Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Global properties of the mapping between local amino acid sequence and local structure in proteins.

Local protein structure prediction efforts have consistently failed to exceed approximately 70% accuracy. We characterize the degeneracy of the mapping from local sequence to local structure responsible for this failure by investigating the extent to which similar sequence segments found in different proteins adopt similar three-dimensional structures. Sequence segments 3-15 residues in length from 154 different protein families are partitioned into neighborhoods containing segments with similar sequences using cluster analysis. The consistency of the sequence-to-structure mapping is assessed by comparing the local structures adopted by sequence segments in the same neighborhood in proteins of known structure. In the 154 families, 45% and 28% of the positions occur in neighborhoods in which one and two local structures predominate, respectively. The sequence patterns that characterize the neighborhoods in the first class probably include virtually all of the short sequence motifs in proteins that consistently occur in a particular local structure. These patterns, many of which occur in transitions between secondary structural elements, are an interesting combination of previously studied and novel motifs. The identification of sequence patterns that consistently occur in one or a small number of local structures in proteins should contribute to the prediction of protein structure from sequence.

Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is expressed in the adrenal cortex and gonads and regulates the expression of several P450 steroid hydroxylases in vitro. We examined the role of SF-1 in the adrenal glands and gonads in vivo by a targeted disruption of the mouse SF-1 gene. All SF-1-deficient mice died shortly after delivery. Their adrenal glands and gonads were absent, and persistent Mullerian structures were found in all genotypic males. While serum levels of corticosterone in SF-1-deficient mice were diminished, levels of adrenocorticotropic hormone (ACTH) were elevated, consistent with intact pituitary corticotrophs. Intrauterine survival of SF-1-deficient mice appeared normal, and they had normal serum level of corticosterone and ACTH, probably reflecting transplacental passage of maternal steroids. We tested whether SF-1 is required for P450 side-chain-cleavage enzyme (P450scc) expression in the placenta, which expresses both SF-1 and P450scc, and found that in contrast to its strong activation of the P450scc gene promoter in vitro, the absence of SF-1 had no effect on P450scc mRNA levels in vivo. Although the region targeted by our disruption is shared by SF-1 and by embryonal long terminal repeat-binding protein (ELP), a hypothesized alternatively spliced product, we believe that the observed phenotype reflects absent SF-1 alone, as PCR analysis failed to detect ELP transcripts in any mouse tissue, and sequences corresponding to ELP are not conserved across species. These results confirm that SF-1 is an important regulator of adrenal and gonadal development, but its regulation of steroid hydroxylase expression in vivo remains to be established.

Cryptorchidism and homeotic transformations of spinal nerves and vertebrae in Hoxa-10 mutant mice.

Homozygous mice mutated by homologous recombination for the AbdB-related Hoxa-10 gene are viable but display homeotic transformations of vertebrae and lumbar spinal nerves. Mutant males exhibit unilateral or bilateral criptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and sterility. These results reveal an important role of Hoxa-10 in patterning posterior body regions and suggest that Hox genes are involved in specifying regional identity of both segmented and nonovertly segmented structures of the developing body.

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Molecular cloning and characterization of chitinase genes from Candida albicans.

Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.

Volunteer Engagement: Does Engagement Predict the Degree of Satisfaction among New Volunteers and the Commitment of Those who have been Active Longer?

This study examines the concept of engagement in samples of volunteers from different non-profit organisations. Study 1 analyzes the psychometric properties of the abbreviated version of the Utrecht Work Engagement Scale (UWES) (Schaufeli, Bakker, & Salanova, 2006a). Two factorial structures are examined: one-dimensional and three-dimensional structures. Based on the Three-Stage Model of Volunteers’ Duration of Service (Chacón, Vecina, & Dávila, 2007), Study 2 investigates the relationship between engagement, volunteer satisfaction, and intention to remain in a sample of new volunteers and the relationship between engagement, organisational commitment, and intention to remain in a sample of veteran volunteers. Moderated mediation analysis is provided using duration of service as a moderator in order to set a splitting point between new and veteran volunteers. The results of the confirmatory factor analysis suggest that the three-factor model fits better to the data. Regarding the structural models, the first one shows that engagement is crucial to volunteer satisfaction during the first stage, while volunteer satisfaction is the key variable in explaining intention to continue. The second structural model shows that engagement reinforces the participant’s commitment to the organisation, while organizational commitment predicts intention to continue. Both models demonstrate a notable decline when samples are changed.

Contribuição ao estudo de instabilidade em túneis não revestidos da estrada de ferro Vitória-Minas através da teoria dos blocos-chave e caracterização da rocha através de ensaios laboratoriais e de campo.

No país existem inúmeras estruturas e obras civis que estão em operação a dezenas de anos e necessitam de monitoramento periódico devido a sua importância. Por este motivo, a dissertação apresenta um caso de um túnel antigo com problema de queda de bloco e visa instigar novas pesquisas e aumentar o conhecimento sobre o tema. Foram realizadas inspeções em campo em alguns túneis não revestidos da Estrada de Ferro Vitória-Minas (EFVM), bem como os ensaios em laboratório e in situ realizados nas amostras e no maciço rochoso para caracterizar o problema. Para o estudo foi escolhido o túnel Monte Seco Linha 1 e Linha 2 nos quais foram realizadas sondagens rotativas inclinadas e orientadas próximas ao eixo para investigação dos planos de descontinuidade. Os conceitos da Teoria dos Blocos-Chave foram aplicados às famílias de descontinuidades encontradas nos Túneis Monte Seco L1 e L2 para identificar os possíveis blocos instáveis formados pelas escavações. Para obtenção dos parâmetros geotécnicos de resistência e deformabilidade foram realizados ensaios de compressão uniaxial instrumentados com strain gages. A resistência a tração foi obtida através de Ensaio de Compressão Diametral (ECD). No ensaio de campo foi utilizado o Martelo de Schmidt para avaliação da rocha in situ. Através da análise dos dados foi possível distinguir setores cuja ocorrência de queda de blocos são maiores e a classe do maciço rochoso de acordo com a proposta de Bieniawski.

Use of subpixel techniques in pocket cameras to measure vibrations and displacements

Presentación oral SPIE Photonics Europe, Brussels, 16-19 April 2012.

Micro/Mesoporous Activated Carbons Derived from Polyaniline: Promising Candidates for CO2 Adsorption

A series of activated carbons were prepared by carbonization of polyaniline at different temperatures, using KOH or K2CO3 as activating agent. Pure microporous or micro/mesoporous activated carbons were obtained depending on the preparation conditions. Carbonization temperature has been proven to be a key parameter to define the textural properties of the carbon when using KOH. Low carbonization temperatures (400–650 °C) yield materials with a highly developed micro- and mesoporous structure, whereas high temperatures (800 °C) yield microporous carbons. Some of the materials prepared using KOH exhibit a BET surface area superior to 4000 m2/g, with total pore volume exceeding 2.5 cm3/g, which are among the largest found for activated carbons. On the other hand, microporous materials are obtained when using K2CO3, independently of carbonization temperature. Some of the materials were tested for CO2 capture due to their high microporosity and N content. The adsorption capacity for CO2 at atmospheric pressure and 0 °C achieves a value of ∼7.6 mmol CO2/g, which is among the largest reported in the literature. This study provides guidelines for the design of activated carbons with a proper N/C ratio for CO2 capture at atmospheric pressure.

SMEs and Level 2 BIM, the Way Forward

The UK construction industry comprises a very high proportion of SMEs that is companies employing up to 250. A Department for Business, Innovation and Skills research paper, found that SMEs had a 71.2% share of work in the construction industry. Micro and small firms (i.e. those employing up to 50) had a share of 46.7% of work (Ive and Murray 2013). The Government has high ambitions for UK construction. Having been found by successive government commissioned studies to be inefficient and highly fragmented, ambitious targets have been set for the industry to achieve 33% reduction in costs and 50% faster delivery by 2025. As a significant construction client, the Government has mandated the use of Level 2 BIM from 2016 on publicly funded projects over £5 million. The adoption of BIM plays a key role in the 2025 vision but a lack of clarity persists in the industry over BIM and significant barriers are perceived to its implementation, particularly amongst SMEs. However, industry wide transformation will be challenging without serious consideration of the capabilities of this large majority. Many larger firms, having implemented Level 2 BIM are now working towards Level 3 BIM while many of the smaller firms in the industry have not even heard of BIM. It would seem that fears of a ‘two tier’ industry are perhaps being realised. This paper builds on an earlier one (Mellon & Kouider 2014) and investigates, through field work, the level of Level 2 BIM implementation amongst SMEs compared to a large organisation. Challenges and innovative solutions identified through collected data are fully discussed and compared. It is suggested that where the SME perceives barriers towards adoption of the technologies which underpin BIM, they may consider collaborative methods of working as an interim step in order to work towards realising the efficiencies and benefits that these methods can yield. While the barriers to adoption of BIM are significant, it is suggested that they are not insurmountable for the SME and some recommendations for possible solutions are made.

Collateral and Credit Rationing. The role of collateral in explaining and remediating the limited flow of credit to households and SMEs. ECRI Policy Brief No. 7, February 2014

European-wide data concerning both companies and households indicate that the credit rationing phenomenon, which has been predicted by theory, does in fact occur to a significant degree in the European credit market. Among SMEs, micro companies are most vulnerable and the current economic crisis has only made these concerns more pressing. Top-down use of the monetary transmission mechanism alone is insufficient to counter the problem. The other solution consists of a bottom-up, microeconomic stimulation of lending transactions, by focusing on collateral and guarantees. The data confirm the high importance that lenders – especially individual households and micro companies – attach to collateral and guarantees when making their lending decisions. As a consequence, we would argue that those parts of the law governing security interests and guarantees should be one of the primary targets for government policy aimed at improving credit flows, especially in avoiding a conflict between consumer protection measures and laws on surety and guarantees. This policy brief firstly aims to give an overview of the problem of credit rationing and to show that low-income households and SMEs are most concerned by the phenomenon. Focusing solely on loans as a way of financing and on the issues related to access to finance by micro and small companies as well households, it then sketches possible solutions focused on guarantees. This paper brings together data from the Eurosystem Household Finance and Consumption survey (HFCS), Eurostat, and both the latest wave of the extended biennial EC/ECB Survey on the access to finance of SMEs (EC/ECB SAFE 2013) and the latest wave of the smaller semi-annual ECB SAFE Survey, covering the period between October 2012 and March 2013.

Shifting Between Hegemony and Dominance? A Neo-Gramscian Analysis of the EU as a Structural Foreign Policy Actor: The Singular Case of the Cariforum-EU Economic Partnership Agreement. Bruges Regional Integration & Global Governance Paper 01/2014

The groundbreaking scope of the Economic Partnership Agreement (EPA) between the European Union (EU) and Cariforum (CF) irrefutably marks a substantive shift in trade relations between the regions and also has far-reaching implications across several sectors and levels. Supplementing the framework of analysis of Structural Foreign Policy (SFP) with neo-Gramscian theory allows for a thorough investigation into the details of structural embeddedness based on the EU's historic directionality towards the Caribbean region; notably, encouraging integration into the global capitalist economy by adapting to and adopting the ideals of neoliberal economics. Whilst the Caribbean – as the first and only signatory of a ‘full’ EPA – may be considered the case par excellence of the success of the EPAs, this paper demonstrates that there is no cause-effect relationship between the singular case of the ‘full’ CF-EU EPA and the success of the EPA policy towards the ACP in general. The research detailed throughout this paper responds to two SFP-based questions: (1) To what extent is the EPA a SFP tool aimed at influencing and shaping the structures in the Caribbean? (2) To what extent is the internalisation of this process reflective of the EU as a hegemonic SFP actor vis-à-vis the Caribbean? This paper affirms both the role of the EU as a hegemonic SFP actor and the EPA as a hegemonic SFP tool. Research into the negotiation, agreement and controversy that surrounds every stage of the EPA confirmed that through modern diplomacy and an evolution in relations, consensus is at the fore of contemporary EU-Caribbean relations. Whilst at once dealing with the singular case of the Caribbean, the author offers a nuanced approach beyond 'EU navel-gazing' by incorporating an ‘outside-in’ perspective, which thereafter could be applied to EU-ACP relations and the North-South dialogue in general.

Bosniacki chaos. Zrodla kryzysu politycznego we wspolczesnej Bosni i Hercegowinie. Prace OSW 31/2009. = Bosnia's chaos. Causes of the political crisis in Bosnia and Herzegovina today. OSW Study 31/2009

A new form of 'transformational crisis' has been observed in Bosnia and Herzegovina since at least 2005. Politicians representing the three major ethno-political communities (Bosnians, Croats and Serbs) have successively been raising disputes and have employed various political tools to preserve the conflicts instead of resolving them. As a result, the central state institutions and organisations have been weakened and attempts to replace them with narrower ethnic structures have been made. This is increasingly paralysing the state, thus impeding its everyday operation and preventing its structures and legislation from being modernised; had this been achieved, it would have resulted in a real acceleration of the process of Bosnia's integration with the EU and NATO. The present crisis is also an effect of the disagreement between the key international players - the European Union, the United States and Russia - over the 'plan for Bosnia' and the role and duties of the Office of the High Representative, who acts on behalf of the international community in the country.