a natural parallel in the phenomenon of toxic microalgal blooms. These extremely dense populations of often free-living microalgae destroy populations of competing microalgae and grazing zooplankton that normally control population densities. Bloom populations of planktonic microalgae are unstructured, and seem ill suited for the evolution of cooperation. In this thesis, I have established a new theoretical framework for understanding the evolution of microbial external goods. This framework highlights the importance of cell-level structure in the distribution of these external products, as well as genetic structuring in populations. This perspective informed an investigation into the social niche of a biofilm-dwelling regulatory mutant of the important biocontrol strain Pseudomonas chlororaphis. In the highly self-structured environment of a bacterial biofilm, a surprising mutualistic association between this mutant and the wild type emerged, underscoring the importance of microbial ecology in understanding the evolution of niche construction. Extending these lessons to the evolutionary problem of exotoxins in free-swimming microalgae yields the novel possibility that fluctuations in density of toxic strains shift a cell-level functioning exotoxin into a true public good that may be exploited by cheaters. I show that exotoxicity can serve cell-level functions in Prymnesium parvum. Despite these cell-level benefits, the existence of nontoxic lineages within toxic blooms hints at a complex interaction between rapid evolutionary and ecological changes in toxic blooms.

This thesis proposes the use of compounds that change their colour when reduced by living cells, i.e. electrochromic electron acceptors, in the development of fast and low-cost microbial toxicity bioassays suitable for in situ analysis. Electrochromism enables the monitoring of cell respiration by simple colorimetric measurement or even with the bare eye. Thus, allowing for toxicity determination without the need for complex instrumentation. Hexacyanoferrate compounds have been selected from the wide spectrum of reported electrochromic electron acceptors due to their suitable solubility and stability. To this end, a microbial acute toxicity bioassay was developed based on metabolic reduction of ferricyanide and optical determination with standard laboratory equipment, using Escherichia coli (E. coli) as model bacterium. Interferences in the optical measurement due to biomass light scattering were minimized by dual wavelength detection at 405 nm (ferricyanide absorption and biomass scattering) and 550 nm (biomass scattering). On the other hand, modification of the refractive index (RI) of the medium until matching with refractive index of bacterial cells with (i.e. RI matching) was achieved by addition of 27% (w/v) sucrose, which reduced bacterial light scattering around 50%. The toxic impact of various compounds on E. coli was determined by analysis of ferricyanide reduction kinetics (variation of ferricyanide absorption with time) and single absorbance measurements. Kinetic analysis of bacterial ferricyanide reduction allowed for fast assays (assay time of 10 min) with half maximal effective concentrations (EC50) similar to standard methods (i.e. biolumiscence inhibition test) for organic and inorganic toxic compounds. Technological implementation of the microbial toxicity bioassay was carried out by developing a low-cost miniaturized optofluidic analysis system. The optofluidic system was composed of a poly(methyl methacrylate) (PMMA) optofluidic structure incorporating discrete auxiliary optical elements (i.e. light emitting diodes, LEDs, and detectors) and an electronic circuit enabling for subtraction of ambient light interference. The optical performance of the analytical system for ferricyanide determination was tested. It was insensitive to environmental light changes and compared favourably to commercially available instrumentation. The simplicity, portability and robustness of the analysis system make it suitable for fast and low-cost determination of toxic pollutants in situ. To reduce bioassay instrumentation requirements, we designed a low-cost solid system by trapping bacteria in hygroscopic paper matrices. E. coli cells were stably trapped on low-cost paper matrices (cellulose-based paper discs) and remained viable for long times (1 month when stored at -20 ºC). Apart of acting as bacterial carriers, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Optical properties of individual paper matrices were analysed showing good comparability between them. Chromatic changes associated with bacterial ferricyanide reduction were determined by three different transduction methods, i.e. optical reflectometry, image analysis and visual inspection. Validation of the bioassay was performed by analysis of real samples from natural sources (i.e. wastewater influents/effluents and leachates from contaminated soils) using the mentioned transduction methods and the bioluminiscence inhibition test (Microtox, as standard method). Toxicity values obtained showed good agreement between them and with our reference method (70% of coincidence in toxic samples and 80% in non-toxic samples). The use of a light and inexpensive material and minimum instrumentation requirements of the bioassay make it a true low-cost method for in-situ assessment of toxic water pollution.

Some yeasts secrete polypeptide toxins, which are lethal to other sensitive yeast cells, gram-positive pathogenic bacteria and pathogenic fungi. Therefore these are designated as killer toxins. Killer toxins are suggested as potent antimicrobial agents especially for the protection of fermentation process against contaminating yeasts, biological control of undesirable yeasts in the preservation of foods. Moreover they are promising antimicrobial agents in the medical field
due to immune system suppressing diseases like AIDS, there is an increase in the incidence of fungal diseases and current antimycotics have low selectivity and severe side effects. In this study our aim was to explain the cytocidal effect and enzymatic properties of K5 type yeast killer protein, which is secreted by Pichia anomala NCYC 434 cells, and known to have a broad range of killing spectrum. Competitive inhibition of the toxin with cell wall polysaccharides showed that primary binding site of toxin is &
#946
-1,3-glucans of sensitive cells. Toxin showed exo-&
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-1,3-glucanase activity which causes loss of cell wall rigidity leading cell death. Km and Vmax were found to be 0,3 mg/ml and 372,3 µ
mol/min/mg for laminarin hydrolysis. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Toxin production was found to be dependent on &
#946
-1,3-glucan content of the media. Toxin activity was completely inhibited by Hg+2 ,while several metal ions and DTT increased the activity to different extends. Our findings revealed the characteristics of K5 type killer toxin which will help for its possible uses in near future.

