Nanocomposites as probes for biodetection by SERS

The present work aimed to explore the potential of new nanocomposites based on carbon nanostructures and metal nanoparticles for the detection of biomolecules through surface enhanced Raman scattering (SERS). In a first step, polyvinyl alcohol composites were prepared incorporating silver nanoparticles by two different reduction procedures. At first without introduction of carbon nanostructures. These composites showed good results for the SERS identification of nucleic acids. Next, the synthesis and characterization of graphene oxide was studied to be used in the preparation of silver and gold nanocomposites. The reduction of this nanomaterial with different chemical agents was explored, since its reduction degree may be a determinant factor for the application envisaged (biomolecules interaction). The preparation of the nanocomposites with silver and gold was performed with different reducing agents. The SERS activity of these new nanocomposites was then explored in the presence of different analytes, varying the experimental conditions for Raman spectra acquisition. It was interesting to verify that the silver containing nanocomposites presented the particularity to intensify the graphene D and G bands. It is also important to highlight that a new eco-friendly reducing agent was tested for the synthesis of the graphene oxide composites, an Eucalyptus Globulus extract. Other variable introduced was the preparation of gold nanostars synthesized with hydroxylamine in the presence of graphene oxide, which allowed the preparation of a new nanocomposite with SERS potential. Fibrous membranes were also prepared by electrospinning with the aim to prepare SERS supports with adequate topography and porosity for the formation of nanoparticles agglomerates for the creation of the so-called hot-spots and also to allow the penetration of the analyte molecules. The polymers polyvinyl alcohol and polyacrylonitrile were selected for electrospinning. Using this technique, electrospun mantles with silver and gold nanoparticles and nanocomposites were prepared. Several variables were studied, such as the introduction of the nano-fillers during the electrospinning process, later deposition of the nano-fillers on the simple electrospun polymeric fibres and surface functionalization of the simple polymeric membranes to link the nano-fillers. At last, the potentialities of using carbon nanotubes forests, produced by chemical vapor deposition and coated with gold film by sputtering, as new SERS substrates were explored. It was found that the SERS detection of DNA bases and ADN itself is possible using these substrates.

Structural characterization and biological activity of polysaccharides from infusions used in the popular tradition

The use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.

Surface reactivity enhancement of silica-based glass-ceramic scaffolds

A paradigm shift is taking place from using transplanting tissue and synthetic implants to a tissue engineering approach that aims to regenerate damaged tissues by combining cells from the body with highly porous scaffold biomaterials, which act as templates, guiding the growth of new tissue. The central focus of this thesis was to produce porous glass and glass-ceramic scaffolds that exhibits a bioactive and biocompatible behaviour with specific surface reactivity in synthetic physiological fluids and cell-scaffold interactions, enhanced by composition and thermal treatments applied. Understanding the sintering behaviour and the interaction between the densification and crystallization processes of glass powders was essential for assessing the ideal sintering conditions for obtaining a glass scaffolds for tissue engineering applications. Our main goal was to carry out a comprehensive study of the bioactive glass sintering, identifying the powder size and sintering variables effect, for future design of sintered glass scaffolds with competent microstructures. The developed scaffolds prepared by the salt sintering method using a 3CaO.P2O5 - SiO2 - MgO glass system, with additions of Na2O with a salt, NaCl, exhibit high porosity, interconnectivity, pore size distribution and mechanical strength suitable for bone repair applications. The replacement of 6 % MgO by Na2O in the glass network allowed to tailor the dissolution rate and bioactivity of the glass scaffolds. Regarding the biological assessment, the incorporation of sodium to the composition resulted in an inibition cell response for small periods. Nevertheless it was demonstrated that for 21 days the cells response recovered and are similar for both glass compositions. The in vitro behaviour of the glass scaffolds was tested by introducing scaffolds to simulated body fluid for 21 days. Energy-dispersive Xray spectroscopy and SEM analyses proved the existence of CaP crystals for both compositions. Crystallization forming whitlockite was observed to affect the dissolution behaviour in simulated body fluid. By performing different heat treatments, it was possible to control the bioactivity and biocompatability of the glass scaffolds by means of a controlled crystallization. To recover and tune the bioactivity of the glass-ceramic with 82 % crystalline phase, different methods have been applied including functionalization using 3- aminopropyl-triethoxysilane (APTES). The glass ceramic modified surface exhibited an accelerated crystalline hydroxyapatite layer formation upon immersion in SBF after 21 days while the as prepared glass-ceramic had no detected formation of calcium phosphate up to 5 months. A sufficient mechanical support for bone tissue regeneration that biodegrade later at a tailorable rate was achievable with the glass–ceramic scaffold. Considering the biological assessment, scaffolds demonstrated an inductive effect on the proliferation of cells. The cells showed a normal morphology and high growth rate when compared to standard culture plates. This study opens up new possibilities for using 3CaO.P2O5–SiO2–MgO glass to manufacture various structures, while tailoring their bioactivity by controlling the content of the crystalline phase. Additionally, the in vitro behaviour of these structures suggests the high potential of these materials to be used in the field of tissue regeneration.

