The persistence of biofilms in hospital settings are associated with Healthcare Associated Infections (HCAI), causing increased morbidity, mortality and healthcare costs. The resistance of biofilms against commonly used hospital disinfectants has been well reported. Metal and metal oxide nanoparticles (NP) such as silver (Ag), copper (Cu), zinc oxide (ZnO) and copper oxide (CuO) exhibit antimicrobial properties against various pathogens. Methods: Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus in a Centre for Disease Control (CDC) biofilm reactor and a 96 well plate was compared. A three stage approach including Minimum Biofilm Reduction Concentration (MBRC), R2 values and log(10) reductions was used to assess the efficacy of Ag and ZnO NPs both alone and in combination against P. aeruginosa and S. aureus biofilms. Atomic Absorption Spectroscopy (AAS), Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) was used to further assess the antimicrobial ability of the metal and metal oxide NPs. The prevention of P. aeruginosa and S. aureus adherence on Ag and ZnO thin film coating on silicon (Si) surfaces was also investigated, as well as icaC, ebpS and fnbB gene expression in S. aureus biofilms. Results: The CDC biofilm reactor demonstrated to be the most effective method for P. aeruginosa and S. aureus biofilm production in comparison to 96 well plates, with lower standard errors of the mean (SE) and higher replicability. Individual MBRC of ZnO and Ag NPs in suspension were 256 and 50 µg/ml for P. aeruginosa and 16 and 50 µg/ml for S. aureus respectively. The concentrations in combination were reduced by at least a half, with concentrations of 32/25 µg/ml of ZnO/Ag NPs in suspension resulting in a significant (p ≤0.05) reduction of 3.77 log(10) against P. aeruginosa biofilms and 8/12 µg/ml of ZnO/Ag NPs in suspension resulted in a 3.91 log(10) (p ≤0.05) against S. aureus biofilms. Both combinations showed an additive effect. Time point analysis confirmed that a 24 hour treatment is vital for any significant (p ≤0.05) antimicrobial activity. AAS data suggested that the Ag+ ions quenched Zn2+ ions, therefore the antimicrobial efficacy of the combination is mainly due to Ag+ ions. Damage of the biofilms from Ag and ZnO NPs was observed in the SEM imaging and energy dispersive X-ray (EDX) analysis confirmed the adherence of Zn and Ag within the biofilms. CLSM imaging showed dead (red) cells of P. aeruginosa and S. aureus biofilms throughout the depth of the biofilm. P. aeruginosa formation was reduced by 1.41 log(10) and 1.43 log(10) on Ag and ZnO thin film coatings respectively. For S. aureus, a reduction of 1.82 log(10) and 1.65 log(10) was obtained for Ag and ZnO coating respectively. Only low levels of ribonucleic acid (RNA) were achieved so no further gene analysis could occur. Conclusion: Reductions of ≥3 log(10) were observed for P. aeruginosa and S. aureus biofilm treatment with ZnO/Ag NP suspensions. It can be concluded that the ZnO/Ag NP suspensions had greater antimicrobial activity than Ag and ZnO coated surfaces owing to large concentrations of Ag+ and Zn2+ ions acting upon the biofilms. The slower release of ions from coated surfaces suggest an inadequate concentration of ions in the media, which are therefore unable to prevent biofilm formation as rapidly as NP suspensions, however provide a sustained release of ions over time. The results from this investigation propose that Ag and ZnO NPs in suspension could be a potential alternative to disinfectants for use in nosocomial environments against P. aeruginosa and S. aureus biofilms.

