Diversity of Aeromonas species from different environments in Portugal

Espécies de Aeromonas encontram-se distribuídas por diferentes habitats, estando especialmente relacionadas com ambientes aquáticos. O seu papel em complicações na saúde humana e animal é reconhecido. De facto, não só pelo seu potencial de virulência, mas também pelos determinantes genéticos de resistência a antibióticos que possam conter, estes organismos constituem uma preocupação na medicina humana e veterinária. Assim, é essencial o estudo da diversidade de espécies de Aeromonas bem como explorar as suas características fenotípicas e genéticas que podem conduzir a impactos negativos. A água constitui um importante veículo de transmissão de microrganismos e espécies de Aeromonas estão amplamente distribuídas em águas tratadas e não tratadas. Em Portugal é ainda comum o consumo de águas não tratadas cuja qualidade, na maioria das vezes, não é sujeita a monitorização, como acontece por exemplo, em explorações agrícolas de gestão familiar. Neste estudo, investigou-se a presença de Aeromonas em águas não tratadas para consumo. Estabeleceu-se também uma linha horizontal de colheitas de diferentes amostras de origem agrícola com o intuito de avaliar a possibilidade de a água ser uma das vias de contaminação de culturas agrícolas e animais por espécies de Aeromonas. Obtiveram-se 483 isolados que foram discriminados por RAPD-PCR. 169 estirpes distintas foram identificadas ao nível da espécie por análise filogenética baseada no gene gyrB. Verificou-se uma frequente ocorrência bem como uma diversidade considerável de espécies de Aeromonas. Em alguns casos, as relações genotípicas entre isolados de diferentes amostras eram muito próximas. Adicionalmente, a maioria das amostras continha diferentes espécies e estirpes distintas da mesma espécie. A. media e A. hydrophila foram as espécies mais ocorrentes. Um grupo de isolados apresentou variantes moleculares de gyrB diferente das conhecidas até agora, o que indica que poderão constituir espécies não descritas. O perfil de susceptibilidade da colecção de Aeromonas a diferentes antibióticos foi estabelecido, constituindo um perfil típico do género, com algumas excepções. Estirpes multirresistentes foram encontradas. A presença de genes tet e bla foi investigada por estudos de PCR, hibridação e, em alguns casos, de sequenciação. Como era esperado, cphA/imiS foi o mais detectado. A detecção de integrões fez-se por PCR e hibridação e a sua caracterização foi feita por sequenciação de DNA; a sua ocorrência foi reduzida. A maioria das estirpes sintetizou enzimas extracelulares com actividade lipolítica e proteolítica que potencialmente contribuem para virulência. A análise por PCR e hibridação permitiram a detecção de vários determinantes genéticos que codificam moléculas possivelmente envolvidas em processos patogénicos. Diversas espécies de Aeromonas apresentando características relacionadas com resistência a antibióticos e potencialmente de virulência estão frequentemente presentes em produtos para consumo humano e animal em Portugal. ABSTRACT: Aeromonas spp. are present in a wide range of ecological niches, being mainly related to aquatic environments. Their role in human and animal health complications is recognised. In fact, not only for their putative virulence but also for the antibiotic resistance genetic determinants Aeromonas may harbour, these organisms constitute an issue of concern in human and veterinary medicine. Thus, it is essential to get knowledge on Aeromonas sp. diversity and on their genotypic and phenotypic characteristics that may lead to negative impacts. Water constitutes a good contamination route for microorganisms and Aeromonas are widespread in untreated and treated waters from different sources. In Portugal there is still an extensive use of untreated water which is not regularly monitored for quality. This is often the case in family smallholding farms. In this study untreated drinking and mineral waters were assessed for their content in Aeromonas spp. Furthermore, a sampling scheme was designed to investigate the occurrence and diversity of Aeromonas sp. in different agricultural correlated sources and to assess the possibility of water being the transmission vehicle between those sources. 483 isolates were obtained and discriminated by RAPD-PCR. Identification at the species level for 169 distinct strains was done by gyrB based phylogenetic analysis. Results demonstrated the frequent occurrence and considerable diversity of Aeromonas spp. In some cases, genotypic close relations were found between isolates from different sources. Also, most samples contained different species and distinct strains of the same species. A. media and A. hydrophila were the most occurring. A group of isolates displayed gyrB gene sequences distinct from the previously known, indicating that they may constitute representatives of non-described species. The antibiotic susceptibility profile of the aeromonads collection was established and constituted a typical profile of the genus, although few exceptions. Multiresistance patterns were found. The presence of tet and bla genes was investigated by PCR, hybridisation and, in some cases, sequencing analysis. As expected, cphA/imiS was the most detected. Integrons were screened by PCR and hybridisation and characterised by DNA sequencing; low occurrence was recorded. The bulk of strains was able to produce extracellular enzymes with lipolytic and proteolytic activities, which may contribute to virulence. PCR and hybridisation surveys allowed the detection of distinct genetic determinants coding for molecules putatively involved in pathogenic processes. Diverse Aeromonas sp. presenting distinct antibiotic resistance features and putative virulence traits are frequently present in many sources for human and animal consumption in Portugal.

Adaptation of Cladocerans in metal-polluted heterogeneous environments

No presente trabalho o cladócero Daphnia longispina foi utilizado como organismo modelo para a avaliação dos efeitos ecológicos da adaptação a ambientes contaminados por metais. Foram amostradas populações naturais de D. longispina num local sujeito à contaminação por metais e num local próximo, de referência, ambos localizados no sistema aquático na área envolvente à mina abandonada de São Domingos. Várias linhagens clonais de ambas as populações foram mantidas em laboratório, sob condições controladas, para a execução dos testes. Um dos testes realizados permitiu estudar e quantificar as diferenças na tolerância letal entre as linhagens clonadas de ambas as populações e também avaliar os custos associados. Utilizando vinte linhagens clonais de D. longispina das duas populações verificou-se que apenas clones sensíveis ao cobre estavam presentes na população de referência e clones resistentes ao cobre estavam presentes na população do local contaminado. Os custos associados à tolerância foram ilustrados pela determinação de taxas alimentares mais baixas para a população tolerante quando comparadas com as da população de referência. Outro dos testes realizados permitiu comparar as respostas de clones de populações de ambos os locais – contaminado e referência – à exposição a concentrações sub-letais do metal cobre. A tolerância evidenciada anteriormente ao nível letal foi confirmada ao nível sub-letal, com o clone proveniente da população do local contaminado evidenciando uma maior tolerância ao cobre quando comparado com os restantes clones, para todos os parâmetros analisados (taxas alimentares, consumo de oxigénio, crescimento e reprodução). Os efeitos da aclimatação ao cobre ao longo de várias gerações foram também avaliados num clone de D. longispina. Os resultados evidenciaram a existência de uma adaptação fisiológica ao cobre ao longo das várias gerações que, no entanto, apenas aumentou marginalmente a tolerância a níveis de cobre letais. Para além disso, observou-se também uma grande variação nas respostas do clone de D. longispina estudado, não só entre concentrações de cobre mas também entre gerações. Os resultados obtidos nos vários estudos realizados com linhagens clonais de ambas as populações de D. longispina reforçam a importância de integrar a temática do desenvolvimento de tolerância à poluição aquando da avaliação dos riscos ambientais e ecológicos de compostos químicos, como os metais, no meio ambiente.

Service building in dense service provider environments

Genericamente falando, os serviços sobre redes têm vindo a afastar-se de um modelo monolítico para um modelo de criação de serviços que permite ou - como é mais frequente - requer a cooperação entre vários Provedores de Serviço. A Internet, que tem vindo a forçar a convergência de serviços, mostra que começa a ser virtualmente impossível a um único operador fornecer qualquer serviço com um mínimo de interesse para os utilizadores. Esta tese foca-se em serviços de transporte (e.g., connectividade) e discute o impacto das fronteira que as ofertas de serviços têm com o negócio. A questão central é a seguinte: o que muda quando o mesmo serviço é oferecido não apenas por um mas por mais do que um Provedor de Serviço. Por um lado, esta tese cobre, em abs tracto, a noção de Provedor se Serviço, como evoluiu e em que sentido está a evoluir, particularmente num contexto de muitos Provedores de Serviço. Os primeiros capítulos desta tese analizam e propõem arquitecturas para cooperação inter-Provedor-de-Serviço e para serviços comuns tais como multimédia. Por outro lado, oferece-se soluções práticas, com as respectivas avaliações, para alguns problemas, que ainda hoje se mantêm em aberto, tais como encaminhamento inter-domínio, Qualidade-de-Serviço, Mobilidade e distribuição de conteúdos, tais como as contribuições relacionadas com o impacto da noção administrativa de Sistemas Autónomos sobre encaminhamento inter-domínio, uma arquitectura de transporte inter-domínio e o problema que levanta da ineficiência que decorre do planeamento não- cooperativo de Redes de Entrega de Conteúdos.

Navigation in dynamic environments taking advantage of moving agents

Esta tese propõe uma forma diferente de navegação de robôs em ambientes dinâmicos, onde o robô tira partido do movimento de pedestres, com o objetivo de melhorar as suas capacidades de navegação. A ideia principal é que, ao invés de tratar as pessoas como obstáculos dinâmicos que devem ser evitados, elas devem ser tratadas como agentes especiais com conhecimento avançado em navegação em ambientes dinâmicos. Para se beneficiar do movimento de pedestres, este trabalho propõe que um robô os selecione e siga, de modo que possa mover-se por caminhos ótimos, desviar-se de obstáculos não detetados, melhorar a navegação em ambientes densamente populados e aumentar a sua aceitação por outros humanos. Para atingir estes objetivos, novos métodos são desenvolvidos na área da seleção de líderes, onde duas técnicas são exploradas. A primeira usa métodos de previsão de movimento, enquanto a segunda usa técnicas de aprendizagem por máquina, para avaliar a qualidade de candidatos a líder, onde o treino é feito com exemplos reais. Os métodos de seleção de líder são integrados com algoritmos de planeamento de movimento e experiências são realizadas para validar as técnicas propostas.

Oil hydrocarbon-bacteria interactions in coastal environments

The ability of microorganisms to use oil hydrocarbons as a source of carbon and energy is crucial for environmental oil detoxification. However, there is still a lack of knowledge on fundamental aspects of this process on specific habitats and under different climate scenarios. In the first phase of this work, the culturable fraction of the oil hydrocarbon (OH) degrading bacteria from the sea surface microlayer (SML) of the estuarine system Ria de Aveiro was characterized. In the second phase, the impact of oil contamination on the active bacterial community was studied under climate change scenarios. Pseudomonas emerged as the prevailing genera among OH degrading bacteria in the SML. Moreover, culture-independent methods revealed that the relative abundance and diversity of Gammaproteobacteria, in which Pseudomonas is included, varies along an estuarine gradient of contamination. In order to access the impact of oil contamination on microbial communities under climate change scenarios, an experimental life support system for microcosm experiments (ELLS) was developed and validated for simulation of climate change effects on microbial communities. With the ELSS it is possible to simulate, in controlled conditions, fundamental parameters of the dynamics of coastal and estuarine systems while maintaining community structure in terms of the abundance of the most relevant members of the indigenous bacterial community. A microcosm experiment in which the independent and combined impact of ultraviolet radiation, ocean acidification and oil contamination on microbial communities was conducted. The impact on bacterial communities was accessed with a 16S RNA (cDNA) based barcode pyrosequencing approach. There was a drastic decrease of Desulfobacterales relative abundance after oil contamination under the reduced pH value estimated for 2100, when compared to present values. Since members of this order are known OH degraders, such a significant decrease may have consequences on OH detoxification of contaminated environments under the pH levels of the ocean expected for the future. Metagenome predictions based on the 16S RNA database indicated that several degradation pathways of OH could be affected under oil contamination and reduced water pH. Taken together, the results from this work bring new information on the dynamics of OH degrading bacteria in coastal and estuarine environments under present and future climate scenarios.

Transient transpiration radiometer : development of a heat flux sensor for high aggressivity environments

The development of a new instrument for the measurement of convective and radiative is proposed, based on the transient operation of a transpiration radiometer. Current transpiration radiometers rely on steady state temperature measurements in a porous element crossed by a know gas mass flow. As a consequence of the porous sensing element’s intrinsically high thermal inertia, the instrument’s time constant is in the order of several seconds. The proposed instrument preserves established advantages of transpiration radiometers while incorporating additional features that broaden its applicability range. The most important developments are a significant reduction of the instrument’s response time and the possibility of separating and measuring the convective and radiative components of the heat flux. These objectives are achieved through the analysis of the instrument’s transient response, a pulsed gas flow being used to induce the transient behavior.

Dependable wireless real-time communications for open environments

Wireless communication technologies have become widely adopted, appearing in heterogeneous applications ranging from tracking victims, responders and equipments in disaster scenarios to machine health monitoring in networked manufacturing systems. Very often, applications demand a strictly bounded timing response, which, in distributed systems, is generally highly dependent on the performance of the underlying communication technology. These systems are said to have real-time timeliness requirements since data communication must be conducted within predefined temporal bounds, whose unfulfillment may compromise the correct behavior of the system and cause economic losses or endanger human lives. The potential adoption of wireless technologies for an increasingly broad range of application scenarios has made the operational requirements more complex and heterogeneous than before for wired technologies. On par with this trend, there is an increasing demand for the provision of cost-effective distributed systems with improved deployment, maintenance and adaptation features. These systems tend to require operational flexibility, which can only be ensured if the underlying communication technology provides both time and event triggered data transmission services while supporting on-line, on-the-fly parameter modification. Generally, wireless enabled applications have deployment requirements that can only be addressed through the use of batteries and/or energy harvesting mechanisms for power supply. These applications usually have stringent autonomy requirements and demand a small form factor, which hinders the use of large batteries. As the communication support may represent a significant part of the energy requirements of a station, the use of power-hungry technologies is not adequate. Hence, in such applications, low-range technologies have been widely adopted. In fact, although low range technologies provide smaller data rates, they spend just a fraction of the energy of their higher-power counterparts. The timeliness requirements of data communications, in general, can be met by ensuring the availability of the medium for any station initiating a transmission. In controlled (close) environments this can be guaranteed, as there is a strict regulation of which stations are installed in the area and for which purpose. Nevertheless, in open environments, this is hard to control because no a priori abstract knowledge is available of which stations and technologies may contend for the medium at any given instant. Hence, the support of wireless real-time communications in unmanaged scenarios is a highly challenging task. Wireless low-power technologies have been the focus of a large research effort, for example, in the Wireless Sensor Network domain. Although bringing extended autonomy to battery powered stations, such technologies are known to be negatively influenced by similar technologies contending for the medium and, especially, by technologies using higher power transmissions over the same frequency bands. A frequency band that is becoming increasingly crowded with competing technologies is the 2.4 GHz Industrial, Scientific and Medical band, encompassing, for example, Bluetooth and ZigBee, two lowpower communication standards which are the base of several real-time protocols. Although these technologies employ mechanisms to improve their coexistence, they are still vulnerable to transmissions from uncoordinated stations with similar technologies or to higher power technologies such as Wi- Fi, which hinders the support of wireless dependable real-time communications in open environments. The Wireless Flexible Time-Triggered Protocol (WFTT) is a master/multi-slave protocol that builds on the flexibility and timeliness provided by the FTT paradigm and on the deterministic medium capture and maintenance provided by the bandjacking technique. This dissertation presents the WFTT protocol and argues that it allows supporting wireless real-time communication services with high dependability requirements in open environments where multiple contention-based technologies may dispute the medium access. Besides, it claims that it is feasible to provide flexible and timely wireless communications at the same time in open environments. The WFTT protocol was inspired on the FTT paradigm, from which higher layer services such as, for example, admission control has been ported. After realizing that bandjacking was an effective technique to ensure the medium access and maintenance in open environments crowded with contention-based communication technologies, it was recognized that the mechanism could be used to devise a wireless medium access protocol that could bring the features offered by the FTT paradigm to the wireless domain. The performance of the WFTT protocol is reported in this dissertation with a description of the implemented devices, the test-bed and a discussion of the obtained results.

A creative approach to isometries integrating Geogebra and Italc with ‘paper and pencil’ environments

Creativity is recognized nowadays as a basic skill. However, the educational system fails in promoting their development. On the other hand, a growing acknowledgement of the importance of geometry emerges. Conceptual renewal, namely on isometries, requires new approaches based on mathematically significant tasks. The digital revolution has brought powerful tools but demands changes in the educational process. The use of Dynamic Geometry Environments (DGE), complementing ‘paper and pencil’, can contribute to provide rich learning environments, enhanced by Classroom Management Systems (CMS) such as iTALC. Indeed, the qualitative case study we carried out suggests that: the creation of an "atmosphere" of cooperation, collaboration and sharing seems to increase creativity dimensions; the use of DGE can facilitate the emergence of more creative productions; development of knowledge and geometrical capabilities seems to benefit from a complementary approach that combines DGE and ‘paper and pencil’ environments. Different approaches, with a more technological and exploratory nature seem to promote more favourable attitudes towards mathematics in general, and geometry, in particular.

Seamless integration of heterogeneous networks in multicast environments

Nowadays, communication environments are already characterized by a myriad of competing and complementary technologies that aim to provide an ubiquitous connectivity service. Next Generation Networks need to hide this heterogeneity by providing a new abstraction level, while simultaneously be aware of the underlying technologies to deliver richer service experiences to the end-user. Moreover, the increasing interest for group-based multimedia services followed by their ever growing resource demands and network dynamics, has been boosting the research towards more scalable and exible network control approaches. The work developed in this Thesis enables such abstraction and exploits the prevailing heterogeneity in favor of a context-aware network management and adaptation. In this scope, we introduce a novel hierarchical control framework with self-management capabilities that enables the concept of Abstract Multiparty Trees (AMTs) to ease the control of multiparty content distribution throughout heterogeneous networks. A thorough evaluation of the proposed multiparty transport control framework was performed in the scope of this Thesis, assessing its bene ts in terms of network selection, delivery tree recon guration and resource savings. Moreover, we developed an analytical study to highlight the scalability of the AMT concept as well as its exibility in large scale networks and group sizes. To prove the feasibility and easy deployment characteristic of the proposed control framework, we implemented a proof-of-concept demonstrator that comprehends the main control procedures conceptually introduced. Its outcomes highlight a good performance of the multiparty content distribution tree control, including its local and global recon guration. In order to endow the AMT concept with the ability to guarantee the best service experience by the end-user, we integrate in the control framework two additional QoE enhancement approaches. The rst employs the concept of Network Coding to improve the robustness of the multiparty content delivery, aiming at mitigating the impact of possible packet losses in the end-user service perception. The second approach relies on a machine learning scheme to autonomously determine at each node the expected QoE towards a certain destination. This knowledge is then used by di erent QoE-aware network management schemes that, jointly, maximize the overall users' QoE. The performance and scalability of the control procedures developed, aided by the context and QoE-aware mechanisms, show the advantages of the AMT concept and the proposed hierarchical control strategy for the multiparty content distribution with enhanced service experience. Moreover we also prove the feasibility of the solution in a practical environment, and provide future research directions that bene t the evolved control framework and make it commercially feasible.

Resistance to last-resort antibiotics in natural environments

Last-resort antibiotics are the final line of action for treating serious infections caused by multiresistant strains. Over the years the prevalence of resistant bacteria has been increasing. Natural environments are reservoirs of antibiotic resistance, highly influenced by human-driven activities. The importance of aquatic systems on the evolution of antibiotic resistance is highlighted from the assumption that clinically-relevant resistance genes have originated in strains ubiquitous in these environments. We hypothesize that: a) rivers are reservoirs and disseminators of antibiotic resistance; b) anthropogenic activities potentiate dissemination of resistance to last-resort antibiotics. Hence, the main goal of the work is to compare the last-resort antibiotics resistome, in polluted and unpolluted water. Rivers from the Vouga basin, exposed to different anthropogenic impacts, were sampled. Water quality parameters were determined to classify rivers as unpolluted or polluted. Two bacterial collections were established enclosing bacteria resistant to cefotaxime (3rd generation cephalosporin) and to imipenem (carbapenem). Each collection was characterized regarding: phylogenetic diversity, antibiotic susceptibility, resistance mechanisms and mobile genetic elements. The prevalence of cefotaxime- and imipenem-resistant bacteria was higher in polluted water. Results suggested an important role in the dissemination of antibiotic resistance for Enterobacteriaceae, Pseudomonas and Aeromonas. The occurrence of bacteria resistant to non-beta-lactams was higher among isolates from polluted water as also the number of multiresistant strains. Among strains resistant to cefotaxime, extended-spectrum beta-lactamase (ESBL) genes were detected (predominantly blaCTX-M-like) associated to mobile genetic elements previously described in clinical strains. ESBL-producers were often multiresistant as a result of co-selection mechanisms. Culture-independent methods showed clear differences between blaCTX-M-like sequences found in unpolluted water (similar to ancestral genes) and polluted water (sequences identical to those reported in clinical settings). Carbapenem resistance was mostly related to the presence of intrinsically resistant bacteria. Yet, relevant carbapenemase genes were detected as blaOXA-48-like in Shewanella spp. (the putative origin of these genes), and blaVIM-2 in Pseudomonas spp. isolated from polluted rivers. Culture-independent methods showed an higher than the previously reported diversity of blaOXA-48-like genes in rivers. Overall, clear differences between polluted and unpolluted systems were observed, regarding prevalence, phylogenetic diversity and susceptibility profiles of resistant bacteria and occurrence of clinically relevant antibiotic resistance genes, thus validating our hypotheses. In this way, rivers act as disseminators of resistance genes, and anthropogenic activities potentiate horizontal gene transfer and promote the constitution of genetic platforms that combine several resistance determinants, leading to multiresistance phenotypes that may persist even in the absence of antibiotics.

Integrated coastal geoengineering approach for maritime environments

This dissertation introduces several methodological approaches which integrate a proposed coastal management model in an interdisciplinary perspective. The research presented herein is displayed as a set of publications comprising different thematic outlooks. The thesis develops an integrated coastal geoengineering approach which is intrinsically linked to the studied maritime environments. From sandy coasts and marine works to rocky platforms and sea cliffs, this study includes field work between Caminha – Figueira da Foz (NW Portugal) and Galicia (NW Spain). The research also involves an analysis and geological-geotechnical characterisation of natural rock (armourstone) and artificial units (concrete blocks) applied to coastal structures. The main goal is to contribute to the characterisation and re-evaluation of georesources and to determine armourstone suitability and availability from its source (quarry). It was also important to diagnose the geomaterials in situ concerning their degradation/deterioration level on the basis of the current status of the coastal protection works in order to facilitate more efficient monitoring and maintenance, with economic benefits. In the rocky coast approach the coastal blocks were studied along the platform, but also the geoforms were studied from a coastal morphodynamics point of view. A shoreline evolution analysis was developed for sandy coasts through Digital Shoreline Analysis System (DSAS) extension. In addition, the spatial and statistical analysis applied to sea cliffs allowed the establishment of susceptibility zones to erosion and hazardous areas. All of these studies have different purposes and results however, there is a common denominator – GIS mapping. Hence, apart from the studied coastal environment, there is an integrated system which includes a sequence of procedures and methodologies that persisted during the research period. This is a step forward in the study of different coastal environments by using almost the same methodologies. This will allow the characterisation, monitoring and assessment of coastal protection works, rocky coasts, and shore platforms. With such data, it is possible to propose or recommend strategies for coastal and shoreline management based on several justifications in terms of social, economic, and environmental questions, or even provide a GIS-based planning support system reinforced by geocartographic decisions. Overall the development of the applied cartography embraces six stages which will allow the production of detailed maps of the maritime environment: (1) high-resolution aerial imagery surveys; (2) visual inspection and systematic monitoring; (3) applied field datasheet; (4) in situ evaluation; (5) scanline surveying; and (6) GIS mapping. This thesis covers fundamental matters that were developed over the course of scientific publication and as a consequence they represent the results obtained and discussed. The subjects directly related to the thesis architecture are: (i) cartography applied to coastal dynamics (including an art historical analysis as a tool to comprehend the coastal evolution and the littoral zone); (ii) georesources assessment (the role of cartography in georesources zoning, assessment and armourstone durability); (iii) coastal geoengineering applications and monitoring (Espinho pilot site in NW Portugal as an experimental field); (iv) rocky coast and shore platform studies and characterisation; (v) sandy and mixed environment approaches; (vi) coastal geosciences GIS mapping and photogrammetric surveying (coastal geoengineering); and (vii) shoreline change mapping and coastal management strategies (the CartGalicia Project as an example – NW Spain). Finally, all of these thematic areas were crucial to generate the conceptual models proposed and to shape the future of integrated coastal coastal geoengineering management.