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.

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.