Integrating data from heterogeneous DNA microarray platforms

DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus.

Systems biology approaches for the design of novel Saccharomyces cerevisiae winemaking strains for enhanced flavour compounds synthesis

Tese de Doutoramento em Biologia Ambiental e Molecular

HOXA9 as a key regulator in glioma initiation, aggressiveness and response to therapy

Tese de Doutoramento em Ciências da Saúde

Changes in reproductive roles are associated with changes in gene expression in fire ant queens.

Abstract In species with social hierarchies, the death of dominant individuals typically upheaves the social hierarchy and provides an opportunity for subordinate individuals to become reproductives. Such a phenomenon occurs in the monogyne form of the fire ant, Solenopsis invicta, where colonies typically contain a single wingless reproductive queen, thousands of workers and hundreds of winged nonreproductive virgin queens. Upon the death of the mother queen, many virgin queens shed their wings and initiate reproductive development instead of departing on a mating flight. Workers progressively execute almost all of them over the following weeks. To identify the molecular changes that occur in virgin queens as they perceive the loss of their mother queen and begin to compete for reproductive dominance, we collected virgin queens before the loss of their mother queen, 6 h after orphaning and 24 h after orphaning. Their RNA was extracted and hybridized against microarrays to examine the expression levels of approximately 10 000 genes. We identified 297 genes that were consistently differentially expressed after orphaning. These include genes that are putatively involved in the signalling and onset of reproductive development, as well as genes underlying major physiological changes in the young queens.

Routine use of point-of-care tests: usefulness and application in clinical microbiology.

Point-of-care (POC) tests offer potentially substantial benefits for the management of infectious diseases, mainly by shortening the time to result and by making the test available at the bedside or at remote care centres. Commercial POC tests are already widely available for the diagnosis of bacterial and viral infections and for parasitic diseases, including malaria. Infectious diseases specialists and clinical microbiologists should be aware of the indications and limitations of each rapid test, so that they can use them appropriately and correctly interpret their results. The clinical applications and performance of the most relevant and commonly used POC tests are reviewed. Some of these tests exhibit insufficient sensitivity, and should therefore be coupled to confirmatory tests when the results are negative (e.g. Streptococcus pyogenes rapid antigen detection test), whereas the results of others need to be confirmed when positive (e.g. malaria). New molecular-based tests exhibit better sensitivity and specificity than former immunochromatographic assays (e.g. Streptococcus agalactiae detection). In the coming years, further evolution of POC tests may lead to new diagnostic approaches, such as panel testing, targeting not just a single pathogen, but all possible agents suspected in a specific clinical setting. To reach this goal, the development of serology-based and/or molecular-based microarrays/multiplexed tests will be needed. The availability of modern technology and new microfluidic devices will provide clinical microbiologists with the opportunity to be back at the bedside, proposing a large variety of POC tests that will allow quicker diagnosis and improved patient care.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Transmission disequilibrium of small CNVs in simplex autism.

We searched for disruptive, genic rare copy-number variants (CNVs) among 411 families affected by sporadic autism spectrum disorder (ASD) from the Simons Simplex Collection by using available exome sequence data and CoNIFER (Copy Number Inference from Exome Reads). Compared to high-density SNP microarrays, our approach yielded ∼2× more smaller genic rare CNVs. We found that affected probands inherited more CNVs than did their siblings (453 versus 394, p = 0.004; odds ratio [OR] = 1.19) and that the probands' CNVs affected more genes (921 versus 726, p = 0.02; OR = 1.30). These smaller CNVs (median size 18 kb) were transmitted preferentially from the mother (136 maternal versus 100 paternal, p = 0.02), although this bias occurred irrespective of affected status. The excess burden of inherited CNVs among probands was driven primarily by sibling pairs with discordant social-behavior phenotypes (p < 0.0002, measured by Social Responsiveness Scale [SRS] score), which contrasts with families where the phenotypes were more closely matched or less extreme (p > 0.5). Finally, we found enrichment of brain-expressed genes unique to probands, especially in the SRS-discordant group (p = 0.0035). In a combined model, our inherited CNVs, de novo CNVs, and de novo single-nucleotide variants all independently contributed to the risk of autism (p < 0.05). Taken together, these results suggest that small transmitted rare CNVs play a role in the etiology of simplex autism. Importantly, the small size of these variants aids in the identification of specific genes as additional risk factors associated with ASD.

