Missense, Nonsense, and Neutral Mutations Define Juxtaposed Regulatory Elements of Splicing in Cystic Fibrosis Transmembrane Regulator Exon 9

Exonic sequence variations may induce exon inclusion or exclusion from the mature mRNA by disrupting exonic regulatory elements and/or by affecting a nuclear reading frame scanning mechanism. We have carried out a systematic study of the effect on cystic fibrosis transmembrane regulator exon 9 splicing of natural and site-directed sequence mutations. We have observed that changes in the splicing pattern were not related to the creation of premature termination codons, a fact that indicates the lack of a significant nuclear check of the reading frame in this system. In addition, the splice pattern could not be predicted by available Ser/Arg protein matrices score analysis. An extensive site-directed mutagenesis of the 3' portion of the exon has identified two juxtaposed splicing enhancer and silencer elements. The study of double mutants at these regulatory elements showed a complex regulatory activity. For example, one natural mutation (146C) enhances exon inclusion and overrides all of the downstream silencing mutations except for a C to G transversion (155G). This unusual effect is explained by the creation of a specific binding site for the inhibitory splicing factor hnRNPH. In fact, on the double mutant 146C-155G, the silencing effect is dominant. These results indicate a strict dependence between the two juxtaposed enhancer and silencer sequences and show that many point mutations in these elements cause changes in splicing efficiency by different mechanisms

Condensado do ar exalado como bioindicador humano de exposição ocupacional a chumbo

Tese de mestrado, Biologia (Biologia Humana e Ambiente), 2009, Universidade de Lisboa, Faculdade de CiênciasA recolha do condensado do ar exalado (EBC, do inglês exhaled breath condensate) é um método não invasivo que pode fornecer informações sobre as vias respiratórias. O presente trabalho pretendeu avaliar se a análise da composição do EBC poderia constituir um procedimento não invasivo de avaliação da exposição individual a poluentes em ambiente industrial. Foi estudada a qualidade do EBC como bioindicador de exposição a poeiras e gases de chumbo no ambiente de trabalho em duas fábricas em que este metal é utilizado. Foi recolhido EBC dos trabalhadores expostos para determinação dos níveis de chumbo e de outros metais em fases sucessivas da semana de trabalho, no início e no fim do turno. Os trabalhadores tiveram ainda uma avaliação funcional respiratória, por espirometria. O nível de partículas inaláveis no ambiente de trabalho foi avaliado com amostradores pessoais e os seus níveis de metais foram registados com amostradores fixos em locais seleccionados. A análise elementar das partículas em suspensão no ar foi efectuada por INAA e PIXE, e do EBC por espectrometria de massa (ICP-MS). As metodologias de recolha, armazenamento e análise do EBC foram avaliadas tendo em conta o tempo de recolha, a homogeneidade da amostra e contaminações resultantes do manuseamento e armazenagem. A determinação elementar no EBC foi validada por inter-comparação dos resultados com a técnica certificada de TXRF. Verificou-se que as concentrações elementares no EBC de trabalhadores expostos aumentaram ao longo da semana de trabalho (com excepção do zinco) nas duas fábricas, embora os níveis de concentração diferissem significativamente nas duas instalações fabris, reflectindo as diferenças entre os dois ambientes de trabalho. Verificou-se também uma correlação positiva entre as concentrações de chumbo no EBC e no sangue dos trabalhadores expostos. Este trabalho contribuiu para o estabelecimento de uma metodologia de recolha e análise do EBC como bioindicador de exposição ocupacional.The collection of exhaled breath condensate (EBC) samples is a noninvasive method that can provide information about the respiratory pathways. This work intended to evaluate if the analysis of the EBC composition may be applied as a noninvasive procedure to assess the individual exposure to pollutants in an industrial environment. Therefore, the exhaled air quality was studied as a bioindicator of exposure to lead dust and aerosols in the working environment, where this metal is used. The EBC was collect from exposed workers to assess the levels of lead and other metals at different stages of the work week, according to their shifts. The workers' respiratory function was also assessed by spirometry. The level of inhalable airborn particulate matter (APM) was evaluated at the work place by personal samplers and their metal concentration was assessed through steady samplers in selected places. The elemental analysis of APM was performed by INAA and PIXE, and the EBC was studied by mass spectrometry (ICP-MS). The EBC sampling methodologies, storage and analysis were evaluated regarding the collection time, samples' homogeneity and contaminations resulting from the handling and storage. The elemental determination of EBC was validated by an inter-comparison of results with TXRF (a certified technique). It was verified that the elemental concentrations of EBC in exposed workers tended to increase throughout the work week (except for zinc) in both industries, although the concentration levels were significantly different. This reflects the differences between the two working environments. It was also found a positive correlation between the concentrations of lead in EBC and in blood, in the exposed workers. This work contributed to the establishment of a sampling and analysis methodology of EBC as a bioindicator of occupational exposure

Alternative Intelligent Material for Packaging Using Chitosan Films as Colorimetric Temperature Indicators

Chlorophyll was incorporated into chitosan films, forming intelligent systems able to detect variations in temperature. Chitosan is a biodegradable polymer that forms flexible, resistant films with an efficient oxygen barrier. Chlorophyll was added (0.25 g/100 g) into chitosan suspension (2.00 g/100 g) and intelligent films (FIQ) were cast. the effects of temperature (10 degrees C to 50 degrees C) and luminosity (0 to 1000 lx) on the films were studied using an experimental design with two variables, analyzing the color parameters L*, a*, b* and mechanical properties. the films were characterized according to their homogeneity, flexibility, color and handling. Chitosan suspensions containing chlorophyll were also applied as coating on the surface of pieces of card-paper, forming a card paper film system (S-FP). By visual inspection we observed the color of the S-FP changing irreversibly from green to yellow when the temperature was above 50 degrees C. Therefore, the S-FP system can potentially be used as a colorimetric indicator for the 50 degrees C to 70 degrees C range, being advantageous owing to the ease of fabrication, biodegradability, safe use for food and low cost.Clorofila, um pigmento natural termossensível, foi incorporado à matriz de filmes de quitosana visando a obter sistemas inteligentes de indicação de variação de temperatura. A quitosana é um polímero biodegradável que forma filmes flexíveis com eficiente barreira ao oxigênio, podendo ser alternativa ao uso de polímeros sintéticos. Clorofila foi adicionada (0,25 g/100 g) à suspensão de quitosana (2,00 g/100 g) formando filmes inteligentes de quitosana (FIQ). Os efeitos da temperatura (10 °C a 50 °C) e luminosidade (0 a 1000 lx) foram estudados utilizando um planejamento experimental, avaliando os parâmetros de cor (L*, a*, b*) e propriedades mecânicas. As suspensões de quitosana contendo clorofila foram aplicadas como revestimento em superfície de papel cartão formando o sistema de material flexível filme-papel cartão (S-FP), reduzindo significativamente o tempo de secagem. Os filmes caracterizaram-se pela homogeneidade, flexibilidade, coloração esverdeada e fácil manuseio. A variação de cor foi visualmente observada no sistema S-FP, alterando irreversivelmente de verde para amarelo quando submetido a temperaturas acima de 50 °C, independentemente da luminosidade. Desta forma, o sistema proposto tem potencial de aplicação como indicador colorimétrico de temperatura na faixa de 50 °C a 75 °C, com a vantagem de simples fabricação, biodegradabilidade e uso de materiais seguros para aplicação em contato direto com alimentos e fármacos, além do baixo custo.Univ Estadual Campinas, UNICAMP, Fac Engn Quim, BR-13083852 Campinas, SP, BrazilUNIFESP, Dept Ciencias Exatas & Terra, Campinas, SP, BrazilUNIFESP, Dept Ciencias Exatas & Terra, Campinas, SP, BrazilWeb of Scienc

An Intronic Polypyrimidine-rich Element Downstream of the Donor Site Modulates Cystic Fibrosis Transmembrane Conductance Regulator Exon 9 Alternative Splicing *

Two intronic elements, a polymorphic TGmTn locus at the end of intron 8 and an intronic splicing silencer in intron 9, regulate aberrant splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Previous studies (Pagani, F., Buratti, E., Stuani, C., Romano, M., Zuccato, E., Niksic, M., Giglio, L., Faraguna, D., and Baralle, F. E. (2000) J. Biol. Chem. 275, 21041–21047 and Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) Embo J. 20, 1774–1784) have demonstrated that trans-acting factors that bind to these sequences, TDP43 and Ser/Arg-rich proteins, respectively, mediate splicing inhibition. Here, we report the identification of two polypyrimidine-binding proteins, TIA-1 and polypyrimidine tract-binding protein (PTB), as novel players in the regulation of CFTR exon 9 splicing. In hybrid minigene experiments, TIA-1 induced exon inclusion, whereas PTB induced exon skipping. TIA-1 bound specifically to a polypyrimidine-rich controlling element (PCE) located between the weak 5′-splice site (ss) and the intronic splicing silencer. Mutants of the PCE polypyrimidine motifs did not bind TIA-1 and, in a splicing assay, did not respond to TIA-1 splicing enhancement. PTB antagonized in vitro TIA-1 binding to the PCE, but its splicing inhibition was independent of its binding to the PCE. Recruitment of U1 small nuclear RNA to the weak 5′-ss by complementarity also induced exon 9 inclusion, consistent with the facilitating role of TIA-1 in weak 5′-ss recognition by U1 small nuclear ribonucleoprotein. Interestingly, in the presence of a high number of TG repeats and a low number of T repeats in the TGmTn locus, TIA-1 activated a cryptic exonic 3′-ss. This effect was independent of both TIA-1 binding to the PCE and U1 small nuclear RNA recruitment to the 5′-ss. Moreover, it was abolished by deletion of either the TG or T sequence. These data indicate that, in CFTR exon 9, TIA-1 binding to the PCE recruits U1 small nuclear ribonucleoprotein to the weak 5′-ss and induces exon inclusion. The TIA-1-mediated alternative usage of the 3′-splice sites, which depends on the composition of the unusual TGmTn element, represents a new mechanism of splicing regulation by TIA-1

Autophagy and rheumatoid arthritis: Current knowledges and future perspectives

Autophagy is a degradation mechanism by which cells recycle cytoplasmic components to generate energy. By influencing lymphocyte development, survival, and proliferation, autophagy regulates the immune responses against self and non-self antigens. Deregulation of autophagic pathway has recently been implicated in the pathogenesis of several autoimmune diseases, including rheumatoid arthritis (RA). Indeed, autophagy seems to be involved in the generation of citrullinated peptides, and also in apoptosis resistance in RA. In this review, we summarize the current knowledge on the role of autophagy in RA and discuss the possibility of a clinical application of autophagy modulation in this disease

Electrostatic Self-Assembled Chitosan-Pectin Nano- and Microparticles for Insulin Delivery

A polyelectrolyte complex system of chitosan-pectin nano- and microparticles was developed to encapsulate the hormone insulin. The aim of this work was to obtain small particles for oral insulin delivery without chemical crosslinkers based on natural and biodegradable polysaccharides. The nano- and microparticles were developed using chitosans (with different degrees of acetylation: 15.0% and 28.8%) and pectin solutions at various charge ratios (n+/n− given by the chitosan/pectin mass ratio) and total charge. Nano- and microparticles were characterized regarding particle size, zeta potential, production yield, encapsulation efficiency, stability in different media, transmission electron microscopy and cytotoxicity assays using Caco-2 cells. The insulin release was evaluated in vitro in simulated gastric and intestinal media. Small-sized particles (~240–~1900 nm) with a maximum production yield of ~34.0% were obtained. The highest encapsulation efficiency (~62.0%) of the system was observed at a charge ratio (n+/n−) 5.00. The system was stable in various media, particularly in simulated gastric fluid (pH 1.2). Transmission electron microscopy (TEM) analysis showed spherical shape particles when insulin was added to the system. In simulated intestinal fluid (pH 6.8), controlled insulin release occurred over 2 h. In vitro tests indicated that the proposed system presents potential as a drug delivery for oral administration of bioactive peptide

Public ground as coastal defence: imagining the Mediterranean beachfront by the Atlantic

The article explores the role of public ground composition design as a defence element for coastal agglomerations vulnerable to mean sea level rise in extreme weather events scenarios. It addresses one of the pilot case studies of the [ENTRA]MAR Urban form intertwined with the sea research project, Quarteira, a coastal city located in the Algarve region of southern Portugal, which is vulnerable to flooding and erosion phenomena. The article provides an opportunity to systematise the reading and interpretation phases and also the research by design phase based on scenario development that was conducted during the fourth-year design studio at the Lisbon School of Architecture of the Universidade de Lisboa and therefore, to share its preliminary results on “imagining the Mediterranean beachfront by the Atlantic”. Attributing Mediterranean values and characteristics to Quarteira involves, as Braudel wrote in “Les Mémoires de la Méditerranée”, observing the coastal landscapes over and over again, until the physical and urban characteristics are eloquently revealed and named. Finally, it is underlined the need to design the space between land and sea based on the inherited memory of the coastal landscape and the operative role the public ground design <may have as coastal defence.The article explores the role of public ground composition design as a defence element for coastal agglomerations vulnerable to sea level rise in extreme weather events scenarios. It addresses one of the pilot case studies of the [ENTRA]MAR Urban form intertwined with the sea research project, Quarteira, a coastal city located in the Algarve region of southern Portugal, which is vulnerable to flooding and erosion phenomena. The article provides an opportunity to systematise the reading and interpretation phases and also the research by-design phase based on scenario development that was conducted during the fourth-year design studio at the Lisbon School of Architecture of the Universidade de Lisboa and therefore, to share its preliminary results on "imagining the Mediterranean beachfront by the Atlantic". Attributing Mediterranean values and characteristics to Quarteira involves, as Braudel wrote in Les Mémoires de la Méditerranée, observing the coastal landscapes over and over again, until the physical and urban characteristics are eloquently revealed and named. Finally, it is underlined the need to design the space between land and sea based on the inherited memory of the coastal landscape and the operative role the public ground design may have as coastal defence.This work is financed by national funds through FCT - Fundação para a Ciência e a Tecnologia, I.P., under the Strategic Project with the references UIDB/04008/2020 and UIDP/04008/2020.Peer Reviewe

A Ideia de Foedus Pacificum em Sebastião de Magalhães Lima

ResumoSebastião de Magalhães Lima foi um verdadeiro apóstolo do ideário federalista republicano e pacifista. Ao longo da sua vida, escreveu inúmeros textos em defesa de uma Federação Ibérica como primeira etapa para a construção dos Estados Unidos da Humanidade e a realização da Paz Universal.O breve estudo que se segue pretende sintetizar o pensamento federalista e pacifista de Magalhães Lima, tendo como base dois textos da sua autoria, publicados no final do século XIX, O Federalismo (1898) e Paz e Arbitragem (1897), embora não descurando outras obras da sua autoria. Atentaremos especialmente à influência do pensamento de Immanuel Kant, Proudhon, Pi y Margal e Teófilo Braga.Palavras-Chaves: Federalismo; República; Paz; Iberismo; Humanidade AbstractSebastião de Magalhães Lima was a true apostle of the republican and pacifist federalist ideology. Throughout his life, he wrote countless texts defending an Iberian Federation as the first step towards the construction of the United States of Humanity and the achievement of Universal Peace.The following brief study intends to synthesize the federalist and pacifist thought of Magalhães Lima, based on two of his texts, published in the late nineteenth century, Federalismo (1898) and Paz e Arbitragem (1897), although not neglecting his other works. We will mainly analyse the influence of the thought of Immanuel Kant, Proudhon, Pi y Margal and Teófilo Braga.Keywords: Federalism; Republic; Peace; Iberianism; Humanit

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV

Prevalence, sensitivity and specificity of antibodies against carbamylated proteins in a monocentric cohort of patients with rheumatoid arthritis and other autoimmune rheumatic diseases

Antibodies against carbamylated proteins (anti-CarP) have been recently identified in the sera of patients with rheumatoid arthritis (RA). The objective of the study was to evaluate the prevalence, sensitivity and specificity of anti-CarP compared to anti-citrullinated peptide antibodies (ACPA) and rheumatoid factor (RF), replicating the existing data in a large cohort of Italian patients with RA and extending the evaluation to other autoimmune rheumatic diseases (AIRDs)