Metalworking fluids (MWFs) are petroleum emulsions employed for metal machining processes as coolants and lubricants. To date, they have been irreplaceable in modern heavy and manufacturing industries, with annual usage exceeding two billion litres worldwide. However, the large amount of MWFs, the highly concentrated complex recalcitrant and toxic petroleum components contained in them continue to cause significant concern in terms of sustainable routes of end-of-life treatment and disposal. Compared with other treatment methods, the anaerobic treatment method has significant advantages, such as the low capital, operating and maintenance costs and energy recovery. This latter factor has the potential benefit of generating bio-energy from waste organic matter whilst aerobic route leads to CO₂ emission. However, the bio-toxicity of MWFs is a huge challenge in terms of employing bio-treatment of waste MWFs. In this study, the anaerobic biodegradability of a typical MWF was investigated employing an activated sludge experimental system. Furthermore, the toxic effects of the MWF on the anaerobic ecosystem, particularly on methanogen species, were investigated using bio-molecular analytical methods and a biosensor. In order to overcome its toxicity, the indigenous anaerobic bacteria isolated from spent MWFs were employed in the treatment of the MWF since they were assumed to be acclimated to the conditions. The major findings include: (1) approximately 80% of the MWF (5,000mgCOD/L) was found to be anaerobically biodegradable, with around 35% of the biodegraded COD could be converted to methane; (2) the MWF appeared to be toxic to the anaerobic ecosystem, especially to methanogen species; and (3) however, treatment employing the anaerobic bacteria successfully reduced the toxicity of the MWF and enhanced the methane production in the process.

The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings. In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.

Doctor of Philosophy
Department of Diagnostic Medicine/Pathobiology
Michael W. Sanderson
Shiga-toxin-producing Escherichia coli (STEC), of which enterohemorrhagic E. coli (EHEC) are a pathogenic sub-group, are foodborne pathogens of significant public health importance in the United States. STEC belong to the family Enterobacteriaceae commonly found in the large intestine of humans and other warm-blooded animals. EHEC harbors shiga toxin (stx1 and/or stx2) and eae genes which confers the ability to cause human illnesses. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven STEC (O26, O45, O103, O111, O121, O145, and O157) as adulterants in ground beef and non-intact beef products to reduce/eliminate the burden of the pathogens in the beef production chain. STEC control efforts in the U.S. include the development of quantitative microbial risk assessment (QMRA) to identify mitigation strategies that are effective and economical in reducing exposure and reduce occurrence and public health risk from STEC in the beef chain. Collection of accurate and unbiased data is critical for the development of a QMRA that is valid for decision making. Determining the prevalence and concentration of the seven STEC in the different cattle types and seasons is valuable for the development a valid QMRA for STEC in beef production in the U.S. Our systematic review and meta-analysis study of the prevalence and concentration of E. coli O157 along the beef production chain indicated differences in the fecal prevalence of E. coli O157 among cattle types and seasons, revealed decreasing prevalence and concentration of E. coli O157 on cattle hides and carcass surfaces from pre-evisceration to the final chilled carcass stage, and identified study setting, detection method, hide or carcass swab area, and study design as significant sources of heterogeneity among studies reporting prevalence of E. coli O157 along the beef production chain. Bayesian estimation of the diagnostic performance of three laboratory methods (culture, conventional PCR [cPCR], and multiplex quantitative PCR [mqPCR]) used for the detection of the seven STEC in the feces of cattle is necessary to estimate true prevalence of EHEC in cattle. The analysis revealed highest sensitivity of mqPCR, followed by cPCR, and culture for the detection of E. coli O157; the cPCR and mqPCR had comparable specificity, but specificity of mqPCR method was heavily dependent on prior specification. The mqPCR method was the most sensitive for the detection O26, O45, and O103 serogroups. The cPCR method was more sensitive than the culture method for serogroups O26, and O121, but comparable for serogroups O45, O103, O111, and O145. The cPCR method showed higher specificity than mqPCR within serogroups O45, O121, and O145 but no apparent differences within serogroups O26, O103, and O111. A second order quantitative microbial risk assessment was developed to quantify the prevalence and concentration of the seven STEC on pre-evisceration beef carcasses and evaluate the impact of peri-harvest interventions. Simulation scenarios of current industry peri-harvest intervention practices showed variable effectiveness in reducing STEC contamination on pre-evisceration beef carcass, however, a scenario of increased adoption of peri-harvest interventions was more effective at reducing STEC contamination. Fecal-to-hide transfer and hide-to-carcass transfer had a large effect on prevalence and concentration of STEC on pre-evisceration carcasses.

