Orientadores: Theo Guenter Kieckbusch, Mariana Altenhofen da Silva / Dissertação (mestrado) - Universidade Estadual de Campínas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-21T16:22:52Z (GMT). No. of bitstreams: 1 Vaz_JulianaMiguel_M.pdf: 25278318 bytes, checksum: 2af6f4b99f503d19043c081e804f17b7 (MD5) Previous issue date: 2012 / Resumo: Alginatos são polissacarídeos compostos pela co-polimerização de unidades de beta-D-manuronato (M) e alfa-L-guluronato (G), arranjados em bloco homopoliméricos (poli-M ou poli-G) ou blocos em sequencia alternada, capazes de formar filmes resistentes quando reticulados com cálcio. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Alginates are linear copolymers of (1,4)-linked beta-D-mannuronate (M) and alfa-L-guluronate (G) units, arranged as homopolymeric blocks (poly-M and poly-G) and blocks with alternating sequence....Note: The complete abstract is available with the full electronic document / Mestrado / Engenharia de Processos / Mestra em Engenharia Química

Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.

The biological treatment of metalworking fluids (MWFs) is a cost effective alternative to conventional waste disposal processes. While research has proven that this process is capable of treating large volumes of wastes with high organic concentrations, there are uncertainties about the mechanisms by which the treatment occurs, and there are limitations that must be overcome. There is a need to understand the importance of the mechanisms by which carbon (and hence COD) is removed from the wastewater. This will allow for waste practitioners to make better decisions for optimizing the process, and for disposing of waste (i.e sludge) that is generated. The biological treatment process is also susceptible to biocides present within formulations. These compounds either need to be removed before the treatment process, or the bioreactors need to be made more resistant to them to ensure that their presence does not hinder the reactor functioning. This study aims to answer the uncertainties about the carbon removal mechanisms involved in the treatment of oil-containing MWFs. In the first experimental chapter, it is shown that the predominant mechanism of carbon removal is oil/water separation induced by emulsifier degradation, and hence the bioprocess treatment rate is significantly affected by the biodegradability of surfactants and by the presence of cations found naturally in the water that used to prepare the emulsions. The study then provides insights into the potential that coagulation and coalescence has for removing inhibitory components commonly found in MWFs. Coagulation and coalescence is shown to effectively remove biocides with low aqueous solubility (iodopropynyl butylcarbamate) and those that partition themselves into the oil phase (o-phenyl phenate and its sodium salt). Finally, to improve the resistance of reactors to inhibitory compounds, factors influencing the development of fixed-film reactors are investigated. A micro-cosmic system is used to study the both physico-chemical effects and nutritional factors on the development of biofilm reactors. It is shown that biofilm yields can be controlled through pH adjustment, and that these yields are maximized with phosphate stimulation and ammonium limitation. It is then shown that fixed-film reactors are able to treat metalworking fluids even under conditions deemed to be inhibitory. In summary, this project provides insights into further understanding and enhancing the biological treatment of MWFs.

The prevalence of oral diseases such as dental caries and periodontitis and the universal need for effective control of oral health has stimulated a great deal of interest in oral hygienic formulations both scientifically and commercially driven. Such formulations are normally deployed as complex formulations commonly containing antimicrobial actives together with excipients, where both classes of ingredients may contribute to the bacteriological effect of the oral hygienic product. However, the mode of action and/or the bacteriological and microecological effects of exposure of microorganisms to oral hygiene products are poorly understood. In this context, this doctoral dissertation represents a series of investigations to contribute to knowledge in the area. The impact of selected oral antimicrobial actives (triclosan, sodium lauryl sulphate, stannous fluoride and zinc lactate) on a key aspect of bacterial cellular membrane function was investigated. This involved measuring major cellular respiratory pathways during exposure to the test agents using two types of tetrazolium dyes possessing different redox potentials as respiration pathway indicators. Spectrophotometric analyses indicated that sub-lethal levels of triclosan and sodium lauryl sulphate act as uncoupling agents, an observation not previously been reported. Sub-lethal concentrations of stannous fluoride and zinc lactate however, blocked cellular respiration with resulting shifts towards glycolytic/fermentative pathways. The contribution of a variety of test agents to the overall antimicrobial effect of a complex formulation (Listerine®) was investigated in order to understand the relative efficacy of the actives. This was achieved by testing the essential oils present in the formulation singly and in combination utilising in vitro models. The use of the hydroxyapatite disc model (HDM) to grow salivary microcosms to test the efficacy of the ingredients revealed hitherto unreported synergistic activity between the active ingredients thymol and menthol. Proprietary dentifrices (Colgate Total® and Crest ProHealth®) containing the antimicrobial agents triclosan or stannous fluoride/zinc lactate, respectively, were comparatively evaluated. This was performed by simultaneously establishing salivary microcosms in Sorbarod Biofilm Devices (SBDs). Following the establishment of dynamic steady-states, paired devices were dosed with each of the two proprietary dentifrices. Bacteriological data generated after multiple dosing indicated that both dentifrices were comparably effective in the reduction of all tested bacterial functional groups in the plaque models. However, data generated using HDM models indicated greater reductions in Gram-negative anaerobes after exposure to Colgate total®. The observations presented in this thesis may contribute to the development of oral formulations with optimised antimicrobial efficacies against adventitious pathogens present in the oral cavity and help in reducing the incidence of oral diseases and potentially related systemic interface.

