Browsing by Author "Fernandes, Lisete"
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- Absence of Gim proteins, but not GimC complex, alters stress-induced transcriptionPublication . Amorim, Ana Fátima; Pinto, Dora; Kuras, Laurent; Fernandes, LiseteSaccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.
- Adaptive mutations in bacteria: high rate and small effectsPublication . Perfeito, Lília; Fernandes, Lisete; Mota, Catarina; Gordo, IsabelEvolution by natural selection is driven by the continuous generation of adaptive mutations. We measured the genomic mutation rate that generates beneficial mutations and their effects on fitness in Escherichia coli under conditions in which the effect of competition between lineages carrying different beneficial mutations is minimized. We found a rate on the order of 10–5 per genome per generation, which is 1000 times as high as previous estimates, and a mean selective advantage of 1%. Such a high rate of adaptive evolution has implications for the evolution of antibiotic resistance and pathogenicity.
- Alternative regimens for prostate cancer treatment using radiation therapyPublication . Oliveira, S. M.; Teixeira, Nuno; Fernandes, Lisete; Brás, S.; Serra, F.Purpose/Objective: The purpose of this work was to determine biologically equivalent alternative regimens for the treatment of prostate cancer using External Beam Radiotherapy (EBRT) and Low Dose-Rate Brachytherapy (LDRBT) with 125I implants and to evaluate the sensitivity of these regimens to different sets of radiobiological parameters of the Linear-Quadratic (LQ) model.
- Alternative regimens for treating prostate cancer using equivalent uniform dose and Monte Carlo methodsPublication . Oliveira, Susana; Rosmanets, Yuriy; Teles, Pedro; Fernandes, Lisete; Teixeira, Nuno; Vaz, PedroIntroduction - Conventional radiotherapy treatments are administrated with 2 Gy external beam radiotherapy (EBRT) fractions. It has been postulated that prostate cancer would respond to radiotherapy as a slowly proliferating late-responding normal tissue, benefiting from hypo-fractionated regimens. Highly conformed brachytherapy is a treatment option either alone or combined with EBRT. Purpose - To identify alternative radiotherapy regimens for treating prostate cancer using EBRT and low dose-rate brachytherapy (LDRBT) with 125I implants, biologically equivalent to conventional treatments in terms of uniform equivalent dose (EUD). Materials and methods - The EUD concept was used, together with Monte Carlo (MC) methods. Two voxel phantoms were segmented from the computed tomography of patients to obtain the energy deposition derived from the MC simulations of EBRT and LDRBT treatments on a voxel-by-voxel basis. The energy deposition was converted in EUD. Equivalent regimens to EUDs of 72 Gy, 80 Gy, 90 Gy, and 100 Gy were determined for increasing fractions of 1.8–5.0 Gy and amounts of LDRBT from 0 Gy (EBRT exclusive) to 145 Gy. The resulting EUD for rectum was also evaluated. Results - Alternative schemes equivalent, in terms of EUD, were obtained. For example, it is equivalent to an EUD of 72 Gy, 38 × 2 Gy, 20 × 3 Gy or 9 × 5 Gy of EBRT, or 6 × 5 Gy of EBRT plus 50 Gy of LDRBT. The rectum benefits of higher amounts of LDRBT for EBRT fractionations <2.5 Gy and larger fractions for LDRBT dose <50 Gy. Conclusion - Alternative regimens for the treatment of prostate cancer with EBRT and LDRBT are proposed. The rational for the use of brachytherapy becomes less relevant with the increasing therapeutic ratio achieved with hypo-fractionated EBRT.
- Dosimetric effect of tissue heterogeneity for 125I prostate implantsPublication . Oliveira, Susana Maria; Teixeira, Nuno; Fernandes, Lisete; Teles, Pedro; Vaz, PedroAim - To use Monte Carlo (MC) together with voxel phantoms to analyze the tissue heterogeneity effect in the dose distributions and equivalent uniform dose (EUD) for (125)I prostate implants. Background - Dose distribution calculations in low dose-rate brachytherapy are based on the dose deposition around a single source in a water phantom. This formalism does not take into account tissue heterogeneities, interseed attenuation, or finite patient dimensions effects. Tissue composition is especially important due to the photoelectric effect. Materials and Methods - The computed tomographies (CT) of two patients with prostate cancer were used to create voxel phantoms for the MC simulations. An elemental composition and density were assigned to each structure. Densities of the prostate, vesicles, rectum and bladder were determined through the CT electronic densities of 100 patients. The same simulations were performed considering the same phantom as pure water. Results were compared via dose-volume histograms and EUD for the prostate and rectum. Results - The mean absorbed doses presented deviations of 3.3-4.0% for the prostate and of 2.3-4.9% for the rectum, when comparing calculations in water with calculations in the heterogeneous phantom. In the calculations in water, the prostate D 90 was overestimated by 2.8-3.9% and the rectum D 0.1cc resulted in dose differences of 6-8%. The EUD resulted in an overestimation of 3.5-3.7% for the prostate and of 7.7-8.3% for the rectum. Conclusions - The deposited dose was consistently overestimated for the simulation in water. In order to increase the accuracy in the determination of dose distributions, especially around the rectum, the introduction of the model-based algorithms is recommended.
- Evaluation of coumarins effect in saccharomyces cerevisiaePublication . Pinto, D.; Amorim, A. F.; Kuras, L.; Soares, M. B. P.; Fernandes, LiseteCoumarin and derivates (coumarins) are phenolic compounds widely distributed in the plant kingdom, as for example in tonka beam and cassia cinnamon. These compounds are involved in various processes such as the defense against phytopathogens, the response to abiotic stress and the regulation of oxidative stress. Coumarins can be produced synthetically and are broadly used as additives in the food, perfumes and cosmetics and pharmaceutical industry due th their vast array of biological activities, including anticoagulant, analgesic, anti-inflammatory and anti-microbial.
- Functional validation of LexADBDGim chimeras in saccharomyces cerevisiaePublication . Amorim, A. F.; Pinto, D.; Fernandes, LiseteGimC/Prefoldin is a hetero-oligomeric complex involved in cytoskeleton biogenesis. In order to identify by two-hybrid system targets that directly interact with Gims and support the stress phenotypes, this work aimed the functional validation of all Gims in saccharomyces cerevisiae.
- OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factorsPublication . Serra, Tânia S.; Figueiredo, Duarte D.; Cordeiro, André M.; Almeida, Diego M.; Lourenço, Tiago; Abreu, Isabel A.; Sebastián, Alvaro; Fernandes, Lisete; Contreras-Moreira, Bruno; Oliveira, M. Margarida; Saibo, Nelson J.High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice.
- Protection against oxidative stress through SUA7/TFIIB regulation in Saccharomyces cerevisiaePublication . Paes de Faria, Joana; Fernandes, LiseteThe 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.
- Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phasePublication . Cirne, Luísa; Martins, L.; Fernandes, Lisete; Marinho, H. SusanaGene 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.