Aboral ectoderm-specific expression and regulation of the LpS1 genes of {\it Lytechinus pictus\/}

The Spec genes serve as molecular markers for examining the ontogeny of the aboral ectoderm lineage of sea urchin embryos. These genes are activated at late-cleavage stage only in cells contributing to the aboral ectoderm of Strongylocentrotus purpuratus and encode 14,000-17,000 Da calcium-binding proteins. A comparative analysis was undertaken to better understand the mechanisms underlying the activation and function of the Spec genes by investigating Spec homologues from Lytechinus pictus, a distantly related sea urchin. Spec antibodies cross-reacted with 34,000 Da proteins in L. pictus embryos that displayed a similar ontogenetic pattern to that of Spec proteins. One cDNA clone, LpS1, was isolated by hybridization to a synthetic oligonucleotide corresponding to a calcium-binding domain or EF-hand. The LpS1 mRNA has developmental properties similar to those of the Spec mRNAs. LpS1 encodes a 34,000 Da protein containing eight EF-hand domains, which share structural homology with the Spec EF-hands; however, little else in the protein sequence is conserved, implying that calcium-binding is important for Spec protein function. Genomic DNA blot analysis showed two LpS1 genes, LpS1$\alpha$ and LpS1$\beta$, in L. pictus. Partial gene structures for both LpS1$\alpha$ and $\beta$ were constructed based on genomic clones isolated from an L. pictus genomic library. These revealed internal duplications of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Sequencing analysis showed there was little in common among the 5$\sp\prime$-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF.^ A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5$\sp\prime$-flanking DNA from the LpS1$\beta$ gene were sufficient for correct temporal and spatial expression of reporter genes in sea urchin embryos. Deletions at the 5$\sp\prime$ end to 511, 368, or 108bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase (CAT) activity and disrupted the restricted activation of the lac Z gene in aboral ectoderm cells.^ A full-length Spec1 protein and a truncated LpS1 protein were induced and partially purified from an in vitro expression system. (Abstract shortened with permission of author.) ^

Isolation, characterization and functional analysis of the {\it xnf7\/} gene from {\it Xenopus laevis\/}

A fundamental problem in developmental biology concerns the mechanisms involved in the establishment of the embryonic axis. We are studying Xenopus nuclear factor 7 (xnf7) which we believe to be involved in dorsal-ventral patterning in Xenopus laevis. Xnf7 is a maternal gene product that is retained in the cytoplasm during early embryogenesis until the mid-blastula transition (MBT) when it reenters the nuclei. It is a member of a novel zinc finger proteins, the B-box family, consisting mainly of transcription factors and protooncogenes.^ The xnf7 gene is reexpressed during embryogenesis at the gastrula-neurula stage of development, with its zygotic expression limited to the central nervous system (CNS). In this study we showed that there are two different cDNAs coding for xnf7, xnf7-O and xnf7-B. They differ by 39 amino acid changes scattered throughout the cDNA. The expression of both forms of xnf7 is limited primarily to the central nervous system (CNS) and dorsal axial structures during later stages of embryogenesis.^ In order to study the spatial and temporal regulation of the gene, we screened a Xenopus genomic library using part of xnf7 cDNA as a probe. A genomic clone corresponding to the xnf7-O type was isolated, its 5$\sp\prime$ putative regulatory region sequenced, and its transcriptional initiation site mapped. The putative promoter region contained binding sites for Sp1, E2F, USF, a Pu box and AP1. CAT/xnf7 fusion genes were constructed containing various 5$\sp\prime$ deleted regions of the xnf7 promoter linked to a CAT (Chloramphenicol Acetyl Transferase) reporter vector. These constructs were injected into Xenopus oocytes and embryos to study the regions of the xnf7 promoter responsible for basal, temporal and spatial regulation of the gene. The activity of the fusion genes was measured by the conversion of chloramphenicol to its acetylated forms, and the spatial distribution of the transcripts by whole mount in situ hybridization. We showed that the elements involved in basal regulation of xnf7 lie within 121 basepairs upstream of the transcriptional inititiation site. A DNase I footprint analysis performed using oocyte extract showed that a E2F and 2 Sp1 sites were protected. During development, the fusion genes were expressed following the MBT, in accordance with the timing of the endogenous xnf7 gene. Spatially, the expression of the fusion gene containing 421 basepairs of the promoter was localized to the dorsal region of the embryo in a pattern that was almost identical to that detected with the endogenous transcripts. Therefore, the elements involved in spatial and temporal regulation of the xnf7 gene during development were contained within 421 basepairs upstream of the transcriptional initiation site. Future work will further define the elements involved in the spatial and temporal regulation and the trans-factors that interact with them. ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

New QR Survey Methodologies to Analyze User Perception of Service Quality in Public Transport: The Experience of Madrid

Customer Satisfaction Surveys (CSS) have become an important tool for public transport planners, as improvements in the perceived quality of service lead to greater use of public transport and lower traffic pollution. Until now, Intelligent Transportation System (ITS) enhancements in public transport have traditionally included fleet management systems based on Automatic Vehicle Location (AVL) technologies, which can be used to optimize routing and scheduling, and to feed real-time information into passenger information channels. However, surveys of public transport users could also benefit from the new information technologies. As most customers carry their smartphones when traveling, Quick Response (QR) codes open up the possibility of conducting these surveys at a lower cost.This paper contributes to the limited existing literature by developing the analysis of QR codes applied to CSS in public transport and highlighting their importance in reducing the cost of data collection and processing. The added value of this research is that it provides the first assessment of a real case study in Madrid (Spain) using QR codes for this purpose. This pilot experience was part of a research project analyzing bus service quality in the same case study, so the QR code survey (155 valid questionnaires) was validated using a conventional face-to-face survey (520 valid questionnaires). The results show clearly that, after overcoming a few teething troubles, this QR code application will ultimately provide transport management with a useful tool to reduce survey costs

c-Myc target gene specificity is determined by a post-DNAbinding mechanism

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

A genomewide survey of basic helix–loop–helix factors in Drosophila

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Sintomas obsessivos e compulsivos em utentes dos cuidados de saúde primários

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Terapêutica da amigdalite aguda em idade pediátrica : estudo transversal sobre antibioterapia nos cuidados de saúde primários e urgência hospitalar em 2012

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Percepção dos usuários sobre o programa de saúde da família : um contributo para a reflexão

A Estratégia Saúde da Família apresenta-se como uma possibilidade de reestruturação da atenção primária, a partir de um conjunto de ações conjugadas em sintonia com os princípios de intersetorialidade, descentralização, equidade, coresponsabilidade e priorização de grupos populacionais com maior risco de adoecerem ou morrerem. Assim, este estudo objetivou caracterizar a percepção dos usuários da Unidade Básica de Saúde da Família (UBSF) sobre o Programa Saúde da Família (PSF). Trata-se de um estudo qualitativo desenvolvido na USF Barra de Sirinhaém, do Estado de Pernanbuco. Para tanto, contou-se com a participação de 54 usuárias do sexo feminino. Estas responderam a um formulário sócio-demográfico e um roteiro de entrevista semi-diretiva. Os discursos foram submetidos a análise de conteúdo à luz da literatura pertinente. Os principais resultados mostraram que a percepção das usuárias acerca do Programa de Saúde Familiar é de que este ainda funciona com abordagem curativa, mas que possuem maior acessibilidade e construíram um vínculo mais efetivo com os profissionais de saúde. Diante do exposto infere-se que a estratégia de saúde da família ainda não se concretiza pela dificuldade das pessoas entenderem a filosofia do programa, ainda polarizarem uma visão hospitalocêntrica da saúde e revelaram um certo alheamento da compreensão do significado do PSF. Palavras-chave: Estratégia Saúde da Família; Percepção; Assistência à Saúde.

Satisfação no trabalho e absentismo dos enfermeiros de um ACeS

A satisfação no trabalho é definida como sendo um fenómeno complexo, subjetivo e multifatorial (Rodrigues, 2011). No entanto a avaliação da satisfação dos profissionais constitui um imperativo para as organizações de saúde (Lei de Bases da Saúde), sendo fundamental para avaliar a qualidade das instituições, o desempenho dos profissionais e a qualidade dos cuidados de saúde prestados aos clientes. Os contextos de trabalho dos enfermeiros são influenciados por uma diversidade de fatores que inevitavelmente se refletem na sua saúde, na satisfação com o trabalho e no absentismo. Integrado no Projeto INTO_SO da Escola Superior de Enfermagem do Porto, o presente estudo, de cariz quantitativo, exploratório, descritivo, correlacional e transversal, teve como objetivos identificar o nível de satisfação e absentismo no trabalho dos enfermeiros; analisar a relação entre a satisfação no trabalho, o absentismo, variáveis sociodemográficas e profissionais e analisar a relação entre a satisfação no trabalho e o absentismo dos enfermeiros. Pretendemos com este estudo contribuir para a promoção da saúde no trabalho dos enfermeiros. Recorremos a um método de amostragem não probabilística. Aplicou-se a uma amostra de conveniência de 109 enfermeiros, um instrumento de recolha de dados constituído por três grupos (caracterização sociodemográfica e profissional; questionário de satisfação no trabalho de Meliá & Peiró (1989) adaptado para a língua portuguesa por Pocinho e Garcia (2008) e questões associadas ao absentismo). Os resultados deste estudo evidenciaram que os enfermeiros percecionam uma satisfação no trabalho (total e fatores) que se situa entre o “indiferente” e o “algo satisfeito”. São os enfermeiros mais velhos, com maior grau académico, sem dependentes a cargo e em que no seu agregado familiar não depende exclusivamente do seu salário, os que percecionam maior satisfação no trabalho com o ambiente físico. Os enfermeiros com parceiro percecionam maior satisfação no trabalho com a 14 participação e os que realizam atividades de lazer percecionam maior satisfação no trabalho com a satisfação intrínseca, sendo as atividades de lazer mais realizadas o ginásio e as caminhadas. Os enfermeiros que realizam horário rotativo percecionam maior satisfação no trabalho total, na satisfação com os benefícios e políticas da organização e na satisfação com a participação. Os enfermeiros das USFs e os que não consideram o seu trabalho stressante estavam significativamente mais satisfeitos com o ambiente físico de trabalho e com a satisfação intrínseca no trabalho, respetivamente. Este estudo sugere ainda que o sexo, ter filhos, ter ou não ajudas para prestar cuidados, o tempo de serviço na instituição, o tipo de vínculo e o desempenhar funções no Serviço de Atendimento a Situações Urgentes (SASU) não influenciam a satisfação no trabalho. Relativamente ao absentismo, os enfermeiros que têm filhos e os que exercem atividade em unidades de saúde familiar (USF) foram os que mais faltaram ao trabalho. A razão mais apontada foi a doença do próprio seguindo-se a doença de familiar (filhos e pais). Os enfermeiros que não faltaram ao serviço foram os que percecionaram maior satisfação no trabalho total e seus fatores. Sugerem-se entre outros, aprofundar o conhecimento do maior nível de absentismo verificado nos enfermeiros das USF e a elaboração de programas de promoção de saúde no local de trabalho alicerçados nos resultados encontrados.