Determinação da curva de resistência à fractura em materiais metálicos: "curva R"

Trabalho final de mestrado para obtenção do grau de mestre em Engenharia Mecânica.No presente trabalho foi abordado a determinação da curva de resistência à fractura (“Curva R”), em materiais metálicos, através dos métodos de curva de resistência e de normalização descritos na ASTM E 1820. O método de curva de resistência consiste na obtenção da curva de resistência à fractura (expressa em integral J ou CTOD), através de uma única amostra de material, recorrendo à técnica de elastic compliance. O método normalização consiste na obtenção da curva de resistência (expressa em integral J) directamente do diagrama de força deslocamento, e da medição do comprimento inicial e final da fenda na superfície de fractura. Foram ensaiados dois conjuntos de provetes (6+8) de dois materiais metálicos distintos. Dois provetes de cada conjunto foram ensaiados através do método de normalização. Os restantes foram submetidos ao método de curva de resistência, através do ensaio desenvolvido no software Wavematrix baseado no procedimento da ASTM E 1820. Foram confrontados os resultados entre cada um dos métodos dentro de cada material e ainda foram comparadas as curvas de resistência entre os dois materiais metálicos. Com as curvas de resistência obtidas foram ainda aplicados os procedimentos do anexo A9 da ASTM E 1820 para se obterem os valores de iniciação à fractura JIC.In the present work, a study is made about the experimental determination of the fracture resistance curve (R curve) of metallic materials, through the methods: resistance curve and normalizantion, present on ASTM E 1820. The resistance curve method consists in obtaining the “R curve” (Expressed in integral J or CTOD) through a single specimen.Within this method, the elastic compliance technique was used to obtain crack extension values. The normalization method consists in obtaining the resistance curve (expressed in integral J) directly from the force vs displacement diagram and the initial and final crack lengths, measured at the fracture surface. Two sets of specimens (6+8) of two metallic materials were tested. Two specimens from each set were submitted to the normalization method, while the remaining were studied with the resistance curve method, developed in the Instron Wavematrix software based on the procedure of ASTM E 1820. Within each material, the respective resistance curves were compared using both methods. The procedures of Annex A9 of ASTM E 1820 were also applied to obtain the fracture initiation values, JIC.N/

Péptido natriurético de tipo B : um (potencial) biomarcador de aterosclerose

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2016A aterosclerose é a principal entidade responsável pela patogénese da doença cardiovascular. É uma afecção sistémica que se manifesta de maneira focal, podendo afectar vários territórios arteriais como o território cerebral, coronário e outros territórios viscerais e periféricos. Clinicamente, doentes que se apresentem com um determinado evento aterotrombótico podem ter risco aumentado de desenvolver um novo evento, quer no mesmo território, quer em leitos arteriais diferentes. Mas como identificar os doentes que, tendo evidência de doença arterial num dado território, têm risco acrescido de ter afecção noutros? À luz do conhecimento actual, é facto que o péptido natriurético de tipo B (BNP) e o seu fragmento terminal (NT-proBNP) não são exclusivamente biomarcadores de insuficiência cardíaca mas sim biomarcadores de stress cardiovascular, o que tem aberto caminho para a pesquisa de novas utilizações clínicas para estes péptidos. O presente trabalho tem como objetivo ser uma revisão da literatura acerca da relação entre o risco de eventos adversos cardiovasculares e os níveis de peptídeos natriuréticos que, de acordo com bibliografia recente, pode ser entendida como uma curva em "U": baixos níveis de NT-proBNP (ou de BNP), poderão ser devidos a variações genéticas que determinem menor produção do péptido e consequentemente menos efeitos benéficos da sua actuação na regulação cardiovascular, com maior risco de desenvolvimento futuro de doença aterosclerótica e insuficiência cardíaca; níveis elevados de peptídeos natriuréticos, ainda que abaixo do limiar utilizado para o diagnóstico de insuficiência cardíaca, reflectem a existência de stress cardiovascular e têm valor potencial como biomarcadores de risco aterotrombótico. No entanto, ainda que estes péptidos sejam potenciais biomarcadores de aterosclerose e possam no futuro constituir uma ferramenta adicional na avaliação do doente em risco, a aplicação prática de tal conhecimento é difícil e mais estudos são necessários para definir as múltiplas questões ainda em aberto.Atherosclerosis is the main responsible for pathogenesis of cardiovascular diseases. It is a systemic entity but clinical manifestations are focal and may affect several arterial territories like cerebral, coronary, visceral and peripheral territories. Theoretically, patients presenting with one atherothrombotic event may have increased risk of developing a new event, either in the same territory or in different arterial beds. How can we descriminate patients risk of having a second atherothrombotic event? Actual knowledge shows that natriuretic peptide type B (BNP) and its terminal fragment (NT-proBNP) are not exclusively biomarkers of cardiac insufficiency but cardiovascular stress biomarkers. This findings are giving new opportunities to studies about clinical importance of this peptides in atherosclerosis. This work aims to be a literature review about the relationship between natriuretic peptide levels and the risk of have adverse cardiovascular events which, in the agreement with recent literature, can be understood as a "U" curve: low levels of NT-proBNP (and BNP) could be influenced by genetic variations that determine a lower production of this peptides and consequently, less beneficial effects of its action in cardiovascular regulation, with increased cardiovascular risk; High levels of natriuretic peptides, even below the threshold used to make the diagnosis of cardiac insufficiency, reflect the existence of cardiovascular stress and have potential value to be atherothrombotic risk biomarkers. Natriuretic peptides are potential biomarkers of atherosclerosis and may constitute in the future an additional tool in risk assessment, but practical application of such knowledge is difficult and further studies are needed to answer some questions who are still open issues

O emprego de computadores na história

Na presente Comunicação, serão focalizados exemplos do emprego de computação eletrônica em Projetos de pesquisa histórica, desenvolvidos no Departamento de História da Universidade Federal do Paraná, e que se vêm utilizando os serviços do computador IBM 1130, e do plotter 1627, do Centro de Computação Eletrônica da Universidade, órgão instalado em setembro de 1969.

Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning

Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis

Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions

BACKGROUND: Mycobacteria inhabit diverse niches and display high metabolic versatility. They can colonise both humans and animals and are also able to survive in the environment. In order to succeed, response to environmental cues via transcriptional regulation is required. In this study we focused on the TetR family of transcriptional regulators (TFTRs) in mycobacteria. RESULTS: We used InterPro to classify the entire complement of transcriptional regulators in 10 mycobacterial species and these analyses showed that TFTRs are the most abundant family of regulators in all species. We identified those TFTRs that are conserved across all species analysed and those that are unique to the pathogens included in the analysis. We examined genomic contexts of 663 of the conserved TFTRs and observed that the majority of TFTRs are separated by 200 bp or less from divergently oriented genes. Analyses of divergent genes indicated that the TFTRs control diverse biochemical functions not limited to efflux pumps. TFTRs typically bind to palindromic motifs and we identified 11 highly significant novel motifs in the upstream regions of divergently oriented TFTRs. The C-terminal ligand binding domain from the TFTR complement in M. tuberculosis showed great diversity in amino acid sequence but with an overall architecture common to other TFTRs. CONCLUSION: This study suggests that mycobacteria depend on TFTRs for the transcriptional control of a number of metabolic functions yet the physiological role of the majority of these regulators remain unknown. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1696-9) contains supplementary material, which is available to authorized users

A novel TetR-like transcriptional regulator is induced in acid-nitrosative stress and controls expression of an efflux pump in mycobacteria

Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its ortholog in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here, we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and used reporter assays to confirm that the MSMEG_3762/63/65 promoter was induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nM). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release

An integrated whole genome analysis of Mycobacterium tuberculosis reveals insights into relationship between its genome, transcriptome and methylome.

Human tuberculosis disease (TB), caused by Mycobacterium tuberculosis (Mtb), is a complex disease, with a spectrum of outcomes. Genomic, transcriptomic and methylation studies have revealed differences between Mtb lineages, likely to impact on transmission, virulence and drug resistance. However, so far no studies have integrated sequence-based genomic, transcriptomic and methylation characterisation across a common set of samples, which is critical to understand how DNA sequence and methylation affect RNA expression and, ultimately, Mtb pathogenesis. Here we perform such an integrated analysis across 22 M. tuberculosis clinical isolates, representing ancient (lineage 1) and modern (lineages 2 and 4) strains. The results confirm the presence of lineage-specific differential gene expression, linked to specific SNP-based expression quantitative trait loci: with 10 eQTLs involving SNPs in promoter regions or transcriptional start sites; and 12 involving potential functional impairment of transcriptional regulators. Methylation status was also found to have a role in transcription, with evidence of differential expression in 50 genes across lineage 4 samples. Lack of methylation was associated with three novel variants in mamA, likely to cause loss of function of this enzyme. Overall, our work shows the relationship of DNA sequence and methylation to RNA expression, and differences between ancient and modern lineages. Further studies are needed to verify the functional consequences of the identified mechanisms of gene expression regulation