Centro de recursos biológicos : novas exigências e oportunidades para a bioeconomia

Desde 1982 que as colecções de culturas microbianas trabalham em conjunto para implementarem uma política comum, partilharem tecnologias e procurarem projectos colaborativos. Adicionalmente, o Grupo de Trabalho em Biotecnologia da Organização para a Cooperação e o Desenvolvimento Económico (OCDE) tem defendido os centros de recursos biológicos (CRBs) como elementos chave na infra-estrutura científica e tecnológica das ciências da vida e biotecnologia. A OCDE, em 2001, publicou um relatório que enfatiza o potencial dos CRBs onde recomenda a criação de uma rede global de CRBs. Na segunda fase desta iniciativa, a OCDE encarregou um grupo de missão com a tarefa de desenvolver um plano de implementação das recomendações enunciadas no relatório. Isto incluiu, (i) normas de funcionamento comum, (ii) normas para ligação e trocas de informação, (iii) acções apropriadas para a segurança, (iv) regulação sobre a gestão da arquitectura institucional e (v) financiamento. Após este trabalho terminado, em 2009 lançou-se o projecto demonstrativo da rede global de CRBs (GBRCN, www.gbrcn.org). Este projeto conta com 15 países e trabalha para aumentar a eficiência das colecções microbianas com vista a garantir padrões de qualidade adequados para que as colecções possam trabalhar em rede. Adicionalmente, a Comissão Europeia apostou no financiamento do projecto europeu do consórcio de centros de recursos microbianos (EMbaRC, www.embarc.eu) como forma de fortalecer a Europa para a bioeconomia e a competitividade. Nesta sequência de esforços e exigências globais e europeias o fornecimento de material biológico de qualidade e com garantia de autenticidade passou a ser, concomitantemente, uma exigência central para as colecções. Em resposta a este contexto, a Micoteca da Universidade do Minho (MUM, www.micoteca.deb.uminho.pt) tem vindo a desenvolver critérios internos de gestão e a aplicar novas tecnologias de identificação, como é o caso do MALDI-TOF ICMS (matrixassisted laser desorptionionisation time-of-flight intact cell mass spectrometry), para garantir uma crescente qualidade e autenticidade dos recursos biológicos que preserva e fornece

Stability based on a polyphasic approach of fungal samples preserved on alginate

Alginate-encapsulation is a commonly used, simple and cost effective method to preserve plant samples. Since alginate has been proven to protect tissues against physical and environmental damage, minimising dehydration, it is considered a good preservation technique. The application of this method for the preservation of filamentous fungi was intended to present an alternative to the commonly used preservation methods, especially for recalcitrant fungi. MALDI-TOF MS emerged in the late 1980s as a sound technique to investigate the mass spectrometry of molecular high-mass of organic compounds through a soft ionisation of molecules resulting in minimum fragmentation. This technique has high potential for the identification of filamentous fungi species and occasionally strains. One of the most interesting advantages of the technique is the analysis of intact fungal cells thereby generating peptides and proteins profiles. A novel technique was applied to the preservation of Botrytis cinerea (MUM 10.163, 10.165, 10.167), Aspergillus ibericus (MUM 04.68) and Aspergillus brasiliensis (MUM 06.181) using alginate encapsulation, under two conditions: distilled water (I) and 10% glycerol (II), both at 4°C for 1 year after which the viabilities were studied. A comparison with Castellani preservation in water (III) was made, using viability test, colonial morphology and MALDI-TOF MS analysis for the evaluation of the preservation methods. The strains preserved by condition (I) presented lower viability than those preserved by condition (II). In addition, when comparing the results from the samples preserved by (III) with the ones encapsulated and maintained in 10% glycerol (II), we noted that the latter presented a higher viability, faster growth and health colony formation. The MALDI-TOF MS analysis indicated that the strains clustered according to species. For B. cinerea only small spectral differences (< 5%) were presented in the percentages of similarity, which is in the tolerance range of the technique, except for MUM 10.167 (>5%) when as (III). In the present study we can conclude that the success of the preser

Assessment of fungi stability by MALDI-TOF ICMS following preservation on alginate encapsulated samples