Diamond microelectrodes for corrosion studies

Este trabalho teve como objetivos a produção, caracterização e aplicação de microelétrodos (MEs) de diamante como sensores amperométricos e potenciométricos em sistemas de corrosão nos quais a agressividade do meio e a presença de produtos de corrosão, constituem obstáculos que podem diminuir o desempenho, ou inviabilizar a utilização, de outros tipos de sensores. Os microeléctrodos são baseados em filmes finos de diamante dopado com boro (BDD – Boron Doped Diamond) depositados sobre fios de tungsténio afiados, através do método de deposição química a partir da fase vapor, assistida por filamento quente (HFCVD – Hot Filament Chemical Vapor Deposition). A otimização das diversas etapas de fabricação dos MEs deu origem ao desenvolvimento de um novo sistema de afiamento eletroquímico para obtenção destes fios e a várias opções para a obtenção dos filmes de diamante condutor e seu isolamento com resinas para exposição apenas da ponta cilíndrica. A qualidade cristalina dos filmes de diamante foi avaliada por espectroscopia de Raman. Esta informação foi complementada com uma caracterização microestrutural dos filmes de diamante por microscopia eletrónica de varrimento (SEM), em que se fez a identificação da tipologia dos cristais como pertencendo às gamas de diamante nanocristalino ou microcristalino. Os filmes de BDD foram utilizados na sua forma não modificada, com terminações em hidrogénio e também com modificação da superfície através de tratamentos de plasma RF de CF4 e O2 indutores de terminações C-F no primeiro caso e de grupos C=O, C-O-C e C-OH no segundo, tal como determinado por XPS. A caracterização eletroquímica dos MEs não modificados revelou uma resposta voltamétrica com elevada razão sinal/ruído e baixa corrente capacitiva, numa gama de polarização quasi-ideal com extensão de 3 V a 4 V, dependente dos parâmetros de crescimento e pós-tratamentos de superfície. Estudou-se a reversibilidade de algumas reações heterogéneas com os pares redox Fe(CN)6 3-/4- e FcOH0/+ e verificou-se que a constante cinética, k0, é mais elevada em elétrodos com terminações em hidrogénio, nos quais não se procedeu a qualquer modificação da superfície. Estes MEs não modificados foram também testados na deteção de Zn2+ onde se observou, por voltametria cíclica, que a detecção da redução deste ião é linear numa escala log-log na gama de 10-5-10-2 M em 5 mM NaCl. Realizaram-se também estudos em sistemas de corrosão modelares, em que os microeléctrodos foram usados como sensores amperométricos para mapear a distribuição de oxigénio e Zn2+ sobre um par galvânico Zn-Fe, com recurso a um sistema SVET (Scanning Vibrating Electrode Technique). Foi possível detetar, com resolução lateral de 100 μm, um decréscimo da concentração de O2 junto a ambos os metais e produção de catiões de zinco no ânodo. Contudo verificou-se uma significativa deposição de zinco metálico na superfície dos ME utilizados. Os MEs com superfície modificada por plasma de CF4 foram testados como sensores de oxigénio dissolvido. A calibração dos microeléctrodos foi efetuada simultaneamente por voltametria cíclica e medição óptica através de um sensor de oxigénio comercial. Determinou-se uma sensibilidade de ~0.1422 nA/μM, com um limite de deteção de 0.63 μM. Os MEs modificados com CF4 foram também testados como sensores amperométricos com os quais se observou sensibilidade ao oxigénio dissolvido em solução, tendo sido igualmente utilizados durante a corrosão galvânica de pares Zn-Fe. Em alguns casos foi conseguida sensibilidade ao ião Zn2+ sem que o efeito da contaminação superficial com zinco metálico se fizesse sentir. Os microeléctrodos tratados em plasma de CF4 permitem uma boa deteção da distribuição de oxigénio, exibindo uma resposta mais rápida que os não tratados além de maior estabilidade de medição e durabilidade. Nos MEs em que a superfície foi modificada com plasma de O2 foi possível detetar, por cronopotenciometria a corrente nula, uma sensibilidade ao pH de ~51 mV/pH numa gama de pH 2 a pH 12. Este comportamento foi associado à contribuição determinante de grupos C-O e C=O, observados por XPS com uma razão O/C de 0,16. Estes MEs foram igualmente testados durante a corrosão galvânica do par Zn-Fe onde foi possível mapear a distribuição de pH associada ao desenvolvimento de regiões alcalinas causadas pela redução do oxigénio, acima da região catódica, e de regiões ácidas decorrentes da dissolução anódica do ânodo de zinco. Com o par galvânico imerso em 50 mM NaCl registou-se uma variação de pH aproximadamente entre 4,8 acima do ânodo de zinco a 9,3 sobre o cátodo de ferro. A utilização pioneira destes MEs como sensores de pH é uma alternativa promissora aos elétrodos baseados em membranas seletivas.

A computational study of ionic liquids used in the production of fuels and biofuels

For the past decades it has been a worldwide concern to reduce the emission of harmful gases released during the combustion of fossil fuels. This goal has been addressed through the reduction of sulfur-containing compounds, and the replacement of fossil fuels by biofuels, such as bioethanol, produced in large scale from biomass. For this purpose, a new class of solvents, the Ionic Liquids (ILs), has been applied, aiming at developing new processes and replacing common organic solvents in the current processes. ILs can be composed by a large number of different combinations of cations and anions, which confer unique but desired properties to ILs. The ability of fine-tuning the properties of ILs to meet the requirements of a specific application range by mixing different cations and anions arises as the most relevant aspect for rendering ILs so attractive to researchers. Nonetheless, due to the huge number of possible combinations between the ions it is required the use of cheap predictive approaches for anticipating how they will act in a given situation. Molecular dynamics (MD) simulation is a statistical mechanics computational approach, based on Newton’s equations of motion, which can be used to study macroscopic systems at the atomic level, through the prediction of their properties, and other structural information. In the case of ILs, MD simulations have been extensively applied. The slow dynamics associated to ILs constitutes a challenge for their correct description that requires improvements and developments of existent force fields, as well as larger computational efforts (longer times of simulation). The present document reports studies based on MD simulations devoted to disclose the mechanisms of interaction established by ILs in systems representative of fuel and biofuels streams, and at biomass pre-treatment process. Hence, MD simulations were used to evaluate different systems composed of ILs and thiophene, benzene, water, ethanol and also glucose molecules. For the latter molecules, it was carried out a study aiming to ascertain the performance of a recently proposed force field (GROMOS 56ACARBO) to reproduce the dynamic behavior of such molecules in aqueous solution. The results here reported reveal that the interactions established by ILs are dependent on the individual characteristics of each IL. Generally, the polar character of ILs is deterministic in their propensity to interact with the other molecules. Although it is unquestionable the advantage of using MD simulations, it is necessary to recognize the need for improvements and developments of force fields, not only for a successful description of ILs, but also for other relevant compounds such as the carbohydrates.