Les biomatériaux insérés dans la cavité orale, tout particulièrement les prothèses dentaires amovibles en résine, constituent des surfaces propices à la formation de biofilms incorporant des levures du genre Candida, à l’origine de candidose. Les levures, comme d’autres micro-organismes, adaptent leur prolifération à l’environnement par des molécules dites du quorum sensing. D’autre part, la contamination du milieu oral est contrôlée par de nombreuses protéines sécrétées par les glandes salivaires, mais leur utilisation en hygiène est peu documentée. Le but de ce travail est d’étudier l’effet anti-biofilm de molécules du quorum sensing et de protéines exocrines, le lysozyme et la lactoperoxydase, dans la perspective de réduire la colonisation des surfaces de prothèses dentaires par des levures du genre Candida et par conséquent de prévenir une stomatite prothétique. A cette fin, des biofilms à Candida ont été produits dans des plaques multi-puits à fond plat en polystyrène et quantifiés par coloration au cristal violet. L’effet de deux molécules du quorum sensing - tyrosol et farnésol - sur la formation des biofilms à Candida a été investigué in vitro sur la souche de référence ATCC 10231 et sur des souches cliniques isolées à partir de prothèses dentaires. Le tyrosol et le farnésol à hautes concentrations (> 6 mM et > 1 mM respectivement) entraînent une limitation de la formation de biofilms sans altérer la croissance fongique tandis que des concentrations plus faibles (~ 1 mM et ~ 1 µM respectivement) favorisent la formation de biofilms. Le lysozyme présente aussi un effet dépendant de la dose sur la formation de biofilms par Candida: promoteur de la formation de biofilms à une concentration de 1000 µg/ml et inhibiteur de la formation de biofilms à des concentrations plus faibles (3 et 10 µg/ml). Les composés hypohalogéneux (> 500 µM) produits par un système peroxydase limitent la croissance des levures et par conséquent leur capacité à former des biofilms in vitro. Un seul trempage ex vivo de prothèses amovibles pendant 5 minutes dans une solution d’ions hypohalogéneux (2000 µM) entraîne une diminution d’au moins 1 unité logarithmique du nombre de Candida dans 58,3 % des cas (N = 23) alors qu’un trempage dans de l’eau n’a aucun effet sur la colonisation de la prothèse :cet effet est statistiquement significatif (Chi carré, p = 0,0006, test de Fisher, p = 0,0009). / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

La technique d’échantillonnage passif DGT (« Diffusive Gradients in Thin Films ») possède de nombreux avantages (intégration des variations temporelles, abaissement des limites de quantification) qui font d’elle une méthode prometteuse pour une utilisation en réseaux de mesure pour quantifier les éléments traces dans les eaux naturelles. Cependant, il existe encore des zones d’ombre qui constituent des freins à son utilisation dans un contexte réglementaire. Ce travail a donc pour objectif d’identifier des biais potentiels et ainsi contribuer à fiabiliser la méthode. Cette étude montre que l’obtention d’un résultat avec une incertitude minimisée doit passer par la détermination expérimentale des facteurs d’élution ; cependant, l’utilisation d’une valeur standard de 0,8 pour le Cr(III) et de 0,85 pour Al(III), Cd(II), Co(II), Cu(II), Ni(II), Pb(II) et Zn(II) est proposée afin d’alléger les manipulations tout en conservant une incertitude raisonnable (<10%). L’étude de l’influence de l’encrassement des dispositifs DGT a montré que la sorption des cations Cd(II), Cu(II) et Pb(II) sur les filtres encrassés affectent respectivement peu, modérément et fortement leur accumulation dans les échantillonneurs et donc leur quantification. Des durées d’exposition de moins d’une semaine sont alors préconisées pour ces éléments. En revanche, l’encrassement a eu un impact négligeable sur le Ni(II) et sur les oxyanions As(V), Cr(VI), Sb(V) et Se(VI). Enfin, une méthode de quantification simultanée du Cr(III), essentiel à la vie, et du Cr(VI), toxique, a été développée en vue