Pèptids biarílics a partir de 4-iodofenilalanina o 3-iodotirosina per borilació i reacció de Suzuki-Miyaura en fase sòlida. Avaluació de l'activitat antimicrobiana

En aquesta tesi doctoral es va estudiar la preparació de pèptids biarílics en fase sòlida. En primer lloc, es varen borilar residus de fenilalanina o tirosina presents a la seqüència peptídica a través d’una reacció de Miyaura. A continuació, es varen arilar els boronats resultants a través d’una reacció de Suzuki-Miyaura sota irradiació de microones, utilitzant diversos halurs d’aril i haloaminoàcids. La metodologia trobada es va estendre a la preparació de pèptids biarílics cíclics. Aquesta aproximació presenta l’avantatge d’evitar la síntesi en dissolució i la purificació del boronoaminoàcid. A més, permet la preparació d’una àmplia diversitat de pèptids biarílics a partir d’un únic boronopèptid. L’avaluació de l’activitat biològica dels pèptids sintetitzats va permetre idenficar seqüències actives enfront dels bacteris Erwinia amylovora, Xanthomonas vesicatoria, i Pseudomonas syringae, que són responsables de malalties greus en plantes d’interès econòmic com pereres i pomeres, i que varen resultar ser molt poc tòxics enfront cèl•lules eucariotes.

Contribución al estudio de la extracción química del Fe3+ contaminante de la madera arqueológica subacuática impregnada con polietilenglicol

Due to the large quantity of contamination by Fe in the waterlogged wood and the composites previously processed with poliethilenglycol, PEG , we have thought the possible extraction and formation of complexes Fe-L (L= 2-Phosphonobuta-1,2,4- tricarboxylic acid, (PBTC)) and their effect on (i) structure of the wood, (ii) structure and physiques characteristics of PEG and (iii) post-treatment contamination by Fe coming from atmospheric agents. This work is a project for to study the formation of these complexes and possible modifications in structures.

Vivienda obrera en Bilbao y el Bajo Nervión: las casas baratas, una nueva forma de alojamiento (1911-1936)

A finales del siglo XIX y principios del siglo XX la provincia de Vizcaya despuntó en el contexto internacional como importe centro extractor de mineral de hierro y como relevante foco productor siderometalúrgico. Las explotaciones mineras se adueñaron de los Montes de Triano y las fábricas conquistaron las márgenes del Río Nervión, arrastrando tras de sí una densa marea humana. Alojar a esa población se convirtió en una cuestión de primer orden. Paralelamente, las instituciones estatales desbordadas por problemas similares en otros puntos de la geografía española, idearon y pusieron en práctica un complejo engranaje administrativo para erigir las primeras viviendas sociales. Así, las denominadas Casas Baratas fueron la respuesta oficial a la insuficiencia de vivienda obrera y Vizcaya fue una de las provincias donde mayor desarrollo tuvieron este tipo de propuestas constructivas. De este modo, la presente investigación comienza indagando sobre el origen de la problemática en Bilbao y la Cuenca del Bajo Nervión, para después repasar las medidas adoptadas por el Estado y la Diputación de Vizcaya en esta materia. Hecho esto, se da paso a un concienzudo repaso de las características principales de la edificación de Casas Baratas en toda la provincia de Vizcaya, descendiendo, posteriormente, a la realidad de Bilbao, Baracaldo, Sestao, Portugalete, Guecho y Erandio, haciendo hincapié en las inquietudes locales. El estudio finaliza con un ejercicio comparativo con las Casas Baratas de Barcelona, Sabadell y Tarrasa, estableciéndose similitudes y divergencias con el foco industrial más importante del momento en el panorama nacional.

A suite of parallel vectors for baculovirus expression

The expression of proteins using recombinant baculoviruses is a mature and widely used technology. However, some aspects of the technology continue to detract from high throughput use and the basis of the final observed expression level is poorly understood. Here, we describe the design and use of a set of vectors developed around a unified cloning strategy that allow parallel expression of target proteins in the baculovirus system as N-terminal or C-terminal fusions. Using several protein kinases as tests we found that amino-terminal fusion to maltose binding protein rescued expression of the poorly expressed human kinase Cot but had only a marginal effect on expression of a well-expressed kinase IKK-2. In addition, MBP fusion proteins were found to be secreted from the expressing cell. Use of a carboxyl-terminal GFP tagging vector showed that fluorescence measurement paralleled expression level and was a convenient readout in the context of insect cell expression, an observation that was further supported with additional non-kinase targets. The expression of the target proteins using the same vectors in vitro showed that differences in expression level were wholly dependent on the environment of the expressing cell and an investigation of the time course of expression showed it could affect substantially the observed expression level for poorly but not well-expressed proteins. Our vector suite approach shows that rapid expression survey can be achieved within the baculovirus system and in addition, goes some way to identifying the underlying basis of the expression level obtained. (c) 2006 Elsevier Inc. All rights reserved.

Alternative syntheses of linear polyurethanes using masked isocyanate monomers

A range of linear polyurethanes featuring aliphatic, aromatic and ether residues have been prepared by co-polymerisation of novel 'masked' isocyanate A(2)-type monomers and diols. The reactive isocyanate monomers were generated in situ via the triphenylphosphine mediated decomposition of the heterocyclic disulfide, 1,2,4-dithiazolidine-3,5-dione. Two different synthetic approaches were examined and assessed for the construction of the novel A(2)-type monomers, which involved either coupling two 1,2,4-dithiazolidine-3,5-diones together through a spacer group or construction of 1,2,4-dithiazolidine-3,5-diones directly from diamines. The resulting polyurethanes were purified via solvent extraction and analysed using GPC, NMR and IR spectroscopic analyses. Molecular weight data were obtained and compared from both GPC and H-1 NMR (via end-group analysis) spectroscopic analysis. The thermal properties of the polyurethanes were determined using DSC and their solubility in common aprotic organic solvents was also assessed and related to their structural composition. (c) 2006 Elsevier B.V. All rights reserved.

Selective catalytic oxidation of alkylaromatic molecules by nanosize water droplets containing Co2+ species in supercritical carbon dioxide fluid

Stabilized nano-sized water droplet carrying water-soluble Co2+ species is employed as a new catalyst system for the oxidation of the alkyl aromatics in the presence of a fluorinated surfactant. This stable system contains no labile C-H structure and can facilitate excellent mixing of catalytic Co(II)/NaBr species, hydrocarbon substrates and oxygen in supercritical carbon dioxide fluid, which is demonstrated to be an excellent alternative solvent system to acetic acid or nitric acid for air oxidation of a number of alkyl aromatic hydrocarbons using Co(II) species at mild conditions. As a result, potential advantages of this 'greener' catalytic method including safer operation, easier separation and purification, higher catalytic activity with selectivity and without using corrosive or oxidation unstable solvent are therefore envisaged.

Volatile flavour profile of goat meat extracted by three widely used techniques

Three procedures for the isolation of volatiles from grilled goat meat were compared: dynamic headspace entrainment on Tenax TA, simultaneous steam distillation-extraction, and solid-phase microextraction. Headspace entrainment on Tenax TA extracted the highest number of Maillard-derived volatile compounds. Two hundred and three volatile components were identified: 159 are reported for the first time in goat meat. Most of the volatiles detected (155) were lipid oxidation products, such as hydrocarbons, aldehydes, alcohols, ketones, carboxylic acids and esters. Forty-eight Maillard-derived compounds were identified. comprising pyrazines, pyrroles, thiophenes, furanthiol derivatives, alkyl and alicyclic sulphides, pyridines, and thiazoles. Some reported character impact compounds of cooked meat, e.g., 12-methyltridecanal, (EE)-2,4-decadienal, methional, and dimethyl trisulphide were identified in the volatile profile of goat meat, together with a series of C-2 to C-5 alkylformylcyclopentenes, which have been reported in cooked chicken, pork, beef and lamb, as being important for the characteristic flavour impression of different animal species. (C) 2009 Published by Elsevier Ltd.

Recovery of astaxanthin using colloidal gas aphrons (CGA): a mechanistic study

The aim of this study is to investigate the mechanism responsible for the recovery of astaxanthin using Colloidal Gas Aphrons (CGA), which are surfactant stabilised microbubbles. The latter were produced using different surfactant solutions (Cetyl Trimethyl Ammonium Bromide (CTAB)-cationic, Sodium Dodecyl Sulfate (SDS)-anionic, TWEEN 60-non-ionic and mixtures of TWEEN 60-SPAN 80- non-ionic with varying hydrophobicity) at stirring speed 8000 rpm and stirring time 5 min. Experiments were carried out at varying pH and volumetric ratios of astaxanthin to CGA, and with two different astaxanthin standard suspensions: (i) astaxanthin dispersed in aqueous solutions and (ii) astaxanthin dispersed in ethanolic/aqueous solutions with different compositions of ethanol (20/80 (v/v) and 40/60 (v/v)). When astaxanthin is dispersed in aqueous solutions the separation seems to occur mainly by electrostatic interactions. Therefore the recoveries are higher in the case of the cationic surfactant when astaxanthin particles are strongly negatively charged, as shown by the zeta potential measurements. When ethanol is present, highest recoveries are achieved with CGA produced from the non-ionic surfactant, which indicates that, under these conditions, separation is driven mainly by hydrophobic interactions. In experiments with ethanolic/aqueous suspensions, when the hydrophobicity of the surfactant was increased by increasing volumes of SPAN 80, the CGA produced were less stable; thus higher recoveries of astaxanthin under conditions that favour hydrophobic interactions were not observed. (C) 2008 Elsevier B.V All rights reserved.

A systematic review of the routine monitoring of growth in children of primary school age to identify growth-related conditions

Objectives: To clarify the role of growth monitoring in primary school children, including obesity, and to examine issues that might impact on the effectiveness and cost-effectiveness of such programmes. Data sources: Electronic databases were searched up to July 2005. Experts in the field were also consulted. Review methods: Data extraction and quality assessment were performed on studies meeting the review's inclusion criteria. The performance of growth monitoring to detect disorders of stature and obesity was evaluated against National Screening Committee (NSC) criteria. Results: In the 31 studies that were included in the review, there were no controlled trials of the impact of growth monitoring and no studies of the diagnostic accuracy of different methods for growth monitoring. Analysis of the studies that presented a 'diagnostic yield' of growth monitoring suggested that one-off screening might identify between 1: 545 and 1: 1793 new cases of potentially treatable conditions. Economic modelling suggested that growth monitoring is associated with health improvements [ incremental cost per quality-adjusted life-year (QALY) of pound 9500] and indicated that monitoring was cost-effective 100% of the time over the given probability distributions for a willingness to pay threshold of pound 30,000 per QALY. Studies of obesity focused on the performance of body mass index against measures of body fat. A number of issues relating to human resources required for growth monitoring were identified, but data on attitudes to growth monitoring were extremely sparse. Preliminary findings from economic modelling suggested that primary prevention may be the most cost-effective approach to obesity management, but the model incorporated a great deal of uncertainty. Conclusions: This review has indicated the potential utility and cost-effectiveness of growth monitoring in terms of increased detection of stature-related disorders. It has also pointed strongly to the need for further research. Growth monitoring does not currently meet all NSC criteria. However, it is questionable whether some of these criteria can be meaningfully applied to growth monitoring given that short stature is not a disease in itself, but is used as a marker for a range of pathologies and as an indicator of general health status. Identification of effective interventions for the treatment of obesity is likely to be considered a prerequisite to any move from monitoring to a screening programme designed to identify individual overweight and obese children. Similarly, further long-term studies of the predictors of obesity-related co-morbidities in adulthood are warranted. A cluster randomised trial comparing growth monitoring strategies with no growth monitoring in the general population would most reliably determine the clinical effectiveness of growth monitoring. Studies of diagnostic accuracy, alongside evidence of effective treatment strategies, could provide an alternative approach. In this context, careful consideration would need to be given to target conditions and intervention thresholds. Diagnostic accuracy studies would require long-term follow-up of both short and normal children to determine sensitivity and specificity of growth monitoring.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Improved process topology for membrane distillation

In membrane distillation in a conventional membrane module, the enthalpies of vaporisation and condensation are supplied and removed by changes in the temperatures of the feed and permeate streams, respectively. Less than 5% of the feed can be distilled in a single pass, because the potential changes in the enthalpies of the liquid streams are much smaller than the enthalpy of vaporisation. Furthermore, the driving force for mass transfer reduces as the feed stream temperature and vapour pressure fall during distillation. These restrictions can be avoided if the enthalpy of vaporisation is uncoupled from the heat capacities of the feed and permeate streams. A specified distillation can then be effected continuously in a single module. Calculations are presented which estimate the performance of a flat plate unit in which the enthalpy of distillation is supplied and removed by the condensing and boiling of thermal fluids in separate circuits, and the imposed temperature difference is independent of position. Because the mass flux through the membrane is dependent on vapour pressure, membrane distillation is suited to applications with a high membrane temperature. The maximum mass flux in the proposed module geometry is predicted to be 30 kg/m2 per h at atmospheric pressure when the membrane temperature is 65°C. Operation at higher membrane temperatures is predicted to raise the mass flux, for example to 85 kg/m2 per h at a membrane temperature of 100°C. This would require pressurisation to 20 bar to prevent boiling at the heating plate of the feed channel. Pre-pressurisation of the membrane pores and control of the dissolved gas concentrations in the feed and the recyled permeate should be investigated as a means to achieve high temperature membrane distillation without pore penetration and wetting.

The separation of americium(III) from europium(III) by two new 6,6'-bistriazinyl-2,2'-bipyridines in different diluents

The synthesis and extraction of americium(III) and europium(III) from aqueous nitric acid solutions by the new BTBP ligands 6,6’-bis(5,5,7,7- tetramethyl-5,7-dihydrofuro[3,4-e]-1,2,4-triazin-3-yl)-2,2’-bipyridine (Cy5-O-Me4-BTBP), and 6,6’-bis(5,5,7,7-tetramethyl-5,7-dihydrothieno[3,4-e]-1,2,4-triazin-3-yl)- 2,2’-bipyridine (Cy5-S-Me4-BTBP) is described. The affinity for Am(III) and the selectivity for Am(III) over Eu(III) of Cy5-S-Me4-BTBP were generally higher than for Cy5-O-Me4-BTBP. For both ligands, the extraction of Am(III) and Eu(III) from 3 M HNO3 into 3 mM organic solutions varied with the diluent used. The highest distribution ratios and separation factors observed were in cyclohexanone and 2-methylcyclohexanone, respectively. For Cy5-S-Me4-BTBP, there is a strong correlation between the distribution ratio for Am(III) and the permittivity of the diluent used. With 1-octanol as the diluent, low distribution ratios (D(Am) < 1) were observed for Cy5-S-Me4-BTBP although this ligand extracts Am(III) selectively (SFAm/Eu = 16-46 from 1-4 M HNO3). For Cy5-S-Me4-BTBP, Am(III) is extracted as the disolvate. The distribution ratios for Am(III), and the separation factors for Am(III) over Eu(III) are both significantly higher for CyMe4-BTBP than they are for Cy5-O-Me4-BTBP and Cy5-S-Me4-BTBP in cyclohexanone. Changing the diluent from cyclohexanone to 2-methylcyclohexanone leads to a decrease in D(Am) but an increase in SFAm/Eu for Cy5-S-Me4-BTBP.

Recovery of gallic acid with colloidal gas aphrons generated from a cationic surfactant

In a previous study we have demonstrated that gallic acid (GA) in its anionic form can be recovered from aqueous solutions using colloidal gas aphrons (CGA) generated from the cationic surfactant cetyltrimethylammonium bromide (CTAB). The aim of the present work is to get a better understanding of the separation mechanism in order to determine the optimum operating conditions to maximise the recovery of GA while preserving its antioxidant properties. Zeta potential measurements were carried out to characterise the surface charge of GA, CTAB and their mixtures at three different pH conditions (both in buffers and in aqueous solutions). GA interacted strongly with CTAB at pH higher than its pKa 3.14 where it is ionised and negatively charged. However, at pH higher than 7 GA becomes oxidised and loses its antioxidant power. GA recovery was mainly affected by pH, ionic strength, surfactant/GA molar ratio, mixing conditions and contact time. Scale-up of the separation using a flotation column resulted in both higher recovery and reproducibility. Preliminary experiments with grape marc extracts confirmed the potential application of this separation for the recovery of polyphenols from complex feedstocks

Optimisation of recombinant production of active human cardiac SERCA2a ATPase

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.