Nas últimas décadas o crescimento industrial decorrente do desenvolvimento tecnológico e outras atividades consideradas indispensáveis à vida humana, estão gerando graves problemas ambientais, que está despertando uma preocupação a nível mundial. Dentre os vários contaminantes os metais tóxicos têm recebido atenção especial, pela sua persistência ao ingressar nos ecossistemas acumulando-se por toda a cadeia alimentar, uma vez que são extremamente tóxicos, mesmo em quantidades muito baixas. No Brasil, a legislação ambiental vigente já estabelece normas bastante rigorosas no que diz respeito ao descarte de águas contaminadas por metais tóxicos. Para que o tratamento deste tipo de efluentes seja feito adequadamente, e para que possam ser impostos limites máximos cada vez mais compatíveis com a sustentabilidade da vida moderna, torna-se relevante a preocupação com novas tecnologias mais eficientes e econômicas que permitam a remoção de metais tóxicos do ambiente contaminado. Hoje o tratamento do ambiente contaminado é realizado empregandose tecnologias convencionais baseadas em princípios físico-químicos, as quais estão sendo consideradas ineficientes e economicamente inviáveis. Alternativamente, uma nova tecnologia, a biorremediação, vem ganhando cada vez mais importância, devido às vantagens que oferece: simplicidade, eficiência e baixo custo. Para isto é necessário ser avaliada a utilização de microorganismos resistentes e eficazes na remoção do metal, pois, nesta fase são necessários que sejam selecionados organismos com características favoráveis ao processo. Dentro deste contexto, a proposta desse projeto foi isolar e caracterizar micro-organismos de uma área de mineração, avaliando os mecanismos e estratégias para o seu uso na remediação de áreas contaminadas a fim de otimizar a sua capacidade de adsorver íons de metais tóxicos, com vistas à utilização destas bactérias para a biossorção de efluentes de mineração. Foram isoladas e identificadas 105 cepas resistentes a cobre, destas 12 cepas foram capazes de crescer à 7.5 mM de cobre. Experimentos de adsorção com metais tóxicos (Cu, Cd e Ni) mostraram que tais cepas são promissoras na ampliação de tecnologias para uma mineração menos impactante, como a exploração de fontes de metais em baixa concentração, transformando o resíduo em fonte de extração economicamente viável e ambientalmente favorável.
Recently, technological development has been resulted in an industrial growth and has been creating serious environmental problems as concern worldwide. Toxic metals have received special attention among many others contaminants, because their persistence in ecosystems and their capability of accumulation throughout the food chain (even in low amounts). Environmental legislation in Brazil has very strict standards regarding discharge of toxic metals contaminated water. For a successful treatment of such effluents, it is relevant to concern about new technologies more efficient and economics to allow the removal of metals heavy from contaminated environment. Today the treatment of contaminated area is carried out conventional technologies based on physicochemical principles, which are inefficient and uneconomical. Alternatively, a new technology, called bioremediation, is being more important due to the advantages it offers: simplicity, efficiency and low cost. Bioremediation may be a viable alternative for areas contaminated by toxic metals. It\'s needed to evaluate the use of resistant and effective microorganisms in removing the metal, because it\'s required to be selected organisms with favorable characteristics for the process. In this context, the aim of this project is isolating and characterizing microorganisms from a mining area, evaluating the mechanisms and strategies for using in the remediation of contaminated areas, in order to optimize its adsorb heavy metal ions ability and use these bacteria for bioremediation of mining effluents. This work isolated and identified 105 strains resistant to copper, 12 of them were able to grow up to 7.5 mM copper, adsorption experiments with toxic metals (Cu, Cd and Ni) showed that these strains are promising in expanding technologies for sustainable mining, such as the exploitation of low concentration metal sources, turning waste into economically viable mining and environmentally friendly.

Un dosage radioimmunologique specifique du zinniol a ete au point; les etapes de ce travail ont ete les suivantes: - synthese d'une molecule immunogene (conjugue zinniol-proteine porteuse). - obtention d'anticorps specifique (4 ci/mmole). - mise au point du dosage radioimmunologique valide du point de vue de sa specificite, de sa reproductibilite et de sa sensibilite (limite de detection 0,14 nn/essai). Cet outil a permis de realiser des dosages sur des plantes infectees. Ces resultats inedits montrent que la molecule est emise tres rapidement dans les tissus infectes (12 h apres inoculation).