Circulating microRNAs as long-term biomarkers for the detection of erythropoiesis-stimulating agent abuse.

MicroRNAs (miRNAs) are small, non-protein coding transcripts involved in many cellular and physiological mechanisms. Recently, a new class of miRNA called 'circulating miRNAs' was found in cell-free body fluids such as plasma and urine. Circulating miRNAs have been shown to be very stable, specific, and sensitive biomarkers. In this paper, we investigate whether circulating miRNAs can serve as biomarkers for erythropoiesis-stimulating agent abuse. To this end, we analyzed miRNA levels in plasma by miRNA microarrays and quantitative real-time polymerase chain reaction (PCR). Plasma samples are derived from a clinical study with healthy subjects injected with erythropoiesis-stimulating agent (C.E.R.A.). Based on microarray results, we observed a significant difference in the levels of miRNAs in plasma after C.E.R.A. injection. We demonstrated that a specific miRNA, miR-144, exhibit a high increase that lasts 27 days after C.E.R.A. stimulation. Considering the fact that miR-144 is an essential erythropoiesis agent in different organisms, these findings suggest the possibility of using miR-144 as a sensitive and informative biomarker to detect C.E.R.A. abuse. Copyright © 2011 John Wiley & Sons, Ltd.

Obtenció de jerarquies d'estats cel·lulars via web

Hi ha diversos mètodes d'anàlisi que duen a terme una agrupació global de la sèries de mostres de microarrays, com SelfOrganizing Maps, o que realitzen agrupaments locals tenint en compte només un subconjunt de gens coexpressats, com Biclustering, entre d'altres. En aquest projecte s'ha desenvolupat una aplicació web: el PCOPSamplecl, és una eina que pertany als mètodes d'agrupació (clustering) local, que no busca subconjunts de gens coexpresats (anàlisi de relacions linials), si no parelles de gens que davant canvis fenotípics, la seva relació d'expressió pateix fluctuacions. El resultats del PCOPSamplecl seràn les diferents distribucions finals de clusters i les parelles de gens involucrades en aquests canvis fenotípics. Aquestes parelles de gens podràn ser estudiades per trobar la causa i efecte del canvi fenotípic. A més, l'eina facilita l'estudi de les dependències entre les diferents distribucions de clusters que proporciona l'aplicació per poder estudiar la intersecció entre clusters o l'aparició de subclusters (2 clusters d'una mateixa agrupació de clusters poden ser subclusters d'altres clusters de diferents distribucions de clusters). L'eina és disponible al servidor: http://revolutionresearch.uab.es/

Bioinformática: interfaz web para estudiar el efecto de diferentes condiciones sobre la expresión de los genes

El trabajo realizado se divide en dos bloques bien diferenciados, ambos relacionados con el análisis de microarrays. El primer bloque consiste en agrupar las condiciones muestrales de todos los genes en grupos o clústers. Estas agrupaciones se obtienen al aplicar directamente sobre la microarray los siguientes algoritmos de agrupación: SOM,PAM,SOTA,HC y al aplicar sobre la microarray escalada con PC y MDS los siguientes algoritmos: SOM,PAM,SOTA,HC y K-MEANS. El segundo bloque consiste en realizar una búsqueda de genes basada en los intervalos de confianza de cada clúster de la agrupación activa. Las condiciones de búsqueda ajustadas por el usuario se validan para cada clúster respecto el valor basal 0 y respecto el resto de clústers, para estas validaciones se usan los intervalos de confianza. Estos dos bloques se integran en una aplicación web ya existente, el applet PCOPGene, alojada en el servidor: http://revolutionresearch.uab.es.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Desenvolupament d'eines d'alta ressolució per al cribatge (screening) de l'activitat de fàrmacs