Alginate-encapsulation is a commonly used, simple and cost effective, method to preserve plant samples. Since alginate has proven to protect tissues against physical and environmental damage, minimising dehydration, it presents as a good preservation technique. The application of this method for the preservation of filamentous fungi was intended to present an alternative for the commonly used preservation methods, especially to be used on recalcitrant fungal strains. Matrix-assisted laser desorption⁄ionisation time-of-flight intact cell mass spectrometry (MALDI-TOF) emerged in the late 1980s as a sound technique to investigate the mass spectrometry of molecular high-mass of organic compounds through a soft ionisation of molecules resulting in minimum fragmentation. This technique has demonstrated its high potentiality for identification of filamentous fungi species and, in some specific cases, for strain identification. One of the most interesting advantages of the technique is the possibility of analysing the intact fungal cell generating peptides and proteins profiles. A novel technique was applied on the preservation of Botrytis cinerea (MUM 10.163, 10.165, 10.167), Aspergillus ibericus (MUM 04.68) and Aspergillus brasiliensis (MUM 06.181) using the methodology of alginate encapsulation, in two different conditions: distilled water (I) and a 10% glycerol solution (II), both at 4 ºC for 1 year; the viability of these strains was studied. The assessment was made by comparison with the method of Castellani preservation in water (III), using morphologic and MALDI-TOF ICMS analyses for the analysis of the 3 preservation methods. The encapsulated samples of the strains preserved in distilled water (I), presented lower viability than those preserved in 10% glycerol (II). However, when comparing the growth from the samples preserved in water (III) with the ones encapsulated and maintained in 10% glycerol (II), we noted that the last ones presented a healthier and faster growth. From the MALDI-TOF ICMS analysis, it was observed that the strains were clustered according to species identification, and for Botrytis cinerea all presented small differences (< 5%) in the percentages of similarity, except for MUM 10.167 (>5%) when preserved in water (III). From our evaluation we were able to conclude that the success of the preservation method is strain dependent.European Community’s Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228310 (EMbaRC project), FCT – Portugal for the scholarship SFRH/BD/64260/2009

The importance of structural diversity of spores in the taxonomy of Aspergillus (section Nigri)

Species of Aspergillus from section Nigri, also known as black aspergilli, are distributed worldwide and have been widely used for purposes of various types such as: biotechnological, industrial, medical, among others. They have been extensively studied for being the causing agents of biodeterioration of commodities and food. Within this section, new species have been recently described and among them Aspergillus ibericus and Aspergillus uvarum were both isolated from grapes. The polyphasic approach used in the analysis of these species, either by morphological techniques as well as by molecular methods, allowed not only their characterisation but also their consequent separation from all others in the section. Microbial taxonomy has the identification of species as one of its fundamental goals. Data such as: morphological characteristics description, physiological and biochemical properties, ecological roles, and societal risks or benefits, are key elements in any fungal identification process. But, this process is subjected to periodic changes due to frequent revisions of the taxonomic schemes, therefore becoming time consuming and more demanding and difficult, even for skilled specialists. Furthermore, each taxonomic group has specialised literature, terminology and characters. This takes place since identifications have difficulties of consensual naming, depending on the criteria used and the amount of information available when producing all data. It is increasingly becoming clear that, to better achieve a more accurate concept of species, microbial identification and authentication require a polyphasic approach to produce consistent, useful and quality data. Characterisation of morphologic and structural aspects of spores from Aspergillus strains, section Nigri, has been carried out using scanning electron microscopy (SEM) and intends to contribute to the associated data of the strains from this section. Colonies from 13 Aspergillus from section Nigri, from the Micoteca da Universidade do Minho (MUM), were grown at 25 ºC for 3 or 4 days in malt extract agar directly mounted in a sterilised SEM stub. The samples were covered with a mixture of gold and platinum (80/20%) and then examined in the SEM [NanoSEM - FEI NovaTM 200 (FEG/SEM); EDAX - Pegasus X4M (EDS/EBSD)]. A stereomicroscope (Leica MZ12.5) was used to have a clear image comparison of the size of the conidial head. Although the analysed strains presented dimensional, morphological and structural diversity, common or typical features could be inferred and related to each taxon like those represented in Fig 1. From the SEM analysis we were able to conclude that in the section Nigri of genus Aspergillus the spore wall ornamentation, and its size and shape continue to present themselves as important primary diagnostic traits for species differentiation

