Neste trabalho foi explorado o potencial das actinobactérias como produtoras de agentes antimicrobianos contra bactérias causadoras da mastite bovina. Para este fim, foram otimizados dois diferentes bioensaios um de antagonismo e o outro de antibiose, contra 13 bactérias Gram-positivas e Gram-negativas relacionadas a mastite bovina. Através do bioensaio de antagonismo 120 actinobactérias foram triadas, sendo 55% delas consideradas ativas. Para o ensaio de antibiose foram triados 70 extratos brutos e 60% mostram a formação de halos de inibição. Os extratos brutos Caat 1-54, Caat 2-63 e Caat 8-25 foram considerados os mais promissores dentre todos os extratos estudados. Esses extratos brutos foram submetidos a estudos de desreplicação empregando LC-PAD-MS/MS e isolamento guiado por bioensaio para a identificação de seus metabolitos ativos. Os extratos Caat 1-54 e Caat 2-63 foram ativos principalmente contra as linhagens de bactérias Gram-positivas e a ação antimicrobiana observada foi atribuída a presença da lisolipina I e actinomicina D e V, respectivamente. O extrato Caat 8-25 foi ativo tanto contra bactérias Gram-positivas como para as Gram-negativas, sendo a oxitetraciclina associada a atividade contra as linhagens Gram-positivas e a herbicidina B ativo contra linhagens Gram-negativas. O estudo de desreplicação por LC-MS/MS também possibilitou a identificação de outros metabolitos secundários presentes nos diferentes extratos estudados. Para o extrato Caat 2-63 foi identificada a presença de outros peptídeos provenientes da via biossintética da actinomicina D e, no extrato bruto Caat 8-25 também foi identificado herbicidina F. Um estudo de mudança no metabolismo também foi realizado para a actinobactérias Caat 1-54 produtora de lisolipina. Através da suplementação de íons brometo no meio de cultivo em diferentes períodos da fermentação foi observada a produção de um análogo da lisolipina com bromo e da lisolipina clorada e bromada. Estudos de DESI-TLC-MS associado a bioautografia em TLC também foram aplicados para análise do extrato bruto Caat 2-63 e mostraram a potencialidade desta metodologia para acelerar a identificação de compostos ativos, pois a análise por espectrometria de massas na região da placa cromatográfica correspondente ao halo de inibição revelou a presença de actinomicina D e V, as quais já haviam sido associadas a atividade antimicrobiana deste extrato bruto. Assim os resultados obtidos nesta tese confirmam o grande potencial das actinobactérias como fonte promissoras de compostos bioativos e que a espectrometria de massas empregada nos seus diferentes modos de análise acelera o processo de identificação desses compostos. / In this work were used two different bioassays to evaluate the antimicrobial potential of some actinomycetes against bacteria that cause bovine mastitis. For this propose an antagonism and antibiose assays were optimized against 13 Gram-positive and Gram-negative bacteria related with bovine mastitis. Through antagonism bioassay 120 actinomycetes were screened and 55% were considered active. In disk diffusion bioassay, 70 crude extracts were screened and 60% showed inhibition zones. The crude extracts Caat 1-54, Caat 2-63 and Caat 8-25 were considered the most promising of all the extracts. Dereplication studies and fractionation guided by bioassay were procedure with this extracts to identify the actives metabolites. The extracts Caat 1-54 and Caat 2-63 were active against strains of Gram-positive bacteria and the antimicrobial effect was related to lisolipin I and actinomycin D and V, respectively. The extract Caat 8-25 was active against both Gram-positive and Gram-negative bacteria. Oxytetracycline were associated with activity against Gram-positives strains and a herbicidin B was active against Gram-negative bacteria. Using dereplication strategies by LC-MS/MS others compounds were characterized in different crude extracts. The extract Caat 2-63 was also identified the presence of other peptides from the same biosynthetic pathway of actinomycin D and for crude extract Caat 8-25 were identified herbicidin F. In addition, studies of the shift in secondary metabolism carried out for actinobacteria Caat 1-54 (lysolipin producer). Culture medium supplementation by bromide ions at different stages of fermentation induce the production of bromine lysolipin analog and a chloro-bromine lysolipin. DESI-MS TLC studies associated with TLC bioautography were used to analyze a crude extract Caat 2-63. This study showed how this method could be used to accelerate the identification of active compounds, because mass spectrometry analysis in situ at bioautigraphy inhibition zone showed the presence of actinomycin D and V, which had been associated with antimicrobial activity of this crude extract. Therefore, the results of this thesis confirm the potential of actinobacteria as bioactives compounds source and the importance of mass spectrometry experiments to accelerate the identification of these compounds.