Harmattan and Dust (sand) storms together with anthropogenic activities including the use of firewood and kerosene as fuel for cooking, and diesel/petrol generators for electricity generation are potential sources of particulate and gaseous pollutants in homes in Damaturu town, Nigeria. Other activities like the burning of locally produced incense and mosquito coils as well as the use of aerosol sprays are further possible sources of indoor pollution, which may result in exposure of people to a range of pollutants through inhalation, by ingestion of settled dusts as well as dermal contact. Local people associate occurrence of dust events with adverse health effects and hence there is a need for an understanding of the composition of the settled and airborne dusts in order to assess the possible associated health risks. The first phase of the study involved selection and development of methods of dust sampling and analysis. For validation of the methods employed and to establish a broad understanding of the characteristics of the settled dusts, an initial survey study was conducted involving the application of thermal desorption/gas chromatography/mass spectrometry (TD/GC/MS) analysis for organic compound analysis, scanning electron microscopy (SEM), inductively coupled plasma-mass spectrometry (ICP-MS) for analysis of metals, and microbiological analysis. Airborne samples were also collected using sorbent tubes to determine organic compounds in air during activities such as cooking with kerosene, gas, and firewood as well as during electricity generation with fossil fuels. Carbon monoxide (CO) and ultrafine particles (UFPs) monitored simultaneously during some of the household activities. The study involved a novel method of extracting organic chemical emissions from dust by heating of the dusts directly in a micro chamber (μ-CTETM) and collection of emissions on sampling tubes. The method provided a relatively quick way of collecting chemical emissions from dusts that are readily available for release. The sampled tubes were analysed by TD/GC/MS. The conventional solvent extraction of the dusts was also carried out and the extracts were analysed by liquid injection-GC/MS and results of the two methods compared. The study determined a number of constituents (metals, SVOCs, phthalates and physical properties) of dusts collected from households in Damaturu during different weather events and from different indoor/outdoor locations; and compared with some UK samples. The samples investigated include dusts deposited; during two notable dusty-weather events (Harmattan and Storm) as well as when there was no notable dust event; during human activities; and dusts from different types of buildings (modern and traditional homes) as well as inside and outside homes. A standard reference material for organic chemicals (SRM 2585) was also analysed. The physical characterization of the settled house dust samples analysed revealed the various shapes and sizes, and elemental composition of the constituents, which included respirable particles. The microbial analysis also indicated the presence of the spores of a host of fungi and bacterial species; and the possible contributions of household activities to the increased production of pollutants (UFP and CO) ascertained. The μ-CTE extraction of the house dusts by heating with TD/GC/MS analysis of the emissions as well as the solvent extraction-GC/MS revealed the presence of many organic chemical compounds with different analytical retention times and varying concentrations in the dust samples. Chemicals of interest quantified: benzene, hexanal, nonanal, diethyl phthalate (DEP), diisobutylphthalate (DIBP), dibutylphthalate (DBP), and diethylhexylphthalate (DEHP). A host of other chemicals commonly present in the analysed samples identified using the NIST library associated with the MS system software. These chemicals included naphthalene and C10-C16 aliphatic and aromatic hydrocarbons, which would need confirmation by running the pure compound samples. There was an observed higher concentration of the chemicals in the solvent extracts than the μ-CTE extracted dust. The higher concentration of the chemicals in the solvent extracts expected due to the aggressive removal of the chemicals by the organic solvent whereas in the case of thermal extraction only the readily available chemicals (loosely bound to the matrix) released by increases in temperature were removed. Generally, the concentrations of the chemicals found were higher in the indoor than in the outdoor dust samples. In the analysis of the dusts collected during weather events; higher chemical concentrations observed in the samples collected during Harmattan period than the other periods. The Harmattan dust period may pose increased exposures to dust and possible health risks. More exposure is expected to occur in the traditional homes compared with the modern homes due to the higher concentrations of the chemicals in both the indoors and the outdoors and this may be especially important to women and children who spend most of their times at home. Metal analysis involved microwave-assisted digestion of the dust samples followed by ICP-MS analysis. The total quant method of metal analysis for a general profiling indicated the presence of more than 50 elemental contaminants in house dust. The results of the quantitative analysis for six target metals: Cd, Cr, Cu, Ni, Pb, and Zn showed their presence in all indoor and the outdoor dust samples. The mean concentrations showed that the metals were in higher concentrations in the indoor dusts than in the outdoor dusts. The quantitative analysis carried out indicated higher metal contents in the storm dusts than the dusts during the other periods. Results of the dusts collected from modern and traditional homes indicated the presence of the metals in higher concentrations in the dusts from traditional homes than the dusts from the modern homes. The estimated mean concentrations of the metals and phthalates inadvertently ingested as a constituent of dust indicated that some of the pollutants could exceed the tolerable daily intake (TDI) due to high exposures to dust expected to be the case in Damaturu. The results of the investigation of the dust composition, combined with information on exposure to dust and pollutants, show that dusts are a risk to the health of people in the Damataru community. Recommendations are made for more studies to provide a better understanding of dust ingestion and exposure to some phthalates and heavy metals in particular and the possible health risks. To the best of my knowledge, this is the first ever research study of airborne and settled dusts undertaken in North-Eastern Nigeria.

Xanthomonas campestris pv. Vasculorum produit in vivo une phytotoxine tres hydrophile. L'ensemble de ce travail a consiste a: isoler et purifier cette toxine, apporter des informations sur sa structure et nature chimique, mettre en evidence sa specificite d'interaction vis-a-vis d'une molecule affine presente chez les plantes hotes; les effets physiologiques qu'elle induit dans les cellules de l'hote. Ces derniers resultats contribuent a mieux approcher son mecanisme biochimique d'action qui se manifeste sur les hotes potentiels, a des niveaux d'organisation differents (tissu foliaire et protoplastes) essentiellement par une augmentation du taux d'acide abscissique

In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced.
Graduation date: 2000