Quality parameters in a culture collection - Micoteca da Universidade do Minho

Tese de doutoramento em Engenharia Química e BiológicaThe biological diversity is quite important to the world, in a social, industrial, economic and scientific point of view. Nowadays it is mandatory to assure and guarantee biodiversity conservation, its success, sustainable use and the equitable share of benefits arising from the use of genetic resources through: ethical sourcing practices, and collaborations between the different Biological Resource Centres (BRCs). It is now the era of awareness of quality assurance. This work presents ways to contribute and increase the quality, knowledge, information, maintenance and preservation of biological resources, applied to filamentous fungi. The achievement of quality within the BRCs and culture collections (CC) context is a dynamic process, always evolving as well as the backlog and build-up of biological resources data that increase with each research made. An option to achieve quality is the implementation of a Quality Management System (QMS) based on the standard ISO 9001:2008, like the one here described and explained on a CC of filamentous fungi: Micoteca da Universidade do Minho (MUM). The QMS implemented at MUM in 2011 and the obtained certification, are a continuous improvement process focused on customer satisfaction. Maintenance of biological resources implies the choice of the best methods and constant search of cheaper, faster and more practical, with assured procedures and validation of preservation success. With this purpose, an assessment of preservation methods was performed, through a polyphasic approach using several techniques of fungal characterisation [macroscopic (photography), microscopic (optical, stereomicroscopy and SEM), mycotoxin screening (HPLC), enzymatic screening (spectrophotometry and specific inducer media), MALDI-TOF MS and molecular biology analysis]. It was found that long-term stored lyophilized samples were not significantly altered making this method one of the most appropriate in the case of filamentous fungi preservation. The minor changes observed were dependent on the strain to preserve. In the same context of the continuous strive for improvement, a proposal to develop new methods for the preservation of strains, specially delicate and/or recalcitrant strains of fungi was made. Perlite and alginate encapsulation were used in a specific group of selected fungal strains. For the conditions chosen, after morphological and MALDI-TOF MS analysis, it was found that perlite does not allow the viability of samples after preservation whether alginate encapsulation proved to be a better alternative to the common and well known Castellani method, i.e., preservation in water.A diversidade biológica é muito importante do ponto de vista social, industrial, económico e científico. Assegurar e garantir a conservação da diversidade biológica, o seu uso sustentável e a repartição equitativa dos benefícios resultantes da utilização dos recursos genéticos, através de: práticas de abastecimento éticas e colaborações entre os diferentes Centros de Recursos Biológicos (BRCs), é um requisito actual. Este trabalho pretende apresentar formas de contribuir e aumentar a qualidade, conhecimento, informação, manutenção e conservação de fungos filamentosos. A obtenção de qualidade dentro do contexto dos BRCs e Colecções de Cultura (CCs) é um processo dinâmico, em constante evolução, bem como o aumento crescente e acumulação de dados sobre os recursos biológicos que aumentam a cada pesquisa feita. Uma opção para alcançar a qualidade é a implementação de um Sistema de Gestão da Qualidade (SGQ) baseado na norma ISO 9001:2008, como o aqui descrito e explicado para uma CC de fungos filamentosos: Micoteca da Universidade do Minho (MUM). O SGQ implementado na MUM em 2011 e a certificação obtida, são um processo de melhoria contínua focado na satisfação do cliente. A manutenção dos recursos biológicos implica a escolha dos melhores métodos e a busca constante de outros mais baratos, rápidos, práticos, com procedimentos assegurados e validados. Com esta finalidade, foi realizada uma avaliação de métodos de preservação, através de uma abordagem polifásica com o uso de várias técnicas de caracterização fúngicas [macroscópica (fotografia), microscópica (óptica, esteromicroscópica e SEM), rastreio de micotoxinas, rastreio de produção enzimática, MALDI-TOF MS e análise de biologia molecular]. Verificou-se que as amostras liofilizadas armazenadas por longos períodos não sofrem grandes alterações o que torna este método como um dos mais apropriados na preservação de fungos filamentosos. As poucas alterações observadas são dependentes da estirpe a preservar. Foi ainda realizada uma proposta de desenvolvimento de novos métodos para a preservação de estirpes fúngicas, delicadas e/ou recalcitrantes. Foram testados: perlite e encapsulamento em alginato. Verificou-se que a perlite não permite a viabilidade das amostras após preservação, mas o encapsulamento em alginato demonstrou, após análise morfológica e por MALDI-TOF, ser uma boa alternativa ao método de preservação em água, ou seja: método Castellani

Avaliação da correlação da interposição lingual e ou hábitos de sucção e maloclusões na dentição decídua e mista : estudo preliminar de uma amostra de pacientes odontopediátricos na Clínica Dentária da UCP

