Public service culture collections of microorganisms: are they important for biotechnology?

Microbiology as a scientific discipline recognised the need to preserve microorganisms for scientific studies establishing from its very beginning research culture collections (CC). Later on, to better serve different scientific fields and bioindustries with the increasing number of strains of scientific, medical, ecological and biotechnological importance public service CC were established with the specific aims to support their user communities. Currently, the more developed public service CC are recognised as microBiological Resources Centres (mBRC). mBRC are considered to be one of the key elements for sustainable international scientific infrastructure, which is necessary to underpin successful delivery of the benefits of biotechnology, whether within the health sector, the industrial sector or other sectors, and in turn ensure that these advances help drive economic growth. In more detail, mBRCs are defined by Organisation for Economic Co-operation and Development (OECD) as service providers and repositories of the living cells, genomes of organisms, and information relating to heredity and functions of biological systems. mBRCs contain collections of culturable organisms (e.g., microorganisms, plant, animal cells), replicable parts of these (e.g. genomes, plasmids, virus, cDNAs), viable but not yet culturable organisms, cells and tissues, as well as database containing molecular, physiological and structural information relevant to these collections and related bioinformatics. Thus mBRCs are fundamental to harnessing and preserving the world’s microbial biodiversity and genetic resources and serve as an essential element of the infrastructure for research and development. mBRCs serve a multitude of functions and assume a range of shapes and forms. Some are large national centres performing a comprehensive role providing access to diverse organisms. Other centres play much narrower, yet important, roles supplying limited but crucial specialised resources. In the era of the knowledge-based bio-economy mBRCs are recognised as vital element to underpinning the biotechnology.

Design and validation of a biomechanical bioreactor for cartilage tissue culture

Specific tissues, such as cartilage undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation - amplitude and frequency - to three dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Osteogenic differentiation of adipose stem cells by endothelial cells co-culture within liquified capsules

Inspired by the native co-existence of multiple cell types and from the concept of deconstructing the stem cell niche, we propose a co-encapsulation strategy within liquified capsules. The present team has already proven the application of liquified capsules as bioencapsulation systems1. Here, we intend to use the optimized system towards osteogenic differentiation. Capsules encapsulating adipose stem cells alone (MONO-capsules) or in co-culture with endothelial cells (CO-capsules) were maintained in endothelial medium with or without osteogenic differentiation factors. The suitability of the capsules for living stem and endothelial cells encapsulation was demonstrated by MTS and DNA assays. The osteogenic differentiation was assessed by quantifying the deposition of calcium and the activity of ALP up to 21 days. CO capsules had an enhanced osteogenic differentiation, even when cultured in the absence of osteogenic factors. Furthermore, osteopontin and CD31 could be detected, which respectively indicate that osteogenic differentiation had occurred and endothelial cells maintained their phenotype. An enhanced osteogenic differentiation by co-encapsulation was also confirmed by the upregulation of osteogenic markers (BMP-2, RUNX2, BSP) while the expression of angiogenic markers (VEGF, vWF, CD31) revealed the presence of endothelial cells. The proposed capsules can also act as a growth factor release system upon implantation, as showed by VEGF and BMP-2 quantification. These findings demonstrate that the co-encapsulation of stem and endothelial cells within liquified injectable capsules provides a promising strategy for bone tissue engineering.  

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage.

Integration of geometric consistency contributory factors in three-leg junctions collision prediction models of Portuguese two-lane national highways

This paper aims at developing a collision prediction model for three-leg junctions located in national roads (NR) in Northern Portugal. The focus is to identify factors that contribute for collision type crashes in those locations, mainly factors related to road geometric consistency, since literature is scarce on those, and to research the impact of three modeling methods: generalized estimating equations, random-effects negative binomial models and random-parameters negative binomial models, on the factors of those models. The database used included data published between 2008 and 2010 of 177 three-leg junctions. It was split in three groups of contributing factors which were tested sequentially for each of the adopted models: at first only traffic, then, traffic and the geometric characteristics of the junctions within their area of influence; and, lastly, factors which show the difference between the geometric characteristics of the segments boarding the junctionsâ area of influence and the segment included in that area were added. The choice of the best modeling technique was supported by the result of a cross validation made to ascertain the best model for the three sets of researched contributing factors. The models fitted with random-parameters negative binomial models had the best performance in the process. In the best models obtained for every modeling technique, the characteristics of the road environment, including proxy measures for the geometric consistency, along with traffic volume, contribute significantly to the number of collisions. Both the variables concerning junctions and the various national highway segments in their area of influence, as well as variations from those characteristics concerning roadway segments which border the already mentioned area of influence have proven their relevance and, therefore, there is a rightful need to incorporate the effect of geometric consistency in the three-leg junctions safety studies.

Assessment of national parks affected by naturbanization processes in Southern Europe

This paper assesses land-use changes related to naturbanization processes on three biosphere reserves in Southern Europe. A comparative analysis has been done on the National Parks in Peneda-Ger^es in North Portugal, C_evennes in South France and Sierra Nevada in South Spain, using Corine Land Cover data from 1990 until 2006. Results indicate that the process of land-use intensification is taking place in the frame of naturbanization dynamics that could jeopardize the role of Protected Areas. Focusing on the trends faced by National Parks and their surrounding territories, the analysis demonstrates, both in quantitative and spatial terms, the intensification processes of land-use changes and how it is important to know them for coping with increasing threats. The article concludes that in the current context of increasing stresses, a broader focus on nature protection, encompassing the wider countryside, is needed if the initiatives for biodiversity protection are to be effective.

Seismic-resistant building practices resulting from local seismic culture

Considering that vernacular architecture may bear important lessons on hazard mitigation, this chapter focuses on the European Mediterranean countries and studies traditional seismic-resistant architectural elements and techniques that local populations developed to prevent or repair earthquake damage. This area was selected as a case study because, as a highly seismic region, it has suffered the effect of many earthquakes along the history and, thus, regions within this area are prone to have developed a Local Seismic Culture. After reviewing seismic resistant construction concepts, a wide range of traditional construction solutions that, in many cases, have shown to improve the seismic performance of vernacular constructions of these regions is presented, as a contribution to the general overview of retrofitting building systems provided in this book. The main motivation is that most of these techniques can be successfully applied to preserve and to retrofit surviving examples without prejudice for their identity.

Quantitative image analysis as a tool for Yarrowia lipolytica dimorphic growth evaluation in different culture media

Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as γ-decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for γ-decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.

O ensino de teatro de animação: contribuições para construção de novos saberes e fazeres acerca da cultura de ensinar e aprender teatro

Tese de Doutoramento em Ciências da Educação - Especialidade de Desenvolvimento Curricular

Changes in institutional governance and management of higher education in Laos: National University of Laos

PhD thesis in Educational Sciences (specialization in Politics of Education).

Adult learning and education policies in Germany, Portugal and Sweden: An analysis of national reports to CONFINTEA VI

The chapter presents a theoretical proposal of three analytical models of Adult Learning and Education (ALE) policies. Some analytical categories and the corresponding dimensions are organised according to the ALE rationale which is typical of each social policy model. Historical, cultural and educational features are mentioned in connexion with the different policy models and its interpretative capacity to making sense of policies and practices implemented in Germany, Portugal and Sweden. !e analysis includes the states of the art and the official representations of ALE produced by the respective national authorities through national reports which were presented to CONFINTEA VI (2009).

Hydrophobic/hydrophilic P(VDF-TrFE)/PHEA polymer blend membranes

Polymer blend membranes have been obtained consisting of a hydrophilic and a hydrophobic polymers distributed in co-continuous phases. In order to obtain stable membranes in aqueous environments, the hydrophilic phase is formed by a poly(hydrohyethyl acrylate), PHEA, network while the hydrophobic phase is formed by poly(vinylidene fluoride-co-trifluoroethylene) P(VDF-TrFE). To obtain the composites, in a first stage, P(VDF-TrFE) is blended with poly(ethylene oxyde) (PEO), the latter used as sacrificial porogen. P(VDF-TrFE)/PEO blend membranes were prepared by solvent casting at 70° followed by cooling to room temperature. Then PEO is removed from the membrane by immersion in water obtaining a P(VDF-TrFE) porous membrane. After removing of the PEO polymer, a P(VDF-TrFE) membrane results in which pores are collapsed. Nevertheless the pores reopen when a mixture of hydroxethyl acrylate (HEA) monomer, ethyleneglycol dimethacrylate (as crosslinker) and ethanol (as diluent) is absorbed in the membrane and subsequent polymerization yields hybrid hydrophilic/hydrophobic membranes with controlled porosity. The membranes are thus suitable for lithium-ion battery separator membranes and/or biostable supports for cell culture in biomedical applications.

Lideranças curriculares intermédias no contexto da avaliação externa das escolas em Portugal

Tese de Doutoramento em Ciências da Educação (área de especialização em Desenvolvimento Curricular).