As propriedades fotofísicas e fotoquímicas de cristal violeta (CV) foram investigadas em soluções isotrópicas e verificou-se que solventes com constante dielétrica pequena favorecem a formação do par iônico, já o aumento na viscosidade do meio restringe a movimentação rotacional dos anéis aromáticos, resultando em um aumento no tempo de vida de fluorescência e, portanto no rendimento quântico de fluorescência (Φf) (Oliveira 2002). Os experimentos com CV foram conduzidos em micelas reversas do tensoativo aniônico bis-2-etilhexil sulfoccinato de sódio (AOT) em isooctano. A localização interfacial do CV nas micelas reversas de AOT em valores da razão molar entre água e surfactante (W0)pequenos e grandes foram encontrados através da técnica de Ressonância Magnética Nuclear (RMN) de próton e de carbono 13. Utilizando-se espectroscopia UV-Vis identificou-se que pares iônicos de contato estão presentes a valores pequenos de W0 e com o aumento do W0 pares iônicos separados por solventes são as espécies que predominam em solução. A comparação da eficiência de fotodegradação de CV em micelas reversas de AOT em função do W0 indicou que a fotoreatividade é maior em baixos valores de W0 . Este efeito deve estar relacionado à restrição da movimentação dos anéis aromáticos de CV devido ao ambiente restrito no qual este se localiza na micela reversa de OAT a W0 pequenos. A formação de intermediários reativos foi verificada através de Fotólise de Relâmpago a Laser e Emissão no infra-vermelho próximo, indicando a presença de espécies triplete, radical e oxigênio singlete com valor de rendimento quântico menor que 1%. Os produtos de fotólise foram identificados por técnicas cromatográficas e espectroscópicas. Na presença de oxigênio, houve maior formação de cetona de Michler. Com baixa concentração de oxigênio, o produto observável foi leuco-CV. Destes estudos propomos o mecanismo de CV neste meio. Após os estudos com micelas reversas, células cancerosas HeLa foram empregadas para comparar fotoatividade do CV com o azul de metileno (MB). As proporções de CV e MB dentro das células são altas, incorporando 70% e 80% da concentração da solução de incubação, respectivamente. Com o aumento da concentração de MB, um favorecimento da formação de dímero foi identificada. Já CV não sofre agregação nas condições estudadas. Nenhum dos fotossensibilizadores estudados tem um efeito danoso sobre as células HeLa em concentrações abaixo de 10µM. Após irradiação, MB causou uma diminuição de cerca de duas vezes maior na taxa de sobrevivência celular comparado com CV. A formação de formação de oxigênio singlete após incorporação dos fotossensibilizadores foi investigada. Há formação de oxigênio singlete em células incubadas com MB, já com CV a geração de oxigênio singlete é pouco significativa sugerindo um mecanismo radicalar. O processo de morte celular foi estudado por citometria de fluxo e verificou-se que MB induz apoptose depois da irradiação em células HeLa. A absorção de luz por ambos fotossensibilizadores é similar, o que indica que a diminuição na sobrevivência não se deve à diferença de absorção luminosa. As diferenças de sobrevivência observadas com células incubadas com CV e MB e irradiadas foram relacionadas às diferenças das propriedades fotoquímicas destes fotossensibilizadores. A localização celular de CV e MB em células foram caracterizadas por microscopia de fluorescência. Verificou-se que ambos localizam-se em mitocôndrias. O aumento na concentração de CV não alterou o seu perfil de localização. Já para MB ao aumentar a concentração de MB, observa-se que o mesmo localiza-se além das mitocôndrias, em lisossomos. A comparação das propriedades fotoquímicas e de localização foram consideradas para explicar as diferenças de atividade fotodinâmica do CV e do MB em células HeLa / The photophysical and photochemical properties of crystal violet (CV) were investigated in isotropic solutions and it was found that solvents with small dielectric constants favor the formation of the ion pair and that the increase in viscosity of the medium restricts the rotational movement of the aromatic rings, resulting in an increase in fluorescent lifetime and therefore in the fluorescence quantum yield (Φf) (Oliveira 2002). CV experiments were conducted in reverse micelles of the anionic tensoactive sodium bis-2-ethylhexyl-sulfosuccinate (AOT) in isooctane. The interfacial localization of CV in the AOT reverse micelles at low and high values of molar ratio between water and surfactant (W0 was found through the proton and carbon 13 Nuclear Magnetic Resonance techniques (NMR). Using UV-Vis spectroscopy, it was identified that contact ion pairs are present in low W0 values and with the increase in the W0 solvent separated ion pairs are the species that predominate in solution. The comparison of the photobleaching efficiency of