d’améliorer l’évaluation de la toxicité d’une eau. Un unique échantillonneur DGT fixe les deux formes tandis qu’elles sont ensuite sélectivement séparées par une étape d’élution. Cette méthode est robuste sur une large gamme de forces ioniques et de concentrations en sulfate mais sur une gamme de pH plus restreinte ne couvrant pas toutes les eaux naturelles (4 à 6). / The passive sampling DGT technique (Diffusive Gradients in Thin Films) has a lot of benefits (time-weighted average concentrations, low limits of quantification) and would therefore be a useful tool for monitoring studies to quantify trace elements in natural water. However, there are still some limitations and grey areas that put the brakes on the development of the method for regulatory applications. The aim of this work is to identify potential biases and contribute to increase the method reliability. This study shows that a minimized uncertainty on results can be obtained only if elution factors are experimentally determined; however, standard values of 0.8 for Cr(III) and 0.85 for Al(III), Cd(II), Co(II), Cu(II), Ni(II), Pb(II) and Zn(II) are suggested to reduce manipulations while keeping reasonable uncertainty (<10%). Studying the influence of fouling developed on DGT devices showed that the sorption of cations Cd(II), Cu(II) and Pb(II) had, respectively, a slight, moderate and strong impact on their accumulation in DGT samplers and therefore on their quantification. Samplers should then be deployed for less than one week. In contrast, fouling had a negligible impact on oxyanions As(V), Cr(VI), Sb(V) and Se(VI). Finally, a method was developed to simultaneously quantify both Cr oxidation states naturally occurring in natural waters, which are Cr(III), essential to life, and Cr(VI), toxic. Both forms are accumulated in a single DGT sampler before being selectively separated during an elution step. This method is robust for wide ranges of ionic strengths and sulfate concentrations but for a narrower range of pH (4 to 6).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The use of plants for medicinal purposes is an ancient practice because vegetables have in its composition chemical structures with important pharmacological activities. In this study we highlight the hyemale Equisetum species, belonging to the phylum Sphenophyta and Equisetaceae family. This is an aerial plant, commonly found in tropical and temperate climates. The infusion of plant stems is popularly used as diuretic, antihypertensive and for the treatment of infectious diseases and inflammations. All species of the genus are known as Horsetail. This study aimed to carry out a phytochemical study and evaluate the antioxidant activity, antimicrobial, and anti-parasitic antibiofilm the crude extract and fractions of E. hyemale. The plant was collected in the municipality of Santa Maria, district of São Martinho da Serra, in November 2013. The voucher specimen of the material was deposited in the Herbarium UFSM Forestry, cataloged under the registration number 6756. The material was macerated with ethanol (70%) and filtered to afford a portion of the aqueous extract was reserved for obtaining the crude extract and ethanol extract was evaporated. The remainder was partitioned into separate vials using increasingly polar solvents: dichloromethane, ethyl acetate and butanol, and the crude extract was obtained by drying the aqueous extract. Assays of flavonoids and polyphenols totas through the colorimetric quantification technique were performed. To determine the antioxidant capacity was used DPPH technique. The phenolic acids were identified and quantified by HPLC-DAD. The antimicrobial activity was evaluated by broth microdilution technique and the reductive capacity of biofilm formation by crystal violet technique. To evaluate the antiparasitic activity was used to microtiter technique. The results obtained in the determinations of total polyphenols and flavonoids show that the levels of phenolic compounds, in the crude extract was higher, being 