Introdução: O crescimento e desenvolvimento craniofacial é resultante da interacção de mecanismos genéticos, hormonais e neurológicos, influenciados por factores locais, como os hábitos orais. A oclusão normal, para além de depender de um adequado crescimento da face, depende também da angulação e morfologia dos dentes, dos músculos da mastigação e dos movimentos funcionais. A influência de factores locais pode originar deformações ósseas, maloclusões e alteração de funções tais como a respiração, a mastigação, a deglutição, a sucção e a dicção. Assim, torna-se importante a prevenção das potenciais alterações advindas dos hábitos orais através do diagnóstico precoce e do tratamento preventivo/interceptivo. O objectivo principal do presente estudo consistiu em detectar a correlação entre os hábitos de sucção e hábitos de interposição e maloclusões numa amostra de pacientes odontopediátricos, no estádio de dentição decídua e mista. Materiais e métodos: Para a realização deste estudo foi seleccionada uma amostra populacional dos pacientes que compareceram às consultas de Odontopediatria da Clínica Dentária da Universidade Católica de Viseu – Pólo de Viseu, entre 14 de Fevereiro e 21 de Junho. Após a aplicação dos critérios de inclusão e exclusão seleccionou-se uma amostra de 68 pacientes. Para a recolha de dados foi desenvolvido um questionário que engloba parâmetros tendentes a estabelecer uma correlação entre variáveis, não comparadas previamente. Com este questionário pretendeu avaliar-se se o paciente foi amamentado e qual a duração da amamentação, se foi alimentado por biberão, se tinha algum hábito de sucção e qual a duração deste hábito, se tinha algum hábito de interposição (interposição lingual, interposição labial e deglutição atípica), o tipo de respiração, a presença de amígdalas e adenóides, o trespasse horizontal e o trespasse vertical, a existência de mordida aberta ou cruzada, a classe de Angle Molar e Canina. A análise estatística dos dados foi realizada utilizando o Teste Qui-Quadrado, o Coeficiente V de Cramer e o Odds Ratio, recorrendo ao programa SPSS 17.0. Foram considerados significativos os valores cujo nível de significância foi inferior a 5% (p <0,05). Resultados e Conclusão: Verificou-se uma correlação estatisticamente significativa entre as variáveis deglutição atípica e trespasse vertical diminuído (p=0,008); amamentação e respiração nasal (p=0,044); duração da amamentação e desenvolvimento de hábitos orais (p=0,003); e respiração nasal e mordida cruzada (p=0,035).Introduction: The craniofacial growth and development is a result of the interaction of genetic, hormonal and neurological mechanisms, influenced by local factors, such as oral habits. Normal occlusion depends on adequate growth of the face as well as teeth angulations and morphology, mastication muscles and functional movements. The influence of local factors can lead to bone deformities, malocclusion, and changes in functions such as breathing, chewing, swallowing, sucking and diction. Thus, it is relevant the prevention of correlated potential deformities by means of early diagnosis and preventive/interceptive treatment. The main aim of this study is the evaluation of a possible correlation between sucking habits and tongue thrusting and malocclusion on the stage of deciduous and mixed dentition. Materials and methods: For this study a sample was selected from the patients who attended to the Pediatric consults of Dental Clinic of Universidade Católica Portuguesa – Pólo de Viseu, between February 14 and June 21. After applying the inclusion and exclusion criteria, was selected a 68 patients sample. For data collection was developed a questionnaire which includes parameters aimed to establish a correlation between variables, which comparison was not described. This questionnaire allowed to assess if patients were breast-fed and for how long, if children were bottle-fed, if had sucking habits and its duration, if they had labial or tongue thrust at rest or during swallow, the breathing pattern, the presence of tonsils and adenoids, overbite and overjet, the presence of openbite or crossbite, and the molar and canine Angle Class. The statistical analysis was performed using the Chi-Square Test, the Cramer’s V Coefficient and the Odds Ratio, using SPSS 17.0 program. Were considered significant the values which the significance level was less than 5% (p< 0,05). Results and Conclusion: There was a statistically significant correlation between tongue thrust during swallow and diminished overbite (p=0,008); breastfeeding and nasal breathing (p=0,044); breastfeeding duration and oral habits development (0,003); and nasal breathing and crossbite (p=0,035)

Micoteca da Universidade do Minho (MUM): uma colecção de fungos filamentosos a olhar o futuro

Em 2001 a OCDE publica o relatório: Biological Resource Centres – Underpinning the Future of Life Sciences and Biotechnology apontando para a necessidade do reforço e modificações dos centros de recursos biológicos no sentido destes estarem à altura das necessidades do século XXI. Os centros de recursos biológicos (CRBs) são entendidos como uma parte essencial da infra­estrutura que suportam as ciências da vida e a biotecnologia. Eles consistem em fornecer serviços e serem depositários de células vivas, de genomas de organismos, e da informação relacionada com a hereditariedade e as funções biológicas dos sistemas. Neste contexto, a Micoteca da Universidade do Minho (MUM – www.micoteca.deb.uminho.pt), fundada em 1996, tem como missão ser uma colecção de fungos filamentosos com o objectivo principal de manter e fornecer linhagens com qualidade e autenticidade para a investigação em biotecnologia e ciências da vida, e laboratórios de ensino, actuando também como um centro de conhecimento, informação e formação na área da micologia, em articulação com outras colecções de culturas nacionais e internacionais. A MUM é membro e acolhe o secretariado da European Culture Collections Organization (ECCO), é membro e está no conselho executivo da World Federation for Culture Collections (WFCC). Tem participado activamente nos grupos de trabalho da OCDE para os assuntos dos CRBs. É parceira do projecto demonstrativo Global Biological Resource Centres Network (GBRCN) e do projecto financiado pela comissão europeia European Consortium of Microbial Resource Centres (EMbaRC). Assim, a introdução de parâmetros de gestão da qualidade e na identificação e autenticação das linhagens conservadas através de abordagens polifásicas, com especial ênfase na biologia molecular e na tipagem espectral com o recurso da técnica de matrix­assisted laser desorption∕ionization timeof­flight mass spectrometry (MALDI­TOF MS), são uma vertente fundamental para a requalificação da colecção. Estas metodologias específicas associadas a novos modelos de gestão são relevantes na adaptação da MUM para o seu futuro dado que as linhagens que fornece terão maior qualidade e competitividade

Structural diversity of aspergillus (section nigri) spores

The taxonomy of fungal species, similar to that of many other microorganisms, suffers frequent revisions due to the discovery of new species and to the development and gathering of characterization data and morphological information. Morpho-taxonomy helps in the identification of many species. This work presents the macro, micro-morphological, and spectral mass analyses for phenotypic characterization of 13 species of Aspergillus section Nigri, showing that the characterization of spores (conidia) by scanning electron microscopy can be used as a tool to discriminate key morphological characteristics and separate closely related fungi. These results were corroborated by colony plates, stereomicroscopy, light microscopy, and spectral mass data.This research has received funding from the European Community's Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under grant No. FP7-228310 (EMbaRC project). M.F. Simoes acknowledges FCT-Portugal for the scholarship SFRH/BD/64260/2009

Aged freeze-dried ampoules of preserved biotechnological important fungi

The implementation of consistent fungal preservation techniques and appropriate quality assurance are key issues for an effective and efficient preservation. The cost and convenience of each method are important aspects to be taken into consideration such as the knowledge of all parameters capable of affecting the procedures [1]. Preservation methods currently used are highly empirical and in many instances, do not provide reliable genetic and phenotypic stability. Freeze-drying is commonly used to preserve fungal strains at room temperature, however, genetic and phenotypic alterations after long term-storage are yet unknown. Therefore, the main goal of the present experimental study is to evaluate the freeze-drying preservation method for the effective long-term preservation of strains belonging to Aspergillus section Nigri. Twenty-one strains representative of Aspergillus section Nigri were selected and preserved by freeze-drying. The strains were subjected to accelerated storage during 4 weeks at 37 ºC. These samples were morphological, physiological and genotypical analysed. In order to detect macro and micro-morphological changes, growth for seven days at 25°C on Potato Dextrose Agar, Malt Extract Agar, Czapek Yeast Extract Agar and Czapek Dox Agar was performed. The physiological changes were monitored for the detection of ochratoxin A and fumonisin B2 as described elsewhere [2,3]. In order to identify genotypic changes, DNA fingerprinting techniques using the oligonucleotides M13 and (GACA)4 were performed. All assays were evaluated at 3 points in time: before preservation (I), 2 (II) and 4 (III) weeks after preservation. For all the methodologies used to evaluate freeze-drying of fungi along time the major results are: 1) no significant changes were observed in the macro and micro-morphological analysis; 2) all strains maintained their mycotoxins production pattern, before and after ageing; 3) after ageing different DNA fingerprinting was observed. In conclusion, freeze-drying can be considered a technique of excellence to be used on the maintenance of biodiversity within the filamentous fungi, and more accurately for Aspergillus section Nigri. However, it is recommended to consider possible genetic changes after long shelf-life periods.info:eu-repo/semantics/publishedVersio