CV in AOT reverse micelles as a function of W0indicated that the photoreactivity is high with low W0 values. This effect must be related to the restrict environment in which CV is located. The reactive intermediate formation was found through the Laser Flash Photolysis and Near Infra-Red Emission, indicating the presence of triplet, radical and singlet oxygen species with a yield quantum of less than 1%. The photolysis products were identified through the chromatographic and spectroscopic techniques. In the oxygen presence, there was high Michler ketone formation. With the low oxygen concentration, the observable product was leuco-CV. With these studies we hypothesized a mechanism of CV in these proposed media. After the reverse micelles studies, the HeLa cancerous cells were used, in order to compare the CV and methylene blue (MB) photoactivity. The CV and MB proportion inside the cell was high, reaching 70% and 80% of concentration of the incubation solution, respectively. With the increase of MB concentration, a favoring of dimmer formation was identified. CV does not suffer aggregation in the studied conditions. None of the studied photosensitizers has a damaging effect upon the HeLa cells in concentrations below 10µM. After irradiation, MB caused a decrease about twice higher in the cellular survival rate compared to CV. The singlet oxygen formation after the photosensitizer incorporation was investigated. There is a singlet oxygen formation in the cells incubated with MB, though with the CV the singlet oxygen generation is significantly low suggesting the radicalar mechanism. The cellular death process was studied by Fluorescence Activated Cell Sorting and MB-induced apoptosis was found after MB irradiation in HeLa cells. The light absorption by both photosensitizers is similar, which means that the survival decrease is not because of the light absorption difference. The survival differences observed with the cells incubated with CV and MB and irradiated were related to the differences in the photosensitizer photochemical properties. The cellular location of the CV and MB in cells were characterized by fluorescence microscopy. Both photosensitizers are located in mitochondrias. An increase in the CV concentration does not alter its local profile. However, with an increase in the MB concentration, MB was located not only in mitochondrias but also in lysosomes. The comparison of the photochemical and localization properties was considered in order to explain the differences in the photodynamic activity of CV and MB in HeLa cells

Neste trabalho foram produzidos compósitos de nanotubos de carbono contendo nanopartículas de prata, os quais foram testados como substratos SERS (Surface-enhanced Raman Spectroscopy) na detecção do cristal violeta. Para obter tais compósitos foram necessárias modificações de nanotubos de carbono através de funcionalizações químicas para inserção de grupos carboxila e tiol, capazes de interferir no crescimento de nanopartículas metálicas através de um processo de redução térmica do acetato de prata sobre a superfície das amostras de nanotubo. Para a preparação de tais compósitos foram utilizadas duas amostras de nanotubos, uma de parede simples (SWNT) e outra de paredes múltiplas (MWNT) a fim de avaliar diferenças nos tamanhos e homogeneidade das nanopartículas formadas. Utilizou-se como ferramenta investigativa a espectroscopia Raman na caracterização destes compósitos, que forneceu informações sobre interação dos nanotubos de carbono com as nanopartículas de prata e mudanças estruturais ocasionadas durante a gama de funcionalizações. Para avaliar a morfologia dos compósitos foi utilizada a microscopia eletrônica de varredura (MEV) e a microscopia eletrônica de transmissão (TEM) que forneceram informações sobre o tamanho e a disposição das nanopartículas formadas através do tratamento térmico dos nanotubos com acetato de prata. Os compósitos preparados foram aplicados como substrato SERS na detecção do cristal violeta. Nesta aplicação foi avaliada a capacidade dos nanotubos em adsorver estas moléculas e o potencial do substrato na intensificação do espectro Raman do analito. Observou-se que a adsorção do cristal violeta sobre uma amostra de SWNT foi máxima após o tempo de 60 minutos de agitação. Já o substrato utilizado (SWNT-COOH@Ag) permitiu a detecção do cristal violeta em solução aquosa com concentração de até 1,0.10-8 mol.L-1. Utilizando o mapeamento Raman foi possível avaliar a presença do analito através do monitoramento de uma banda do espectro vibracional do analito intensificada pelo efeito SERS / In this work, we produced carbon nanotube composites containing silver nanoparticles, which were tested as SERS (Surface-enhanced Raman Spectroscopy) substrates in the detection of crystal violet. For these the synthesis of these composites modifications of the carbon nanotubes surface through chemical functionalizations were necessary for insertion of carboxyl and thiol groups, that can affect the growth of metal nanoparticles in thermal reduction process of silver acetate on the surface of the nanotube samples. For the preparation of such composites have single walled carbon nanotubes (SWNT) and a multi-walled carbon nanotubes (MWNT) to evaluate differences in size and homogeneity of the nanoparticles formed. Raman spectroscopy was used as an investigative tool in the characterization of these composites, which provided information on the interaction of carbon nanotubes with silver nanoparticles and structural changes ocurring during the range of functionalizations. To evaluate the morphology of the composites scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used. These techniques provided information about the size and assembly of the silver nanoparticles formed by heat treatment of the nanotubes with silver acetate. The nanotubes@Ag composites were applied as SERS substrates in the detection of crystal violet. In this application, we evaluated the ability of nanotubes to adsorb these molecules and the substrate potential in enhancing the Raman spectrum of the analyte. It was observed that the adsorption of the crystal violet on a sample of SWNT was maximal after 60 minutes of stirring. Substrate used (SWNT-COOH @ Ag) allowed detection of crystal violet in aqueous solution with a concentration as low as 1,0.10-8 mol.L-1. Using Raman mapping was possible to evaluate the presence of the analyte by monitoring a band of vibrational spectrum of the analyte enhanced by SERS effect.

As cinzas leves e pesadas de carvão foram utilizadas na síntese das zeólitas (ZCL e ZCP) por tratamento hidrotérmico alcalino. As zeólitas modificadas de cinzas leves e pesadas (ZMSL e ZMSP) foram preparadas pela mistura de ZCL e ZCP com o surfactante brometo de hexadeciltrimetilamônio. Os adsorventes foram caracterizados por diferentes técnicas, tais como: métodos físico-químicos, fluorescência de raios-X, espectroscopia na região do infravermelho, difração de raios-X e microscopia eletrônica de varredura. O processo de adsorção dos corantes cristal violeta (CV) e do ácido laranja 8 (AL8) foi realizado em sistema de batelada com o objetivo de investigar o equilíbrio e a dinâmica dos materiais zeolíticos no tratamento de efluentes. Os modelos de cinética de pseudo-primeira-ordem e pseudo-segunda-ordem foram aplicados aos dados experimentais e a cinética de pseudo-segunda-ordem descreveu a adsorção dos corantes sobre os adsorventes. Estudos da difusão intrapartícula revelaram que as velocidades de adsorção não eram controladas somente pela etapa de difusão. O modelo de Freundlich ajustou-se melhor aos dados experimentais para todos os sistemas estudados, com exceção do sistema CV/ZCL, que seguiu o modelo de Langmuir. As capacidades máximas de adsorção das zeólitas modificadas foram maiores do que aquelas obtidas para as zeólitas não modificadas. Este melhor desempenho é devido ao ambiente hidrofóbico proporcionado pela adsorção do surfactante catiônico na superfície do adsorvente. Os parâmetros das isotermas de adsorção foram usados para prever o design do equipamento para a realização de processo de adsorção descontínuo de estágio simples. / The coal fly and bottom ashes were used in the synthesis of zeolites (ZFA and ZBA) by alkaline hydrothermal treatment. The modified zeolites from coal fly ash and botton ash (ZMSF and ZMSB) was performed by mixing ZFA e ZBA with the surfactant hexadecyltrimethylammonium bromide. The adsorbents were characterized by different techniques such as: physico-chemical methods, X-ray fluorescence spectroscopy, infrared, X-ray diffraction and scanning electron microscopy. The adsorption of the dye crystal violet (CV) and acid orange 8 (AO8) was carried out in batch system with the purpose of investigating the equilibrium and dynamics of zeolitic materials in wastewater treatment. Pseudo-first- and second-order kinetic models have been applied to the experimental data and pseudo-second-order kinetic was found to describe the adsorption of the dyes on the adsorbents. Intra-particle diffusion studies revealed that the adsorption rates were not solely controlled by the diffusion step. The Freundlich model was better ajusted to the experimental data for all systems studied, except system CV/FTA, which followed the Langmuir model. The maximum adsorption capacities of the modified zeolites were higher than those obtained for the unmodified zeolites. This best performance is due to the hydrophobic ambient provided by the adsorption of cationic surfactant on the surface of the adsorbent. The parameters of adsorption isotherms were used to predict the design of the equipment for performing adsorption discontinuous single stage.

Les patients mucoviscidosiques sont prédisposés à une colonisation chronique de l’arbre bronchique par P. aeruginosa. Ce pathogène opportuniste se caractérise par sa capacité à adhérer à une surface et à y former un biofilm protecteur, hautement tolérant aux agents antimicrobiens. En routine, les antibiogrammes sont effectués sur des cultures bactériennes planctoniques. L’efficacité des antibiothérapies ainsi sélectionnées est donc peu probante pour l’éradication des biofilms bactériens. La réalisation d’Antibiofilmogrammes® sur des isolats cliniques mucoviscidosiques (nouvel outil évaluant la sensibilité des bactéries sessiles aux antibiotiques) a permis de mettre en évidence des phénomènes d’inhibition et d’induction de la formation du biofilm. Plus précisément, les aminosides sont capables de retarder l’adhérence bactérienne. À l’inverse, la famille des β-lactamines présente la capacité de stimuler l’adhésion précoce des micro-organismes. Ces différents effets de l’antibiothérapie générale sur le comportement microbien se vérifient par l’intermédiaire de techniques conventionnelles in vitro (Cristal Violet, traitement enzymatique à la DNase I) et cellulaires (modèle de co-culture statique cellules eucaryotes/bactéries). La pertinence clinique de l’Antibiofilmogramme® se confirme donc par sa capacité à détecter l’initiation précoce de l’adhésion bactérienne, à sélectionner les molécules l’inhibant et à écarter celles pouvant l’induire. Associée aux antibiogrammes traditionnels, son application peu permettre d’affiner les stratégies thérapeutiques pour le traitement des infections pulmonaires chroniques développées au cours de la mucoviscidose. / Cystic fibrosis (CF) patients are predisposed to chronic colonisation of the upper airways by P. aeruginosa. This opportunist pathogen is characterized by its ability to adhere to a surface and to form a protective biofilm, which is highly tolerant to antimicrobials. In routine, antibiograms are realised on planktonic bacterial cultures. The efficacy of the corresponding antimicrobial therapies appears low for the eradication of bacterial biofilms. The realisation of Antibiofilmograms® on CF clinical isolates (a new tool investigating the susceptibility of sessile bacteria to antibiotics) highlighted phenomena of biofilm formation inhibition and induction. More precisely, aminoglycosides are able to delay the bacterial adherence. Conversely, the β-lactam family shows the ability to stimulate the early adhesion of microorganisms. These different effects of antimicrobials on the bacterial behaviour are confirmed with more conventional in vitro methods (Crystal Violet, enzymatic treatment with DNase I) and a cell model (static co-culture of eukaryotic cells and bacteria). The clinical relevance of the Antibiofilmogram® is reinforced by its ability to detect the initiation of the early bacterial adhesion, to select inhibitor molecules and to avoid the inducer ones. Associated to traditional antibiograms, its application should be pertinent to optimise the CF therapies for the treatment of chronic lung infections.