Chronic kidney disease (CKD) is a general term for disorders affecting kidney structure and function. The progressive loss of renal function leads to the accumulation of toxins, the uremic toxins, normally cleared by the kidneys. It is under these circumstances that the “uremic state” is established. Recent studies relate uremic plasma to impaired intestinal barrier function and to depletion of the tight junctions (TJs) protein constituents. Within the intestinal lumen urea is hydrolyzed by microbial urease forming large quantities of ammonia, the major mediator of intestinal barrier disruption in uremic conditions, causing a depletion of the intestinal epithelial TJs proteins in CKD. When the microbial ecosystem is affected, harmful microbial species may overgrowth, as well their metabolism product, leading to an imbalance of the intestinal microbiome. Recent studies suggest that intestinal microbiome exert an influence over both the production of uremic toxins and the progression of CKD. In CKD, the impairment of the intestinal barrier function may allow the translocation of intestinal microorganisms, endotoxins, antigens and other microbial products from intestinal lumen to systemic circulation, contributing to the pathogenesis of systemic inflammation, cardiovascular risk and progress of CKD. Our main goal was to evaluate the application of two in vitro models of intestinal epithelial barrier for the study of microbial translocation and to evaluate the impact of different uremic conditions present in CKD on this microbial translocation. For that, we analyzed the effect of plasma of CKD patients and the uremic toxin urea on microbial intestinal translocation, as well as on integrity, permeability and localization and quantity of TJs proteins in the in vitro intestinal models, Caco-2 monoculture and Caco-2/HT29-MTX/Raji B triple model. The results showed that the experimental uremic conditions simulated in this study did not potentiate the microbial translocation, although interfered at some extent with the integrity and the permeability of intestinal epithelial barrier models. Microbial translocation was higher in Caco-2 monoculture than in triple model, suggesting that the triple model creates a more effective barrier and, therefore, apparently represents a more robust intestinal model of the human intestine. This study allowed to conclude that the uremic state influences the integrity of intestinal barrier, but this influence could not be directly translated in an increase in the microbial translocation through the intestinal epithelium in the in vitro models studied.
Doença renal crónica (DRC) é um termo geral para distúrbios que afetam a estrutura e a função do rim. A perda progressiva da função renal conduz à acumulação de toxinas, toxinas urémicas, normalmente excretadas pelos rins. É nessas circunstâncias que o "estado urémico" é estabelecido. Estudos recentes relacionam o plasma urémico ao dano da função da barreira intestinal e à depleção dos constituintes proteicos das junções de oclusão (JO). No lúmen intestinal, a ureia é hidrolisada pela urease microbiana, formando grandes quantidades de amónia, o principal mediador da disrupção da barreira intestinal em condições urémicas, causando uma depleção das proteínas das JO epiteliais intestinais na DRC. Quando o ecossistema microbiano é afetado, espécies microbianas prejudiciais podem crescer excessivamente, assim como os seus produtos do metabolismo, conduzindo a um desequilíbrio do microbioma intestinal. Estudos recentes sugerem que o microbioma intestinal exerce influência na produção de toxinas urémicas e na progressão da DRC. Na DRC, o dano da função da barreira intestinal pode permitir a translocação de microrganismos intestinais, endotoxinas, antigénios e outros produtos microbianos do lúmen intestinal para a circulação sistémica, contribuindo para a patogénese de inflamação sistémica, risco cardiovascular e progressão da DRC. O nosso principal objetivo foi avaliar a aplicação de dois modelos in vitro de barreira epitelial intestinal para o estudo da translocação microbiana e avaliar o impacto de diferentes condições urémicas presentes na DRC nessa translocação microbiana. Para isso, analisamos o efeito do plasma de doentes com DRC e da toxina urémica ureia na translocação intestinal microbiana, assim como na integridade, permeabilidade e localização e quantidade das proteínas das JO nos modelos intestinais in vitro, monocultura Caco-2 e modelo triplo Caco-2/HT29-MTX/Raji B. Os resultados mostraram que as condições urémicas experimentais simuladas neste estudo não potenciaram a translocação microbiana, embora tenham interferido em certa medida com a integridade e a permeabilidade dos modelos de barreira epitelial intestinal. A translocação microbiana foi maior na monocultura Caco-2 do que no modelo triplo, sugerindo que o modelo triplo cria uma barreira mais eficaz e, portanto, aparentemente representa um modelo intestinal mais robusto do intestino humano. Este estudo permitiu concluir que o estado urémico influencia a integridade da barreira intestinal, mas que essa influência pode não estar diretamente relacionada com um aumento da translocação microbiana através do epitélio intestinal nos modelos in vitro estudados.
Mestrado em Biomedicina Molecular