Ecological quality assessment in transitional systems

Estuaries are poles of attraction for human settlement which is a source of pressures to surface water bodies. The implementation of the European Water Framework Directive (WDF, 2000/60/EC) has increased the investigation in order to develop methodologies to assess the Ecological Quality Status (EQS) of aquatic ecosystems. Transitional systems are naturally stressed and characterized by highly dynamic physical, chemical and hydro-morphologic conditions and by species with a higher level of tolerance to change, being more difficult to develop suitable quality indicators for these systems. The general purpose of this study is to test the ability of synthesis descriptors, including primary (S, taxa richness) and derived biological variable (H’, Shannon-Wiener diversity), biotic indices (AMBI and M-AMBI), body size properties (abundance distribution by body size classes, length, weight and length-weight relationships) and non-taxonomic indices (ISS), as well as functional indicators related to the decomposition rates of various experimental substrates, a macrophyte (Phragmites australis) and an alga (Fucus vesiculosus), to evaluate the environmental quality in transitional systems. This study was carried out in one of the most pristine channels of the Ria the Aveiro, Mira Channel, along a full salinity gradient and in a metals and metalloid sediment contamination area, the Estarreja Channel, and two reference channels (Canelas and Salreu). In this study were used different sampling techniques, the leaf-bag technique and a hand-held corer. In Mira Channel, the alga and the macrophyte presented an opposite trend in the decomposition rate along the salinity gradient, with the decomposition rates of the alga always higher than those of the macrophyte. The decomposition rates of the macrophyte and the alga were higher in the mid estuary and in higher salinity areas, respectively, corresponding to the preferencial distribution areas of each species. The macrobenthic fauna associated with the decaying and an artificial substrate (control) showed equally well the benthic succession from the marine to the freshwater areas and, despite the strong differences in the decay rates, no significant differences were found between the benthic communities associated with the alga and the macrophyte. The body size properties of the macrobenthic fauna associated with the P. australis leaf-bag (1mm and 5mm) and corer samples were studied along the full salinity gradient. The dominant species of the sub-set of measured specimens were not the same of the original macrobenthic fauna sampled but, despite that, the sub-set of measured specimens was also able to show the benthic succession from the marine to the freshwater areas. The body size abundance distribution of the benthic macroinvertebrates according to the ISS size classes did not show a particular trend in any sampler along the salinity gradient. Significant differences were found in the length, weight and length-weight relationships of Annelids, , Molluscs and even some species along the salinity gradient. No significant differences were found in the AMBI, M-AMBI and ISS values along the salinity gradient for all the samplers. The EQS of the corer samples obtained using the M-AMBI was lower than that of the leaf-bags. The EQS obtained with the ISS was higher than that obtained with the M-AMBI in the leaf-bags but not in the corer samples. The ecological effects of contaminated sediments associated with the industrial chemical effluents discharged in the Estarreja Channel were studied a decade after ceasing the emissions, using the Sediment Quality Triad approach and two reference channels. The results showed that despite the emissions ceased in 2004, the sediment remains polluted with high levels of metals and metalloid, available to bioaccumulation and with severe consequences at the community level. The sediment contamination problem was also studied using the leaf-bag technique with a macrophyte, an alga and a control substrate. The results showed that the decay rates, the associated macrofauna and the application of the AMBI, M-AMBI and ISS indices to the mesh-bag samples were not able to identify the sediment contamination. Contrarily to the AMBI, the M-AMBI and the ISS showed significant differences between the contaminated and the reference channels for the corer samples. Although such statistical significance, the interest of using these complex biotic indices could be questioned, when much simple ones, like the S and H’ allow to reach the same conclusions.

Development of infrared spectroscopy for assessing bacterial quality in foods

Rapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.

Assessing the effectiveness of chemical treatment with nanomaterials in improving the quality of different industrial effluents

Industrial activities are the major sources of pollution in all environments. Depending on the type of industry, various levels of organic and inorganic pollutants are being continuously discharged into the environment. Although, several kinds of physical, chemical, biological or the combination of methods have been proposed and applied to minimize the impact of industrial effluents, few have proved to be totally effective in terms of removal rates of several contaminants, toxicity reduction or amelioration of physical and chemical properties. Hence, it is imperative to develop new and innovative methodologies for industrial wastewater treatment. In this context nanotechnology arises announcing the offer of new possibilities for the treatment of wastewaters mainly based on the enhanced physical and chemical proprieties of nanomaterials (NMs), which can remarkably increase their adsorption and oxidation potential. Although applications of NMs may bring benefits, their widespread use will also contribute for their introduction into the environment and concerns have been raised about the intentional use of these materials. Further, the same properties that make NMs so appealing can also be responsible for producing ecotoxicological effects. In a first stage, with the objective of selecting NMs for the treatment of organic and inorganic effluents we first assessed the potential toxicity of nanoparticles of nickel oxide (NiO) with two different sizes (100 and 10-20 nm), titanium dioxide (TiO2, < 25 nm) and iron oxide (Fe2O3, ≈ 85x425 nm). The ecotoxicological assessment was performed with a battery of assays using aquatic organisms from different trophic levels. Since TiO2 and Fe2O3 were the NMs that presented lower risks to the aquatic systems, they were selected for the second stage of this work. Thus, the two NMs pre-selected were tested for the treatment of olive mill wastewater (OMW). They were used as catalyst in photodegradation systems (TiO2/UV, Fe2O3/UV, TiO2/H2O2/UV and Fe2O3/H2O2/UV). The treatments with TiO2 or Fe2O3 combined with H2O2 were the most efficient in ameliorating some chemical properties of the effluent. Regarding the toxicity to V. fischeri the highest reduction was recorded for the H2O2/UV system, without NMs. Afterwards a sequential treatment using photocatalytic oxidation with NMs and degradation with white-rot fungi was applied to OMW. This new approach increased the reduction of chemical oxygen demand, phenolic content and ecotoxicity to V. fischeri. However, no reduction in color and aromatic compounds was achieved after 21 days of biological treatment. The photodegradation systems were also applied to treat the kraft pulp mill and mining effluents. For the organic effluent the combination NMs and H2O2 had the best performances in reduction the chemical parameters as well in terms of toxicity reduction. However, for the mine effluent the best (TiO2/UV and Fe2O3/UV) were only able to significantly remove three metals (Zn, Al and Cd). Nonetheless the treatments were able of reducing the toxicity of the effluent. As a final stage, the toxicity of solid wastes formed during wastewater treatment with NMs was assessed with Chironomus riparius larvae, a representative species of the sediment compartment. Certain solid wastes showed the potential to negatively affect C. riparius survival and growth, depending on the type of effluent treated. This work also brings new insights to the use of NMs for the treatment of industrial wastewaters. Although some potential applications have been announced, many evaluations have to be performed before the upscaling of the chemical treatments with NMs.

