Analysis of the interaction of polycyclic aromatic compounds in a model organism: integration of genotoxic and histopathological effects

Due to their toxicity, especially their carcinogenic potential, polycyclic aromatic hydrocarbons (PAHs) became priority pollutants in biomonitoring programmes and environmental policy, such as the European Water Framework Directive. The model substances tested in this study, namely benzo[b]fluoranthene (B[b]F), considered potentially carcinogenic to humans and an effector carcinogenic PAH to wildlife, and phenanthrene (Phe), deemed a non-carcinogenic PAH, are common PAHs in coastal waters, owning distinct properties reflected in different, albeit overlapping, mechanisms of toxicity. Still, as for similar PAHs, their interaction effects remain largely unknown. In order to study the genotoxic effects of caused by the interaction of carcinogenic and non-carcinogenic PAHs, and their relation to histopathological alterations, juvenile sea basses, Dicentrarchus labrax, a highly ecologically- and economically-relevant marine fish, were injected with different doses (5 and 10 μg.g-1 fish ww) of the two PAHs, isolated or in mixture, and incubated for 48 h. Individuals injected with B[b]F and the PAH mixture exhibited higher clastogenic/aneugenic effects and DNA strand breakage in blood cells, determined through the erythrocytic nuclear abnormalities (ENA) and Comet assays, respectively. Also, hepatic histopathological alterations were found in all animals, especially those injected with B[b]F and the PAH mixture, relating especially to inflammation. Still, Phe also exhibited genotoxic effects in sea bass, especially in higher doses, revealing a very significant acute effect that was accordant with the Microtox test performed undergone in parallel. Overall, sea bass was sensitive to B[b]F (a higher molecular weight PAH), likely due to efficient bioactivation of the pollutant (yielding genotoxic metabolites and reactive oxygen species), when compared to Phe, the latter revealing a more significant acute effect. The results indicate no significant additive effect between the substances, under the current experimental conditions. The present study highlights the importance of understanding PAH interactions in aquatic organisms, since they are usually present in the aquatic environment in complex mixtures.

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

Fiber functionalization for biomedical applications

Deep-eutectic solvents (DES) are considered novel renewable and biodegradable solvents, with a cheap and easy synthesis, without waste production. Later it was discovered a new subclass of DES that even can be biocompatible, since their synthesis uses primary metabolites such as amino acids, organic acids and sugars, from organisms. This subclass was named natural deep-eutectic solvents (NADES). Due to their properties it was tried to study the interaction between these solvents and biopolymers, in order to produce functionalized fibers for biomedical applications. In this way, fibers were produced by using the electrospinning technique. However, it was first necessary to study some physical properties of NADES, as well as the influence of water in their properties. It has been concluded that the water has a high influence on NADES properties, which can be seen on the results obtained from the rheology and viscosity studies. The fluid dynamics had changed, as well as the viscosity. Afterwards, it was tested the viability of using a starch blend. First it was tested the dissolution of these biopolymers into NADES, in order to study the viability of their application in electrospinning. However the results obtained were not satisfactory, since the starch polymers studied did not presented any dissolution in any NADES, or even in organic solvents. In this way it was changed the approach, and it was used other biocompatible polymers. Poly(ethylene oxide), poly(vinyl alcohol) and gelatin were the others biopolymers tested for the electrospinning, with NADES. All polymers show good results, since it was possible to obtain fibers. However for gelatin it was used only eutectic mixtures, containing active pharmaceutical ingredients (API’s), instead of NADES. For this case it was used mandelic acid (antimicrobial properties), choline chloride, ibuprofen (anti-inflammatory properties) and menthol (analgesic properties). The polymers and the produced fibers were characterized by scanning electron microscope (SEM), Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With the help of these techniques it was possible to conclude that it was possible to encapsulate NADES within the fibers. Rheology it was also study for poly(ethylene oxide) and poly(vinyl alcohol), in a way to understand the influence of polymer concentration, on the electrospinning technique. For the gelatin, among the characterization techniques, it was also performed cytotoxicity and drug release studies. The gelatin membranes did not show any toxicity for the cells, since their viability was maintained. Regarding the controlled release profile experiment no conclusion could be drawn from the experiments, due to the rapid and complete dissolution of the gelatin in the buffer solution. However it was possible to quantify the mixture of choline chloride with mandelic acid, allowing thus to complete, and confirm, the information already obtained for the others characterization technique.

Hygrothermal behaviour of earthen plasters for sustainable housing construction

A ready-mixed and several laboratory formulated mortars were produced and tested in fresh state and after hardening, simulating a masonry plaster for indoor application. All the mortars used a clayish earth from the same region and different compositions of aggregates, eventually including fibres and a phase change material. All the formulated mortars were composed by 1:3 volumetric proportions of earth and aggregate. Tests were developed for consistency, fresh bulk density, thermal conductivity, capillary absorption and drying, water vapour permeability and sorption-desorption. The use of PCM changed drastically the workability of the mortars and increased their capillary absorption. The use of fibres and variations on particle size distribution of the mixtures of sand that were used had no significant influence on tested properties. But particularly the good workability of these mortars and the high capacity of sorption and desorption was highlighted. With this capacity plasters made with these mortars are able to adsorb water vapour from indoor atmosphere when high levels of relative humidity exist and release water vapour when the indoor atmosphere became too dry. This fact makes them able to contribute passively for a healthier indoor environment. The technical, ecological and environmental advantages of the application of plasters with this type of mortars are emphasized, with the aim of contributing for an increased use for new or existent housing.

Design and characterization of novel biopolymeric structure using biocompatible ionic liquids

The main objective of this thesis was the development of polymeric structures from the dissolution of FucoPol, a bacterial exopolysaccharide (EPS), in a biocompatible ionic liquid, choline acetate. The FucoPol was produced by the bacteria Enterobacter A47 using glycerol as carbon source at controlled temperature and pH (30ºC and 7, respectively). At the end of 3 days it was produced 7 g/L of FucoPol. The net yield of Fucopol in glycerol (YP/S) was 0.22 g/g and the maximum productivity 2.37 g/L.d This polymer was characterized about its composition in sugars and acyl groups (by High-Performance Liquid Chromatography - HPLC), containing fucose (35 % mol), galactose (21 % mol), glucose (29 % mol), rhamnose (3% mol) and glucuronic acid (12% mol) as well as acetate (14.28 % mol), pyruvate (2.15 % mol) and succinate (1.80 % mol). Its content of water and ash was 15% p/p and 2% p/p, respectively, and the chemical bonds (determined by Infrared Spectroscopy - FT-IR) are consistent to the literature reports. However, due to limitations in Differential Scanning Calorimetry (DSC) equipment it was not possible to determine the glass transition temperature. In turn, the ionic liquid showed the typical behavior of a Newtonian fluid, glass transition temperature (determined by DSC) -98.03ºC and density 1.1031 g/cm3. The study of chemical bonds by FT-IR showed that amount of water (8.80%) influenced the visualization of the bands predicted to in view of their chemical structure. After the dissolution of the FucoPol in the ionic liquid at different temperatures (50, 60, 80 and 100 ° C) it was promoted the removal of this by the phase inversion method using deionized water as a solvent, followed by drying in an oven at 70 ° C. The mixtures before and after the phase inversion method were characterized through the studies mentioned above. In order to explore possible application field’s biocompatibility assays and collage on balsa wood tests were performed. It was found that the process of washing with water by the phase inversion method was not totally effective in removing the biocompatible ionic liquid, since all FucoPol – IL mixtures still contained ionic liquid in their composition as can be seen by the DSC results and FT-IR. In addition, washing the mixtures with water significantly altered the composition of FucoPol. However, these mixtures, that developed a viscous behavior typical of a non-Newtonian fluid (shear-thinning), have the potential to be applied in the biomedical field as well as biological glues.

Why further investigation of brazilian Strychnos?

During the past fifteen years, African Strychnos species have been pharmacologically and chemically screened by some European Universities, sometimes with the collaboration of some African Institutes. Strychnos usambarensis will be discussed here because of our interest in it, after the discovery of its use in the preparation arrow poison with curarizing activity.The European studies have show thah the African species of Strychnoschemically are very similar to certain South-American species. Indeed, the occurrence in the African species of curarizing quaternary bases, and in the American species of tetanizing tertiary bases, such as akagerine, emphasizes the unity of genus Strychnos in terms of it alkaloids. Many biological activities (antmicrobial, hypotensive, smasmolytic, amoebicial...) have been detected.Finally, the antimitotic properties of some alkaloids will be presented. Besides know antitumor drugs, e.g. alstonine and ellipticine, African species possess new antimitotic products, e.g. strychnopentamine present in Strychnos usambarensis.Because of these findings, the main reasons why further investigate Brazilian Strychnos species are the lack of knowledge about:- the distribution on the species in the southern part of the great Amazon Basin.Accordin to Krukoff, it is possible that new species may be found there.- the distribution of alkaloids in many of know species. Indeed, there is little information on the variation in compposition of the alkaloidal mixtures in the different parts of the plants.- the biological of alkaloids. In the past, interest has been focused too narrowly on possible curarizing properties.Why should Brazilian species not cantain alkaloids with properties similar to those found in the African species?

