Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Synthesis of silica films at the air/water interface: Effect of template chain length and ionic strength

We have grown surfactant-templated silicate films at the air-water interface using n-alkyltrimethylammonium bromide and chloride in an acid synthesis with tetraethyl orthosilicate as the silicate source. The films have been grown with and without added salt (sodium chloride, sodium bromide) and with n-alkyl chain lengths from 12 to 18, the growth process being monitored by X-ray reflectometry. Glassy, hexagonal, and lamellar structures have been produced in ways that are predictable from the pure surfactant-water phase diagrams. The synthesis appears to proceed initially through an induction period characterized by the accumulation of silica-coated spherical micelles near the surface. All syntheses, except those involving C(12)TACl, show a sudden transformation of the spherical micellar phase to a hexagonal phase. This occurs when the gradually increasing ionic strength and/or changing ethanol concentration is sufficient to change the position of boundaries within the phase diagram. A possible mechanism for this to occur may be to induce a sphere to rod transition in the micellar structure. This transformation, as predicted from the surfactant-water phase diagram, can be induced by addition of salts and is slower for chloride than bromide counteranions. The hexagonal materials change in cell dimension as the chain length is changed in a way consistent with theoretical model predictions. All the materials have sufficiently flexible silica frameworks that phase interconversion is observed both from glassy to hexagonal and from hexagonal, to lamellar and vice versa in those surfactant systems where multiple phases are found to exist.

In vivo protein cyclization promoted by a circularly permuted Synechocystis sp PCC6803 DnaB mini-intein

A synthetic Synechocystis sp. PCC6803 DnaB split mini-intein gene was constructed for the in vivo cyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coli DnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures similar to14 degreesC higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2 and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (DeltaDeltaG approximate to 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

The crystal structure of free human hypoxanthineguanine phosphoribosyltransferase reveals extensive conformational plasticity throughout the catalytic cycle

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved.

Molecular biology in tropical dermatoses

Initially, basic concepts are presented concerning the cell, genetic code and protein synthesis, and some techniques of molecular biology, such as PCR, PCR-RFLP, DNA sequencing, RT-PCR and immunoblotting. Protocols of nucleotides and of proteins extraction are supplied, such as salting out in peripheral blood allied to phenol-chloroform and trizol methods in skin samples. To proceed, commented examples of application of those techniques of molecular biology for the etiologic diagnosis and for research in tropical dermatoses, with emphasis to American tegumentary leishmaniasis and leprosy are presented.

Comparison of visual inspection, radiographic examination, laser fluorescence and their combinations on treatment decisions for occlusal surfaces

Purpose: To compare visual inspection (VI), radiographic examination (RX) and the laser fluorescence device DIAGNOdent (L), as well as their combinations in vitro regarding treatment decisions for occlusal surfaces. Methods: 72 extracted human permanent teeth (molars and premolars) were used. Treatment decisions were recorded by three calibrated examiners, and the options available were fissure sealant and conservative restoration. For validation of treatment decisions, the teeth were sectioned and examined in a stereomicroscope. Thereafter, dental slices were scanned and the images were edited to facilitate classification of existing carious lesions. Intra and inter-examiner reproducibility for the determination of treatment plans were calculated using Cohen`s kappa test (95%-CI). Sensitivity, specificity, positive and negative predictive values, and the area under the ROC curve were also calculated. Results: VI and L provided on average the greatest intra- and inter-examiner reproducibility, respectively. Although the combination of diagnostic methods may decrease both intra- and inter examiners reproducibility, combination of VI, L and RX resulted in the greatest sensitivity, being statistically superior to RX and L. There was more inter-examiner agreement for the option of restorative treatment, while the use of sealants as a treatment option yielded the lowest values. Negative predictive values were numerically inferior to positive predictive values, indicating that the examiners preferred not to restore a carious tooth than to proceed operatively in an intact tooth. The combination of the three methods studied showed the best results in determining treatment plans for occlusal surfaces, when compared to the other types of exams. On the other hand, radiographic examination and laser fluorescence were less efficient when used alone.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

Guilty? An explanation of the Lockerbie trial. (Case notes)

The Lockerbie Trial, concerning the explosion of Pan Am flight 103 over Lockerbie, Scotland, in 1988, took place in the Netherlands because of the fear that pre-trial publicity would make a fair trial impossible and also fears for the physical safety of the accused - case for the prosecution - application of Scottish law - report of an observer at the trial, Professor Kochler - the International Criminal Court - leave to appeal granted.