Síntese de receptores para o reconhecimento e transporte de aniões

Nesta tese, realizada no âmbito do Programa Doutoral em Química da Universidade de Aveiro, foram desenvolvidas duas famílias de receptores sintéticos: macrocíclicos baseados na plataforma tetraazacalix[2]areno[2]triazina; e acíclicos construídos a partir de diaminas simples. A plataforma macrocíclica foi decorada nos átomos de azoto em ponte com unidades de reconhecimento molecular contendo fragmentos com grupos amida para o reconhecimento de aniões ou com grupos ácidos carboxílicos para a coordenação de metais de transição. Os receptores acíclicos foram obtidos por acoplamento de diaminas (etilenodiamina, orto-fenilenodiamina ou 2-aminobenzilamina) com uma unidade lipofílica incorporando um anel heterocíclico (derivados de oxadiazole ou furano) e com um derivado isocianato. Estas moléculas assimétricas com um grupo amida e um grupo ureia como unidades de reconhecimento molecular foram avaliadas como receptores e transportadores transmembranares de aniões biologicamente relevantes (Cl- e HCO3-). Os resultados experimentais obtidos serão descritos ao longo de três capítulos, após um primeiro capítulo bibliográfico. No Capítulo 1 começa-se por fazer uma revisão bibliográfica sucinta sobre o desenvolvimento recente de receptores funcionais baseados em azacalixarenos bem como das suas aplicações, designadamente no reconhecimento molecular. Numa segunda parte apresenta-se uma revisão sucinta de receptores derivados de (tio)ureias, relacionados com os receptores sintetizados no âmbito desta tese e com propriedades de reconhecimento e transporte transmembranar de aniões. No Capítulo 2 reporta-se uma série de macrociclos novos com os átomos de azoto em ponte de tetraazacalix[2]areno[2]triazina funcionalizados com bromoacetato de metilo. Foram preparados três novos macrociclos com quatro grupos éster, como braços pendentes, a partir de percursores tetraazacalix[2]areno[2]triazina com os anéis de triazina substituídos com cloro, metilamina ou hexilamina. Os grupos acetato foram hidrolisados em condições básicas, tendo cada um dos derivados dialquilamina originado um composto com quatro grupo carboxílicos, enquanto o análogo diclorado originou uma mistura de compostos com dois grupos carboxílico e com os átomos de cloro substituídos por grupos hidroxilo. Subsequentemente, as propriedades de coordenação dos derivados alquilamina para cobre(II) foram avaliadas por espectroscopia de UV-Vis, tendo-se obtido constantes de estabilidades semelhantes (logk ≈ 6,7). No Capítulo 3 descrevem-se três macrociclos obtidos através da funcionalização dos átomos de azoto em ponte de tetraazacalix[2]areno[2]triazina com grupos amida derivados de N-Boc-etilenodiamina, benzilamina e (S)-metilbenzilamina. A afinidade destes receptores para a série de aniões carboxilato (oxalato, malonato, succinato, glutarato, diglicolato, pimelato, suberato, fumarato, maleato, ftalato e isoftalato) e inorgânicos (Cl-, H2PO4- e SO42-) por titulação de RMN de 1H, foi avaliada. Estes macrociclos conjuntamente com os descritos no Capítulo 2 são os primeiros exemplos reportados na literatura de receptores sintéticos baseados na plataforma de tetraazacalix[2]areno[2]triazina com grupos funcionais nos azotos em ponte. O receptor derivado de N-Boc-etilenodiamina, com oito grupos N-H, entre os três receptores, é o que apresenta maior afinidade para os aniões estudados. No Capítulo 4 é descrita a síntese 59 compostos acíclicos (vide supra) obtidos em três passos de síntese com bons rendimentos. No design desta biblioteca de moléculas a afinidade para aniões dos grupos ureia foi modelada pela inserção de diferentes substituintes arilo ou alquilo, com propriedades electrónicas distintas. A introdução destes grupos em conjugação com um anel de oxadiazole ou furano permitiu também modelar a lipofília destes compostos. A afinidade destes receptores para aniões cloreto e bicarbonato, e em alguns casos para fumarato e maleato, foi investigada por titulação de RMN de 1H. Estes compostos apresentaram constantes de associações compatíveis com o transporte transmembranar de cloreto. Por outro lado estes receptores apresentaram afinidades elevadas para fumarato e maleato, com seletividade para este último. São também discutidos os resultados dos ensaios de transporte de cloreto por estes receptores através de vesículas de em POPC. No Capítulo 5 encontram-se as conclusões gerais desta tese de Doutoramento. No Capitulo 6 encontram-se os dados espectroscópicos e os restantes detalhes experimentais para todos os compostos sintetizados.

Cultivo da Salicornia ramosissima da Ilha dos Puxadoiros®

O estágio realizado teve como premissa principal perceber o comportamento da planta face ao seu crescimento em altura da própria planta e em número em função de diferentes épocas de sementeira, e de condições de cultivo, nomeadamente de tipos de solo. O trabalho realizado inclui todo o processo desde a apanha, preparação das sementes e dos campos, e a sementeira propriamente dita. Foi acompanhado o crescimento das plantas em 3 alturas diferentes, por contagem do número de plantas e do comprimento das plantas aéreas. Igualmente foram realizadas análises da granulometria dos solos. Determinaram-se os metais pesados nos solos e nas plantas, assim como os açúcares após a hidrólise e derivatização das amostras liofilizadas, tanto da raíz como da parte aérea. Foi possível concluir que a planta nasce com maior abundância em solos franco-siltosos do que em solos franco-arenosos, onde o seu crescimento é muito pobre ou mesmo nulo. Os valores de cádmio e chumbo encontram-se a níveis adequados para o consumo humano. Coloca-se a hipótese da existência de arabinoglucoronoxilanas como constituinte da raiz desta planta.