Phenanthroimidazoles as fluorimetric and colourimetric chemosensors in aqueous solution

2,4,5-Triaryl-imidazoles are versatile compounds with application in medicine, due to their biological activity, and materials sciences, for their interesting optical properties. These properties can be tuned by careful selection of substituents at positions 2, 4 and 5: replacement of the aryl group by an heterocyclic group results in larger π-conjugated systems with improved optical properties for application in nonlinear optics, OLEDs, DNA intercalators, and chemosensors. Moreover, it is expected that introducing more conjugation and rigidity into the resulting system will further improve its properties. The development of chromo/fluorescent probes that are capable of detecting ions with high sensitivity and selectivity in aqueous media is currently a topic of strong interest and the design of heteroditopic receptors that contain two or more different binding sites for the simultaneous complexation of cationic and anionic guests is a emerging field of supramolecular chemistry. In this communication, we report the synthesis of new phenanthroimidazoles substituted at position 2 with arylthienyl or arylfuryl moieties possessing substituents of different electronic character, in order to tune the chromo/fluoro response in the presence of relevant anions and metal cations. Their photophysical properties and chemosensory ability were studied in acetonitrile and mixtures of acetonitrile and water, and selective detection of cyanide was achieved in aqueous mixtures for some of the derivatives.

Development of foam one-part geopolymers with enhanced thermal insulation performance and low carbon dioxide emissions

Buildings are responsible for more than 40% of the energy consumption and greenhouse gas emissions. Thus, increasing building energy efficiency is one the most cost-effective ways to reduce emissions. The use of thermal insulation materials could constitute the most effective way of reducing heat losses in buildings by minimising heat energy needs. These materials have a thermal conductivity factor, k (W/m.K) lower than 0.065 while other insulation materials such as aerated concrete can go up to 0.11. Current insulation materials are associated with negative impacts in terms of toxicity. Polystyrene, for example contains anti-oxidant additives and ignition retardants. In addition, its production involves the generation of benzene and chlorofluorocarbons. Polyurethane is obtained from isocyanates, which are widely known for their tragic association with the Bhopal disaster. Besides current insulation materials releases toxic fumes when subjected to fire. This paper presents experimental results on one-part geopolymers. It also includes global warming potential assessment and cost analysis. The results show that only the use of aluminium powder allows the production mixtures with a high compressive strength however its high cost means they are commercially useless when facing the competition of commercial cellular concrete. The results also show that one-part geopolymer mixtures based on 26%OPC +58.3%FA +8%CS +7.7%CH and 3.5% hydrogen peroxide constitute a promising cost efficient (67 euro/m3), thermal insulation solution for floor heating systems with low global warming potential of 443 KgCO2eq/m3.

Plastic waste use as aggregate and binder modifier in open-graded asphalts

Road pavements are very important infrastructures for the Society, but they can cause serious environmental impacts during construction, operation and rehabilitation phases. Thus, it is essential to develop surface paving solutions that promote not only the durability but also a comfortable and safe use. In fact, this work aims to study the properties of new opengraded mixtures for surface layers produced with plastic wastes. First, HDPE and EVA wastes were used as bitumen modifiers, and then another plastic waste (PEX) replaced part of the aggregates. After studying the modified binders, the open-graded mixtures were designed, and then they were tested concerning their particle loss, rutting resistance, surface texture and damping effect. It was concluded that both ways of using the plastic wastes can improve the mechanical and functional properties of the open-graded mixtures related to the pavement performance.

Effect of incorporating different waste materials in bitumen

The increasing environmental concern about waste materials and the necessity of improving the performance of asphalt mixtures prompted the study of incorporating different waste materials in conventional bitumen. The reuse of waste materials can present benefits at an environmental and economic level, and some wastes can be used to improve the pavement performance. Thus, the purpose of this study is to evaluate the incorporation of different waste materials in bitumen, namely waste motor oil and different polymers. In order to accomplish this goal, 10% of waste motor oil and 5% of polymers (high density polyethylene, crumb rubber and styrene-butadiene-styrene) were added to a conventional bitumen and the resulting modified bitumens were characterized through basic and rheological tests. From this work, it can be concluded that the incorporation of different waste materials improve some important properties of the conventional bitumen. Such improvements might indicate a good behaviour at medium/high temperatures and an increase of fatigue and rutting resistance. Therefore, these modified bitumens with waste materials can contribute to a sustainable development of road paving industry due to their performance and environmental advantages.

