Compósitos CNTs/biocerâmico para a estimulação elétrica óssea in situ

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of ~500 mg/day. The deposition parameters were optimised to obtain high pure CNTs ~99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.

The comparative in situ hygrothermal performance of Hemp and Stone Wool insulations in vapour open timber frame wall panels

An in situ experiment in a full scale timber frame test building was carried out to compare the hygrothermal performance of Hemp and Stone Wool insulations of identical thermal conductivity. Hemp and Stone Wool insulations were installed in timber frame wall panels without vapour barrier. The comparison was made in terms of heat transfer properties, likelihood of mould growth and condensation. Step changes in internal relative humidity were performed to explore the effect of high and normal internal moisture load on the wall panels. No significant difference between the average equivalent thermal transmittance (U-values) of the panels incorporating Hemp and Stone Wool insulations was observed. The average equivalent U-values of the panels were closer to the calculated U-values of the panels based on the manufacturers’ declared thermal conductivity of Hemp and Stone Wool insulations. It was observed that the placement of heat flux sensor along the depth of the insulation had significant influence on the measured equivalent U-value of the panels during high internal moisture load. The frequency and likelihood of condensation was higher in the interface of Stone Wool and Oriented Strand Board (OSB). In terms of the parametric assessment of mould germination potential, relative humidity, temperature and exposure conditions in the insulation-OSB interfaces were found to be favourable to germination of mould spore. However, when the insulations were dismantled, no mould was visually detected.

Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

In-situ development of self-defensive antibacterial biomaterials: phenol-g-keratin-EC based bio-composites with characteristics for biomedical applications

Recently, the development of highly inspired biomaterials with multi-functional characteristics has gained considerable attention, especially in biomedical, and other health-related areas of the modern world. It is well-known that the lack of antibacterial potential has significantly limited biomaterials for many challenging applications such as infection free wound healing and/or tissue engineering etc. In this perspective, herein, a series of novel bio-composites with natural phenols as functional entities and keratin-EC as a base material were synthesised by laccase-assisted grafting. Subsequently, the resulting composites were removed from their respective casting surfaces, critically evaluated for their antibacterial and biocompatibility features and information is also given on their soil burial degradation profile. In-situ synthesised phenol-g-keratin-EC bio-composites possess strong anti-bacterial activity against Gram-positive and Gram-negative bacterial strains i.e., B. subtilis NCTC 3610, P. aeruginosa NCTC 10662, E. coli NTCT 10418 and S. aureus NCTC 6571. More specifically, 10HBA-g-keratin-EC and 20T-g-keratin-EC composites were 100% resistant to colonisation against all of the aforementioned bacterial strains, whereas, 15CA-g-keratin-EC and 15GA-g-keratin-EC showed almost negligible colonisation up to a variable extent. Moreover, at various phenolic concentrations used, the newly synthesised composites remained cytocompatible with human keratinocyte-like HaCaT, as an obvious cell ingrowth tendency was observed and indicated by the neutral red dye uptake assay. From the degradation point of view, an increase in the degradation rate was recorded during their soil burial analyses. Our investigations could encourage greater utilisation of natural materials to develop bio-composites with novel and sophisticated characteristics for potential applications.

Assessment of the environmental radiation by in situ gamma ray spectrometry

Naturally occurring radioactive materials (NORM) under certain conditions can reach hazardous radiological levels contributing to an additional exposure dose to ionizing radiation. Most environmental concerns are associated with uranium mining and milling sites, but the same concerns should be addressed to natural near surface occurrences of uranium as well as man-made sources such as technologically enhanced naturally occurring radioactive materials (TENORM) resulting from phosphates industry, ceramic industry and energy production activities, in particular from coal-fired power plants which is one of the major sources of increased exposure to man from enhanced naturally occurring materials. This work describes the methodology developed to assess the environmental radiation by in situ gamma spectrometry in the vicinity of a Portuguese coal fired power plant. The current investigation is part of a research project that is undergoing in the vicinity of Sines Coal-Fired Power Plant (south of Portugal) until the end of 2013.

Semi-destructive in situ tests as support to the assessment of a conservation process

This paper refers to the assessment on site by semi-destructive testing (SDT) methods of the consolidation efficiency of a conservation process developed by Henriques (2011) for structural and non-structural pine wood elements in service. This study was applied on scots pine wood (Pinus sylvestris L.) degraded by fungi after treatment with a biocidal product followed by consolidation with a polymeric product. This solution avoids substitutions of wood moderately degraded by fungi, improving its physical and mechanical characteristics. The consolidation efficiency was assessed on site by methods of drill resistance and penetration resistance. The SDT methods used showed good sensitivity to the conservation process and could evaluate their effectiveness. (C) 2015 Elsevier Ltd. All rights reserved.

Recycling old screen-printed electrodes with newly designed plastic antibodies on the wall of carbon nanotubes as sensory element for in situ detection of bacterial toxins in water

Using low cost portable devices that enable a single analytical step for screening environmental contaminants is today a demanding issue. This concept is here tried out by recycling screen-printed electrodes that were to be disposed of and by choosing as sensory element a low cost material offering specific response for an environmental contaminant. Microcystins (MCs) were used as target analyte, for being dangerous toxins produced by cyanobacteria released into water bodies. The sensory element was a plastic antibody designed by surface imprinting with carefully selected monomers to ensure a specific response. These were designed on the wall of carbon nanotubes, taking advantage of their exceptional electrical properties. The stereochemical ability of the sensory material to detect MCs was checked by preparing blank materials where the imprinting stage was made without the template molecule. The novel sensory material for MCs was introduced in a polymeric matrix and evaluated against potentiometric measurements. Nernstian response was observed from 7.24 × 10−10 to 1.28 × 10−9 M in buffer solution (10 mM HEPES, 150 mM NaCl, pH 6.6), with average slopes of −62 mVdecade−1 and detection capabilities below 1 nM. The blank materials were unable to provide a linear response against log(concentration), showing only a slight potential change towards more positive potentials with increasing concentrations (while that ofthe plastic antibodies moved to more negative values), with a maximum rate of +33 mVdecade−1. The sensors presented good selectivity towards sulphate, iron and ammonium ions, and also chloroform and tetrachloroethylene (TCE) and fast response (<20 s). This concept was successfully tested on the analysis of spiked environmental water samples. The sensors were further applied onto recycled chips, comprehending one site for the reference electrode and two sites for different selective membranes, in a biparametric approach for “in situ” analysis.

Dimensionamento de lajes aligeiradas betonadas "in situ"

A conjuntura económica da construção não se encontra numa fase de prosperidade, existindo a necessidade de criar soluções construtivas que permitam a redução de custos dos materiais a nível de obra. As lajes de betão armado, devido à sua grande área de implantação, possuem uma parcela importante no orçamento final de qualquer obra de edifícios correntes. Sabendo das possibilidades de aligeiramento de lajes, torna-se necessária a realização de um estudo pormenorizado e comparativo entre soluções de aligeiramento. O desenvolvimento deste relatório reflete todo o trabalho efetuado durante a realização de um estágio curricular desenvolvido na empresa FercaNorte – Estruturas, Lajes e Cofragens, Lda. e foca-se num dimensionamento e estudo comparativo de soluções de lajes fungiformes aligeiradas, aplicadas a dois casos de estudo e também num dimensionamento e estudo comparativo de soluções de lajes aligeiradas unidirecionais mas apenas aplicadas a um caso de estudo. No primeiro caso de estudo, laje fungiforme com um vão de 6 metros nas duas direções, são dimensionadas e comparadas quatro soluções: laje fungiforme maciça; laje fungiforme aligeirada com blocos elipsoides perdidos dispostos de forma ortogonal e envolvidos totalmente por betão; laje fungiforme aligeirada com blocos elipsoides perdidos dispostos em formato de favo de mel e envolvidos totalmente por betão; e laje fungiforme nervurada com moldes recuperáveis. No segundo caso de estudo, laje fungiforme com um vão de 7,04 metros nas duas direções, são dimensionadas e comparadas cinco soluções: laje fungiforme maciça, laje fungiforme aligeirada com blocos cúbicos perdidos dispostos de forma ortogonal e envolvidos totalmente por betão; laje fungiforme aligeirada, com blocos elipsoides perdidos dispostos de forma ortogonal e envolvidos totalmente por betão; laje fungiforme aligeirada com blocos elipsoides perdidos dispostos em formato de favo de mel e envolvidos totalmente por betão; e laje fungiforme nervurada com moldes recuperáveis. No terceiro caso de estudo, laje unidirecional com um vão de 8 metros apoiada em vigas com vão de 5 metros, são dimensionadas e comparadas três soluções: laje maciça; laje aligeirada com perfis tubulares com secção transversal reta oval; e laje nervurada com moldes recuperáveis. No final é apresentada uma estimativa orçamental para cada solução analisada nos três casos de estudo abordados.

The Discrimination of Colored Acrylic, Cotton, and Wool Textile Fibers Using Micro-Raman Spectroscopy. Part 1 : In Situ Detection and Characterization of Dyes

Raman spectroscopy has been applied to characterize fiber dyes and determine the discriminating ability of the method. Black, blue, and red acrylic, cotton, and wool samples were analyzed. Four excitation sources were used to obtain complementary responses in the case of fluorescent samples. Fibers that did not provide informative spectra using a given laser were usually detected using another wavelength. For any colored acrylic, the 633-nm laser did not provide Raman information. The 514-nm laser provided the highest discrimination for blue and black cotton, but half of the blue cottons produced noninformative spectra. The 830-nm laser exhibited the highest discrimination for red cotton. Both visible lasers provided the highest discrimination for black and blue wool, and NIR lasers produced remarkable separation for red and black wool. This study shows that the discriminating ability of Raman spectroscopy depends on the fiber type, color, and the laser wavelength.

Modeling of in-situ crystallization processes in the Permian mafic layered intrusion of Mont Collon (Dent Blanche nappe, western Alps)

The Mont Collon mafic complex is one of the best preserved examples of the Early Permian magmatism in the Central Alps, related to the intra-continental collapse of the Variscan belt. It mostly consists (> 95 vol.%) of ol+hy-nonnative plagioclase-wehrlites, olivine- and cpx-gabbros with cumulitic structures, crosscut by acid dikes. Pegmatitic gabbros, troctolites and anorthosites outcrop locally. A well-preserved cumulative, sequence is exposed in the Dents de Bertol area (center of intrusion). PT-calculations indicate that this layered magma chamber emplaced at mid-crustal levels at about 0.5 GPa and 1100 degrees C. The Mont Collon cumulitic rocks record little magmatic differentiation, as illustrated by the restricted range of clinopyroxene mg-number (Mg#(cpx)=83-89). Whole-rock incompatible trace-element contents (e.g. Nb, Zr, Ba) vary largely and without correlation with major-element composition. These features are characteristic of an in-situ crystallization process with variable amounts of interstitial liquid L trapped between the cumulus mineral phases. LA-ICPMS measurements show that trace-element distribution in the latter is homogeneous, pointing to subsolidus re-equilibration between crystals and interstitial melts. A quantitative modeling based on Langmuir's in-situ crystallization equation successfully duplicated the REE concentrations in cumulitic minerals of all rock facies of the intrusion. The calculated amounts of interstitial liquid L vary between 0 and 35% for degrees of differentiation F of 0 to 20%, relative to the least evolved facies of the intrusion. L values are well correlated with the modal proportions of interstitial amphibole and whole-rock incompatible trace-element concentrations (e.g. Zr, Nb) of the tested samples. However, the in-situ crystallization model reaches its limitations with rock containing high modal content of REE-bearing minerals (i.e. zircon), such as pegmatitic gabbros. Dikes of anorthositic composition, locally crosscutting the layered lithologies, evidence that the Mont Collon rocks evolved in open system with mixing of intercumulus liquids of different origins and possibly contrasting compositions. The proposed model is not able to resolve these complex open systems, but migrating liquids could be partly responsible for the observed dispersion of points in some correlation diagrams. Absence of significant differentiation with recurrent lithologies in the cumulitic pile of Dents de Bertol points to an efficiently convective magma chamber, with possible periodic replenishment, (c) 2005 Elsevier B.V. All rights reserved.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

Resultados preliminares de las mediciones de fuerza de blanco in situ de las principales especies pelágicas. Crucero BIC Humboldt 9803-05 de Tumbes a Tacna

Se han utilizado los resultados de 144 lances ejecutados en el transcurso del Crucero 9803-05. Se compararon los histogramas de TS y los de tallas en búsqueda de correlación entre ambas a fin de determinar el factor b20 de la Ecuación de Fuerza de Blanco (20 log (LT,cm) + b20). Se han determinado nuevas ecuaciones de TS para anchoveta (38 y 120 kHz), sardina (120 kHz), jurel (38 kHz) y caballa (38 y 120 kHz). Se deberá continuar con estas mediciones puesto que un cambio en las propuestas reflectivas de los peces puede afectar la correcta obtención de los estimados de biomasa.

Mediciones in situ de TS de diversas especies a finales del invierno de 1998. Crucero BIC Humboldt 9808-09

Se efectuaron mediciones de TS (Fuerza de Blanco, Target Strenght) durante los 108 lances de comprobación de ecotrazos realizados por el BIC Humboldt en el marco del Crucero 9808-09 de Evaluación Hidroacústica de Recursos Pelágicos. Se consideran para el análisis únicamente aquellos lances donde más del 90% de la captura perteneció a una sola especie. Los factores b20 de la ecuación de TS (TS=20 log L - b20) que han sido determinados, las cuales deberán ser considerados como provisionales, son las siguientes: Pez cinta (Trichiurus lepturus) = 70,95 (120 kHz); Vinciguerria o pez linterna (Vinciguerria lucetia pacifici) = 83,29 (120 kHz); Samasa (Anchoa nasus) = 86,57 (120 kHz); Caballa (Scomber japonicus) = 83,09 (120 kHz); Pez cinta (Trichiurus lepturus) = 71,41 (38 kHz); y Vinciguerria o pez linterna (Vinciguerria lucetia pacifici)= 82,04 (38kHz).