Projecte de recerca elaborat a partir d’una estada a la Satandford University, EEUU, entre 2007 i 2009. Els darrers anys, hi ha hagut un avanç espectacular en la tecnologia aplicada a l’anàlisi del genoma i del proteoma (microarrays, PCR quantitativa real time, electroforesis dos dimensions, espectroscòpia de masses, etc.) permetent la resolució de mostres complexes i la detecció quantitativa de diferents gens i proteïnes en un sol experiment. A més a més, la seva importància radica en la capacitat d’identificar potencials dianes terapèutiques i possibles fàrmacs, així com la seva aplicació en el disseny i desenvolupament de noves eines de diagnòstic. L’aplicabilitat de les tècniques actuals, però, està limitada al nivell al que el teixit pot ser disseccionat. Si bé donen valuosa informació sobre expressió de gens i proteïnes implicades en una malaltia o en resposta a un fàrmac per exemple, en cap cas, s’obté una informació in situ ni es pot obtenir informació espacial o una resolució temporal, així com tampoc s’obté informació de sistemes in vivo. L’objectiu d’aquest projecte és desenvolupar i validar un nou microscopi, d’alta resolució, ultrasensible i de fàcil ús, que permeti tant la detecció de metabòlits, gens o proteïnes a la cèl•lula viva en temps real com l’estudi de la seva funció. Obtenint així una descripció detallada de les interaccions entre proteïnes/gens que es donen dins la cèl•lula. Aquest microscopi serà un instrument sensible, selectiu, ràpid, robust, automatitzat i de cost moderat que realitzarà processos de cribatge d’alt rendiment (High throughput screening) genètics, mèdics, químics i farmacèutics (per aplicacions diagnòstiques i de identificació i selecció de compostos actius) de manera més eficient. Per poder realitzar aquest objectius el microscopi farà ús de les més noves tecnologies: 1)la microscopia òptica i d’imatge, per millorar la visualització espaial i la sensibilitat de l’imatge; 2) la utilització de nous mètodes de detecció incloent els més moderns avanços en nanopartícules; 3) la creació de mètodes informàtics per adquirir, emmagatzemar i processar les imatges obtingudes.

MIMAS 3.0 is a Multiomics Information Management and Annotation System.

BACKGROUND: DNA sequence integrity, mRNA concentrations and protein-DNA interactions have been subject to genome-wide analyses based on microarrays with ever increasing efficiency and reliability over the past fifteen years. However, very recently novel technologies for Ultra High-Throughput DNA Sequencing (UHTS) have been harnessed to study these phenomena with unprecedented precision. As a consequence, the extensive bioinformatics environment available for array data management, analysis, interpretation and publication must be extended to include these novel sequencing data types. DESCRIPTION: MIMAS was originally conceived as a simple, convenient and local Microarray Information Management and Annotation System focused on GeneChips for expression profiling studies. MIMAS 3.0 enables users to manage data from high-density oligonucleotide SNP Chips, expression arrays (both 3'UTR and tiling) and promoter arrays, BeadArrays as well as UHTS data using MIAME-compliant standardized vocabulary. Importantly, researchers can export data in MAGE-TAB format and upload them to the EBI's ArrayExpress certified data repository using a one-step procedure. CONCLUSION: We have vastly extended the capability of the system such that it processes the data output of six types of GeneChips (Affymetrix), two different BeadArrays for mRNA and miRNA (Illumina) and the Genome Analyzer (a popular Ultra-High Throughput DNA Sequencer, Illumina), without compromising on its flexibility and user-friendliness. MIMAS, appropriately renamed into Multiomics Information Management and Annotation System, is currently used by scientists working in approximately 50 academic laboratories and genomics platforms in Switzerland and France. MIMAS 3.0 is freely available via http://multiomics.sourceforge.net/.

Interfaz web para mostrar los genes según las relaciones que mantienen sus expresiones

La investigació entre les relacions dels nivells d’expressió dels gens aporta molta informació sobre els processos biològics i patològics. Mitjançant la tècnica de les microarrays es possibilita la investigació de les relacions d’expressió de milers de gens a la vegada. La finalitat d’aquest projecte es fent ús de l’aplicatiu web PCOPGene-Net, permetre la identificació dels gens per les relacions d’expressió no lineals que tenen amb la resta de gens i permetre també la identificació de les relacions d’expressió no lineals entre els gens d’una microarray.