A cebola comum Allium cepa L. (2n=16), devido à sua excelente condição cromossômica, tem sido largamente utilizada como um sistema de testes para se examinar os efeitos citogenéticos causados por diversos compostos químicos em material biológico. Em estudos de monitoramento de riscos ambientas, observou-se limitação no crescimento das raízes quando as cebolas são expostas a aguas residuárias provenientes de indústrias químicas. Nesse estudo, nós avaliamos este sistema de testes escolhendo alguns compostos de metais pesados, cujos efeitos em outros sistemas de testes já se encontram descritos. Além disso, foram estuados vários fatores que poderiam interferir nos resultados tais como: temperatura; luminosidade; qualidade da água e qualidade das cebolas. As concentrações mais baixas empregadas (1,5 mg/l) nos testes, de cádmio e cobre demostraram alta capacidade inibidora sobre o crescimento das raízes. O zinco apresentou toxicidade moderada a 1,5 mg/l e crescente às concentrações sucessivas mais elevadas. Efluentes industriais, contendo misturas de níquel e sulfatos ou cobre e cianeto, demonstraram-se altamente inibidores às concentrações mais baixas empregadas (0,7%), resultados que demonstram a alta sensibilidade do teste aos sais de metais pesados. Quanto às condições experimentais que levaram a esses resultados, os testes revelaram que não existe influência pronunciada dos fatores luminosidade e temperatura. Quanto à água para controle e diluição, não deve ser de torneira devido ao efeito inibidor do zinco e do cobre. Os resultados foram contraditórios em relação a um possível efeito osmótico da água destilada, sendo conveniente adicionar entre 100 a 250 mg/l de NaCl. A melhor resposta foi obtida com cebolas de origem argentina de 3,5 a 4,0 cm de diâmetro sendo preferível a manutenção de cascas para evitar murchamento. / The common onion Allium cepa. L., due to its excellent chromosome conditions, has been widely used as a test system for examining the citogenetic effects of several chemical compounds on biological material. It has been observerd, in monitoring environmental studies, some growth restriction on Allium roots when they are exposed to wastewater from chemical factories. In this study, we evaluate these test systems by choosing some hard metals which harmful effects on biological materials has been already described. Furthermore, some factors that could interfere on the results, such as: temperature; light; water quality and onion quality were studied. The lowest concentration (1.5 mg/l) for cadmium and cupper were found to have hard capacity to inhibit root growth. The zinc showed moderate toxicity at 1.5 mg/l and this effect was more evident at increased concentrations. The industrial wastewater that contains mixture of nickel and sulphate or cupper and cyanate inhibited root growth at lower concentration (0.7%). These results showed that the test system has high sensitivity for hard metals. There was no direct influence of temperature and light on the experiment conditions. With regard to the type of water, tap water should not be used because of inhibition on the root growth caused by zinc and copper. The results contradicted a potential osmotic effect of distillated water, being convenient to add 100 - 250 mg/l of NaCl. The best response was obtained with onions from Argentina with 3.5 - 4.0 cm of diameter being recommended to keep the onion peel for avoiding dryness.