97.76 ± 0.65 mg / g for polyphenols and 43.65 ± 1.24 mg / g for flavonoids. The DPPH assay revealed an intermediate capacity reduction for activated free radical, having an IC50 of 86.55 ug / ml for crude extracts. The HPLC analyzes revealed the presence of rosmarinic acid in dichloromethane fraction (7.38 ± 0.08mg / g FS) and cloregênico acid in ethyl acetate fraction (8.4 ± 0.26 mg / g FS). The crude extract and fractions showed antimicrobial activity, being capable of inhibiting growth of E. faecalis ATCC 29212 S. epidermidis ATCC35985, S. saprophyticus ATCC 15305, S. aureus ATCC 33691, S. aureu resistant to methicillin (clinical isolate) L. monocytogenes ATCC 7644, E. coli ATCC 25922, S. enteritidis ATCC 13076, ATCC 12022323A flexineri S., P. aeruginosa ATCC 27853, P. aeruginosa PA01, K. pneumoniae ATCC 1705, at concentrations of 52.4 mg / ml and 3.27 mg / ml. Head Candida species inhibitory concentrations ranged from 52.4 mg / ml to 6.5 mg / ml, and in front Mycobacterium species in the smaller values concentrations able to inhibit growth of the microorganism were between 2.5 mg / ml and 0.625 mg / ml. As regards the reduction of biofilm formation testing the largest percentage of reduction can be seen in dichloromethane fraction, which was the highest concentration able to reduce 83% of the biofilm formation by P. aeruginosa and 51% of C. albicans biofilm formed. To test the antiparasitic, can be observed the decrease of T. evansi 100% after 9h, when exposed to crude extract and N-butanol fraction. This study demonstrates for the first time antiparasitic activity and antibiofilm the crude extract and fractions of rods E.hyemale. / A utilização de plantas para fins medicinais é uma prática milenar, pois os vegetais possuem em sua composição estruturas químicas com importantes atividades farmacológicas. Neste estudo destacamos a espécie Equisetum hyemale, pertencente ao filo Sphenophyta e família Equisetaceae. Trata-se de uma planta aérea, comumente encontrada em climas tropicais e temperados. A infusão das hastes da planta é utilizada popularmente como diurético, anti-hipertensivo e para tratamento de doenças infecciosas e inflamações. Todas as espécies do gênero são conhecidas como Cavalinha. O presente trabalho teve como objetivo realizar um estudo fitoquímico bem como avaliar as atividades antioxidante, antimicrobiana, antibiofilme e antiparasítária do extrato bruto e frações da E. hyemale. A planta foi coletada no município de Santa Maria, distrito de São Martinho da Serra, em novembro de 2013. A exsicata do material foi depositado no Herbário da Engenharia Florestal da UFSM, catalogado sob o número de registro 6756. O material foi macerado com etanol (70%) e filtrado, obtendo-se o extrato aquoso uma parte foi reservada para a obtenção do extrato bruto e o extrato etanolico foi evaporado. O restante foi particionado em ampolas de separação, utilizando solventes de polaridade crescente: diclorometano, acetato de etila e butanol, sendo que o extrato bruto foi obtido pela secura do extrato aquoso. Foram realizados os doseamentos de flavonóides e polifenóis totas através da técnica de quantificação por colorimetria. Para determinar a capacidade antioxidante foi utilizada a técnica de DPPH. Os ácidos fenólicos foram identificados e quantificados por CLAE-DAD. A atividade antimicrobiana foi avaliada através da técnica de microdiluição em caldo e a capacidade redutora de formação de biofilme através da técnica de cristal violeta. Para a avaliação da atividade antiparasitária foi utilizada a técnica de microtitulação. Os resultados obtidos nos doseamentos de polifenóis totais e flavonóides mostram que os teores de compostos fenólicos, foram maiores no extrato bruto, sendo de 97,76 ± 0,65 mg/g para polifenóis e 43,65 ± 1,24 mg/g para flavonóides. O ensaio de DPPH revelou uma capacidade intermediária, para ativada redutora de radicais livres, apresentando um IC50 86,55 μg/ ml para o extrato bruto. As análises por CLAE revelaram a presença de ácido rosmarínico na fração diclorometano (7,38 ± 0,08mg/g FS) e ácido cloregênico na fração acetato de etila (8,4± 0,26 mg/g FS). O extrato bruto e as frações apresentaram atividade antimicrobiana, sendo capazes de inibir o crescimento de E. faecalis ATCC 29212, S. epidermidis ATCC35985, S. saprophyticus ATCC 15305, S. aureus ATCC 33691, S. aureu resistente a meticilina (isolado clínico), L. monocytogenes ATCC 7644, E. coli ATCC 25922, S. enteritidis ATCC 13076, S. flexineri ATCC 12022323A, P. aeruginosa ATCC 27853, P. aeruginosa PA01, K. pneumoniae ATCC 1705, em concentrações entre 52,4 mg/ml e 3,27 mg/ml. Frente a espécies de Candida as concentrações inibitórias variaram entre 52,4 mg/ ml a 6,5 mg/ml, e frente a espécies de Mycobacterium os valores das menores concentrações capazes de inibir o crescimento do microrganismo ficaram entre 2,5 mg/ml e 0,625 mg/ml. Quanto aos ensaios de redução da formação de biofilmes o maior percentual de redução pode ser observado na fração diclorometano, que na maior concentração foi capaz de reduzir 83% do biofilme formado por P. aeruginosa e 51% do biofilme formado por C. albicans. Para o teste antiparasitário, pode ser observado a redução de 100% do T. evansi, após 9h, quando expostos ao extrato bruto e a fração N-butanol. Este estudo demonstra pela primeira vez a atividade antiparasitária e antibiofilme do extrato bruto e frações das hastes da E.hyemale.

In Brazil, agricultural activities have a high potential for contamination of water resources, especially by pesticides use. This potential is highest in river basins where agriculture is developed out of agricultural aptitude and without adequate land use planning. However, the severity of this context has been disregarded for two reasons: i. by the understanding that no-tillage system (SPD), as adopted in Rio Grande do Sul, is effective in controlling pollution; and ii. due to technical limitations and/or absence of hydrographic basin monitoring systems. Recently, the development of POCIS (Polar Organic Chemical Integrative Sampler) and use of epilithic biofilms has sought to improve monitoring systems. However, neither POCIS was tested in Brazilian tropical conditions, nor biofilms were used in agrochemical monitoring. In this context, this doctorate thesis aims to monitor the presence of active pesticides in river waters of river basin in state of Rio Grande do Sul (RS). Monitoring was performed in the Guaporé River Basin because it was representative of use conditions and management of the physiographic region of Plateau–RS. Same was done by three sampling methods: active sampling, passive sampling with POCIS and passive sampling with biofilms, at 08 points under the influence of SPD, 09 points in conventional planting areas and 1 point in forest area. In the three sampling methodologies the occurrence and concentration of pesticides, associated or not with the systems of use and management practiced in the basin, were verified. In active sampling, 11 active ingredients were detected from the 79 analysis. This methodology was unable to detect the active ingradients of high polarity (2,4-D) and those of lower polarity epoxiconazole and tebuconazole and presented no statistical difference between management systems. The adoption of POCIS in less frequent frequency of use and application, allowed the detection of compounds that had not been detected in the active sampling, besides increasing the frequency of detection of each active ingradient. The management systems were differentiated by the higher concentration of fungicides, epoxiconazole and tebuconazole, used in soybean crops in SPD. Sampling using epilite biofilms detected a greater number of active ingradients in the Guaporé River Basin, and the frequency of detection of each pesticide was higher than the other sampling methodologies. Regarding land use and management systems, biofilms confirmed that the main difference between them is related to the higher concentration of fungicides in areas under SPD. In three monitoring systems it was verified that all the points sampled, regardless of the system of use and management, are contaminated by agrochemicals, even the forest area. In addition, the highest concentrations of agrochemicals in the Guaporé river system are located on its main river and follow the flow direction of water. Epilithic biofilms were more promising to detect contamination than the active and POCIS sampling techniques, since they captured active pesticides indistinctly from their polarizations, even in the period of less application. / No Brasil as atividades agrícolas apresentam elevado potencial de contaminação dos recursos hídricos, em especial pelo uso de agrotóxicos. Esse potencial é mais elevado nas bacias hidrográficas nas quais a agricultura é desenvolvida fora da aptidão agrícola e sem o adequado ordenamento do uso do solo. A gravidade desse contexto tem sido desconsiderada por dois motivos: (i) pela compreensão de que o sistema plantio direto (SPD), tal como adotado no Rio Grande do Sul é eficaz no controle da poluição, e (ii) pelas limitações técnicas e/ou ausência do sistema de monitoramento das águas em bacias hidrográficas. Recentemente, o desenvolvimento de amostradores passivos POCIS (Polar Organic Chemical Integrative Sampler) e a adoção dos biofilmes epilíticos têm buscado aprimorar os sistemas de monitoramento. Porém, nem o POCIS foi experimentado nas condições tropicais brasileiras, nem tão pouco os biofilmes foram utilizados no monitoramento de agrotóxicos. Nesse contexto, a presente tese de doutorado visa monitorar a presença de princípios ativos de agrotóxicos nas águas da rede fluvial de uma bacia hidrográfica rural do Estado do Rio Grande do Sul (RS). O monitoramento foi executado na bacia do Rio Guaporé por ser representativa das condições de uso e manejo da região fisiográfica do Planalto – RS em duas épocas (primavera e outono). Foram utilizados três métodos de amostragem: amostragem ativa, amostragem passiva com POCIS e amostragem passiva com biofilmes. Foram selecionados 18 pontos de amostragem em função do uso e manejo do solo, desde áreas com maior até menor proporção de lavouras. Nas três metodologias de amostragem verificou-se a ocorrência e concentração dos agrotóxicos, associados ou não aos sistemas de uso e manejo praticados na bacia. Na amostragem ativa foram detectados 11 princípios ativos dos 79 analisados. Essa metodologia foi incapaz de detectar os princípios ativos de polaridades extremas, tais como 2,4-D, epoxiconazol e tebuconazol, e não apresentou diferença estatística entre as regiões Norte e Sul da bacia hidrográfica. A utilização do POCIS, na época de menor frequência de uso e aplicação de agrotóxicos, possibilitou a detecção de compostos que não tinham sido detectados na amostragem ativa, além de aumentar a frequência de detecção dos princípios ativos. Os sistemas de manejo foram diferenciados pela maior concentração dos fungicidas epoxiconazol e tebuconazol utilizados nas lavouras de soja sob plantio direto na região Norte. Já a amostragem utilizando biofilmes epilíticos detectou maior número de princípios ativos (n = 21) na bacia do Rio Guaporé. A frequência de detecção dos princípios ativos foi maior com biofilmes do que com amostragem ativa e via POCIS. Quanto aos sistemas de uso e manejo do solo, os biofilmes ratificaram que a principal diferença entre eles está relacionada à maior concentração de fungicidas na região Norte. Nos três sistemas de monitoramento verificou-se que todos os pontos amostrados, independente do sistema de uso e manejo, estão contaminados por agrotóxicos, mesmo na área de floresta revegetada. Além disso, as maiores concentrações de agrotóxicos na rede fluvial do Guaporé estão localizadas no seu rio principal e seguem o sentido do fluxo da água. Os biofilmes epilíticos foram mais promissores em detectar a contaminação do que a técnica da amostragem ativa e amostragem passiva via POCIS, visto que captaram princípios ativos de agrotóxicos indistintamente das suas polaridades, mesmo no período de menor utilização de agrotóxicos na bacia hidrográfica.

Lorsqu’un morceau de bois atteint le fond de l’océan, il provoque la mise en place d’un écosystème capable de se développer en absence de lumière. Cet écosystème est qualifié de chimiosynthétique du fait de la présence d’une faune pouvant fixer le carbone inorganique présent dans l’eau de mer. De plus, ce système attire une faune ultra-specialisée qui utilise des symbiontes bactériens pour digèrer le bois. Avant ces travaux, la plupart des études s’interressaient principalement à la macrofaune et le rôle des microorganismes libres demeurait inconnu. Nous avons pu démontrer dans cette thèse le rôle essentiel que jouent les microorganismes libres dans la mise en place de cet écosystème. Nous avons prouvé que des communautés de microorganismes se succédaient au cours de la première année de colonisation et que cette succession était influencée par le type de bois et l’environment dans lequel il se trouve. La première phase de cette succession aboutit au développement après un mois, d’une population de bactéries sulfato-réductrices produisant de l’hydrogène sulfuré et ce, même en l’absence d’organismes foreurs. Cette production d’hydrogène sulfuré est à la base (1) du développement rapide d’un biofilm chimiolithoautotrophe et (2) du recrutement d’espèces possédants des symbiontes chimiosynthétiques. Nos résultats ont permis d’aboutir à la proposition d’une succession d’étapes clés liées permettant la transformation d’un substrat térrigène en un écosysteme qui, il y a plusieurs millions d’années, aurait permis à la faune chimiosynthétique de coloniser les grands fonds. / When wood sinks to the deep-sea floor it creates a new ecosystem that does not depend directly on energy from sunlight. This ecosystem is called chemosynthetic because of the presence of a fauna associated with symbiotic bacteria that can assimilate inorganic carbon from seawater. Furthermore this system is colonized by specialized fauna that use symbiotic bacteria to digest the wood matrix. Previous studies mostly focused on these symbiotic macroorganisms and the role played by non-symbiotic microorganisms in the sunken wood ecosystem remains unknown. We demonstrate in this thesis the important role played by non symbiotic microorganisms during the sunken wood ecosystem establishment. We reveal the ecological succession of microorganisms driven by time and wood structure. The first step of this succession is characterized by a microbial population able to produce hydrogen sulfide after one month of immersion. This hydrogen sulfide production is the basis for (1) a chemolithoautotroph biofilm development on the wood surface and (2) a recruitment of species associated with chemoautotrophic bacteria. Our results suggest a succession of different phases that transform a terrigeneous substrate into an environment that may have helped, million years ago, the colonization of the deep sea by chemosynthetic species.

Biofilms are surface-bound bacterial colonies that are held together by a self-produced extracellular polymeric matrix. They are highly resistant to antibiotics and host defence mechanisms, and are known to be the cause of persistent infections. Biofilm-degrading enzymes have been shown to prevent biofilm formation, remove mature biofilm, and enhance the efficacy of antibiotics. This study investigated the antibacterial and antibiofilm actions of the commercial enzyme Pectinex Ultra SP-L (Pectinex), alone and in combination with antibiotics, on standard and clinical cultures of Staphylococcus aureus and Pseudomonas aeruginosa. The cytotoxicity of Pectinex was determined on human cell cultures in vitro. Pectinex (7.42 – 950 PGU/ml) was not bactericidal, and had no effect on the antibacterial efficacy of amoxicillin-clavulanate and ciprofloxacin in cultures of S. aureus (ATCC 12600) and P. aeruginosa (ATCC 9027), respectively. However, in clinical cultures of P. aeruginosa, Pectinex caused an 89.0% (from 1.0 to 1.89 μg/ml) and 92.8% (from 1.67 to 3.22 μg/ml) increase in the MIC and MBC of ciprofloxacin, respectively. In clinical cultures of S. aureus, both bactericidal indices of amoxicillin-clavulanate were increased by 28.0% (from 2.0 to 2.56 μg/ml). In all bacterial cultures, low concentrations of Pectinex (≤ 118.75 PGU/ml) and prolonged incubation periods (≥ 6 h) were both associated with increased viability and biofilm biomass. Over a short incubation period (≤ 6 h), higher concentrations of Pectinex (237.5 – 950 PGU/ml) effectively inhibited biofilm formation in P. aeruginosa ATCC (237.5 – 950 PGU/ml) and clinical (950 PGU/ml) strains but not in S. aureus cultures. Pectinex (237.5 – 950 PGU/ml) was cytotoxic to HeLa cells, lymphocytes and neutrophils, and induced morphological features that included shrunken rounded cells, blebs, apoptotic bodies, cytoplasmic vacuoles and cell debris. The effects at 475 and 950 PGU/ml were comparable to mitomycin C 10 μg/ml and staurosporine 1 μg/ml. Pectinex was shown to either enhance or reduce biofilm biomass and cell viability in cultures of S. aureus and P. aeruginosa. The manifested effects depended on the concentration of the enzyme, the specific bacterial species and strain, and the maturity of the biofilms. Further studies are still needed in order to determine the actions of Pectinex on other clinical pathogens. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Pharmacology / PhD / Unrestricted