Single and joint effects of natural and chemical stressors to the terrestrial isopod Porcellionides pruinosus

Environmental contamination and climate changes constitute two of the most serious problems affecting soil ecosystems in agricultural fields. Agriculture is nowadays a highly optimized process that strongly relies on the application of multiple pesticides to reduce losses and increase yield production. Although constituting, per se, a serious problem to soil biota, pesticide mixtures can assume an even higher relevance in a context of unfavourable environmental conditions. Surprisingly, frameworks currently established for environmental risk assessments keep not considering environmental stressors, such as temperature, soil moisture or UV radiation, as factors liable to influence the susceptibility of organisms to pesticides, or pesticide mixtures, which is raising increasing apprehension regarding their adequacy to actually estimate the risks posed by these compounds to the environment. Albeit the higher attention received on the last few years, the influence of environmental stressors on the behaviour and toxicity of chemical mixtures remains still poorly understood. Aiming to contribute for this discussion, the main goal of the present thesis was to evaluate the single and joint effects of natural stressors and pesticides to the terrestrial isopod Porcellionides pruinosus. The first approach consisted on evaluating the effects of several abiotic factors (temperature, soil moisture and UV radiation) on the performance of P. pruinosus using several endpoints: survival, feeding parameters, locomotor activity and avoidance behaviour. Results showed that these stressors might indeed affect P. pruinosus at relevant environmental conditions, thus suggesting the relevance of their consideration in ecotoxicological assays. At next, a multiple biomarker approach was used to have a closer insight into the pathways of damage of UV radiation and a broad spectrum of processes showed to be involved (i.e. oxidative stress, neurotoxicity, energy). Furthermore, UV effects showed to vary with the environment medium and growth-stage. A similar biomarker approach was employed to assess the single and joint effects of the pesticides chlorpyrifos and mancozeb to P. pruinosus. Energy-related biomarkers showed to be the most differentiating parameters since age-classes seemed to respond differently to contamination stress and to have different metabolic costs associated. Finally, the influence of temperature and soil moisture on the toxicity of pesticide mixtures was evaluated using survival and feeding parameters as endpoints. Pesticide-induced mortality was found to be oppositely affected by temperature, either in single or mixture treatments. Whereas chlorpyrifos acute toxicity was raised under higher temperatures the toxicity of mancozeb was more prominent at lower temperatures. By the opposite, soil moisture showed no effects on the pesticide-induced mortality of isopods. Contrary to survival, both temperature and soil moisture showed to interact with pesticides to influence isopods’ feeding parameters. Nonetheless, was however the most common pattern. In brief, findings reported on this thesis demonstrated why the negligence of natural stressors, or multiple stressors in general, is not a good solution for risk assessment frameworks.

Development of polymeric materials to inhibit bacterial quorum sensing

Bacterial infections are an increasing problem for human health. In fact, an increasing number of infections are caused by bacteria that are resistant to most antibiotics and their combinations. Therefore, the scientific community is currently searching for new solutions to fight bacteria and infectious diseases, without promoting antimicrobial resistance. One of the most promising strategies is the disruption or attenuation of bacterial Quorum Sensing (QS), a refined system that bacteria use to communicate. In a QS event, bacteria produce and release specific small chemicals, signal molecules - autoinducers (AIs) - into the environment. At the same time that bacterial population grows, the concentration of AIs in the bacterial environment increases. When a threshold concentration of AIs is reached, bacterial cells respond to it by altering their gene expression profile. AIs regulate gene expression as a function of cell population density. Phenotypes mediated by QS (QSphenotypes) include virulence factors, toxin production, antibiotic resistance and biofilm formation. In this work, two polymeric materials (linear polymers and molecularly imprinted nanoparticles) were developed and their ability to attenuate QS was evaluated. Both types of polymers should to be able to adsorb bacterial signal molecules, limiting their availability in the extracellular environment, with expected disruption of QS. Linear polymers were composed by one of two monomers (itaconic acid and methacrylic acid), which are known to possess strong interactions with the bacterial signal molecules. Molecularly imprinted polymer nanoparticles (MIP NPs) are particles with recognition capabilities for the analyte of interest. This ability is attained by including the target analyte at the synthesis stage. Vibrio fischeri and Aeromonas hydrophila were used as model species for the study. Both the linear polymers and MIP NPs, tested free in solutions and coated to surfaces, showed ability to disrupt QS by decreasing bioluminescence of V. fischeri and biofilm formation of A. hydrophila. No significant effect on bacterial growth was detected. The cytotoxicity of the two types of polymers to a fibroblast-like cell line (Vero cells) was also tested in order to evaluate their safety. The results showed that both the linear polymers and MIP NPs were not cytotoxic in the testing conditions. In conclusion, the results reported in this thesis, show that the polymers developed are a promising strategy to disrupt QS and reduce bacterial infection and resistance. In addition, due to their low toxicity, solubility and easy integration by surface coating, the polymers have potential for applications in scenarios where bacterial infection is a problem: medicine, pharmaceutical, food industry and in agriculture or aquaculture.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5ºC in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the “interactive” nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

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.

Effect of photodynamic therapy on the virulence factors of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria who integrate the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation process (PDI) is based in the combined use of a light source, an oxidizing agent like oxygen and an intermediary agent (a photosensitizer). These three components interact to form cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. In fact, PDI is being shown to be a promising alternative to the antibiotic approach in the inactivation of pathogenic microorganisms. However, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus. For this, as photosensitizer the 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus (one reference strain, one strain with 1 enterotoxin, two strains with 3 enterotoxins and two strains resistant to methicillin, MRSA – one with 5 enterotoxins and the other without enterotoxins) were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production and resistance to methicillin was assessed. The results indicate that the expression of some virulence factors in the cells subjected to this therapy is affected. Additionally the susceptibility of the strains to PDI did not decrease upon successive treatments.

Aplicação da terapia fotodinâmica no processo de esterilização de sangue

A importância médica do sangue associada ao risco de doenças infeciosas levou a um melhoramento das técnicas de rastreio de patogénicos no sangue doado. No entanto, devido aos períodos de "janela", durante o qual os agentes infeciosos não podem ser detetados, a desinfeção de sangue e seus derivados assume uma importância vital. Considerando que as técnicas convencionais de desinfeção (tratamento com solvente-detergente ou irradiação com UV ou radiação gama) pode ser empregue em concentrados de plasma ou de proteínas, o efeito colateral associado aos respetivos tratamentos não permite a sua utilização em frações celulares. Consequentemente, é necessário o desenvolvimento de uma nova alternativa eficaz para inativar microrganismos em sangue. Uma boa estratégia que merece ser considerada baseia-se na terapia fotodinâmica antimicrobiana (aPDT). aPDT envolve a interação entre a luz e um fotossensibilizador (PS) na presença de oxigénio molecular. Esta interação produz espécies reativas de oxigénio (ROS), que causam danos oxidativos às moléculas microbianas necessárias à sobrevivência do microrganismo. Em alguns países, esta metodologia já está aprovada para descontaminação de plasma, utilizando azul de metileno ou psoraleno como PSs. O objetivo deste estudo foi avaliar a adequação de de estrutura do tipo ftalocianina (Pc) e porfirina (Por) para desinfeção fotodinâmica de hemoderivados. Plasma e sangue total foram infetados com 108 unidades formadoras de colónias (CFU) / mL de Escherichia coli e após incubação com os derivados Pc e Por em estudo, expostos respetivamente a luz vermelha ou a luz branca com uma irradiância de 150 W/m2durante 270 min. As concentrações de E. coli viáveis foram determinadas a 0, 30, 60, 90, 180 e 270 min e comparadas com as obtidas nos controlos claro (amostras irradiadas na ausência de PS) e controlos escuro (amostras incubadas com PS mas não irradiadas). O efeito do tratamento aPDT nas células do sangue (glóbulos vermelhos e brancos) também foi avaliado. Os resultados obtidos mostram que, em todos os componentes do sangue, a Por em estudo é mais eficaz na inativação de E. coli que o derivado Pc. Após o tratamento aPDT, o número de células vermelhas e brancas no sangue é semelhante aos valores observados nas amostras de controlo. A eficiente inativação de células de E. coli e a ausência de efeito sobre as células de sangue transformam os derivados porfirínicos e ftalocianinas potenciais candidatos a serem utilizados com fotossensibilizadores na desinfeção fotodinâmica de produtos derivados do sangue.

Genetic and bacterial programming for B-Spline neural networks design

The design phase of B-spline neural networks is a highly computationally complex task. Existent heuristics have been found to be highly dependent on the initial conditions employed. Increasing interest in biologically inspired learning algorithms for control techniques such as Artificial Neural Networks and Fuzzy Systems is in progress. In this paper, the Bacterial Programming approach is presented, which is based on the replication of the microbial evolution phenomenon. This technique produces an efficient topology search, obtaining additionally more consistent solutions.

Antioxidantes em frutos e vegetais

O oxigénio é um elemento necessário à sobrevivência da maioria dos seres vivos. Contudo, este pode revelar-se tóxico, provocando a oxidação e a destruição celular. Em consequência desta oxidação formam–se, a partir do oxigénio molecular (O2), grupos de átomos extremamente reactivos – os radicais livres. A oxidação, tanto dos tecidos animais como dos tecidos vegetais, pode ocorrer tanto como resultado do simples metabolismo celular aeróbio formando os designados radicais livres endógenos, como também pelas agressões externas ao organismo que levam à formação dos radicais livres exógenos (Plummer, 1989).

Design of B-spline neural networks using a bacterial programming approach

The design phase of B-spline neural networks represents a very high computational task. For this purpose, heuristics have been developed, but have been shown to be dependent on the initial conditions employed. In this paper a new technique, Bacterial Programming, is proposed, whose principles are based on the replication of the microbial evolution phenomenon. The performance of this approach is illustrated and compared with existing alternatives.

Efeito de um concentrado de microalgas liofilizadas na larvicultura de dourada, Sparus aurata

Dissesrtação de Mestrado, Biologia Marinha, Especialização em Pescas e Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Efeito da proteína anticongelante tipo I (AFP I) na criopreservação de embriões e blastómeros de teleósteos

Dissertação de mest., Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2007

Characterisation of physiological conditions and evaluation of well-being in two species of bivalve molluscs (Ruditapes philippinarum and Chamelea gallina) intensively exploited in coastal and lagoon areas of the Northern Adriatic Sea (Italy)

Tese de Doutoramento, Ecologia, Especialidade de Ecofisiologia, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2007

Aplicação de técnicas de conservação in vitro para a conservação de espécies ameaçadas

Dissertação mest., Engenharia Biológica, Universidade do Algarve, 2008

Effects of stocking density on A. tonsa (Copepoda: Calanoida) culture

Dissertação de Mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009