Bioprivileged Fluorinated Ionic Liquids in the Purification of Proteins: Towards Enhanced Tunability of Aqueous Biphasic Systems

Proteins are high value biomolecules with application in several fields, therefore the study of novel extraction methods using more biocompatible techniques is in large extent. Aqueous Biphasic Systems are increasingly being studied for that purpose, due to presenting two aqueous phases, usually protein-friendly. Fluorinated Ionic Liquids (FILs) are an emerging category of Ionic Liquids due to their unique properties, namely self-aggregation behavior, and already showing protein benign behavior. In this work, Lysozyme was used as model protein, and used to be partitioned by systems composed by IL and FIL, besides several salting-out agents, specifically inorganic salt, carbohydrates and a dihydrogen phosphate choline ([N1112OH][H2PO4]). These studies revealed favored partition to FIL-aqueous rich phase comparing with IL-rich phase. Lysozyme activity was tested for each component and after partition protocol, and maintained for most cases. Turbidimetry was used to infer the presence of some interactions between the protein and some systems elements; Tryptophan Intrinsic Fluorescence studies were carried to verify some conformation changes in the presence of IL, FIL and N1112OH][H2PO4]. Then, nano-Differential Scanning Calorimetry (nano-DSC) was used to confer the maintenance of thermal stability of Lysozyme, and determination of melting temperature (Tm). These studies have revealed the presence of some interaction between IL/FIL and protein, besides different conformational changes in Lysozyme provoked by these compounds compared to reference. Also, was observed a decrease on Tm with increasing IL and FIL concentration, however at partition conditions, FIL presented more Tm conservation than IL. This work permitted a higher insight on FIL application in ABS for protein extraction, and reveled their promising assets for this end; however more studies relatively to Lysozyme structure and thermodynamic parameters in the presence of IL/FIL are needed.As proteínas são biomoléculas de elevado valor e aplicabilidade em diversas áres, desta forma o estudo de novas formas para a sua extração usado metodologias mais biocompatíveis é cada vez mais aprofundado. Os Sistemas Aquosos Bifásicos são cada vez mais estudados para este efeito, por apresentarem duas fases aquosas, tornando-os sistemas de extração mais biocompatíveis. Os Líquidos Iónicos Fluorados (FIL) são uma categoria de Líquidos Iónicos (IL) emergente devido às suas características únicas, nomeadamente a sua capacidade de fazerem “self-aggregations” e demonstrarem propriedades compatíveis com as proteínas. Neste trabalho, a Lisozima foi usada como modelo e usada para estudo da partição em sistemas aquosos bifásicos compostos por IL, para além de uma variedade de agentes de “salting-out”, especificamente sais inorgânicos, carbohidratos e colina dihidrogenofostato ([N1112OH][H2PO4]). Estes estudos revelaram uma partição maioritária para as fases ricas em FIL, em comparação com as fases ricas em IL. A atividade enzimática foi também testada para cada componente e após o procedimento de partição, e mantida para a maioria dos casos. Turbidimetria foi usada para aferir a presença de interações entre a proteína e alguns compostos usados na partição; Fluorescência Intrínseca do Triptofano foi usado para verificar algumas alterações conformacionais na presença de IL, FIL e [N1112OH][H2PO4]. Em seguida, nano-Calorimetria de Varrimento Diferencial (nano-DSC) foi usada para conferir a manutenção da estabilidade térmica da Lisozima provocada por estes componentes e determinação da Temperatura de Fusão (Tm). Estes estudos revelaram a presença de interações entre o IL/FIL e a proteína, para além de diferentes alterações na conformação provocados por estes compostos, comparando com a referência em água. Foi também verificada uma diminuição de Tm com o aumento da concentração de IL e FIL, contudo às condições de partição, o FIL apresentava maior conservação da Tm. Este trabalho permitiu uma maior análise da aplicação de FILs em ABS para extração das proteínas, e revelou características únicas para este fim; contudo são necessários mais estudos relativamente à estrutura da Lisozima e aos parâmetros termodinâmicos na presença de IL e FIL

Charcoal canker (Biscogniauxia mediterranea) in cork oak decline in Portugal

Doutoramento em Engenharia Florestal e dos Recursos Naturais - Instituto Superior de AgronomiaCharcoal canker is a disease caused by Biscogniauxia mediterranea that affects Quercus suber and other hardwood species. The fungus develops endophytically in the hosts becoming pathogenic under stress conditions. However, it has become more frequent, particularly in young trees expressing atypical symptoms. This work intends to clarify the agent involved in these disease outbreaks and its variability in different situations, as well as dispersion in natural conditions. The fungal anamorph was identified as Nodulisporium sp. in young hosts. In the stands, ascospores airborne dispersal is predominant, occurring after precipitation periods, and vertical contamination of plants doesn’t happen. A collection of isolates obtained from cork oak in Portugal and other Mediterranean countries, from other hosts and from trees with different ages and disease expression were analyzed by microsatellite-primed PCR, multigene sequencing, morpho-cultural characteristics and growth rates at different temperatures. All the approaches revealed high level of intraspecific polymorphism among isolates, not allowing relating the disease development with any considered feature. The results highlighted the variability and adaptation ability of this fungus in special in the Mediterranea region, in the present worrying scenario of climatic change. All the conditions are gathered to favor the aggravation of the disease in cork oak stand

Multiyear surveillance of Influenza A virus in wild birds in Portugal

This report presents the results of a multiyear (2005 to 2009) study of avian influenza virus (AIV) occurrence in wild birds in Portugal. A total of 5691 samples from wild birds belonging to 13 different orders were examined. Ninety-three samples tested positive for AIV by matrix reverse transcriptase-polymerase chain reaction, giving a total prevalence of 1.63%. Twenty-one viruses were successfully cultured in embryonated chicken eggs, which represent a rate of viral infectivity of 22.6% in the samples. Nine subtypes of haemagglutinin (H1, H3 to H7, H9 to H11) and eight subtypes of neuraminidase (N1 to N4, N6 to N9) were identified in 20 different combinations. The most prevalent subtypes of haemagglutinin detected were H5, H1 and H4, while for neuraminidase subtypes N2 and N6 were the most common. The subtype combinations H4N6 and H1N1 were predominant (15.1%). All H5 and H7 viruses detected in the present study were low pathogenic for poultry as determined by the sequence of amino acids at the cleavage site of haemagglutinin. The full-length nucleotide sequences of five H5, one H7 and five N3 genes were analysed phylogenetically. The Bayesian analysis revealed that all but one of the strains analysed were closely related to isolates detected in the same period in North and Central European countries. Three H5N3 isolates, all from 2007, formed a separate cluster in both H5 and N3 phylogenetic trees. This study provides evidence that various subtypes of AIV, including subtypes H5 and H7, circulate in Portugal, which may pose a risk to industrial poultry

Developmental recovery after adoption: child and adoptive family’s characteristics

Clínicos e investigadores têm alertado para os efeitos problemáticos das experiências precoces adversas, nomeadamente a da institucionalização, no crescimento físico, no desenvolvimento cognitivo e sócio-emocional, bem como nas relações de apego da criança. Após a adoção, a recuperação nestes domínios parece ser evidente, observando-se uma curiosa heterogeneidade nos resultados desenvolvimentais. Este artigo de revisão apresenta uma sistematização de resultados de estudos que analisaram a influência das características da criança e da família adotiva naquela recuperação. Pesquisou-se nas principais bases de dados eletrônicas e foram identificados estudos originais, de revisão e capítulos de livros. Concluiu-se que um entendimento da recuperação exige a exploração dos fatores genéticos, das experiências pré-natais e pré-institucionalização, e das vivências de cuidados anteriores e posteriores à adoção.Clinicians and researchers have been warning about the harmful effects of a history marked by early adverse experiences in shelter institutions in child’s health, cognitive and socioemotional development as well as attachment. After adoption, the recovery in these domains seems to be clear and it is also observed a curious heterogeneity in developmental results. This paper review presents a summary of the main empirical fi ndings about the characteristics of the child and of the adoptive family involved in the recovery process. The search was done in the major electronic databases and original studies, review articles, and book chapters were identifi ed. It is concluded that the recovery requires the identifi cation of genetic, prenatal and pre-institutionalization factors, and also the care experiences before and after the adoptionFundação para a Ciência e a Tecnologia (FCT).PTDC/PSI-PCL/101506/2008; Fundação Bial – Bolsa 13/06