The investigation presented in this thesis examined the microbial and functional diversity of the meltwater ponds Fresh, Orange and Salt Ponds on the McMurdo Ice Shelf, near Bratina Island, Antarctica. These sites were chosen because of the ecological importance and absence of detailed characterisations of their diversity and function as part of Antarctica?s largest wetland. Particular focus was on cyanobacterial diversity, nitrogen fixation and secondary metabolite production. Using 16S rRNA gene and morphological analysis a large diversity of cyanobacteria (more than 22 phylotypes) was identified with high phylogenetic similarities (up to 99% sequence identity) to cyanobacteria from mats in other regions of Antarctica. In addition biogeographical distributions were identified including potentially endemic and cosmopolitan cyanobacteria. High salinities were also connected to the change and reduction of diversity. Lipid marker analyses were performed targeting hydrocarbons, ether-linked hydrocarbons, methylated fatty acid esters (FAME), wax esters, hopanols and sterols. Lipid biomarker profiles were similar to typical cyanobacteria dominated mats with major input from microorganisms including oxygenic and anoxygenic phototrophs, obligate aerobic and anaerobic heterotrophs that conduct the metabolic processes of fermentation, sulphate reduction, sulphate and iron-oxidation, methanogeneses. Signature lipids indicative of Chloroflexus and archaea, as well as branched aliphatic alkanes with quaternary substituted carbon atoms (BAQCs), were identified for the first time in Fresh, Orange and Salt Ponds. Based on nifH gene analysis, the nitrogen fixing diversity characterised in Orange Pond consisted of cyanobacterial Nostoc sp. as well as firmicutes, beta-, gamma- and delta-proteobacteria. Acetylene reduction assays and nifH gene RNA transcript diversity identified Nostoc sp. as a main contributor of nitrogenase activity in these ponds. Furthermore, analytical methods were used to identify the cyanobacterial secondary metabolites microcystins, although the genetic basis for this production and the toxin producer could not been identified. However non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) genes were identified which could be the genetic basis for novel bioactives. The use of a multi-disciplinary approach synthesis and subsequent results significantly increased our understanding of the diversity and function of microbial mat communities in the unique meltwater ponds of the McMurdo Ice shelf, Antarctica.

246 samples of bulk and packaged spices from retail stores in the western, southeastern, southern, midwestern, and northeastern areas of the U.S. were examined for the presence of Clostridium -perfringens. Isolates were checked for the presence of the lecithinase gene (cpa) and enterotoxin genes (cpe) by PCR. Enterotoxin formation during sporulation was investigated using the Oxoid Toxin Detection Kit. Forty-three confirmed isolates (from 17% of total samples) were cpa-positive. Of those, 27 were cpe-positive. Together, levels of C. perfringens spores ranged from 3.6-2400/gm. The amount of enterotoxin in cell extracts ranged from 2-16 ng/ml. Some of the SEM images of isolated spore (# 78) and one plasmid-borne ent control (FD-153) showed an organized surface structure termed “candy-wrapper”. This extracellular structure remained after treatment with 0.1 % SDS for 1 hr, suggesting it was not composed of membrane debris from the mother cell. The D values of spores ranged from 1.19- 3.31 min. The addition of lysozyme in the plating medium elevated the recovery rate of heat-treated spores. The growth rate of a cocktail of spores from spices (# 31, # 32, # 45) between 4 to 5 hr after inoculation was determined with a doubling time of 6.82 min in hamburger. A cocktail of spores of plasmid-borne ent control showed an optimum growth rate between 5 to 8 hr after inoculation with doubling time of 15.98 min. However; spice isolate cocktail, plasmid-borne ent control cocktail (FD-5603 and FD-153), and a chromosome-borne ent control (NTCT 8239) were unable to germinate and outgrowth at 20oC. Inoculation in laboratory medium FTG indicated the same result as hamburger at 20oC. The ability of C. perfringens spores in spices to potentially survive cooking procedures can be followed by germination and growth of vegetative cells during improper cooling to levels associated with foodborne illness caused by this organism. Our results suggest that retail spices are potential vehicles of transmission of enterotoxin-positive C. perfringens.

Indiana University-Purdue University Indianapolis (IUPUI)
In this research, Environmental Fluid Dynamic Code (EFDC) and Adaptive- Networkbased Fuzzy Inference System Models (ANFIS) were developed and implemented to determine the spatial-temporal distribution of cyanobacterial metabolites: 2-MIB and geosmin, in Eagle Creek Reservoir, IN. The research is based on the current need for understanding algae dynamics and developing prediction methods for algal taste and odor release events. In this research the methodology for prediction of 2-MIB and geosmin production was explored. The approach incorporated a combination of numerical and heuristic modeling to show its capabilities in prediction of cyanobacteria metabolites. The reservoir’s variable data measured at monitoring stations and consisting of chemical/physical and biological parameters with the addition of calculated mixing conditions within the reservoir were used to train and validate the models. The Adaptive – Network based Fuzzy Inference System performed satisfactorily in predicting the metabolites, in spite of multiple model constraints. The predictions followed the generally observed trends of algal metabolites during the three seasons over three years (2008-2010). The randomly selected data pairs for geosmin for validation achieved coefficient of determination of 0.78, while 2-MIB validation was not accepted due to large differences between two observations and their model prediction. Although, these ANFIS results were accepted, the further application of the ANFIS model coupled with the numerical models to predict spatio-temporal distribution of metabolites showed serious limitations, due to numerical model calibration errors. The EFDC-ANFIS model over-predicted Pseudanabaena spp. biovolumes for selected stations. The predicted value was 18,386,540 mm3/m3, while observed values were 942,478 mm3/m3. The model simulating Planktothrix agardhii gave negative biovolumes, which were assumed to represent zero values observed at the station. The taste and odor metabolite, geosmin, was under-predicted as the predicted v concentration was 3.43 ng/L in comparison to observed value of 11.35 ng/l. The 2-MIB model did not validate during EFDC to ANFIS model evaluation. The proposed approach and developed methodology could be used for future applications if the limitations are appropriately addressed.

Indiana University-Purdue University Indianapolis (IUPUI)
Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.

The North American Great Lakes are a vital source on a global scale, as they hold ~18 % of the potable water resources on our planet. Cyanobacteria of the genus Microcystis are commonly found in fresh water environments around the world, and since the mid-1990s also in Lake Erie. The reasons for the success for these potentially toxic cyanobacteria in Lake Erie are not completely understood. In this study we have applied modern molecular tools to analyze field samples to provide an insight into the genotypic composition and diversity of the Microcystis community in the past and present day Lake Erie. We have also analyzed a three-year data set to identify specific environmental factors that contribute to the abundance of Microcystis genotypes and microcystin production. In addition, in a laboratory-based study we examined the effect of nutrients on transcriptional activity of the microcystin synthetase gene mcyD. The results of this study suggest that, although toxic Microcystis form < 10 % of the total cyanobacterial population in Lake Erie, the toxin-producing Microcystis community in Lake Erie is diverse, and that these populations are stabile on a time scale of decades. Sediments acting as a reservoir of Microcystis are likely contributing to the persistence of the population. Although Microcystis is the dominant microcystin producer in the lake, other microcystin-producing cyanobacteria were also found in spatially isolated regions of the lake. While microcystin concentration in Lake Erie is correlated positively with total phosphorus (P<0.001) and surface reactive phosphorus (P<0.001), and negatively with the molar ratio total nitrogen to total phosphorus (P<0.001); toxic Microcystis abundance correlates negatively with NO3 concentration (P=0.04) and positively with surface water temperatures (ranging from 20.8 °C to 27.4 °C) (P=0.03). These observations, along with findings from culture based experiments, suggest decoupling of the factors governing proliferation of toxic cells and toxin production. Culture based experiments also suggested that the chemical form of phosphorus may be an important factor in regulating microcystin biosynthesis in Microcystis based on monitoring relative transcriptional activity of the mcyD gene. The transcriptional activity of mcyD was higher (P=0.118) in cells grown in BG11-medium containing 2.3 μM organic phosphorus (glycerol 2-phosphate disodium salt hydrate) than in cells grown in BG11-medium containing 2.3 μM inorganic phosphorus (K2HPO4).

The degradation of toxic and volatile contaminants in aqueous streams is considered a challenge using conventional bioremediation strategies. At moderate concentrations, toxic contaminants induce microbial inhibition, which results in an overall decrease of reaction rates. On the other hand, volatile compounds are often stripped out of solution into the atmosphere during aeration in conventional wastewater treatments, and are not treated. The addition of a second non-aqueous phase with affinities for the contaminants can reduce aqueous concentrations to sub-inhibitory levels and also decrease contaminant volatilization, while still allowing controlled release of contaminants back to the microbial population; such systems have been denoted as Two Phase Partitioning Bioreactor (TPPB). The current work examined and compared the performance of solid-liquid TPPB to a liquid-liquid TPPB and a single phase system. The systems were compared in the simultaneous degradation of phenol and butyl acetate, two substrates known for their relatively high levels of toxicity and volatility, respectively. The solid-liquid TPPB, using 2 polymers selected heuristically, showed an improvement of 40 and 54 % in phenol degradation rates compared to the single phase and the liquid-liquid systems. Additionally, the solid-liquid system presented a 55 and 11 % enhancement in the amount of butyl acetate degraded. At higher initial substrate concentration the solid-liquid TPPB showed an improvement in the phenol degradation rate and the amount of butyl acetate degraded of 44 and 94 % respectively, compared to the single phase system. In order to rationalize polymer screening for solid-liquid TPPBs, selection criteria based on first principles were developed, and were based on consideration of polymer accessibility and polymer-solute thermodynamic affinity. Polymer accessibility was evaluated by considering glass transition temperature (Tg) and degree of crystallinity, while polymer-solute thermodynamic affinity was assessed using three different methods, Hildebrand solubility parameters, Hansen iii Solubility Parameters (HSP) and activity coefficients at infinite dilution. It was found that the HSP method gave the best trends and its predictions had better agreement with the experimental results. Consequent biodegradation experiments with a single, rationally selected polymer, and a mixture of waste polymers, demonstrated the superior performance of rational selected polymers.
Thesis (Master, Chemical Engineering) -- Queen's University, 2013-05-02 16:24:39.655

