Caracterização petrográfica e geoquímica dos magmatitos da região do Sardoal, Abrantes, e seu enquadramento geodinâmico

Os magmatitos do Sardoal estão inseridos na Unidade dos Ortognaisses de Mouriscas, considerada de idade proterozóica. Apresentam intercalações mesocratas, com estruturação idêntica à dos gnaisses e intercalações máficas com diferente estruturação. Os Gnaisses do Sardoal exibem deformação variável, desde texturas com foliação milonítica a texturas metaígneas. Correspondem a rochas ácidas, sub-alcalinas, com assinaturas geoquímicas características de ambiente de arco continental. As Rochas Intercaladas apresentam foliação bem desenvolvida, composição básica (as máficas) e intermédia (as mesocratas) e assinaturas geoquímicas indicativas de diferentes fontes. Sugere-se que as Rochas Intercaladas correspondam a termos de sequências sub-alcalinas de diferente natureza - as intermédias relacionadas com ambiente de arco continental e as máficas, ou inseridas em ambiente do mesmo tipo ou de intraplaca continental. Estes dados permitiram correlacionar as rochas desta região com o arco vulcânico já definido no NE da ZOM e que deverá estar relacionado com o fecho da orogenia cadomiana

Cloning and expression of a recombinant human CBD (carbohydrate binding domain) : a comparison between two expression models

Genetic engineering is a method that provides the production of recombinant proteins for different purposes. Nowadays there are several and commercially available expression systems aiming the production of these proteins. However, in same cases, the solubility and stability of the produced protein can be a problem, especially for eukaryotic proteins, which need post translational modifications to be biologically active. In the present work, different strategies were tested to increase the solubility of a human carbohydrate binding domain (CBD), present in laforin phosphatase. Namely, different expression systems, different hosts and fermentation conditions, the presence of additives and detergents during lyses, were used. The DNA coding sequence was cloned by PCR into three prokaryotic expression systems: pET 29a, pET 25b; and two eukaryotic systems: pGAPZaC and pPICZaC. The CBD was expressed at high level in pET system. In pET29a the CBD protein was obtained in inclusion bodies. In pET25b, a small amount of soluble protein was obtained in the presence of arginine and CHAPS, in the lyses buffer. Although a soluble recombinant protein was obtained, it was not stable in solution, aggregating easily. On the other hand, the utilisation of two expression systems of Pichia pastoris led to the production of soluble and stable CBD in extra cellular medium; however, this CBD was obtained at low expression level and its activity was not confirmed

Improving the solubility of a recombinant human CBD (carbohydrate binding domain)

Different expression systems have been developed, and are commercially available, aiming at producing recombinant proteins from different organisms, ranging from bacteria to man. However, in same cases, the solubility and stability of the produced protein can be a problem, especially for eukaryotic proteins, which need post translational modifications to be biologically active. In the present work, different strategies were tested to increase the solubility of a human carbohydrate binding domain (CBD), present in laforin phosphatase. Namely, different expression systems, different hosts and fermentation conditions, the presence of additives and detergents during lyses, were used. The DNA coding sequence was cloned by PCR into three prokaryotic expression systems: pET 29a, pET 25b; and two eukaryotic systems: pGAPZaC and pPICZaC. The CBD was expressed at high level in pET system. In pET29a the CBD protein was obtained in inclusion bodies. In pET25b, a small amount of soluble protein was obtained in the presence of 0,6M of arginine, in the lyses buffer. Although a functional recombinant protein was obtained, it was not stable in solution, aggregating easily. On the other hand, the utilisation of two expression systems of Pichia pastoris led to the production of soluble and stable CBD in extra cellular medium; however, this CBD was obtained at low expression level and its activity was not confirmed. Glycosilation of the expressed protein may explain the increased stability, at the expense of reduced functionality. Studies are underway to confirm this hypothesis

The importance of proteins of the RNase II/RNB-family in pathogenic bacteria

WOS: 84907164467publishersversionpublishe

Characterization of dextrin-based hydrogels : rheology, biocompatibility, and degradation

