Genome wide mining of alternative splicing in metazoan model organisms

Tese de doutoramento, Ciências Biomédicas (Ciências Morfológicas), Universidade de Lisboa, Faculdade de Medicina, 2009Background: Mining current mRNA and EST databases for novel alternatively spliced isoforms is of paramount importance for shedding light on the way in which the maturation of RNA is used to regulate gene expression. Preliminary observations revealed a tendency for greater amounts of potentially non protein-coding alternative transcripts in human genes than in orthologous genes from other organisms. However, many of these isoforms did not appear in recently published alternative splicing databases on account of constraints imposed in the selection of transcripts. This prompted us to develop a less constrained database with the aim of contributing to the identification of the full repertoire of splice variants in the transcriptome of different organisms. Given that mechanisms of control of gene expression involving non-protein-coding splice variants have been described in a variety of genes, this information may be crucial to deciphering more intricate layers of gene regulation in complex organisms brought about by alternative splicing. Description: An algorithm was developed to cluster mRNA and EST BLAT alignments to annotated gene regions. Consensus splice sites were the main requirement imposed on the selection of transcripts. The method was applied to thirteen model organisms. The alternative splicing information generated has been incorporated into a database with clear graphical displays representing the splicing patterns and is available from the ExonMine website (http://www.imm.fm.ul.pt/exonmine). It incorporates information on constitutive exons, poly-A signals, open reading frames and translation, expression specificity of any exon or splicing pattern relative to biological source of mRNA/EST, alternative splicing events and respective exon and junction sequences for microarray probe design. The ExonMine interface also provides several tools to support laboratory validation of splicing patterns. Conclusions: ExonMine detects a higher percentage of spliced genes and isoforms than currently available alternative splicing databases. The analysis reveals a marked increase, in complex organisms, of splice variants with either retained introns or incorporating novel exons with no apparent protein-coding potential. About 18% of unannotated exons detected in ExonMine were found expressed in primary human cells using tiling arrays. Validation of some of these results for the U2AF family of splicing factors was successfully performed in collaboration with members of the lab revealing primate specific transcripts and an alternatively spliced transcript carrying a microRNA. The database was also successfully used for genome wide analysis of sequence elements involved in the regulation of alternative splicing and for custom alternative splicing microarray design. Matching of ExonMine data to a commercial exon microarray platform covering the majority of human exons was also performed and will assist in large-scale analysis of alternative splicing data. The algorithm developed also provides for easy updatability, taking only 48 hours to generate data for the whole human genome and far less time for less complex organisms. In conclusion, ExonMine represents a new useful resource for future research on alternative splicing and gene regulation.Muscular Dystrophy Association (MDA3662), European Commission (LSHG-CT-2005-518238, EURASNET) and Fundação para a Ciência e Tecnologia, Portugal (PTDC/SAU-GMG/69739/2006)

Photophysics of single nitrogen-vacancy centers in diamond nanocrystals

A study of the photophysical properties of nitrogen-vacancy (NV) color centers in diamond nanocrystals of size of 50~nm or below is carried out by means of second-order time-intensity photon correlation and cross-correlation measurements as a function of the excitation power for both pure charge states, neutral and negatively charged, as well as for the photochromic state, where the center switches between both states at any power. A dedicated three-level model implying a shelving level is developed to extract the relevant photophysical parameters coupling all three levels. Our analysis confirms the very existence of the shelving level for the neutral NV center. It is found that it plays a negligible role on the photophysics of this center, whereas it is responsible for an increasing photon bunching behavior of the negative NV center with increasing power. From the photophysical parameters, we infer a quantum efficiency for both centers, showing that it remains close to unity for the neutral center over the entire power range, whereas it drops with increasing power from near unity to approximately 0.5 for the negative center. The photophysics of the photochromic center reveals a rich phenomenology that is to a large extent dominated by that of the negative state, in agreement with the excess charge release of the negative center being much slower than the photon emission process

SyTroN: a virtual classroom for collaborative and distant e-learning systemby teleoperating real devices

Distant E-learning is a main issue nowadays, and it is strongly motivated by social and economical considerations. The increased people mobility and the reduction of educational costs push to develop ad hoc solutions enabling to access to knowledge regardless to geographical situation and economical capabilities. These parameters should not be limits for good training: learning material's pertinence and efficiency have to remain the core of educational activities. In this paper we address the problem through SyTroN: a tele-learning system. This system combines virtual reality and teleoperation techniques to offer an open platform with two main objectives. The first one is to propose intuitive virtual classrooms/desks, including a real teacher supervision and supporting collaborative and individual distant learning. The second goal is to place learners in real conditions with remote connections to real devices allowing distant experimentations. Both goals participate to increase learning impacts and to reduce costs, that is, sharing costly real devices from anywhere at any time. After 5 years of development, our work has been validated by an extensive use at a high engineering school. In situ tests and learning impact studies have been done. They show some advantages and some drawbacks of our global solution

Regulation of bacterial sugar-H+ symport by phosphoenolpyruvate-dependent enzyme I/HPr-mediated phosphorylation

The lactose-H+ symport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein(s) domains of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). C-terminal truncation mutants were constructed and expressed in Escherichia coli and their properties were analyzed. Remarkably, the entire IIA domain (160 amino acids) could be deleted without significant effect on lactose-H+ symport and galactoside equilibrium exchange. Analysis of the LacS mutants in S. thermophilus cells suggested that transport is affected by PTS-mediated phosphorylation of the IIA domain. For further studies, membrane vesicles of S. thermophilus were fused with cytochrome c oxidase-containing liposomes, and, when appropriate, phosphoenolpyruvate (PEP) plus purified enzyme I and heat-stable protein HPr were incorporated into the hybrid membranes. Generation of a protonmotive force (Delta p) in the hybrid membranes resulted in accumulation of lactose, whereas uptake of the PTS sugar sucrose was not observed. With PEP and the energy-coupling proteins enzyme I and HPr of the PTS on the inside, high rates of sucrose uptake were observed, whereas Delta-driven lactose uptake by wild-type LacS was inhibited. This inhibition was not observed with LacS(Delta 160) and LacS(H552R), indicating that PEP-dependent enzyme I/HPr-mediated phosphorylation of the IIA domain (possibly the conserved His-552 residue) modulates lactose-H+ symport activity.</p

Endothelial cell processing and alternatively spliced transcripts of factor VIII: potential implications for coagulation cascades and pulmonary hypertension.

BACKGROUND: Coagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by RT-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab-reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 5 exons within the int22h-1 repeat responsible for intron 22 inversions in 40% of severe haemophiliacs. A reciprocal relationship between the presence of short isoforms and full-length FVIII transcript suggested potential splice-switching mechanisms. CONCLUSIONS/SIGNIFICANCE: The pulmonary endothelium is confirmed as a site of FVIII secretion, with evidence of synthesis, cell surface expression, and coexpression with vWF. There is complex alternate transcription initiation from the FVIII gene. These findings provide a framework for future research on the regulation and perturbation of FVIII synthesis, and of potential relevance to haemophilia, thromboses, and pulmonary hypertensive states

Evaluation of Foliar Insecticides for the Control of Western Bean Cutworm in Field Corn, 2016

The western bean cutworm (WBC) is an important pest of corn and dry beans. In addition to yield loss due to direct feeding on developing kernels in the ear, WBC infestation can also lead to secondary fungal infections. This study was conducted within the historic range of WBC in western Nebraska; however, it has undergone a rapid range expansion into the eastern Corn Belt within the last 16 years. This field trial was established to evaluate the efficacy of a single application of foliar insecticides against this pest to prevent feeding damage to non-Bt corn ears. The trial was located at the University of Nebraska-Lincoln’s Stumpf International Wheat Center in Perkins County, Nebraska, USA (40.856805° N, –101.701594° W). A RCB design with 10 treatments (including an untreated check) and 4 replications was used. Each plot was eight rows by 35 ft. The trial was planted on 13 May using a small plot research planter at 32,000 seeds/acre at an approximate depth of 1.4– 1.75 inch in 30 inch rows. The hybrid planted was DKC62-95 (Monsanto Company, St. Louis, MO) non-Bt with RR2 herbicide tolerance. The plots received irrigation, fertilization, and weed management inputs following standard agronomic practices for the region, with no insecticide applications other than the experimental treatments

Cell sorting in a Petri dish controlled by computer vision.

Fluorescence-activated cell sorting (FACS) applying flow cytometry to separate cells on a molecular basis is a widespread method. We demonstrate that both fluorescent and unlabeled live cells in a Petri dish observed with a microscope can be automatically recognized by computer vision and picked up by a computer-controlled micropipette. This method can be routinely applied as a FACS down to the single cell level with a very high selectivity. Sorting resolution, i.e., the minimum distance between two cells from which one could be selectively removed was 50-70 micrometers. Survival rate with a low number of 3T3 mouse fibroblasts and NE-4C neuroectodermal mouse stem cells was 66 +/- 12% and 88 +/- 16%, respectively. Purity of sorted cultures and rate of survival using NE-4C/NE-GFP-4C co-cultures were 95 +/- 2% and 62 +/- 7%, respectively. Hydrodynamic simulations confirmed the experimental sorting efficiency and a cell damage risk similar to that of normal FACS

Contamination de l’environnement et de la faune par des polluants industriels : les diphényles polychlorés (P.C.B.)

Richou-Bac Lucien, Cumont G., Mollet M. F., Pantaléon Jean. Contamination de l’environnement et de la faune par des polluants industriels : les diphenyles polychlorés (P.C.B.). In: Bulletin de l'Académie Vétérinaire de France tome 125 n°6, 1972. pp. 293-303