Study of the interaction between asphalt and plastic wastes in new polymer modified binders (PMB)

The aim of this study is evaluating the interaction between several base pen grade asphalt binders (35/50, 50/70, 70/100, 160/220) and two different plastic wastes (EVA and HDPE), for a set of new polymer modified binders produced with different amounts of both plastic wastes. After analysing the results obtained for the several polymer modified binders evaluated in this study, including a commercial modified binder, it can be concluded that the new PMBs produced with the base bitumen 70/100 and 5% of each plastic waste (HDPE or EVA) results in binders with very good performance, similar to that of the commercial modified binder.

Assessing the production of jet mix columns using alkali activated waste based on mechanical and financial performance and CO2 (eq) emissions

Recent research has proved the potential of alkaline activated fly-ash for soil stabilisation. However, such studies have not focused on the link between financial, mechanical and environmental aspects of this solution, but only on their absolute mechanical properties. The present paper characterises the mechanical behaviour of a large spectrum of activator-ash-soil combinations used to build jet mixing columns, analysing also the cost and CO2 (eq) emissions. The concern with these two vectors forced a decrease in the quantity of stabilising agent added to the soil, relatively to previous research, and the effects of such low quantities have not yet been published. However, the results clearly showed a significant improve in strength, still well above the average values expected when improving the stressstrain behaviour of a weak soil. Uniaxial compressive strength tests were used to assess the effects of the fly-ash percentage, the alkalieash ratio and the water content. The carbon calculator recently developed by the European Federation of Foundation Contractors and the Deep Foundations Institute was used to quantify the CO2 (eq) emissions associated with this technique. The financial cost was estimated based on the experience of a major Portuguese contractor. For comparison purposes, soil cement mixtures were also analysed, using similar conditions and tools used for the soil-ash analysis. Results showed that the cement and ash solutions are very similar in terms of overall performance, with some advantage of the former regarding financial cost, and a significant advantage of the latter regarding the CO2 (eq) emissions. This new grout, although it is in an embryonic stage, it has the potential for broader developments in the field.

Pultrusion of fibre reinforced thermoplastic pre-impregnated materials

Fibre reinforced thermoplastic pre impregnated materials produced continuously by diverse methods and processing conditions were used to produce composites using pultrusion. The processing windows used to produce these materials and composites profiles were optimized by using the Taguchi / DOE (Design of Experiments) methods. Composites were manufactured by pultrusion and compression moulding and subsequently submitted to mechanical testing and microscopy analysis. The obtained results were compared with the expected theoretical ones predicted from the Rule of Mixtures (ROM) and with those of similar engineering conventional available materials. The results obtained shown that produced composites have adequate properties for applications in common and structural engineering markets.

Cost-efficient one-part alkali-activated mortars with low global warming potential for floor heating systems applications

Increasing building energy efficiency is one the most cost-effective ways to reduce emissions. The use of thermal insulation materials mitigates heat loss in buildings, therefore minimising heat energy needs. In recent years, several papers were published on the subject of foam alkali-activated cements with enhanced thermal conductivity. However, on those papers cost analysis was strangely avoided. This paper presents experimental results on one-part alkali-activated cements. It also includes global warming potential assessment and cost analysis. Foam one-part alkali-activated cements cost simulations considering two carbon dioxide social costs scenarios are also included. The results show that one-part alkali-activated cements mixtures based on 26%OPC + 58.3%FA + 8%CS + 7.7%CH and 3.5% hydrogen peroxide constitute a promising cost-efficient (67 euro/m3), thermal insulation solution for floor heating systems. This mixture presents a low global warming potential of 443 KgCO2eq/m3. The results confirm that in both carbon dioxide social cost scenarios the mixture 26 OPC + 58.3 FA + 8 CS + 7.7 CH with 3.5% hydrogen peroxide foaming agent is still the most cost efficient.

Using polymers to improve the rutting resistance of asphalt concrete

This study assesses rutting on two types of modified asphalt mixtures containing: (i) amorphous polyolefin polymer and (ii) a particular polymer obtained by combining LDPE (low density polyethylene) and EVA (ethyl-vinyl-acetate). Rutting tests were performed by a wheel tracking device. Stiffness and fatigue tests were carried out to confirm the performance of the asphalt mixtures. The testing showed that polymer modification in this study improved rut resistance without compromising the stiffness and fatigue behavior. The rutting results were fit in the NCHRP 1-37A model and the in situ rutting performance of asphalt mixtures can be predicted.