Analysis of the relationship between transcription and alternative splicing of eukaryotic mRNAs

En el presente trabajo de tesis nos hemos propuesto buscar evidencias sobre la coordinación entre los procesos de transcripción y splicing alternativo. Nuestro modelo de estudio consistió en construir una batería de minigenes α-globina/ fibronectina que incluyen al exón EDI de la fibronectina capaz de sufrir splicing alternativo, clonados río abajo de diferentes promotores. Con estos minigenes se transfectaron líneas celulares en cultivo, analizándose luego el patrón de splicing alternativo por medio de las técnicas de Northern blot y/o RTPCR. Encontramos así que el tipo de promotor determina el patrón de splicing alternativo. Este efecto del promotor sobre el splicing alternativo no depende de la fuerza del promotor ni de la secuencia de la región 5' no codificante de los mRNAs, sino de la calidad de factores de transcripción reclutados en el promotor. Posteriormente intentamos dilucidar el mecanismo subyacente a este fenómeno. Para ello sobreexpresamos proteínas que participan del splicing general y/o alternativo en otros modelos. Encontramos que sólo las proteínas SF2/ASF, SRp40, SRp55 y SC-35 son capaces de modificar el patrón de inclusión de EDI, mientras que las proteínas SRp20, SRp30c y Tra2βno tienen efecto alguno. El efecto de SF2/ASF, la proteína con mayor actividad estimulatoria, es específico de promotor y opera a través de las secuencias regulatorias en cis presentes en EDI, tal como lo demuestran los experimentos con minigenes mutantes en estas regiones. Los resultados mencionados constituyen la primera evidencia de coordinación entre transcripción y splicing alternativo. A partir de los mismos proponemos un modelo de interacción entre el dominio carboxiterminal (CTD ) de la RNA polimerasa II y los factores de splicing, compatible con los resultados observados en nuestro laboratorio y en otros grupos de investigación.In this work we searched for evidence of coupling between transcription and alternative splicing. Our model consisted in the construction of a series of alphaglobin/ fibronectin minigenes including the fibronectin alternative ED1 exon, cloned downstream of different promoters. Cell lines were transfected with the minigenes and the alternative splicing pattern was analyzed either by Northern blot and/or RT-PCR. We found that the type of promoter controls the pattern of alternative splicing. This effect is neither dependent on promoter strength nor on the 5' non-coding region of the mRNAs, but on the quality of the transcription factors recruited by the promoter. We then tried to elucidate the mechanism underlying this phenomenon. For this purpose, we overexpressed splicing factors involved in general and/or alternative splicing in other models. We found that SF2/ASF, SRp40, SRp55 y SC-35 were the only the splicing factors with ability to stimulate ED1 inclusion, whereas SRp20, SRp30c y Tra2β had no effect. The effect yielded by SF2/ASF, the protein with the highest stimulation activity, is promoter specific and is mediated by exon regulatory sequences, as revealed by experiments performed with mutants in these regions. The mentioned results comprise the first evidence of coupling between transcription and alternative splicing. Based on our results and evidences presented by other laboratories, we propose a model in which the carboxyterminal domain (CTD) of the RNA polymerase II interacts with the splicing factors.Fil:Cramer, Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Impacts of urbanization around Mediterranean cities : changes in ecosystem service supply

Unidad de excelencia María de Maeztu MdM-2015-0552Urbanization is an important driver of changes in land cover in the Mediterranean Basin and it is likely to impact the supply and demand of ecosystem services (ES). The most significant land cover changes occur in the peri-urban zone, but little is known about how these changes affect the ES supply. For eight European and four North African cities, we have quantified changes in peri-urban land cover, for periods of sixteen years (1990-2006) in the Northern African, and twenty-two years (1990-2012) in the European cities, respectively. Using an expert-based method, we derived quantitative estimates of the dynamics in the supply of twenty-seven ES. The nature of land cover changes slightly differed between European and North African Mediterranean cities, but overall it increased in urban areas and decreased in agricultural land. The capacity of the peri-urban areas of Mediterranean cities to supply ES generally reduced over the last 20-30 years. For nine ES the potential supply actually increased for all four North African cities and three out of the eight European cities. Across all cities, the ES timber, wood fuel and religious and spiritual experience increased. Given the expected increase of urban population in the Mediterranean Basin and the current knowledge of ES deficits in urban areas, the overall decrease in ES supply capacity of peri-urban areas is a risk for human well-being in the Mediterranean and poses a serious challenge for the Sustainable Development Goals in the Mediterranean basin

A inserção do feminino no universo masculino: representações da educação superior

Objetivo deste trabalho consistiu em desvendar as representações das relações de gênero socialmente construídas no espaço organizacional de uma universidade pública especializada em ciências agrárias. Para tanto, tomou-se como referência a teoria das representações sociais e as contribuições pós-estruturalistas sobre rela- ções de gênero. Por meio de uma perspectiva metodológica qualitativa, obteve-se acesso, mediante a análise dos discursos das professoras e professores entrevistados, à construção ou desconstrução das relações vigentes no contexto histórico - social - cultural e psicológico. Essas relações foram distribuídas em categorias como: a relação família-trabalho; a profissionalização como marco de referência na diferenciação de gênero; gênero e magistério superior; e relações de gênero, desempenho no trabalho e ascensão na carreira, possibilitando inferir que nessas instâncias ainda existem assimetrias, apesar do discurso de igualdade promulgado pelos sujeitos organizacionais

Impact of land cover change on ecosystem service supply in mountain systems: a case study in the Cantabrian Mountains (NW of Spain)

P. 529–542Land abandonment and the loss of traditional farming practices are thought to control land cover dynamics, and hence the ecosystem service supply in traditionally managed mountain landscapes. We evaluate the impact of land cover changes in Cantabrian Mountains (NW Spain), over 1990–2012, on the potential supply capacity of ecosystem services (regulating, provisioning, and cultural) at both regional and local scales. We also analyze trends in the use of ecosystem services at the local scale. Land cover changes were estimated from CORINE Land Cover database. Patterns of potential ecosystem service supply were assessed by applying an ecosystem service supply capacity matrix and trends in their actual use by using field data. Main trajectories of land cover change encompassed woody vegetation spread in semi-natural open systems and agricultural expansion in the most suitable areas. The capacity of landscape to provide ecosystem services improved during 1990–2012 at both scales. We detected trade-offs between the potential supply of ecosystem services associated to natural systems and those linked to traditional land uses, at both regional and local scales. Changes in the potential supply of ecosystem services matched trends in ecosystem service use. This study could help develop future scenarios to address upcoming challenges in ecosystem service supplyS

Orientação à saúde dos viajantes : o papel das agências de viagens em Porto Alegre

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Final Results of a Randomized, Phase III Study of Rituximab With or Without Idelalisib Followed by Open-Label Idelalisib in Patients With Relapsed Chronic Lymphocytic Leukemia

PURPOSE A randomized, double-blind, phase III study of idelalisib (IDELA) plus rituximab versus placebo plus rituximab in patients with relapsed chronic lymphocytic leukemia (CLL) was terminated early because of superior efficacy of the IDELA-plus-rituximab (IDELA/R) arm. Patients in either arm could then enroll in an extension study to receive IDELA monotherapy. Here, we report the long-term efficacy and safety data for IDELA-treated patients across the primary and extension studies. PATIENTS AND METHODS Patients were randomly assigned to receive rituximab in combination with either IDELA 150 mg twice daily (IDELA/R; n = 110) or placebo (placebo/R; n = 110). Key end points were progression-free survival (PFS), overall response rate (ORR), overall survival (OS), and safety. RESULTS The long-term efficacy and safety of treatment with IDELA was assessed in 110 patients who received at least one dose of IDELA in the primary study, 75 of whom enrolled in the extension study. The IDELA/R-to-IDELA group had a median PFS of 20.3 months (95% CI, 17.3 to 26.3 months) after a median follow-up time of 18 months (range, 0.3 to 67.6 months). The ORR was 85.5% (94 of 110 patients; n = 1 complete response). The median OS was 40.6 months (95% CI, 28.5 to 57.3 months) and 34.6 months (95% CI, 16.0 months to not reached) for patients randomly assigned to the IDELA/R and placebo/R groups, respectively. Prolonged exposure to IDELA increased the incidence of all-grade, grade 2, and grade 3 or greater diarrhea (46.4%, 17.3%, and 16.4%, respectively), all-grade and grade 3 or greater colitis (10.9% and 8.2%, respectively) and all-grade and grade 3 or greater pneumonitis (10.0% and 6.4%, respectively) but did not increase the incidence of elevated hepatic aminotransferases. CONCLUSION IDELA improved PFS and OS compared with rituximab alone in patients with relapsed CLL. Long-term IDELA was effective and had an expected safety profile. No new IDELA-related adverse events were identified with longer exposure

Interplay between TCR Affinity and Necessity of Coreceptor Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack of CD8 Engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement

The Structure of an RNAi Polymerase Links RNA Silencing and Transcription

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-Å resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world

Consensus recommendations for a standardized Brain Tumor Imaging Protocol in clinical trials

A recent joint meeting was held on January 30, 2014, with the US Food and Drug Administration (FDA), National Cancer Institute (NCI), clinical scientists, imaging experts, pharmaceutical and biotech companies, clinical trials cooperative groups, and patient advocate groups to discuss imaging endpoints for clinical trials in glioblastoma. This workshop developed a set of priorities and action items including the creation of a standardized MRI protocol for multicenter studies. The current document outlines consensus recommendations for a standardized Brain Tumor Imaging Protocol (BTIP), along with the scientific and practical justifications for these recommendations, resulting from a series of discussions between various experts involved in aspects of neuro-oncology neuroimaging for clinical trials. The minimum recommended sequences include: (i) parameter-matched precontrast and postcontrast inversion recovery-prepared, isotropic 3D T1-weighted gradient-recalled echo; (ii) axial 2D T2-weighted turbo spin-echo acquired after contrast injection and before postcontrast 3D T1-weighted images to control timing of images after contrast administration; (iii) precontrast, axial 2D T2-weighted fluid-attenuated inversion recovery; and (iv) precontrast, axial 2D, 3-directional diffusion-weighted images. Recommended ranges of sequence parameters are provided for both 1.5 T and 3 T MR system