Factors affecting the rate of phosphocreatine resynthesis following intense exercise

Within the skeletal muscle cell at the onset of muscular contraction, phosphocreatine (PCr) represents the most immediate reserve for the rephosphorylation of adenosine triphosphate (ATP). As a result, its concentration can be reduced to less than 30% of resting levels during intense exercise. As a fall in the level of PCr appears to adversely affect muscle contraction, and therefore power output in a subsequent bout, maximising the rate of PCr resynthesis during a brief recovery period will be of benefit to an athlete involved in activities which demand intermittent exercise. Although this resynthesis process simply involves the rephosphorylation of creatine by aerobically produced ATP (with the release of protons), it has both a fast and slow component, each proceeding at a rate that is controlled by different components of the creatine kinase equilibrium. The initial fast phase appears to proceed at a rate independent of muscle pH. Instead, its rate appears to be controlled by adenosine diphosphate (ADP) levels; either directly through its free cytosolic concentration, or indirectly, through its effect on the free energy of ATP hydrolysis. Once this fast phase of recovery is complete, there is a secondary slower phase that appears almost certainly rate-dependant on the return of the muscle cell to homeostatic intracellular pH. Given the importance of oxidative phosphorylation in this resynthesis process, those individuals with an elevated aerobic power should be able to resynthesise PCr at a more rapid rate than their sedentary counterparts. However, results from studies that have used phosphorus nuclear magnetic resonance (P-31-NMR) spectroscopy, have been somewhat inconsistent with respect to the relationship between aerobic power and PCr recovery following intense exercise. Because of the methodological constraints that appear to have limited a number of these studies, further research in this area is warranted.

Evaluation of exposure parameters in plain radiography: a comparative study with european guidelines

Typical distribution of exposure parameters in plain radiography is unknown in Portugal. This study aims to identify exposure parameters that are being used in plain radiography in the Lisbon area and to compare the collected data with European references [Commission of European Communities (CEC) guidelines]. The results show that in four examinations (skull, chest, lumbar spine and pelvis), there is a strong tendency of using exposure times above the European recommendation. The X-ray tube potential values (in kV) are below the recommended values from CEC guidelines. This study shows that at a local level (Lisbon region), radiographic practice does not comply with CEC guidelines concerning exposure techniques. Further national/local studies are recommended with the objective to improve exposure optimisation and technical procedures in plain radiography. This study also suggests the need to establish national/local diagnostic reference levels and to proceed to effective measurements for exposure optimisation.

Identificação dos parâmetros técnicos de exposição em exames radiológicos convencionais na região de Lisboa: uma comparação com a referência europeia

Em Portugal não são conhecidos estudos publicados que identifiquem com clareza as distribuições típicas dos valores associados aos parâmetros técnicos de exposição utilizados nos exames radiológicos mais comuns. Este estudo tem como objectivos identificar os parâmetros técnicos utilizados em exames radiológicos convencionais na região de Lisboa e comparar os dados com a referência europeia CEC. Os resultados obtidos evidenciam que nas projecções estudadas existe uma predominância para o uso de termos de exposição acima da recomendação CEC e de valores de potencial da ampola (kV) abaixo da recomendação. Esta investigação sugere a necessidade de fixar os NRD nacionais, e/ou locais, e efectuar as respectivas medições, dado que a optimização da exposição é um mecanismo de controlo fundamental para limitar as exposições desnecessárias dos pacientes às radiações ionizantes. Conclui-se que a nível local (região da Grande Lisboa) existe um desconhecimento da prática radiológica enquadrada nos referenciais europeus de boa prática radiológica. Desconhecendo-se a situação a nível nacional, sugerem-se estudos no sentido de identificar os padrões de prática radiológica a nível do País e de promover a optimização dos procedimentos em radiologia convencional. ABSTRACT - Typical distribution of exposure parameters in plain radiography is unknown in Portugal. This study aims to identify exposure parameters that are being used in plain radiography in Lisbon area and to compare collected data with European references (CEC guidelines). Results show that in the four anatomic regions there is a strong tendency of using exposure times above the European recommendation. We also found that x-ray tube potential values (kV) are below the recommended values from CEC guidelines. This study shows that at a local level (Lisbon region) radiographic practice does not fit with CEC guidelines concerning exposure techniques. We suggest continuing national/local studies with the objective to improve exposure optimization and technical procedures in plain radiography. This study also suggests the need to establish national/local DRL’s and to proceed to effective measurements for exposure optimization.

Projecto de entreposto frigorífico a R-744/R-717 para produtos congelados

Pretende-se com este trabalho de Projecto de Mestrado conceber uma câmara frigorífica de 20000 m3 para armazenar produtos congelados e estudar um sistema frigorífico com dois fluidos frigorigénios que serão o CO2 (R-744) e o NH3 (R-717) O trabalho inicia-se com a definição dos objectivos principais para o projecto de um sistema frigorífico deste tipo. Após a definição dos objectivos, o projecto propõe um estudo termodinâmico do CO2 (R-744) como fluido frigorigénio, definindo se o seu historial de utilização, características principais, diagrama pressão-entalpia com a distinção das diversas fases do fluído, comparação em diversos parâmetros com outros fluidos, o porquê da utilização deste fluído, problemas comuns em sistemas com a presença deste fluído, entre outros parâmetros de estudo. De seguida será feito o dimensionamento de uma câmara frigorífica de 20000 m3 para armazenar produtos congelados paletizados através de um balanço térmico manual e um balanço térmico informático através do programa da Centauro comparando no final o resultado dos dois métodos. Será feito uma descrição das características básicas da câmara frigorífica com um esquema simples do edifício. Será abordado de seguida o sistema frigorífico a CO2 (R-744) e a NH3 (R-717) nas suas diversas características, nomeadamente no cálculo de caudais, diagrama pressão-entalpia, dimensionamento de tubagens e finalmente selecção de equipamento. Procede-se assim também ao estudo um sistema apenas a NH3 (R-717) com as suas diversas características nomeadamente no cálculo de caudais, diagrama pressão-entalpia, dimensionamento de tubagens e finalmente selecção dos equipamentos mais importantes, para que desta forma se proceda à comparação dos dois sistemas a nível energético, consumo eléctrico e manutenção de equipamentos. Finalmente proceder-se-á interpretação de resultados com o objectivo final de escolher a melhor solução nos vários parâmetros de comparação, para o esquema em questão.

Desenvolvimento de um plano de manutenção preventiva na rede de distribuição de água de Lisboa

O presente relatório de estágio enquadra-se no âmbito do trabalho final de Mestrado do curso de Engenharia Civil, área de especialização em Hidráulica, do Instituto Superior de Engenharia de Lisboa, e incide na temática dos sistemas de distribuição de água. O estágio decorreu na empresa EPAL – Empresa Portuguesa das Águas Livres, S.A., e teve como objecto o desenvolvimento de um plano de manutenção preventiva para a rede de distribuição de água de Lisboa. Devido à crescente complexidade dos sistemas de abastecimento de água e à grande dispersão territorial dos diversos órgãos integrados na rede, surge a necessidade de desenvolver um sistema que permita simplificar o planeamento e a gestão das intervenções de inspecção e manutenção preventiva dos órgãos. Para esse efeito, recorreu-se a um software SIG (Sistema de Informação Geográfica) para proceder à sectorização da rede de distribuição de água em pequenos grupos (Polígonos), constituídos por um número determinado de órgãos de manobra e segurança. Esta metodologia permite organizar as intervenções em pequenos grupos e optimizar as rotinas de Inspecção diárias. No decorrer do estágio efectuou-se ainda um levantamento dos procedimentos de inspecção e manutenção dos diversos órgãos integrados na rede de distribuição de água de Lisboa, quer por acompanhamento de actividades com as equipas de inspecção, quer por recolha de recomendações junto dos fabricantes dos órgãos. Este levantamento permitiu elaborar fichas de procedimento para as actividades de inspecção e manutenção preventiva dos diversos órgãos, afim de serem usadas como referência pelos trabalhadores.

Estudo de caso. A implementação do projeto de internalização do RX do Centro de Diagnóstico Pneumológico do Porto

Portugal atravessa uma crise económico-financeira. A austeridade económica é prioritária, e a área de saúde não é exceção. Nesta conjuntura justifica-se de todo a realização de análises económicas na saúde. Este estudo é uma análise custo - mínimo ao Projeto de Internalização do RX do Centro Diagnóstico Pneumológico (CDP) do Porto com o objetivo de rentabilizar o equipamento radiológico instalado no CDP do Porto, pretende-se usar a capacidade máxima instalada e permitida pelo equipamento para produção de radiografias a utentes provenientes dos Agrupamentos de Centros de Saúde (ACES) do Porto. O Projeto de Internalização do RX do CDP do Porto é um projeto da Administração Regional de Saúde (ARS) do Norte, que pretende encaminhar os utentes dos ACES do Porto para realização de exames radiográficos no RX do CDP do Porto. Para proceder à análise económica foram identificados os custos e os benefícios relevantes do RX do CDP do Porto e elaboradas 3 estimativas: 1. Realização dos exames radiográficos do CDP do Porto a utentes do CDP do Porto e realização de exames radiográficos a utentes dos ACES do Porto nos gabinetes de radiologia convencionados pela ARSNorte; 2. Projeto de Internalização do RX do CDP do Porto com realização dos exames radiográficos até esgotar a capacidade máxima instalada do equipamento radiológico com recurso a utentes dos ACES do Porto; 3. Realização dos exames radiográficos nos ACES do Porto e dos exames do CDP do Porto nos gabinetes de radiologia convencionados. Os resultados da análise levaram à conclusão que a implementação do Projeto de Internalização do RX do Porto é rentável, ou seja, o projeto permitirá ganhos financeiros para a ARSNorte se for implementado na sua plenitude.

Cyber-ambient intelligent training of operators in power systems control centres

Cyber-Physical Systems and Ambient Intelligence are two of the most important and emerging paradigms of our days. The introduction of renewable sources gave origin to a completely different dimension of the distribution generation problem. On the other hand, Electricity Markets introduced a different dimension in the complexity, the economic dimension. Our goal is to study how to proceed with the Intelligent Training of Operators in Power Systems Control Centres, considering the new reality of Renewable Sources, Distributed Generation, and Electricity Markets, under the emerging paradigms of Cyber-Physical Systems and Ambient Intelligence. We propose Intelligent Tutoring Systems as the approach to deal with the intelligent training of operators in these new circumstances.