A esquistossomose é incidente em países tropicais e subtropicais e o uso de moluscicidas é adequado para prevenir a infecção de pessoas. O objetivo deste estudo foi buscar compostos em Piper ativos em Biomphalaria glabrata e avaliar a atividade esquistossomicida e toxicidade do composto mais ativo. O fracionamento biomonitorado de Piper diospyrifolium resultou no isolamento da flavocavaína A e ácido 4-hidroxi-3-[3,7,11-trimetildodeca-2,6,10-trienil]benzoico. A busca de compostos também foi realizada em amidas e chalconas e dos oito compostos avaliados, quatro foram ativos, sendo a piplartina mais ativa. A piplartina não foi letal a miracídios e cercárias de Schistosoma mansoni e foi tóxica em Daphnia similis e Danio rerio, contudo ainda foi menos tóxica que a niclosamida. Neste estudo, os compostos moluscicidas foram obtidos pelo fracionamento biomonitorado de P. diospyrifolium e pela avaliação da atividade de amidas e chalconas. Ambas as metodologias foram adequadas e a análise de componente principal mostrou ser uma ferramenta viável para a busca de compostos. / Schistosomiasis is a parasitic disease and the use of molluscicides has been considered an appropriate method to prevent human infection. The aim of this study was to search for compounds in Piper active in B. glabrata and evaluate schistosomicidal activity and toxicity of the most active compound. The P. diospyrifolium bioguided fractionation resulted in flavokavain A and 4-hydroxy-3-[3,7,trimetildodeca-11-2,6,10-trienil]benzoic acid isolation. The search for active compounds was carried out in amides and chalcones. Eight compounds were evaluated, four were active and piplartine was the most active. There was no mortality of miracidia and cercariae exposed to piplartine. It was classified as toxic to D. similis and D. rerio; nevertheless was less toxic than niclosamide. In this study, molluscicidal compounds were obtained from bioguided fractionation of P.diospyrifolium and by evaluating the activity of amides and chalcones. Both methods were suitable to obtain active compounds and principal component analysis also proved to be a viable tool for obtain compounds.

<p>The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atoms and substitution patterns. These compounds tend to be biomagnified in foodwebs and have been shown to induce an array of effects in exposed organisms. The structural characteristics of the PCBs influence their potency as well as mechanism of action. In order to assess the biological potency of these compounds a multi-step quantitative structure-activity relationship (QSAR) procedure was used in the project described in this thesis.</p><p>The ultraviolet absorption (UV) spectra were measured for all 209 PCBs, and digitised for use as physico-chemical descriptors. Interpretations of the spectra using principal component analysis (PCA) showed the number of ortho chlorine atoms and para-para substitution patterns to be significant. Additional physico-chemical descriptors were derived from semi-empirical calculations. These included various molecular energies, the ionisation potential, electron affinity, dipole moments, and the internal barrier of rotation. The internal barrier of rotation was especially useful for describing the conformation of the PCBs on a continuous scale.</p><p>In total 52 physico-chemical descriptors were compiled and analysed by PCA for the tetra- to hepta-chlorinated congeners. The structural variation within these compounds was condensed into four principal properties derived from a PCA for use as design variables in a statistical design to select congeners representative for these homologue-groups. The 20 selected PCBs have been applied to study structure-specific biochemical responses in a number of bioassays, and to study the biomagnification of the PCBs in various fish species.</p><p>QSARs were established using partial least squares projections to latent structures (PLS) for the PCBs potency to inhibit intercellular communication, activate respiratory burst, inhibit dopamine uptake in synaptic vesicles, compete with estradiol for binding to estrogen receptors, and induce cytochrome P4501A (CYP1A) related activities. By the systematic use of the designed set of PCBs the biological potency was screened over the chemical domain of the class of compounds. Further, sub-regions of highly potent PCBs were identified for each response measured. For risk assessment of the PCBs potency to induce dioxin-like activities the predicted induction potencies (PIPs) were calculated. In addition, two sets of PCBs were presented that specifically represent congeners of environmental relevance in combination with predicted potency to induce estrogenic and CYP1A related activities.</p>

The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atoms and substitution patterns. These compounds tend to be biomagnified in foodwebs and have been shown to induce an array of effects in exposed organisms. The structural characteristics of the PCBs influence their potency as well as mechanism of action. In order to assess the biological potency of these compounds a multi-step quantitative structure-activity relationship (QSAR) procedure was used in the project described in this thesis. The ultraviolet absorption (UV) spectra were measured for all 209 PCBs, and digitised for use as physico-chemical descriptors. Interpretations of the spectra using principal component analysis (PCA) showed the number of ortho chlorine atoms and para-para substitution patterns to be significant. Additional physico-chemical descriptors were derived from semi-empirical calculations. These included various molecular energies, the ionisation potential, electron affinity, dipole moments, and the internal barrier of rotation. The internal barrier of rotation was especially useful for describing the conformation of the PCBs on a continuous scale. In total 52 physico-chemical descriptors were compiled and analysed by PCA for the tetra- to hepta-chlorinated congeners. The structural variation within these compounds was condensed into four principal properties derived from a PCA for use as design variables in a statistical design to select congeners representative for these homologue-groups. The 20 selected PCBs have been applied to study structure-specific biochemical responses in a number of bioassays, and to study the biomagnification of the PCBs in various fish species. QSARs were established using partial least squares projections to latent structures (PLS) for the PCBs potency to inhibit intercellular communication, activate respiratory burst, inhibit dopamine uptake in synaptic vesicles, compete with estradiol for binding to estrogen receptors, and induce cytochrome P4501A (CYP1A) related activities. By the systematic use of the designed set of PCBs the biological potency was screened over the chemical domain of the class of compounds. Further, sub-regions of highly potent PCBs were identified for each response measured. For risk assessment of the PCBs potency to induce dioxin-like activities the predicted induction potencies (PIPs) were calculated. In addition, two sets of PCBs were presented that specifically represent congeners of environmental relevance in combination with predicted potency to induce estrogenic and CYP1A related activities.

The risks associated with a soil contaminated by polycyclic aromatic hydrocarbons (PAHs) are generally assessed by measuring individual PAHs in the soil and correlating the obtained amounts to known adverse biological effects of the PAHs. The validity of such a risk estimation is dependent on the presence of additional compounds, the availability of the compounds (including the PAHs), and the methods used to correlate the measured chemical data and biological effects. In the work underlying this thesis the availability, chemical composition and biological effects of PAHs in samples of soils from PAH-contaminated environments were examined. It can be concluded from the results presented in the included papers that the PAHs in the studied soils from industrial sites were not generally physically trapped in soil material, indicating that the availability of the PAHs was not restricted in this sense. However, the bioavailable fraction of the PAHs, as assessed by bioassays with the earthworm Eisenia Fetida, could not be assessed by a number of abiotic techniques (including: solid phase micro extraction, SPME; use of semi-permeable membrane devices, SPMDs; leaching with various solvent mixtures, leaching using additives, and sequential leaching) and it seems to be difficult to find a chemical method that can accurately assess the bioavailability of PAHs. Furthermore, it was shown that PAH-polluted samples may be extensively chemically characterized by GC-TOFMS using peak deconvolution, and over 900 components can be resolved in a single run. The chemical characterization also revealed that samples that appeared to be similar in terms of their PAH composition were heterogeneous in terms of their overall composition. Finally, single compounds from this large set of compounds, which correlated with different biological effects, could be identified using the multivariate technique partial least squares projections to latent structures (PLS). This indicates that PLS may provide a valid alternative to Effect Directed Analysis (EDA), an established method for finding single compounds that correlate to the toxicity of environmental samples. Thus, the instrumentation and data evaluation tools used in this thesis are clearly capable of providing a broad chemical characterization as well as linking the obtained chemical data to results from bioassays. However, the link between the chemical analyses and the biological tests could be improved as as an organic solvent that solubilised virtually all of the contaminants was used during the chemical analysis while the biological tests were performed in an aqueous solution with limited solubility for a number of compounds. Consequently the compounds probably have a different impact in the biological tests than their relative abundance in profiles obtained by standard chemical analyses suggests. The availability and bioavailability of contaminants in soil also has to be studied further, and such future studies should focus on the molecular interactions between the contaminants and different compartments of the soil. By doing so, detailed knowledge could be obtained which could be applied to a number of different contaminants and soil types. Such studies would generate the data needed for molecular-based modelling of availability and bioavailability, which would be a big step forward compared to current risk assessment practices.

In this dissertation we first consider a parallel linear model with correlated dual responses on a symmetric compact design region and construct locally optimal designs for estimating the location-shift parameter. These locally optimal designs are variant under linear transformation of the design space and depend on the correlation between the dual responses in an interesting and sensitive way. Subsequently, minimax and maximin efficient designs for estimating the location-shift parameter are derived. A comparison of the behavior of efficiencies between the minimax and maximin efficient designs relative to locally optimal designs is also provided. Both minimax or maximin efficient designs have advantage in terms of estimating efficiencies in different situations. Thirdly, we consider a linear regression model with a one-dimensional control variable x and an m-dimensional response variable y=(y_1,...,y_m). The components of y are correlated with a known covariance matrix. The calibration problem discussed here is based on the assumed regression model. It is of interest to obtain a suitable estimation of the corresponding x for a given target T=(T_1,...,T_m) on the expected responses. Due to the fact that there is more than one target value to be achieved in the multiresponse case, the m expected responses may meet their target values at different respective control values. Consideration includes the deviation of the expected response E(y_i) from its corresponding target value T_i for each component and the optimal value of calibration point x, say x_0, is considered to be the one which minimizes the weighted sum of squares of such deviations within the range of x. The objective of this study is to find a locally optimal design for estimating x_0, which minimizes the mean square error of the difference between x_0 and its estimator. It shows the optimality criterion is approximately equivalent to a c-criterion under certain conditions and explicit solutions with dual responses under linear and quadratic polynomial regressions are obtained.

Es wird eine Bioassay-Analyse von 168 Testsubstanzen gegenüber Tetrahymena pyriformis beschrieben. Mithilfe dieses In-vitro-Ansatzes konnte eine erste prognostische Aussage über die toxische Wirkung unbekannter Stoffe getroffen werden. Aus den Konzentrations-Wirkungs-Beziehungen erfolgte eine Bestimmung der Toxizitätserhöhung gegenüber der Narkoselevel-Toxizität. Dadurch war eine mechanistische Interpretation der Wirkstärke der Fremdstoffe möglich. Die Wirkung der Fremdstoffe basiert auf direkten toxikologisch relevanten Reaktionsmechanismen mit nukleophilen Biomolekülen oder nach entsprechender enzymatischer Aktivierung und ermöglichte die Aufstellung von Strukturalarmen. Aus jeder Stoffklasse waren Vertreter direkt oder nach enzymatischer Biotransformation erhöht toxisch. Somit ist mithilfe der Ciliaten eine Vorhersage der direkten reaktiven Toxizität möglich. Zudem enthält es für eine metabolische Aktivierung bedeutsame Enzymsysteme. Damit ist es für eine prognostische Vorhersage der Toxizität unbekannter Stoffe anwendbar.

Southwestern Indiana has numerous lakes developed in abandoned coal mine spoils which support recreational sports fisheries. Some lakes, due to exposure to acid mine drainage from coal wastes and pyritic spoils, are unsuitable habitats for fisheries development. This study examines a publicly owned acid mine lake with an area of approximately 51 ha, following reclamation and elimination of acid producing areas in its drainage basin. Fifteen physico-chemical sample collections were made over a thirteen month period (1991-1992). Parameters sampled included pH, total acidity, iron, manganese, and aluminum. Comparisons were made to historic pre-reclamation water quality data and to established models of acid mine lake recovery. Due to the local topography and exposure to prevailing winds, the lake was generally well mixed throughout the study. Virtually no summer stratification was found, but typical winter season stratification occurred. The water column was well oxygenated throughout the study. Secchi disk transparency varied from 2.5 m to clear to lake bottom (6 m). This study found no significant change in lake water pH (2.9-3.0 to 3.0-3.2 s.u.) since reclamation activities in 1988. However, changes in total acidity and total metal concentrations had occurred since reclamation which suggested that the lake was in early recovery stages. No trends in water quality improvement were determined which could assist in planning toward the eventual establishment of a sports fishery. / Department of Biology

The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.