A procedure to track vibration modes under changing external factors: application to a pedestrian bridge

A low-cost vibration monitoring system has been developed and installed on an urban steel- plated stress-ribbon footbridge. The system continuously measures: the acceleration (using 18 triaxial MEMS accelerometers distributed along the structure), the ambient temperature and the wind velocity and direction. Automated output-only modal parameter estimation based on the Stochastic Subspace Identification (SSI) is carried out in order to extract the modal parameters, i.e., the natural frequencies, damping ratios and modal shapes. Thus, this paper analyzes the time evolution of the modal parameters over a whole-year data monitoring. Firstly, for similar environmental/operational factors, the uncertainties associated to the time window size used are studied and quantified. Secondly, a methodology to track the vibration modes has been established since several of them with closely-spaced natural frequencies are identified. Thirdly, the modal parameters have been correlated against external factors. It has been shown that this stress-ribbon structure is highly sensitive to temperature variation (frequency changes of more than 20%) with strongly seasonal and daily trends

The deferral option in the collective dismissal procedure. La opción de aplazamiento en los expedientes de regulación de empleo.

The work in this paper focuses on the integration of the real options theory for organizational projects in the management of Human Resources, and particularly on the inclusion of the deferral option in collective dismissal procedures. This option has been studied and developed to be applied to ?Expediente de regulación de empleo?, which is the legal form existing in Spain for the collective termination of employment contracts and which organizations turn to when confronted with a negative financial situation, as a way of maintaining their viability. Two main issues which it is hoped to resolve are examined: the search for a source of uncertainty to make the deferral option viable for this type of projects, and the development of a procedure to obtain the value of the option and therefore facilitate decision making. The analysis performed has enabled us to state that the volatility of demand is the source of uncertainty that makes the option viable. The procedure developed by the binomial tree, which is determined by the evolution of demand, is the tool that enables the value of the option to be found.

Topographical and mechanical characterization of living eukaryotic cells on opaque substrates: development of a general procedure and its application to the study of non-adherent lymphocytes

The mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy (AFM). A robust experimental procedure was developed to overcome some features of lymphocytes, in particular their spherical shape and non-adherent character. The procedure included the immobilization of the lymphocytes on amine-functionalized substrates, the use of hydrodynamic effects on the deflection of the AFM cantilever to monitor the approaching, and the use of the jumping mode for obtaining the images. Indentation curves were analyzed according to Hertz's model for contact mechanics. The calculated values of the elastic modulus are consistent both when considering the results obtained from a single lymphocyte and when comparing the curves recorded from cells of different specimens

Reciprocal subtraction differential RNA display: An efficient and rapid procedure for isolating differentially expressed gene sequences

A reciprocal subtraction differential RNA display (RSDD) approach has been developed that permits the rapid and efficient identification and cloning of both abundant and rare differentially expressed genes. RSDD comprises reciprocal subtraction of cDNA libraries followed by differential RNA display. The RSDD strategy was applied to analyze the gene expression alterations resulting during cancer progression as adenovirus-transformed rodent cells developed an aggressive transformed state, as documented by elevated anchorage-independence and enhanced in vivo oncogenesis in nude mice. This approach resulted in the identification and cloning of both known and a high proportion (>65%) of unknown sequences, including cDNAs displaying elevated expression as a function of progression (progression-elevated gene) and cDNAs displaying suppressed expression as a function of progression (progression-suppressed gene). Sixteen differentially expressed genes, including five unknown progression-elevated genes and six unknown progression-suppressed genes, have been characterized. The RSDD scheme should find wide application for the effective detection and isolation of differentially expressed genes.

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal 125I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect 125I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when 125I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired 125I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.

A general procedure for locating and analyzing protein-binding sequence motifs in nucleic acids

In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.

How Escherichia coli can bias the results of molecular cloning: Preferential selection of defective genomes of hepatitis C virus during the cloning procedure

Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression. These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected. The aim of the present study was to express HCV structural proteins. However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins. The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription–PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector. The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products. Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis. The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72–92%). Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.

Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a γ-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a β-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in γ-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

Lyophilization-induced reversible changes in the secondary structure of proteins.

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.

Otimização do método lignina brometo de acetila na determinação do teor de lignina em plantas forrageiras e comparação com os métodos lignina detergente ácido e lignina Klason

Técnicas analíticas empregadas para a quantificação do teor de lignina em plantas forrageiras, atualmente em uso, são questionáveis quanto às suas acurácias. O método lignina detergente ácido (LDA), que é um dos métodos mais utilizado em Ciência Animal e Agronomia, apresenta algumas falhas, particularmente devido à parcial solubilização da lignina durante a preparação da fibra em detergente ácido (FDA). A lignina Klason (LK), outro método muito usado, apresenta o inconveniente de mensurar a proteína da parede celular como sendo lignina. Em ambos os procedimentos recomenda-se também mensurar cinzas nos resíduos de lignina. A quantificação da concentração de lignina pelo método espectrofotométrico lignina brometo de acetila (LBA) vem ganhando interesse de pesquisadores no Brasil e no exterior. Nesta metodologia, a lignina da planta contida na preparação parede celular (PC) é solubilizada numa solução a 25% de brometo de acetila em ácido acético e a absorbância mensurada é com luz UV a 280 nm. O valor da absorbância é inserido numa equação de regressão e a concentração de lignina é obtida. Para que esta técnica analítica seja mais aceita pelos pesquisadores, ela deve ser, obviamente, convincente e atrativa. O presente trabalho analisou alguns parâmetros relacionados à LBA em 7 gramíneas e 6 leguminosas, em dois estádios de maturidade. Dentre as diferentes temperaturas de pré-secagem, os resultados indicaram que os procedimentos de 55°C com ventilação e liofilização podem ser utilizados com a mesma eficácia. As temperaturas de 55°C sem ventilação e 80°C sem ventilação não são recomendadas, pois aumentaram os valores de FDA e LDA, possivelmente devido ao surgimento de artefatos de técnica como os compostos de Maillard. No método LBA os valores menores das amostras de leguminosas chamaram a atenção e colocaram em questão se a lignina destas plantas seria menos solúvel no reagente brometo de acetila. Dentre algumas alterações na metodologia da técnica LBA, a utilização do moinho de bolas (para diminuir o tamanho particular) nas amostras de PC não mostrou efeito; a hipótese era melhorar a solubilização da lignina usando partículas menores. O uso de um ultrasonicador, que aumenta a vibração das moléculas e assim, facilitaria a solubilização da lignina no reagente brometo de acetila, melhorou a solubilização da lignina em cerca de 10%, tanto nas gramíneas como nas leguminosas. Foi acoplado um ensaio biológico como referência, a degradabilidade in vitro da matéria seca (DIVMS); e como a lignina está intimamente associada à estrutura fibrosa da parede celular, também foi feito um ensaio de degradabilidade in vitro da fibra em detergente neutro (DIVFDN). Os resultados confirmaram o efeito da maturidade, reduzindo a degradabilidade nas plantas mais maduras, e que o teor de lignina de leguminosas é realmente inferior ao de gramíneas. Os resultados de degradabilidade apresentaram coeficientes de correlação mais elevados com o método LBA, quando foi empregada a técnica do ultrasom; o método LK mostrou os menores coeficientes. Também testou-se, com sucesso, a utilização da FDN, como preparação fibrosa, ao invés de PC. A razão é simples: enquanto que a FDN é amplamente conhecida, a preparação PC não o é. Inquestionável que esta manobra facilitará substancialmente a divulgação desse método, tornando-a mais aceitável pela comunidade científica

ϕ-Divergence Based Procedure for Parametric Change-Point Problems

This paper studies the change-point problem for a general parametric, univariate or multivariate family of distributions. An information theoretic procedure is developed which is based on general divergence measures for testing the hypothesis of the existence of a change. For comparing the exact sizes of the new test-statistic using the criterion proposed in Dale (J R Stat Soc B 48–59, 1986), a simulation study is performed for the special case of exponentially distributed random variables. A complete study of powers of the test-statistics and their corresponding relative local efficiencies, is also considered.

Applying Translation Quality Standards to a Certification Procedure for U.S. Language Service Providers

Many translation quality standards have been implemented to regulate the provision and procurement of language services. However, in the absence of a standardized procedure to certify U.S. language service providers (LSPs), the industry lacks consensus with regard to requirements, procedures, and expectations. This project establishes the need for such a procedure and proposes an LSP Certification Procedure based on existing quality standards. Through a review and analysis of existing translation quality standards, an interview with a key stakeholder, and the presentation of an LSP Certification Procedure, this project concludes that the U.S. language services industry requires a procedure to certify LSPs and that such a procedure may be designed and implemented based on existing standards.

Assessment of a simplified experimental procedure to evaluate impact sound reduction of floor coverings

The impact noise reduction provided by floor coverings is usually obtained in laboratory, using the methodology described in the standard EN ISO 140-8, which requires the use of standard acoustic chambers. The construction of such chambers, following the requirements described in the EN ISO 140-1, implies a significant investment, and therefore only a limited number exists in each country. Alternatives to these standard methodologies, that allow a sufficiently accurate evaluation and require lower resources, have been interesting many researchers and manufacturers. In this paper, one such strategy is discussed, where a reduced sized slab is used to determine the noise reduction provided by floor coverings, following the procedure described in the ISO/CD 16251-1 technical document. Several resilient coverings, floating floors and floating slabs are tested and the results are compared with those obtained using the procedures described in the standards EN ISO 140-8 and EN ISO 717-2.

An improved procedure for obtaining and maintaining well characterized partial water saturation states on concrete samples to be used for mass transport tests

A conditioning procedure is proposed allowing to install into the concrete specimens any selected value of water saturation degree with homogeneous moisture distribution. This is achieved within the least time and the minimum alteration of the concrete specimens. The protocol has the following steps: obtaining basic drying data at 50 °C (water absorption capacity and drying curves); unidirectional drying of the specimens at 50 °C until reaching the target saturation degree values; redistribution phase in closed containers at 50 °C (with measurement of the quasi-equilibrium relative humidities); storage into controlled environment chambers until and during mass transport tests, if necessary. A water transport model is used to derive transport parameters of the tested materials from the drying data, i.e., relative permeabilities and apparent water diffusion coefficients. The model also allows calculating moisture profiles during isothermal drying and redistribution phases, thus allowing optimization of the redistribution times for obtaining homogeneous moisture distributions.

Procedure for calculating the chloride diffusion coefficient and surface concentration from a profile having a maximum beyond the concrete surface

Chlorides induce local corrosion in the steel reinforcements when reaching the bar surface. The measurement of the rate of ingress of these ions, is made by mathematically fitting the so called “error function equation” into the chloride concentration profile, obtaining so the diffusion coefficient and the chloride concentration at the concrete surface. However, the chloride profiles do not always follow Fick’s law by having the maximum concentration at the concrete surface, but often the profile shows a maximum concentration more in the interior, which indicates a different composition and performance of the most external concrete layer with respect to the internal zones. The paper presents a procedure prepared during the time of the RILEM TC 178-TMC: “Testing and modeling chloride penetration in concrete”, which suggests neglecting the external layer where the chloride concentration increases and using the maximum as an “apparent” surface concentration, called C max and to fit the error function equation into the decreasing concentration profile towards the interior. The prediction of evolution should be made also from the maximum.