Fungal stains on paper: Melanins produced by fungi

Books, prints, drawings, watercolours, engravings as well as all other works of art based on paper comprise a great portion of our cultural heritage. Therefore, its preservation is a matter of great concern. Paper can be deteriorated due to physical, chemical and biological agents. Fungi are among the most common biodeteriogens affecting paper-based collections, causing severe material and information losses. This work focused on fungal stains on paper which are often coloured. These interfere with the readability of the artefacts diminishing their artistic and monetary value. Up to now, there is still no definitive answer for this problem. The successful cleaning of fungal stains from paper is a mandatory conservation task, considered a priority by paper conservators. However, most of the authors refer the stain colour and patterns but they do not indicate the colourant or colourants (or chemical compound) responsible for the stain. Black stains on paper are of major concern because, not only they are very frequent, as well as its dark colour leads to a great loss of visibility. Therefore, this work focused primarily in the extraction and characterization of fungal melanins from three different species: Aspergillus niger, Chaetomium globosum and Cladosporium cladosporioides, known to be responsible for black staining on paper and melanin production. UV-Vis, μ-FTIR and μ-Raman analyses were carried out for all three fungi melanin extracts. UV-Vis, μ-FTIR and μ-Raman results show that, after extraction and purification, purified melanin samples were obtained from the three-fungal species. Moreover, SSNMR allowed to characterize A. niger’s melanin as a L-DOPA type melanin, and Cl. cladosporioides as a DHN type melanin, by comparison with the synthetic L-DOPA melanin (Sigma-Aldrich) and the literature. This will allow for a colourant-specific testing of newly developed cleaning methods, considering the base structure of the polymer (melanin) to be removed from the paper

Production of Hydroxyapatite Nanoparticles for cosmetic and orthopaedic applications : effect of chelants and dispersants

Estágio realizado na Fluidinova-Engenharia de Fluídos e orientado pelo Eng.º José Carlos Britpo LopesTese de mestrado integrado. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 200

Analysis of the physical-chemical and sensorial properties of Maria type cookies

Given the importance of the cookies of type Maria worldwide, and considering the absence of any scientific study setting out their main features, it becomes important to identify the differentiating characteristics of several commercialized brands, in particular related to the chemical, physical and sensory characteristics. In this way, the aim of this work was to study and compare eight different brands of cookies of type Maria. The elemental chemical analysis (moisture, ash, protein, fat, fibre and carbohydrates contents), determination of physical parameters (volume, density, texture and colour) and sensory evaluation of studied cookies were performed. Multivariate statistical methods (Pearson correlation, principal component analysis and cluster analysis) were applied to estimating relationships in analysed data. The results for the elemental analysis showed that the samples were very similar in terms of some components, like for example ashes, while quite different in terms of other components, such as moisture and fat contents. With respect to texture and colour the samples showed, in general, some important differences. In terms of sensory evaluation, the sample C was the one that in most sensory tests gathered the preference of the panellists. The cluster analysis showed that the sample A was much different from the other samples. The results of principal component analysis showed that the main component explains 32.6 % of the total variance, and is strongly related to variables associated to colour

Um Estudo sobre a Ludoterapia nas Dificuldades de Aprendizagem: estudo de caso

O objectivo deste estudo centra-se na análise reflexiva da importância do papel da técnica de Ludoterapia no apaziguamento das Dificuldades de Aprendizagem de uma criança. Para tal, debruçamo-nos num estudo de caso de uma criança de seis anos de idade, no qual, metodologicamente, é aplicada uma bateria de instrumentos em dois momentos distintos. Foram realizadas, no total, 25 sessões de Ludoterapia e de apoio psicopedagógico. A bateria de testes é composta pelas provas Bar-Ilan, Escala de Inteligência de Wechsler para a Idade Pré-Escolar e Primária e pela Bateria de Provas Fonológicas. Ao fim de 25 sessões de Ludoterapia, no segundo momento de avaliação verificou-se um desenvolvimento cognitivo adequado para a sua idade, ao contrário dos resultados do primeiro momento de avaliação

Assignment of novel functions to Helicobacter pylori 26695’s genome and reconstruction of a genome-scale metabolic model

Helicobacter pylori is a pathogenic organism associated with human gastric diseases. The development of mathematical models of metabolism is now considered a fundamental part of the study of the cell. For the particular case of microorganisms associated with human diseases, information on metabolic and regulatory networks can be used to understand the molecular factors of the microorganism that are likely to interact with the host and cause diseases. The availability of the genome sequence of H. pylori 26695 and its annotation has allowed in the past the construction of a metabolic model for this organism. The first genome-scale metabolic model for H. pylori 26695 was published in 2002 (iCS291) and a corrected reconstruction was published in 2005 (iIT341 GSM/GPR). The main goal of the present work was to update H. pylori’s genome-scale metabolic model based on the new information made available. For that purposes, using new annotation methodologies and data available in databases, an assignment of novel functions to H. pylori 26695’s genome was performed. For a total of 510 “hypothetical proteins” (almost 1/3 of the genes) identified in the last re-annotation, 137 new functions were attributed. A total of 581 E.C. numbers were assigned to CDS, being 528 complete E.C. numbers. This new information was used as the basis of the model reconstruction. In addition, transport reactions in the model were updated. The biomass equation was reviewed and H. pylori biomass coefficients and composition were adjusted. The obtained model successfully predicted the nutritional requirements and amino acids essentialities, which were experimentally validated. As a result, the present work presents a new H. pylori 26695 genome-scale metabolic model with more accurate and reliable predictions and can be used to identify potential targets for designing more effective drugs for H. pylori inactivation

Assignment of novel functions to Helicobacter pylori 26695’s genome

Helicobacter pylori is a pathogenic bacterium that colonizes the human epithelia, causing duodenal and gastric ulcers as well as gastric cancer. The genome of H. pylori 26695 has been sequenced and annotated. In addition, two genome-scale metabolic models have been developed. In order to maintain accurate and relevant information on coding sequences (CDS) and to retrieve new information, the assignment of new functions to Helicobacter pylori 26695’s genes was performed. The use of software tools, on-line databases and an annotation pipeline for inspecting each gene allowed the attribution of validated E.C. numbers to metabolic genes, and the assignment of 177 new functions to the CDS of this bacterium. This information provides relevant biological information for the scientific community dealing with this organism and can be used as the basis for a new metabolic model reconstruction.(undefined

Challenges in the heterologous production of furanocoumarins in Escherichia coli

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020. D.G. acknowledges her grant SFRH/BD/04433/2020.info:eu-repo/semantics/publishedVersio

Combinatorial approaches for de novo production of flavonoids inEscherichia coli

Polyphenols are plant secondary metabolites that are produced in response to biotic and abiotic stress. Flavonoids, one of the most representative group of these metabolites, includes at least 9000 compounds. Among them, naringenin has been widely studied due to its interesting biological activities, namely anticancer, anti-inflammatory, and antioxidant. Due to its potential applications and to the attempt to satisfy the industrial demand, there has been an increased interest in the heterologous production of this compound in a microbial chassis. The production of naringenin by a microorganism involves a first step of conversion of tyrosine into coumaric acid by tyrosine ammonia-lyase (TAL). Afterwards, coumaric acid is converted into coumaroyl-CoA by 4-coumarate-CoA ligase (4CL). Coumaroyl-CoA is further converted into naringenin chalcone by chalcone synthase (CHS) and then into naringenin by chalcone isomerase (CHI). In this work, we aimed to design, construct and validate an efficient biosynthetic pathway to produce naringenin in Escherichia coli by performing a step-by-step optimization. To construct the biosynthetic pathway to produce naringenin, TAL, 4CL, CHS, and CHI genes from different organisms were selected. Initially, TAL from Rhodotorula glutanis (RgTAL) and TAL from Flavobacterium johnsoniae (FjTAL) were cloned into the pRSFDuet-1 vector and were further expressed in three different E. coli strains (E. coli BL21, K12 MG1655 and M-PAR-121) to select the best enzyme and strain to produce coumaric acid. The highest production was obtained in the E. coli M-PAR-121 strain expressing FjTAL (2.54 g/L of coumaric acid from 40 g/L glucose). E. coli M-PAR-121 is a tyrosine-overproducing strain and it can produce high amounts of tyrosine from glucose that can be converted into coumaric acid. Thus, this strain and enzyme were chosen to construct the complete biosynthetic pathway. Afterwards, 4CL and CHS steps were constructed and validated. Specifically, 4CL from Arabidopsis thaliana (At4CL), 4CL from Vitis Vinifera (Vv4CL), and 4CL from Petroselinum crispum (Pc4CL) were cloned into the pACYCDuet-1 vector. Moreover, CHS from A. thaliana (AtCHS), CHS from Petunia hybrida (PhCHS), and CHS from Curcubita maxima (CmCHS) were cloned into pCDFDuet-1 vector. Twelve different combinations of the 4CL and CHS genes were expressed in the best strain able to produce coumaric acid and the naringenin chalcone production from glucose was evaluated. The best naringenin chalcone production was obtained in the E. coli M-PAR-121 strain expressing pRSFDuet_FjTAL, pACYCDuet_At4CL and pCDFDuet_CmCHS (311.0 mg/L). Aiming to increase the productivity of the engineered strain, the metabolic burden of the cells was reduced by cloning the three genes in only two plasmids. From the different combinations tested, E. coli M-PAR-121 strain holding pRSFDuet_FjTAL_CmCHS and pACYCDuet_At4CL has reached the highest production of naringenin chalcone (560.2 mg/L). Afterwards, the last step of the biosynthetic pathway was validated. At this point, CHI from A. thaliana, CHI from M. sativa and CHI from C. maxima were tested. The maximum production was achieved in the E. coli M-PAR-121 strain expressing pRSFDuet_FjTAL_CmCHS and pACYCDuet_At4CL_AtCHI (366.6 mg/L). This production was one of the highest reported so far in shake-flask experiments using glucose as substrate.Daniela Gomes acknowledges SFRH/BD/04433/2020 grant, from Portuguese Foundation for Science and Technology (FCT). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Evaluation of antibiotics residues levels in Portuguese honey: a concerted study with the Portuguese Beekeepers Associations

The antibiotics residual presence in honey is a current problem with negative implications, mainly commercial since according to European legislation antibiotic occurrence in honey samples is forbidden