A new class of degradable dextrin-based hydrogels (dextrin-HEMA) was developed. The hydroxyethyl methacrylate ester (HEMA) hydroxyl groups were activated with N,N' carbonyldiimidazole (CDI), followed by their coupling to dextrin, yielding a derivatized material that can be polymerized in aqueous solution to form hydrogels. A comparative study of the stability of the dextrin-HEMA hydrogels and dextrin-vinyl acrylate (dextrin-VA, produced in previous work) revealed that only the firsts are effectively hydrolyzed under physiological conditions. A severe mass loss of dextrin-HEMA gels occurs over time, culminating in the complete dissolution of the gels. Rheologic analysis confirmed that physical structuring is less pronounced when dextrin is modified with methacrylate side groups. The biocompatibility results revealed that the dextrin hydrogels have negligible cell toxicity, irrespective of the hydrogel type (HEMA and VA), allowing cell adhesion and proliferation. Gathering the biocompatibility and the ability to tailor the release profiles, we consider dextrin a promising biomaterial for biomedical applications, namely for controlled release

Studies on the cellulose-binding domains adsorption to cellulose

Cellulose-binding domains (CBD) are modular peptides, present in many glycanases, which anchor these enzymes to the substrate. In this work, the effect of CBD adsorption on the surface properties of a model cellulose, Whatman CF11, was studied. The methods applied include inverse gas chromatography (IGC), ESCA, X-ray diffraction, and scanning electron microscopy (SEM). The CBD partition affinity (0.85 L/g) was calculated from adsorption isotherms. However, true adsorption equilibrium does not exist, since CBDs are apparently irreversibly adsorbed to the fibers. Both IGC and ESCA showed that fibers with adsorbed CBD have a lower acidic character and also a slightly higher affinity toward aliphatic molecules. This may however be a consequence of an increased surface area, a hypothesis that is supported by microscopic observations. The crystallinity index was not affected by CBD treatment.Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/6934/2001

UCP2 and ANT differently modulate proton-leak in brain mitochondria of long-term hyperglycemic and recurrent hypoglycemic rats

A growing body of evidence suggests that mitochondrial proton-leak functions as a regulator of reactive oxygen species production and its modulation may limit oxidative injury to tissues. The main purpose of this work was to characterize the proton-leak of brain cortical mitochondria from long-term hyperglycemic and insulininduced recurrent hypoglycemic rats through the modulation of the uncoupling protein 2 (UCP2) and adenine nucleotide translocator (ANT). Streptozotocin-induced diabetic rats were treated subcutaneously with twice-daily insulin injections during 2 weeks to induce the hypoglycemic episodes. No differences in the basal proton-leak, UCP2 and ANT protein levels were observed between the experimental groups. Mitochondria from recurrent hypoglycemic rats presented a decrease in proton-leak in the presence of GDP, a specific UCP2 inhibitor, while an increase in proton-leak was observed in the presence of linoleic acid, a proton-leak activator, this effect being reverted by the simultaneous addition of GDP. Mitochondria from longterm hyperglycemic rats showed an enhanced susceptibility to ANT modulation as demonstrated by the complete inhibition of basal and linoleic acid-induced proton-leak caused by the ANT specific inhibitor carboxyatractyloside. Our results show that recurrent-hypoglycemia renders mitochondria more susceptible to UCPs modulation while the protonleak of long-term hyperglycemic rats is mainly modulated by ANT, which suggest that brain cortical mitochondria have distinct adaptation mechanisms in face of different metabolic insults.The authors’ work is supported by the Fundação para a Ciência e a Tecnologia (FCT) (PTDC/SAU-NEU/103325/2008) co-funded by Fundo Europeu de Desenvolvimento Regional (FEDER) via Programa Operacional Factores de Competitividade (COMPETE). Susana Cardoso has a PhD fellowship from the Portuguese Foundation for Science and Technology (SFRH/BD/43968/2008)

Production of the human carbohydrate binding module from laforin protein : a comparative study between bacterial and yeast expression systems

Fundação para a Ciência e a Tecnologia (FCT) - POCTI/BIO/45356/2002 ; (SFRH/BD/18418/2004)

Escherichia coli expression, refolding and characterization of human laforin

Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5–7 mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI