Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Solid-state Marx based two-switch voltage modulator for the On-Line Isotope Mass Separator accelerator at the European Organization for Nuclear Research

A new circuit topology is proposed to replace the actual pulse transformer and thyratron based resonant modulator that supplies the 60 kV target potential for the ion acceleration of the On-Line Isotope Mass Separator accelerator, the stability of which is critical for the mass resolution downstream separator, at the European Organization for Nuclear Research. The improved modulator uses two solid-state switches working together, each one based on the Marx generator concept, operating as series and parallel switches, reducing the stress on the series stacked semiconductors, and also as auxiliary pulse generator in order to fulfill the target requirements. Preliminary results of a 10 kV prototype, using 1200 V insulated gate bipolar transistors and capacitors in the solid-state Marx circuits, ten stages each, with an electrical equivalent circuit of the target, are presented, demonstrating both the improved voltage stability and pulse flexibility potential wanted for this new modulator.

Repetitive Solid State Pulse Modulator Based on a DC Voltage Multiplier

A newly developed solid-state repetitive high-voltage (HV) pulse modulator topology created from the mature concept of the d.c. voltage multiplier (VM) is described. The proposed circuit is based in a voltage multiplier type circuit, where a number of d.c. capacitors share a common connection with different voltage rating in each one. Hence, besides the standard VM rectifier and coupling diodes, two solid-state on/off switches are used, in each stage, to switch from the typical charging VM mode to a pulse mode with the d.c. capacitors connected in series with the load. Due to the on/off semiconductor configuration, in half-bridge structures, the maximum voltage blocked by each one is the d.c. capacitor voltage in each stage. A 2 kV prototype is described and the results are compared with PSPICE simulations.

Levantamento das metodologias (ensaios e medições acústicas) utilizadas por entidades acreditadas: estado da arte

O ruído é, por definição, um som desagradável ou indesejável que perturba o ambiente, contribuindo para o mal-estar físico e psíquico, podendo pôr em causa a saúde do ser humano. As situações de exposição nos espaços de repouso, lazer e trabalho a valores sonoros elevados, especialmente nos meios urbanos e suburbanos, têm-se multiplicado. Por outro lado, o desenvolvimento cultural dos cidadãos, cada vez mais cientes do direito à qualidade de vida, tem originado um aumento das exigências de conforto, influenciado directamente pela qualidade dos edifícios que habitam. Através da publicação do Regulamento Geral Sobre o Ruído – RGR – (aprovado pelo Decreto Lei nº251/87 de 24 de Junho) bem como o Regulamento dos Requisitos Acústicos dos Edifícios – RRAE - (aprovado pelo Decreto Lei nº129/02 de 11 de Maio), com as devidas alterações no Decreto-lei nº96/08, de 9 de Junho, foi permitido relacionar um conjunto de disposições normativas e legais, permitindo o estabelecimento de condições para a verificação das exigências fundamentais associadas ao conforto acústico dos edifícios (integração urbanística, isolamento sonoro a sons aéreos, isolamento a sons de percussão, exposição ao ruído durante o trabalho), justificando assim a necessidade da existência de regulamentação pelo facto da saúde dos indivíduos também se encontrar relacionada ao conforto acústico. Na presente dissertação, pretende-se dar um contributo para a elaboração da avaliação e certificação do comportamento acústico de edifícios de habitação através da definição dos principais aspectos a avaliar neste processo. Para tal, foi retratado o actual estado da arte e foi feito um levantamento das metodologias utilizadas pelas entidades acreditadas para os ensaios de acústica, tendo em conta a normalização e regulamentação existente, de maneira a obter o panorama destes diagnósticos de conforto acústico para a verificação da sua conformidade com as exigências regulamentares.

Solid-State Bipolar Marx Generator with Voltage Droop Compensation

This paper addresses the voltage droop compensation associated with long pulses generated by solid-stated based high-voltage Marx topologies. In particular a novel design scheme for voltage droop compensation in solid-state based bipolar Marx generators, using low-cost circuitry design and control, is described. The compensation consists of adding one auxiliary PWM stage to the existing Marx stages, without changing the modularity and topology of the circuit, which controls the output voltage and a LC filter that smoothes the voltage droop in both the positive and negative output pulses. Simulation results are presented for 5 stages Marx circuit using 1 kV per stage, with 1 kHz repetition rate and 10% duty cycle.

Solid-State Bipolar Marx Converter with Output Transformer and Energy Recovery

The purpose of this paper is to present and discuss a general HV topology of the solid-state Marx modulator, for unipolar or bipolar generation connected with a step-up transformer to increase the output voltage applied to a resistive load. Due to the use of an output transformer, discussion about the reset of the transformer is made to guarantee zero average voltage applied to the primary. It is also discussed the transformer magnetizing energy recovering back to the energy storage capacitors. Simulation results for a circuit that generates 100 kV pulses using 1000 V semiconductors are presented and discussed regarding the voltage and current stress on the semiconductors and result obtained.

IOPT Petri Net State Space Generation Algorithm with Maximal-Step Execution Semantics

This paper presents an algorithm to efficiently generate the state-space of systems specified using the IOPT Petri-net modeling formalism. IOPT nets are a non-autonomous Petri-net class, based on Place-Transition nets with an extended set of features designed to allow the rapid prototyping and synthesis of system controllers through an existing hardware-software co-design framework. To obtain coherent and deterministic operation, IOPT nets use a maximal-step execution semantics where, in a single execution step, all enabled transitions will fire simultaneously. This fact increases the resulting state-space complexity and can cause an arc "explosion" effect. Real-world applications, with several million states, will reach a higher order of magnitude number of arcs, leading to the need for high performance state-space generator algorithms. The proposed algorithm applies a compilation approach to read a PNML file containing one IOPT model and automatically generate an optimized C program to calculate the corresponding state-space.

Magnetized color flavor locked state and compact stars

The stability of the color flavor locked phase in the presence of a strong magnetic field is investigated within the phenomenological MIT bag model, taking into account the variation of the strange quark mass, the baryon density, the magnetic field, as well as the bag and gap parameters. It is found that the minimum value of the energy per baryon in a color flavor locked state at vanishing pressure is lower than the corresponding one for unpaired magnetized strange quark matter and, as the magnetic field increases, the energy per baryon decreases. This implies that magnetized colorflavor locked matter is more stable and could become the ground state inside neutron stars. The mass-radius relation for such stars is also studied.

Solid State Marx Modulator with Blumlein Stack for Bipolar Pulse Generation

Sub-nanosecond bipolar high voltage pulses are a very important tool for food processing, medical treatment, waste water and exhaust gas processing. A Hybrid Modulator for sub-microsecond bipolar pulse generation, comprising an unipolar solid-state Marx generator connected to a load through a stack Blumlein system that produces bipolar pulses and further multiplies the pulse voltage amplitude, is presented. Experimental results from an assembled prototype show the generation of 1000 V amplitude bipolar pulses with 100 ns of pulse width and 1 kHz repetition rate.

Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were aurprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Protection against oxidative stress through SUA7/TFIIB regulation in Saccharomyces cerevisiae

The general transcription factor TFIIB, encoded by SUA7 in Saccharomyces cerevisiae, is required for transcription activation but apparently of a specific subset of genes, for example, linked with mitochondrial activity and hence with oxidative environments. Therefore, studying SUA7/TFIIB as a potential target of oxidative stress is fundamental. We found that controlled SUA7 expression under oxidative conditions occurs at transcriptional and mRNA stability levels. Both regulatory events are associated with the transcription activator Yap1 in distinct ways: Yap1 affects SUA7 transcription up regulation in exponentially growing cells facing oxidative signals; the absence of this activator per se contributes to increase SUA7 mRNA stability. However, unlike SUA7 mRNA, TFIIB abundance is not altered on oxidative signals. The biological impact of this preferential regulation of SUA7 mRNA pool is revealed by the partial suppression of cellular oxidative sensitivity by SUA7 overexpression, and supported by the insights on the existence of a novel RNA-binding factor, acting as an oxidative sensor, which regulates mRNA stability. Taken together the results point out a primarily cellular commitment to guarantee SUA7 mRNA levels under oxidative environments.

Cellular polarity in aging: role of redox regulation and nutrition

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.

Gene expression regulation and lineage evolution: the North and South tale of the hybrid polyploid Squalius alburnoides complex

The evolution of hybrid polyploid vertebrates, their viability and their perpetuation over evolutionary time have always been questions of great interest. However, little is known about the impact of hybridization and polyploidization on the regulatory networks that guarantee the appropriate quantitative and qualitative gene expression programme. The Squalius alburnoides complex of hybrid fish is an attractive system to address these questions, as it includes a wide variety of diploid and polyploid forms, and intricate systems of genetic exchange. Through the study of genome-specific allele expression of seven housekeeping and tissue-specific genes, we found that a gene copy silencing mechanism of dosage compensation exists throughout the distribution range of the complex. Here we show that the allele-specific patterns of silencing vary within the complex, according to the geographical origin and the type of genome involved in the hybridization process. In southern populations, triploids of S. alburnoides show an overall tendency for silencing the allele from the minority genome, while northern population polyploids exhibit preferential biallelic gene expression patterns, irrespective of genomic composition. The present findings further suggest that gene copy silencing and variable expression of specific allele combinations may be important processes in vertebrate polyploid evolution.

Materiais de mudança de fase em revestimentos

Dissertação para obtenção do grau de Mestre em Engenharia Civil na Área de especialização em Edificações

Characterization of a polyubiquitin gene in T. thermophila and of ubiquitin gene expression during sexual reproduction and under stress conditions

A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.