Industrialization, urbanization and technological developments have improved the standard of living for humans on the one hand, but they also have resulted in the generation of wastes containing toxic heavy metals that are detrimental to the ecosystem on the other hand. Therefore, the treatment of waste water containing toxic heavy metals before discharging into the environment has become imperative. Though, the conventional waste water treatment methods like adsorption, electrochemical process, ion-exchange, precipitation, solvent extraction, etc. have served the purpose of removing toxic heavy metals, they have certain limitations such as formation of secondary sludge, inefficiency in removing lower metal concentration, high cost, to name a few. Thus, it becomes of interest to explore alternative cost effective methods capable of removing lower concentrations of heavy metals from waste water. The method of bioremediation which uses microorganisms for toxic heavy metal removal has gained significance. Various combinations of microorganisms and heavy metals have been researched to assess the abilities of the selected microorganisms in removing the considered metals. Majority of the research studies have focused on the biosorption of metals by the whole cells. However, there is a paucity of research on the role played by the individual cell wall components in metal removal. In addition to remediation, the detection of heavy metals in waste waters is also of equal importance. In the present research study, the significance of bacteria of Pseudomonas species namely P. putida, P. aeruginosa and P. fluorescens for lead remediation have been assessed. Further, detailed studies have been carried out to elucidate the mechanisms of lead removal through the assessment of the roles played by the individual cell wall components. The lead removal capacities of the individual EPS components purified from the Pseudomonas sp. have been determined. Various strategies have been adopted to enhance the lead removal capacities of the three Pseudomonas sp. by thermolysis. For the biosorption studies, the parameters namely pH, time of contact, biomass loading and lead concentration have been optimized to obtain the maximum lead binding. Apart from lead removal, biologically modified carbon paste electrodes (CPEs) have been developed using the Pseudomonas sp. cells and their EPS components. The major objectives of this research work are enumerated as follows: 1. Study of the bioremediation of lead from aqueous solutions using cells of P. putida, P. aeruginosa and P. fluorescens. 2. Understanding the role of bacterial cell wall and its components in lead uptake. 3. Effect of thermolysis of the chosen bacterial cells on lead uptake 4. Determination of the lead uptake capacity of extracellular polymeric substances (EPS) of the selected Pseudomonas sp. namely, proteins, polysaccharides, biosurfactants and DNA. 5. Enrichment of lead binding proteins from total bacterial protein and examination of the lead binding capacity of the purified protein. 6. Comparison of the protein profiles of the three Pseudomonas sp. in the absence and presence of lead. 7. Detection of Pb (II) ions in aqueous solutions using carbon paste electrodes modified with Pseudomonas sp. cells and their EPS components using an electro-analytical technique. The key findings of the research work are summarized below: The fully grown cells of Pseudomonas sp. harvested from the nutrient medium have been used for the experiments. The lead biosorption studies using the Pseudomonas sp. show substantial lead biosorption by all the three bacteria chosen namely, P. putida, P. aeruginosa and P. fluorescens in independent studies. The three Pseudomonas sp. however show a variation in their lead removal capacities. The highest lead removal is obtained when P. putida is used as the biosorbent. The characterization studies using FTIR, EDAX and zeta potential have been carried out for the Pseudomonas sp. cells before and after interaction with lead. The EDAX studies confirm the presence of lead ions on the Pseudomonas sp. surface. Electro-kinetic studies indicate that the negatively charged bacterial surface, become less electronegative after interaction with lead. The carboxyl and phosphate groups are found to play major role in lead binding by P. putida and P. fluorescens. In addition to the carboxyl and phosphate groups, amide group also play role in lead binding on the P. aeruginosa cell. The lead biosorption using all the three Pseudomonas sp. adhere to the Langmuirian isotherm model and follow the pseudo second order kinetics. The lead removal by the Pseudomonas sp. is further improved after thermolysis presumably due to the exposure of more lead binding sites. After thermolysis, the lead uptake is found to increase by about 27 % in the case of P. putida, about 18 % in the case of P. aeruginosa and about 26 % in the case of P. fluorescens. Taking into consideration that the intact and thermolysed Pseudomonas sp. are effective in removing lead, further studies have been carried out to understand which of the individual cell wall components namely DNA, protein, polysaccharide or lipid play a role in lead uptake. The biosorption studies carried out after digesting the cell wall components one at a time using specific enzymes have shown that the lead uptake differs for each component, both in the case of the intact and the thermolysed cells. Though all of the major cell wall components are found to be responsible for lead removal, a greater reduction in lead removal is observed, when the polysaccharide component of Pseudomonas sp. is digested and used as a biosorbent. When the individual cell wall components namely DNA, protein, polysaccharide and biosurfactant are studied for their lead binding capacities in their purified forms, the purified protein from all the three Pseudomonas sp. are found to remove a higher percentage of lead, compared to the other purified components. In the case of DNA, the lead biosorption has been studied using both ssDNA and dsDNA. Amongst the two forms of DNA, ssDNA shows a better lead uptake vis-a-vis dsDNA. This is possibly due to the exposure of more lead binding sites in the case of ssDNA which are otherwise masked in the double helical structure of dsDNA. Further, the hydrophilic part of the DNA is found to play a major role in lead binding compared to the hydrophobic part. Recognizing that the purified total protein is found to be capable of removing more lead compared to the other components, an affinity column chromatography technique has been used to enrich the lead binding protein from the total proteins. The SDS-PAGE documentation of the total and enriched proteins has confirmed the enrichment of specific proteins. The enriched protein fractions exhibit 65 to 95 % of the lead binding capacities of their corresponding total proteins in all the three species of Pseudomonas studied. The SDS-PAGE analysis of protein profile in the absence and presence of lead have shown significant differences consequent to lead binding. Extensive lead detection studies carried out using carbon paste electrode (CPE) modified with the Pseudomonas sp. cells and their purified EPS components have highlighted the potential of the biomass modified CPEs for lead detection. The lowest limit of detection (LLOD) for lead has been estimated for each of the modified CPEs studied. The estimated LLODs show that amongst the many biomass modified CPEs studied, the CPE modified using whole cells indicate a better detection of lead compared to the individual purified EPS components. Amongst the CPEs modified using whole cells, the one modified by blending the lyophilized cells with carbon paste is capable of detecting lower concentrations of lead than the one modified with drop coated cells.

The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations.