A primer for use of genetic tools in selecting and testing the suitability of set-aside sites protected from deep-sea seafloor massive sulfide mining activities

Seafloor massive sulfide (SMS) mining will likely occur at hydrothermal systems in the near future. Alongside their mineral wealth, SMS deposits also have considerable biological value. Active SMS deposits host endemic hydrothermal vent communities, whilst inactive deposits support communities of deep water corals and other suspension feeders. Mining activities are expected to remove all large organisms and suitable habitat in the immediate area, making vent endemic organisms particularly at risk from habitat loss and localised extinction. As part of environmental management strategies designed to mitigate the effects of mining, areas of seabed need to be protected to preserve biodiversity that is lost at the mine site and to preserve communities that support connectivity among populations of vent animals in the surrounding region. These "set-aside" areas need to be biologically similar to the mine site and be suitably connected, mostly by transport of larvae, to neighbouring sites to ensure exchange of genetic material among remaining populations. Establishing suitable set-asides can be a formidable task for environmental managers, however the application of genetic approaches can aid set-aside identification, suitability assessment and monitoring. There are many genetic tools available, including analysis of mitochondrial DNA (mtDNA) sequences (e.g. COI or other suitable mtDNA genes) and appropriate nuclear DNA markers (e.g. microsatellites, single nucleotide polymorphisms), environmental DNA (eDNA) techniques and microbial metagenomics. When used in concert with traditional biological survey techniques, these tools can help to identify species, assess the genetic connectivity among populations and assess the diversity of communities. How these techniques can be applied to set-aside decision making is discussed and recommendations are made for the genetic characteristics of set-aside sites. A checklist for environmental regulators forms a guide to aid decision making on the suitability of set-aside design and assessment using genetic tools. This non-technical primer document represents the views of participants in the VentBase 2014 workshop.

Chemical composition and pharmacological activities of essential oils of Lavandula spp.

Lavandula spp. belong to the family Lamiatae and some species are often used in popular medicine and have been used for centuries in a large number of medical applications and in aromatherapy. Although similar ethnobotanical properties of Lavandula spp., its essential oils, general chemical composition and therapeutic applications differ from different species. Lavandula stoechas L. subsps. luisieri (Rozeira) Rozeira and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Southern Alentejo and Algarve. The aim of our study was evaluate the chemical composition and toxicological and pharmacological activities of leaves essential oils of spontaneous plants of L. stoechas L. subsps. luisieri (Alentejo) and L. viridis (Algarve). The essential oils of these wild plants, collected in spring, were obtained by hydrodistillation in a Clevenger-type apparatus and its chemical composition was evaluated by GC/FID. The acute toxicity of essential oils was evaluated "in vitro" using brine shrimp (LC50) and "in vivo" using Swiss mice (DL50). The analgesic and anti-inflammatory pharmacological properties of L. stoechas subsp. luisieri essential oil were evaluated in mouse or rats by the Amour-Smith and carrageen-induced paw edema tests, respectively. Results showed important differences in chemical composition of essential oils from two species analyzed either to diversity and proportion of its constituents. The essentials oils showed citotoxicity against Artemia salina and a DL50 higher than 2000 mg/kg for mice. The analgesic and anti-inflammatory activities of essential oils were exhibit for the doses of 100 and 200 mg/kg.

CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Pine wilt disease and the pinewood nematode

Pine wilt disease (PWD) is one of the most damaging events affecting conifer forests (in particular Pinus spp.), in the Far East (Japan, China and Korea), North America (USA and Canada) and, more recently, in the European Union (Portugal). In Japan it became catastrophic, damaging native pine species (Pinus thunbergii and P. densiflora), and becoming the main forest problem, forcing some areas to be totally replaced by other tree species. The pine wilt nematode (PWN) Bursaphelenchus xylophilus, endemic, with minor damage, to North America, was introduced in Japan in the early XX century and then spread to Asia (China and Korea) in the 1980s. In 1999 it was detected for the first time in Portugal, where, due to timely detection and immediate government action, it was initially (1999-2008) contained to a small area 30 km SE of Lisbon. In 2008, the PWN spread again to central Portugal, the entire country now being classified as “affected area”. Being an A1 quarantine pest, the EU acted to avoid further PWN spreading and to eradicate it, by actions including financial support for surveyes and eradication, annual inspections and research programs. Experience from control actions in Japan included aerial spraying of insecticides to control the insect vector (the Cerambycid beetle Monochamus alternatus), injection of nematicides to the trunk of infected trees, slashing and burning of large areas out of control, beetle traps, biological control and tree breeding programs. These actions allowed some positive results, but also unsuccessful cases due to the PWN spread and virulence. Other Asian countries also followed similar strategies, but the nematode is still spreading in many regions. In Portugal, despite lower damage than Asia, PWD is still significant with high losses to the forestry industry. New ways of containing PWD include preventing movement of contaminated wood, cutting symptomatic trees and monitoring. Despite a national and EU legislative body, no successful strategy to control and eventually eradicate the nematode and the disease will prevail without sound scientific studies regarding the nematode and vector(s) bioecology and genetics, the ecology and ecophysiology of the pine tree species, P. pinaster and P. pinea , as well as the genomics and proteomics of pathogenicity (resistance/ susceptibility).

Estratégias para a valorização do coberto vegetal da Ilha de Porto Santo

No presente trabalho desenvolveram-se estudos visando a valorização do coberto vegetal da Ilha de Porto Santo, através de duas metodologias de investigação complementares: a) preservação e reintrodução na Ilha de uma espécie endémica e em risco do Arquipélago da Madeira (Olea maderensis) e de uma espécie naturalizada (Olea europaea ssp. europaea var. sylvestris), recorrendo para o efeito a técnicas de biotecnologia (micropropagação); b) análise da percepção da comunidade local e visitante sobre o fenómeno da desertificação e a valorização do coberto vegetal bem como a sua aceitação relativamente à aplicação de técnicas de biotecnologia (para micropropagar e reintroduzir espécies de oliveira na Ilha) para minimização do processo de desertificação. A dissertação estrutura-se em quatro partes principais. A Parte I caracteriza a Ilha de Porto Santo em termos geográficos, geológicos, climáticos, sócio-economicos e do uso do solo. Enquadra, ainda, o problema da desertificação, através da caracterização/evolução do coberto vegetal ao longo dos anos. Finalmente apresentam-se os objectivos gerais deste estudo. A Parte II centra-se no desenvolvimento de metodologias no âmbito da biotecnologia vegetal para propagação de espécies de O. maderensis e O. europaea ssp. europaea var. sylvestris. em larga escala. Esta parte está dividida em seis capítulos. O Capítulo II.1 aborda a distribuição geográfica das espécies de oliveira e faz uma revisão bibliográfica dos aspectos mais importantes da micropropagação de oliveira (O. europaea L.), principalmente através da micropropagação por estimulação de gomos axilares. No final deste capítulo apresentam-se os objectivos específicos desta investigação. No Capítulo II.2 faz-se a caracterização genética de genótipos de O. maderensis do Arquipélago da Madeira através da análise da ploidia e do conteúdo em DNA por citometria de fluxo (FCM) e através da detecção de polimorfismos por análise de microssatélites (SSR). Nesta análise usaram-se ainda outros genótipos, nomeadamente: O. europaea ssp. europaea var. sylvestris, O. cerasiformes e O. europaea ssp. europaea var. europaea. Este estudo contribuiu para uma melhor caracterização desta espécie e permitiu a detecção de um nível de ploidia novo no género Olea (tetraploidia). O Capítulo II.3 descreve a optimização das condições de cultura in vitro (e.g. desinfecção, meio de cultura e enraizamento) para propagar e preservar a O. maderensis. Avalia-se ainda a “performance” dos rebentos in vitro (taxas de crescimento, avaliação da aparência das folhas e estudos fisiológicos), de modo a confirmar a optimização das condições de propagação. Neste capítulo define-se um meio novo (OMG) para propagação desta espécie endémica. O Capítulo II.4 descreve dois protocolos de micropropagação e aclimatização de ambas as espécies (O. maderensis e O. europaea ssp. europaea var. sylvestris) e a qualidade das plantas (“true-to-type”) é avaliada através da possível ocorrência de variabilidade genética através de FCM (ploidia) e SSRs. O Capítulo II.5 descreve um protocolo eficiente de aclimatização ao campo de O. maderensis e avalia a “performance” das plantas micropropagadas no campo através da análise de parâmetros fisiológicos durante o processo. O Capítulo II.6 apresenta os estudos em curso relativamente às plantas de O. maderensis em aclimatização no campo, bem como a introdução de plantas micropropagadas num outro local da Ilha com um maior grau de degradação dos solos. Estas estratégias estão a ser aplicadas juntamente com a DRFRAM, no âmbito de programas de florestação em curso. Finalmente é realçada a necessidade de estudos semelhantes com outras espécies nativas. Na Parte III, são apresentados os estudos sobre a percepção da comunidade local relativamente à valorização do coberto vegetal para a minimização dos processos de degradação dos solos/desertificação. A introdução faz o enquadramento teórico sobre o fenómeno da desertificação, particularmente na Ilha de Porto Santo e sobre a percepção social da desertificação. São ainda apresentados os objectivos específicos desta investigação. A metodologia adoptada recorreu à aplicação de inquéritos por questionário à população residente e aos visitantes da Ilha de Porto Santo e ainda a realização de inquéritos por entrevista a algumas entidades e especialistas. Estes estudos permitiram verificar que existe uma nítida consciência da situação de risco da ilha, das medidas tomadas e a tomar e da premência da resolução do problema. Face ao recurso de estratégias alternativas envolvendo biotecnologia, e apesar de existir algum desconhecimento, concluiu-se ainda que a população manifesta aceitação, desde que essas estratégias valorizem o coberto vegetal e, assim, ajudem a combater a degradação biofísica dos solos. Finalmente são apresentadas as conclusões e algumas recomendações. Na Parte IV apresentam-se as conclusões gerais e perspectivas futuras, onde o potencial ambiental destas oliveiras bravas micropropagadas é destacado, bem como é considerado o alargamento da aplicação destas estratégias a outras espécies indígenas em risco, nesta Ilha (e noutros locais). São ainda resumidas as principais visões da população e das entidades e dos especialistas que poderão contribuir para apoiar a elaboração de medidas de mitigação e prevenção no combate ao processo de degradação dos solos/desertificação em curso.

Assessment of the genetic diversity of the pinewood nematode, Bursaphelenchus xylophilus, in Portugal

O nemátode da madeira do pinheiro (NMP), Bursaphelenchus xylophiius, tem uma extensa distribuição na América do Norte, e encontra-se atualmente distribuído ao longo da maioria dos territórios de Canadá e dos Estados Unidos. Durante o último século, esta espécie foi transportada pelo Homem para outras regiões do mundo (não-nativas), associadas com o comércio e o fluxo global de produtos de origem florestal. Atualmente, esta espécie invasiva está reportada para algumas regiões do SE asiático (China, Japão, Coreia e Taiwan) e mais recentemente para a Europa (Portugal). Devido ao impacto que este organismo agente da doença da murchidão dos pinheiros causa nas florestas nativas destas regiões esta espécie assume uma elevada importância económica a nível mundial Em Portugal, a distribuição do NMP encontra-se confinada a uma área restrita e limitada (500 000 ha), a sul de Lisboa (península de Setúbal); contudo, constitui uma das maiores ameaças às florestas de pinheiro do país e da UE. Ate recentemente, nenhum consenso existia quanto à origem do NMP em Portugal. Diversas hipóteses têm sido colocadas para explicar esta introdução, nomeadamente a partir de zonas onde o nematode ocorre naturalmente (América do Norte), ou de outras áreas (não-nativas) onde o nematode se comporta como uma espécie invasiva (Leste da Ásia). A fim de avaliar a variabilidade genética do NMP proveniente da área afetada em Portugal, foram utilizadas várias técnicas moleculares, designadamente o random amplified polymorphic DNA (RAPD-PCR) e o satellite DNA (satDNA). No caso do RAPD-PCR, foram utilizados 24 isolados do NMP provenientes de Portugal, 1 proveniente da América do Norte e 1 da Ásia, tendo sido utilizado como out-group um isolado de B. mucronatus. A partir dos 28 RAPD primers utilizados obtiveram-se 640 fragmentos. No caso do satDNA, foram utilizados 21 isolados do NMP provenientes de Portugal, obtendo-se no total 206 sequências da família MspI. Ambos os métodos revelaram uma elevada similaridade genética entre os vários isolados do NMP da área afetada em Portugal O nível reduzido de diversidade genética obtido entre os isolados portugueses do NMP, permite concluir que se trata de uma única introdução deste organismo em Portugal, e proveniente de uma região asiática. A inexistência de uma de correlação entre a variabilidade genética e a distribuição geográfica do NMP dentro da área afetada em Portugal, indica que o NMP se encontra distribuído de forma uniforme ao longo de toda a área afetada, provavelmente relacionado com a distribuição e a expansão natural do inseto vector. The pinewood nematode (PWN), Bursaphelenchus xylophilus, has a wide distribution in North America, and is present throughout most of the territories of Canada and the United Stata. During the last century, this species has been transported by man to several non-native regions of the world, associated with trade and the global flow of forest products. Up to date, this invasive species has been reported from Asia (PR China, Japan, Korea and Taiwan) and more recently in Europe (Portugal). Due to the impact on native pine forests of these regions, this nematode species, the causal agent of pine wilt disease, is of great economic importance worldwide. In Portugal, the distribution of the PWN has been constrained to a relatively small area (500 000 ha) in the south of Lisbon (Setúbal Peninsula); however, it has become the most serious threat to pine forests in the country. Until recently, no consensus had emerged on the possible pathway of the PWN introduction in Portugal. Several hypotheses have been put forward to explain this introduction, such as an origin from endemic areas where the nematode naturally occurs (North America), or non-endemic areas where the nematode behaves as an exotic pest (East Asia). Random amplified polymorphic DNA (RAPD-PCR) and satellite DNA (satDNA) techniques were used in order to assess the level of genetic variability and genetic relationships, among several isolates of the PWN, representative of the entire affected area in Portugal. In the case of RAPD-PCR, 24 Portuguese isolates, plus two additional isolates of B. xylophilus, representing North America and East Asia were included. B. mucronatus was used as an out-group. Twenty-eight random primers generated a total of 640 DNA fragments. With satDNA, 206 Mspl sequence repeats were obtained from 21 Portuguese isolates of B. xylophilus. Both molecular methods revealed a high genetic similarity among the Portuguese isolates, and the low level of genetic diversity strongly suggests that they were dispersed recently from a single introduction, and from East Asia. The lack of apparent relationship between the genetic variability and the geographic distribution of the PWN within the affected area, suggests that the recent introduction of this pest (and pathogen) in Portugal has been uniformly distributed since its establishment, probably following the natural distribution and expansion of the insect vector.

Carcinoma da nasofaringe: aspectos epidemiológicos clínicos e moleculares

Após uma revisão de aspectos filogénicos, ontogénicos e embriológicos contribuindo para a estruturação anatómica e fisiológica da nasofaringe, é efectuada uma análise das características clínicas do carcinoma da nasofaringe numa população portuguesa, comparando-as com a literatura. É efectuada análise de taxa de incidência da doença, razão de géneros, frequência relativa dos diversos subtipos de acordo com a classificação OMS, acuidade relativa dos estadiamentos TMN e sobrevivência em função do tratamento. A relação entre carcinoma da nasofaringe e infecção pelo vírus de Epstein – Barr em Portugal é estudada através da análise de detecção de DNA de EBV em tecido tumoral da nasofaringe e sangue periférico de doentes com NPC e em indivíduos saudáveis. São também efectuados estudos caso-controlo no sentido de perspectivar a relevância de dois polimorfismos genéticos na susceptibilidade genética para a doença. Esta Dissertação pretende ainda contribuir para a compreensão dos mecanismos biológicos de CN e a sua relação com o EBV numa região não endémica de baixo risco, como é Portugal, realçando a relevância da definição de um perfil biológico preditivo para o desenvolvimento de CN na população portuguesa.

Relapsing Fever Borreliae: A Global Review

Relapsing fever borreliae were notorious and feared infectious agents that earned their place in history through their devastating impact as causes of both epidemic and endemic infection. More recently they are considered more as an oddity and their burden of infection is largely overshadowed by other infections such as malaria, which presents in a similar clinical way. Despite this, they remain the most common bacterial infection in some developing countries. Transmitted by soft ticks or lice, these fascinating spirochaetes have evolved a myriad of mechanisms to survive within their diverse environments.

A Case of Canine Borreliosis in Iran Caused by Borrelia persica

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by B. persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with Borrelia persica sequences derived from clinical cases or from Ornithodoros tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbour minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks.

Temperatura e luz na germinação do endemismo algarvio Tuberaria major

Dissertação mest., Gestão Sustentável de Espaços Rurais, Universidade do Algarve, 2009

Biocaumulação de alumínio em rebentos micropropagados de Plantago sp.

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2009

Biogeography of the fire salamander (Salamandra salamandra, L.) in Portugal

Dissertação de mest., Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Assessing the effects of pseudo-absences on predictive distribution model performance

Modelling species distributions with presence data from atlases, museum collections and databases is challenging. In this paper, we compare seven procedures to generate pseudoabsence data, which in turn are used to generate GLM-logistic regressed models when reliable absence data are not available. We use pseudo-absences selected randomly or by means of presence-only methods (ENFA and MDE) to model the distribution of a threatened endemic Iberian moth species (Graellsia isabelae). The results show that the pseudo-absence selection method greatly influences the percentage of explained variability, the scores of the accuracy measures and, most importantly, the degree of constraint in the distribution estimated. As we extract pseudo-absences from environmental regions further from the optimum established by presence data, the models generated obtain better accuracy scores, and over-prediction increases. When variables other than environmental ones influence the distribution of the species (i.e., non-equilibrium state) and precise information on absences is non-existent, the random selection of pseudo-absences or their selection from environmental localities similar to those of species presence data generates the most constrained predictive distribution maps, because pseudo-absences can be located within environmentally suitable areas. This study showsthat ifwe do not have reliable absence data, the method of pseudo-absence selection strongly conditions the obtained model, generating different model predictions in the gradient between potential and realized distributions.

In vitro spore germination of Polystichum drepanum, a threatened fern from Madeira Island

Polystichum drepanum (Sw) C. Presl is a threatened fern endemic to a few forest areas in the north-west of Madeira Island. The aims of this work were to establish suitable culture conditions for in vitro germination of spores, and to evaluate short-term storage conditions for P drepanum spores. The highest frequency of germination was obtained in Murishage and Skoog (MS) liquid medium, without agitation. However, gametophytes maintained in MS liquid medium did not grow and, after 4 weeks, became anoxic and died. Thus, after germination in liquid medium, gametophytes were transferred to an MS double-phase culture system for further growth. The effects of storage period, temperature, and relative humidity during storage on in vitro spore germination were studied. Spore viability was assessed after 2, 4 and 6 months, and high viability (> 94%) was observed in all the assays. However, germination capability decreased with increased storage periods. The number of sporophytes obtained also decreased with prolonged storage periods. The results indicate that spores of R drepanum stored for 4 months at 21 degrees C maintain high viability and high germination frequency. ne sporophytes obtained were acclimatised in a mixture of peat and vermiculite [2:1 (v/v)] under high relative humidity (90-95%). Seventy-five sporophytes were successfully acclimatised to ex vitro conditions and showed active growth in the glasshouse.

Synthesis and computer-assisted design of mitochondrial electron transport-chain inhibitors as antimalarial agents

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014