Hyper-Raman-Streuung folgt anderen Symmetrieauswahlregeln als Raman-Streuung und profitiert als nicht-linearer Zweiphotonenprozess noch mehr von verstärkten elektromagnetischen Feldern an der Oberfläche plasmonischer Nanostrukturen. Damit könnte die oberflächenverstärkte Hyper-Raman-Streuung (SEHRS) praktische Bedeutung in der Spektroskopie erlangen. Durch die Kombination von SEHRS und oberflächenverstärkter Raman-Streuung (SERS) können komplementäre Strukturinformationen erhalten werden. Diese eignen sich aufgrund der Lokalisierung der Verstärkung auf die unmittelbare Umgebung der Nanostrukturen besonders für die Charakterisierung der Wechselwirkung zwischen Molekülen und Metalloberflächen. Ziel dieser Arbeit war es, ein tieferes Verständnis des SEHRS-Effekts zu erlangen und dessen Anwendbarkeit für analytische Fragestellungen einzuschätzen. Dazu wurden SEHRS-Experimente mit Anregung bei 1064 nm und SERS-Experimente mit Anregung bei derselben Wellenlänge sowie mit Anregung bei 532 nm - für eine Detektion von SEHRS und SERS im gleichen Spektralbereich - durchgeführt. Als Beispiel für nicht-resonante Anregung wurden die vom pH-Wert abhängigen SEHRS- und SERS-Spektren von para-Mercaptobenzoesäure untersucht. Mit diesen Spektren wurde die Wechselwirkung verschiedener Silbernanostrukturen mit den Molekülen charakterisiert. Anhand von beta-Carotin wurden Einflüsse von Resonanzverstärkung im SEHRS-Experiment durch die gleichzeitige Anregung eines molekularen elektronischen Übergangs untersucht. Dabei wurde durch eine Thiolfunktionalisierung des Carotins eine intensivere Wechselwirkung mit der Silberoberfläche erzielt, sodass nicht nur resonante SEHRS- und SERS-Spektren, sondern auch nicht-resonante SERS-Spektren von Carotin erhalten werden konnten. Die Anwendbarkeit von SEHRS für hyperspektrale Kartierung in Verbindung mit Mikrospektroskopie wurde durch die Untersuchung von Verteilungen verschiedener Farbstoffe auf strukturierten plasmonischen Oberflächen demonstriert. / Hyper-Raman scattering follows different symmetry selection rules than Raman scattering and, as a non-linear two-photon process, profits even more than Raman scattering from enhanced electromagnetic fields at the surface of plasmonic nanostructures. Surface-enhanced hyper-Raman scattering (SEHRS) could thus gain practical importance for spectroscopy. The combination of SEHRS and surface-enhanced Raman scattering (SERS) offers complementary structural information. Specifically, due to the localization of the enhancement to the close proximity of the nanostructures, this information can be utilized for the characterization of the interaction between molecules and metal surfaces. The aim of this work was to increase the understanding of the SEHRS effect and to assess its applicability to answer analytical questions. For that purpose, SEHRS experiments with excitation at 1064 nm and SERS experiments with excitation at the same wavelength, as well as with excitation at 532 nm - to detect SEHRS and SERS in the same spectral region - were conducted. As an example for non-resonant excitation, pH-dependent SEHRS and SERS spectra of para-mercaptobenzoic acid were examined. Based on these spectra, the interaction of different silver nanostructures with the molecules was characterized. beta-Carotene was used to study the influence of resonance enhancement by the excitation of a molecular electronic transition during SEHRS experiments. By the thiol-functionalization of carotene, a more intense interaction with the silver surface was achieved, which enables to obtain not only resonant SEHRS and SERS but also non-resonant SERS spectra of carotene. Hyperspectral SEHRS imaging in combination with microspectroscopy was demonstrated by analyzing the distribution of different dyes on structured plasmonic surfaces.